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"Mareks disease MD is a chicken neoplastic disease which brings huge economic losses to theglobal poultry industry The wild type p53 a tumor suppressor gene plays a key role in blocking cell cyclepromoting apoptosis and maintaining the stability of the genome However the mutant p53 losses its tumorinhibitory role and become an oncogene when a mutation has happenedResults The mutation rate of p53 was in the experimentally and naturally infected chickens The mutationsincluded pointmutations and deletions and mostly located in the DNAbinding domain The mutated p53 wasexpressed in various tumor tissues in an infected chicken The mutant P53 proteins were notably accumulated inthe cytoplasm due to the loss in the function of nuclear localization Unlike the study on human cancer theconcentrations of P53 in the serums of MD infected chicken were significantly lower than the control groupConclusions The p53 mutations were apparent in the development of MD P53 and P53 antibody level in serumcould be a useful marker in the diagnosis and surveillance of MDKeywords Mareks disease p53 P53 antibodyBackgroundMareks disease MD is a lymphoproliferative neoplasticdisease caused by the chicken Mareks disease virusMDV or named Gallid alphaherpesvirus The infection caused by this virus may lead to lymphocyte proliferation tumor formation immunosuppression paralysisand mononuclear cell infiltration in peripheral nervesgonads and immune ans [ ] As one of the mosthighly contagious tumor diseases in chickens the data ofOIE [] reported by about half of the world has shownthat this disease accounts for the loss of up to billionUS dollars annually in the global poultry industry [] Correspondence liusidsdaueducn1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong ChinaFull list of author information is available at the end of the Described often as the guardian of the genome [] andthe cellular gatekeeper [] p53 is the most relevantand important tumor suppressor gene with the highestmutation frequency in human and animal tumor diseases [] The chicken p53 gene has a fulllength reading frame and untranslated regions and a polyadenylation signal which encodes amino acidsThese amino acids share a homology to the aminoacids of human P53 []p53 is divided into the wild type and the mutant typeThe wild type is a normal tumor suppressor gene whilethe mutant p53 is an oncogene transformed from atumor suppressor gene due to spatial conformationalchange As a result the mutant P53 protein losses itsability in regulating cell growth apoptosis and DNA repair [] which often a prerequisite for tumorigenesis and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhang BMC Veterinary Research Page of disease progression [] The proportion of p53 mutations in tumor tissue varies between []The wild type P53 has a very short halflife while themutant P53 protein is prolonged This abnormality ofP53 can lead to the accumulation of P53 antibody inserum as well as the P53 protein in tumor tissue []Several studies have demonstrated that the P53 antibodyplays a predictive role in tumorigenesis and its manifestation in serum is an early event in the development ofmalignant tumors in humans [] The level of P53antibody in serum correlates significantly with commonclinical neoplastic diseases but it has been barely detectable in the serum of healthy subjects [] This correlation may also exist in chicken and currently there is aknowledge gap concerning the role of mutant p53 inMareks disease in chicken Therefore this study aimedto investigate the role of p53 as a tumor marker inassisting the clinical diagnosis and prognosis of MDResultsHistopathological and immunohistochemical analysis ofp53 expressionHistopathological examination revealed the evidence ofliver cells undergoing necrosis in the infected SPFchicken Such liver tissues demonstrated focal infiltration and hyperplasia of lymphoid tumor cells Fig 1aIn addition the lymphoid follicle in the bursa of Fabricius was atrophied diffuse infiltration and proliferationof lymphoid tumor cells between the follicles were observed Fig 1b In the livers of the clinically infectedchickens the cytoplasm of the lymphoid tumor cellsunderwent multifocal proliferation which was stainedbrown by the immunohistochemicalFig 2aPositive staining was also detected in the tumor cells inIHCthe spleen and the bursa of Fabricius tissues Fig 2b and cIn contrast no positive stained cells were observed in thecontrol group Fig 2dMutations in the p53The mutation rate of p53 was in the infectedpoultry There were two types of p53 mutations deletions and point mutations detected which was consistent with the results reported in a previous study []Among the mutated p53 genes the base sites withhigh mutation frequency were and There was no mutation found in the control groupMost of the mutations were located in the core domainand the Cterminal domain The mutation analyses wereshown in Table P53 antigen and P53 antibody levels for MDThe concentrations of the P53 antigen of the clinicaland experimental MDVinfected group were significantlylower than the control group However the P53 antibody levels in the experimental infection group were significantly higher than the control group These analyseswere shown in Fig DiscussionThe human P53 is widely acknowledged as an intranuclear phosphorylated protein The wide type P53 normally exists in the nucleus for an extremely short period[] On the other hand the mutated P53 has a prolonged halflife to h as it is not digested quicklyand therefore accumulates inside tumor cells [] Thisallows the detection of mutant P53 protein via IHC forFig Histopathological observation of diseased chickens infected with MDV a Different sizes and shapes of focal infiltration and hyperplasia oflymphoid tumor cells were observed in the liver tissues HE b Diffuse infiltration and proliferation of lymphoid tumor cells between thefollicles were observed in the bursa of Fabricius HE 0cZhang BMC Veterinary Research Page of Fig Immunohistochemical staining of p53 in infected chickens a Liver lymphoid tumor cells with cytoplasm expression HE b Spleen lymphoid tumor cells with cytoplasm expression HE c Bursa of Fabricius lymphoid tumor cells with cytoplasm expression HE d Liver Negative controls were incubated with PBS HE which IHC has now become an important modality forthe detection of various tumor biomarkers P53 proteinrepresents an effective substitute marker for TP53 mutation status [] but at present P53 IHC is mainly applied for tumor diseases in humans P53 IHC allows theassessment of the stage and grade of cancers with only afew studies reported in poultry tumor diseasesIn this study the immunohistochemical analysis revealed that P53 was present in the liver spleen and thebursa of Fabricius of infected chicken with MDV Interestingly P53 was notably expressed in the cytoplasmThe potential explanation of this phenomenon could bethat the mutated P53 protein lost its function in nuclearlocalizationtheaccumulatedinandeventuallycytoplasm Furthermore p53 has been regarded as thehotspots given that p53 mutations have been frequently detected in a variety of tumors [] In thisstudy several types of p53 mutations were demonstratedin natural and experimental infections of MDV The fivemost frequent mutation sites were and The altered codons of mutant p53 comparedwith the wildtype were ACGACA GCAGCC CGCCGG GCCGCA and ACCACA but these were all synonymous mutationsIn our study a short form of p53 transcript was detected in a clinical case The deleted sequence was located at the bp in the reading frame of thereference sequence resulting in missense mutations fromTable Primers used toamplify chicken p53 cDNAPrimerp531Nucleotide sequenceGTGGCCGTCTATAAGAAATCAGA3AAAAAGGGGGCGTGGTCAGT3p532Annealing temperature Size of fragments amplifiedlocation bp 0cZhang BMC Veterinary Research Page of Fig Levels of p53 antigen and antibody in the serum of study groups The Yaxis represented the concentrations of antigen or antibody andthe Xaxis represented the different groups of samples Each group consisted of seven samples Statistical significance was designated as p or p the position to the termination The short form ofp53 transcript was also found in the cases infected byavian leukosis virus [] A series of missense mutationsfrom the position to the termination due to a base deletion was found in an experimentally infected chicken Inaddition this study found that the p53 was mutated in of poultry oncology with the mutation region concentrated mainly in the DBD The DBD allows the specificrecognition of target sequences [] Once the p53 is deleted or point mutations in this region occursit mayaffect the formation of tetramers which in turn leads tothe conversion of wild type P53 into a mutant type resulting in the loss of normal functionThe conformation of mutant P53 extends its halflifeto several hours in humans The accumulated mutantP53 then acts as a target antigen which elicits an autoimmune response [] However in our study the levelsof serum P53 antigen in clinical and experimentalMDVinfected groups were significantly lower than thecontrol group contrary to the findings in human cancerresearch Only the serum P53 antibody concentrationsof the experimentalinfected group were significantlyhigher than the control group This might be that P53as a tumor suppressor was largely consumed in response to the occurrence of MD Moreover tumorigenesis promoted mutation of p53 and further reduced P53concentrationConclusionsOur study revealed p53 was mutated and expressed inMDV infection which suggested that these mutationswere playing an important role in the development ofMD The mutant p53 was expressed in the tumor cellsof various tissues of the infected chicken Mutant P53protein lost its nuclear localization function and transferred from the nucleus to the cytoplasm Unlike studiesin human cancer the concentration of P53 was significantly lower in the natural and experimental MDV infectionnovelinnovations for the diagnosis and monitoring of MD inpoultryresults maygroup OurprovideMethodsNatural infection of MDV in chickensSeven 160dayold egglaying hens were obtained from alocal flock These hens were confirmed to have acquiredMDV infection naturally with the absence of other common viral diseases by PCR detection Their serums werecollected for the detection of the P53 antigen and antibody Their tissue samples including liver spleen pancreas and bursa of Fabricius were collected and fixedwith formaldehyde for h before histopathologicaland immunohistochemical studiesExperimental infection of chickens with MDVThe number of experimental animal was referred to thenumber of clinical samples Twenty 1dayold SPF chickens were obtained from the Shandong Academy of Agricultural Sciences Poultry Institute SPF Chicken ResearchCenter Jinan China They were randomly divided intotwo equal groups as an infection group and a controlgroup The two groups of chickens were raised separately in isolators with filtered air under positive pressureOn the first day of the experiment each of the chickenin the infection group was inoculated intraabdominallyia with plaqueforming units PFU of vvMDV 0cZhang BMC Veterinary Research Page of GX0101 which normally cause tumors at the tenthweek The chickens in the control group were inoculatedwith PBS In the tenth week serums were collected fromseven chickens in each group The experiment endedafter ten weeks and all chickens were put into a euthanasia box Thirty percent of carbon dioxide CO2 by volume was infused into the euthanasia box per minuteuntil all chickens lost their consciousness Then theCO2 flow rate was increased to for one minuteand the euthanasia box was kept airtight for ten minutesWhen all chickens were confirmed dead they were dissected and their livers and spleens were collected forhistopathology and immunohistochemistry studyHistopathology and ImmunohistochemistryThe fixed liver spleen pancreas and bursa of Fabriciuswere dehydrated waxed and cutinto µm slicesfollowed by Haematoxylin and eosin HE staining for ahistopathology examination In IHC processing tissueswere cut into sections of µm thickness and mountedon microslides treated with polyLlysine The sections were deparaffinized in xylene and rehydrated in agraded series of ethanol solutions into PBS then werepretreated in citrate buffer molL pH °Cfor antigen retrieval by microwaving and cooled at roomtemperature for min [] Endogenous alkaline peroxidase was quenched with hydrogen peroxide solutionin methanol for min [] Nonspecific antibody binding sites were obliterated by incubating the sections with fetal bovine serum for min at room temperatureFollowing this the antiP53 polyclonal antibody BOSTER China as a primary antibody was diluted into and immersed overnight at °C in a black humidchamber The secondary antibodies were HRP horseradish peroxidaseconjugated goatIgGCWBIO China Immunoreactivity was then visualizedwith diaminobenzidine DAB staining The sectionswere counterstained with hematoxylin and mountedNegative controls were incubated with PBS instead ofthe primary antibody in the immunohistochemicalanalysisantimouseTotal RNA isolation and reverse transcriptionThe total RNA ofliver and spleen tissue samples mgtissue were isolated by using TRIzol reagentTakara Japan according to the manufacturers instructions Extracted total RNA 1ug was reverse transcribedto cDNA with PrimeScript¢ RT Reagent Kit RocheSwitzerland According to the published complementaryDNA cDNA sequence of the chicken p53 GeneBankaccession number nm205264 specific primers were designed to amplify the DNAbinding domain DBD ofp53 as shown in Table Using PCR Polymerase ChainReaction techniques p53 cDNA genes sequences wereamplified The PCR reaction was performed by using athermal cycler Takara Japan under the following conditions for min followed by cycles of for s for s for s and a final Table Mutations of p53 genes in MDSample InfoNCMutation analysisBase mutation sitesNDMD CMD CMD CMD CMD CMD CMD EMD EMD EMD EMD EMD E TC AC C G AG AC gA CG CA AC CG CA CG CA delete TC TC AC CG CG CA delete CA CA TG AC CG CA gA CA CA AG AC CG CA CT gA AC CG CA gA gT CT CG CA gANC was SPF chicken without diseases E was experimental chicken C was clinical chickenAmino acid mutationsSite from toNDMutation area Y A E G R HNDND Stop StopNDND N S T I G DND R L V M E Y H YCore domainCterminal domainCore domainCore domainCore domainCore domainCterminal domainCore domainC terminal domain 0cZhang BMC Veterinary Research Page of for min extension cycle DNA templates that were acquired from the MDV positive chickens were amplifiedusing primer pair p5312 The SPF chickens wereregarded as negative controls DNA fragments were successfully amplified with sizes of bp The target fragment was gel extracted and connected to the pEASYT1vector Then the recombinant plasmid was transformedinto bacteria and sequencing analysis was made Finallythe sequencing results were compared with the wild typep53 cDNA sequences reported previouslyEnzymelinked immunosorbent assay ELISA for P53 andP53 antibodyThe P53 antigen and antibody levels of chicken weremonitored by ELISA Mlbio China In order to eliminate subjective interference the assessors were blinded tothe background of the samples The detection rangeswere pgml for P53 antigen and pgmlfor P53 antibody Samples were diluted five time with aspecial diluent and all processes were implemented inaccordance with the instructions The absorbance ODof each sample was finally measured at a wavelength of nm The sample concentration was calculated depending on the standard curveStatistical analysisStatistical analyses were conducted with GraphPadPrism Version San Diego CA USA The differences in the levels of P53 antigen and antibody were antwotailed Students Ttestalyzed by usingStatistical significance was designated as p or p theAbbreviationscDNA Complementary DNA DAB Diaminobenzidine DBD DNAbindingdomain ELISA Enzymelinked immunosorbent assay HE Haematoxylin andeosin IHC Immunohistochemical HRP Horse radish peroxidasePCR Polymerase chain reaction PFU Plaque forming unitsAcknowledgementsNot applicableAuthors contributionsHXZ carried out laboratory work wrote the manuscript and performed thedata analyses MDL designed the experiment wrote the manuscript andperformed the data analyses HZ SLC YL SNJ and YNS carried out laboratorywork SDL conceived and supervised this work revised manuscript All authorsread and approved the final manuscriptFundingThe work was supported by grants from the National natural sciencefoundation project NO The funding body was solely involved infunding and had no role in the design of the study the collection analysisand interpretation of the data or in writing the manuscriptEthics approval and consent to participateThe sample were collected and handled in accordance with the goodanimal practices required by the Animal Ethics Procedures and Guidelines ofthe Peoples Republic of China Informed consent to participate wasobtained from the chicken farmer owner All animal protocols andprocedures were performed according to the Chinese Regulations ofLaboratory Animals and were approved by the Animal Ethics Committee ofShandong Agricultural UniversityConsent for publicationNot applicableAvailability of data and materialsThe data supporting the findings are included in the manuscriptCompeting interestsThe authors declare that they have no competing interestsAuthor details1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong China 2China AnimalHealth and Epidemiology Center Nanjing Road QingdaoShandong ChinaReceived January Accepted August ReferencesJarosinski KW Tischer BK Trapp S Mareks disease virus lytic replicationoncogenesis and control Expert Rev Vaccines Reddy MS Book Review Mareks Disease An Evolving Problem Vet PatholBoodhoo N Gurung A Sharif S Behboudi S Mareks disease in chickens areview with focus on immunology Vet Res Lane DP p53 guardian of the genome Nature Bertzbach LD Kheimar A Ali FAZ Kaufer BB Viral Factors Involved inMareks Disease Virus MDV Pathogenesis Curr Clin Microbiol Rep Levine AJ p53 the cellular gatekeeper for growth and division Cell Soussi T B¨gue A Kress M Stehelin D May P Nucleotide sequence of acDNA encoding the chicken p53 nuclear oncoprotein Nucleic Acids ResZilfou JT Lowe SW Tumor suppressive functions of p53 Review ColdSpring Harb Perspect Biol 20091a001883Stiewe T Haran TE How mutations shape p53 interactions with thegenome to promote tumorigenesis and drug resistance Drug Resist UpdatK¶bel M Piskorz AM Lee S Lui S LePage C Marass F Rosenfeld N MesMasson AM Brenton JD Optimized p53 immunohistochemistry is anaccurate predictor of TP53 mutation in ovarian carcinoma J Pathol Clin Res Zekiye H B¼lent T Taner E p53 antibody is it an indicator ofdedifferentiated thyroid cancer Ann Nucl Med Balogh GA Mailo D Nardi H Corte MM Vincent E Barutta E Lizarraga GLizarraga P Montero H Gentili R Serological levels of mutated p53 proteinare highly detected at early stages in breast cancer patients Exp Ther MedSabapathy K Lane DP Understanding p53 functions through p53antibodies J Mol Cell Biol Kunizaki M Fukuda A Wakata K Tominaga T Nonaka T Miyazaki TMatsumoto K Sumida Y Hidaka S Yasutake T Sawai T Hamamoto RNanashima A Nagayasu T Clinical Significance of Serum p53 Antibody inthe Early Detection and Poor Prognosis of Gastric Cancer Anticancer Res Yue Q Yulong G Liting Q Shuai Y Delong L Yubao L Lili J Sidang LXiaomei W Mutations in and Expression of the Tumor Suppressor Gene p53in EggType Chickens Infected With Subgroup J Avian Leukosis Virus VetPathol Bykov VJN Eriksson SE Bianchi J Wiman KG Targeting mutant p53 forefficient cancer therapy Nat Rev Cancer Yemelyanova A Vang R Kshirsagar M Lu D Marks MA Shih IeM Kurman RJImmunohistochemical staining patterns of p53 can serve as a surrogatemarker for TP53 mutations in ovarian carcinoma an immunohistochemicaland nucleotide sequencing analysis Mod Pathol 0cZhang BMC Veterinary Research Page of Takagi M Ohashi K Morimura T Sugimoto C Onuma M The presence ofthe p53 transcripts with truncated reading frames in Mareks diseasetumorderived cell lines Leuk Res Arlt C Ihling CH Sinz A Structure of fulllength p53 tumor suppressorprobed by chemical crosslinking and mass spectrometry Proteomics Thierry S Analysis of p53 Gene Alterations in Cancer A Critical View Years of p53 Research Stiasny A Freier CP Kuhn C Schulze S Mayr D Alexiou C Janko C Wiest IDannecker C Jeschke U Kost BP The involvement of E6 p53 p16 MDM2and Gal3 in the clinical outcome of patients with cervical cancer OncolLett Shen FX Ma GP Cheng AC Wang MS Li CF Sun KF Chang H Zhu DK JiaRY Chen XY Sun T Development and application of an indirectimmunohistochemical method for the detection of duck plague virusvaccine antigens in paraffin sections and localization in the vaccinatedduckling tissues Poult Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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colorectal carcinoma crc as one of the most commongastrointestinal malignancies has developed the worldsfourth most deadly cancer with a high rate of incidence andmortality [ ] liver metastasis which is the most commonform for crc metastasis is the leading cause of high mortality for this severe malignancy however few clinical prevention and treatment measures could be available for tumormetastasis therefore it is really urgent to develop new biomarkers and explore the underlying mechanism for crcmetastasis and eventually develop new therapeutic strategiesfor crc patientsduring cancer progression metabolic reprograming withincreasing glucose utilization is termed as warburg aï¬ectwhich is accompanied by altered pyruvate and mitochondrialmetabolism the fate of pyruvate is the core manifestationto distinguish normal cells and tumor cells through metabolism in normal cells pyruvate was used for eï¬cient atpproduction directly into mitochondria however pyruvatewas converted into lactate in cytosol despite of normoxicand hypoxic conditions in cancer cells this may be dueto the impaired process of pyruvate from the cytosol intothe mitochondrial matrix which is a critical metabolic steplinking glycolysis and mitochondrial oxidative phosphorylation the mitochondrial pyruvate carriermpc a 0c of immunology researchmultimeric complex containing two distinct proteins mpc1and mpc2 which is located in the inner mitochondrialmembrane is responsible for eï¬cient mitochondrial pyruvate uptake loss of mpc expression or activity blockspyruvate entry into the tca cycle which results in a metabolism switch to increase glycolysis and the compensatoryusage of glutamine [ ]existed studies have reported that mpc1 was related withimmunoregulation stemness metabolism cellular morphology etc [ ] currently the important role of mpc1was uncovered in several tumors in crc and esophagealsquamous cell carcinoma decreased mpc1 results in accelerated aerobic glycolysis and malignant progression [ ] inlung adenocarcinoma mpc1 deï¬ciency accelerates lung adenocarcinoma progression through the stat3 pathway in prostate cancer mpc1 was reported to be involved instemness and metabolism which regulated by couptfii[ ] in renal cell carcinoma hypoxiainduced loss ofmpc1 enhanced the expression of mmp7 and mmp9 to promote cell invasion collectively these data suggestedthat mpc1 maybe serves as a suppressor to disrupt tumormalignancy however whether mpc1 is involved in crcmetastasis and the underlying mechanisms remain to beillustratedin the present study we ï¬gured out the relationshipbetween the mpc1 expression and crc liver metastasiswe identiï¬ed that decreased mpc1 was closely correlatedwith patients metastasis as well as led to poor prognosisfunctionally mpc1 overexpression could attenuate themigration and invasion capacities of crc cells both in vitroand in vivo mechanically mpc1 suppressed crc metastasisthrough mediating the wntcatenin signaling thus ourï¬nding ï¬rstly revealed a critical role of mpc1 in crc livermetastasis materials and methods data mining seven geo datasets gse21510 gse5206gse20916 gse9348 gse89393 gse67675 and gse4183and tcga were used to analyze the mpc1 expression pattern in crc the primary data for tcga datasets weredownloaded wwwcancergov the primary data feo datasets were downloaded at wwwncbinlmnihgovgeo the oncolnc database httpwwwoncolnc was used to detect the prognostic value of mpc1 inthe tcga cohort patients and clinical specimens in our study a total of patients containing paired crc tissues and adjacentnontumor tissues were enrolled from the department of gastrointestinal surgery renji hospital school of medicineshanghai jiao tong university among them cases ofliver metastases were collected and only cases wereavailable with complete followup data for survival analysisinformed consents were signed by all patients the researchwas approved by the research ethics committee of renjihospital and carried out in accordance with ethical standardsas formulated in the helsinki declarationtable sequences of primers used for realtime pcrprimermmp7 forwardmmp7 reverseecadherin forwardecadherin reversesnail1 forwardsnail1 reversemyc forwardmyc reversegapdh forwardgapdh reversesequence ²²gagtgagctacagtgggaacactatgacgcgggagtttaacatatgagtgtcccccggtatcttcacgagcagagaatcataaggcggtatccagagctgtttggaaacattttcctcccaggccatcacagccctcactcacacagattccacaaggtgcctgggctacactgagcaccaagtggtcgttgagggcaatg cell culture and cell transduction human crc celllines hct116 ht29 sw620 rko sw480 and lovoand mouse crc cell line mc38 were gained from the cellbank of the chinese academy of sciences shanghai chinaall cells were cultured in dmem medium supplemented°with fetal bovine serum and antibiotics at c in ahumidiï¬ed incubator with co2mc38 cells were transfected with lentivirus containing aluciferase reporter plasmid stable transfection cells werescreened with μgml blastisidin for days which termedmc38luc in addition one short hairpin rna shrnasequence against mpc1 was packaged as lentivirus andtransfected into mc38luc cells lovo and sw480 were transfected with lentivirus containing fulllength human mpc1cdna or empty vehicle control stable transfection cells werescreened under μgml puromycin for days and veriï¬edby western blot in those assays all lentiviral transfectionswere performed in the presence of μgml polybrene immunohistochemical ihc the protocol of this assayand quantify the mpc1 protein expression level were performed according to previously reported primary antibodies used as follows mmp7 yt2663 immunoway ecadherin cst snail1 ab53519 abcam and mycyp0861 immunoway cellular immunoï¬uorescence assays were performedaccording to the previous description brieï¬y cells wereincubated with antibodies against catenin ab32572abcam and incubated with alexa 488conjugated secondaryantibody the nuclei were stained with ²6diamidino2phenylindole dapi western blotting wholecell lysates or nuclear proteinwas extracted using a protein extraction buï¬er beyotimeshanghai china or nucleoprotein extraction kit sangonbiotech c500009 respectively proteins were resolved bysdspage and transferred onto nitrocellulose nc membranes using standard methods primary antibodies used asfollows mpc1 ab74871 abcam catenin ab32572abcamlamin ac ab8984 abcam and gapdhab9485 abcam speciesspeciï¬c secondary antibodies used 0c of immunology researchtable related enzyme and carrier of pyruvate analyzed in this studygenepdha1pdhbpdk4pdhxmpc2pdk2pdk1pdk3mpc1pcpdp1pdprpdha2pdp2pklrdescriptionpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase lipoamide betapyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase complex component xmitochondrial pyruvate carrier pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme mitochondrial pyruvate carrier pyruvate carboxylasepyruvate dehydrogenase phosphatase catalytic subunit pyruvate dehydrogenase phosphatase regulatory subunitpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase phosphatase catalytic subunit pyruvate kinase liver and rbclocationmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrionas follows irdye goat antimouse igg licor andirdye goat antirabbit igg licorttest was used for comparison between groups p wasconsidered as statistically significantsynthesized using the primescript realtime pcr total rna was extracted using trizoltakara according to the manufacturersinstructionscdna wasreversetranscriptionpolymerasechain reaction rtpcr kittakara the qpcr was performed using sybr green masct method was used to analyze theter mix roche the data and gapdh was used as a loading control the primersequences are listed in table liver metastasis model this study was performed inaccordance with the recommendations in the guide for thecare and use of laboratory animals and relevant chineselaws and regulations the protocol was approved by theinstitutional animal care and use committee iacucof shanghai jiao tong university the mc38 cells transplanting luciferaseexpressing were injected into the spleensof c57bl6n mice n with a concentration of cellsmouse two weeks later the mice were killed andthe liver metastasis tissues were harvested luciferase reporter assay the protocols of this assaywere operated in accordance with previous reported ng top reporter plasmid wntcatenin signalingor ng fop reporter plasmid negative control ofwntcatenin signaling and ng renilla were mixedand transfected into crc cells using lipofectamine dualluciferase reporter assay system promega was usedto detect the ï¬reï¬y and renilla luciferase activities statistical analysesdata are shown as means ± sd spss chicago il usaand graphpad prism software were used to manipulate statistical analyses kaplanmeier method was used to calculatecumulative survival time the chisquare test or students results mpc1 expression was aberrantly decreased in crcpyruvate is a pivotal intermediate in the process of cellmetabolism which connects glycolysis and the tca cycleto determine the potential maladjustment genes involvedin pyruvate metabolism which is located in mitochondriaas shown in table we analyzed the tcga dataset containing crc and their normal counterparts the resultsshowed that multiple genes are significantly upregulated ordownregulated in crc t tissues compared to normal colonn notably mpc1 had a log2 fold change less than figures 1a and 1b as known to us pyruvate translocation from the cytoplasm to the mitochondria is the ï¬rst stepinto the tca cycle which needs mpc1mpc2 heterodimerin the analysis no significant change was found in mpc2therefore mpc1 was selected for further study the expression pattern of mpc1 was further analyzed in ï¬ve independent geo datasetsgse21510 gse5206 gse20916gse9348 and gse4183 consistently we found thatmpc1 was downregulated with statistical diï¬erence in crctissues figure 1c and inï¬ammatory tissue supplementary figure in comparison to their normal counterpartsmeanwhile we found decreased mpc1 expression inhuman crc tissues compared to their normal counterpartsfigure 1d in addition a similar phenomenon wasrevealed in aomdss induced mouse crc modelsfigure 1e then we evaluated the protein level of mpc1used a tissue microarray containing matching cancerand corresponding adjacent nontumortissues whichsubjected ihc staining the expression of mpc1 wasscored as based on the staining area andintensity figure 1f we found that more lower mpc1expression score as and was presented in crc 0c of immunology researcheulavp goltcga t nmpc1mpc2log2 fold changenoisserpxe cpm evitalertcgan normaltumornoisserpxe cpm evitalergeo datasetsgse21510 gse5206 gse20916 gse9348normaltumorabhuman samplecrcnormalaom dssnormalccrcesacesacesacesacdempc1 lower expressionmpc1 higher expressionf egatnecrepromutlamrongfigure expression pattern of mpc1 in crc a volcano plot showed fold changes xaxis and corresponding p values log10 yaxis ofpyruvate metabolismrelated genes located in mitochondria analyzed in the tcga dataset between paired normal and crc samplesstudents ttest b the comparison of mpc1 expression in tumor and matched normal tissues using the tcga dataset students ttest p c expression analysis of mpc1 in tumors and corresponding normal tissue using four independent geo datasetsgse21510 gse5206 gse20916 and gse9348 students ttest p d ihc staining of mpc1 expression in matched crctumor and nontumor tissues scale bar μm e ihc staining of mpc1 expression in the aomdss induced crc mouse models andcontrol animal scale bar μm f representative ihc staining of mpc1 expression in tissue microarray from the ren ji cohortwhich contained crc patients and paired adjacent normal tissue n the tumor tissues were divided into two groups based on theexpression level which scored as scale bar μm g the percentage of tissue displaying diï¬erent expression level ofmpc1 in crc tumor and adjacent nontumor tissues fishers exact test p tissues figure 1g overall these results revealed thatmpc1 was disrupted during crc decreased mpc1 enhanced tumor metastasis capabilityand predicated poor prognosis in crc it is well known thatcrc is one of the most malignant tumors given its strongmetastasis ability so we tried to ï¬gure out whether thempc1 expression was correlated with metastasis we foundthat lower mpc1 expression was closely correlated withmetastasis p lymph node invasion p and tnm stage p which revealed by the analysisbetween the clinical signiï¬cance and mpc1 expression incrc table next to illuminate the expression patternof mpc1 in diï¬erent process of crc data mining was carried out by two independent geo datasets gse21510 andgse89393 which contained normal tissue primary crcand metastatic lesion in the liver mcrc as shown infigures 2a and 2b mpc1 expression was gradually downregulated in patients with an increase in metastasis ability asimilar result was found in mice cells ct26 with high livermetastasis hmct26 or poor liver metastasis pmct26figure 2c which isolated by in vivo selection in an orthotopic mouse model of colon cancer metastasis to the liverfurther analysis showed that mpc1 protein expression was 0c of immunology researchtable clinicopathological correlation of mpc1 expression in theren ji crc cohortclinicopathological featureexpression ofmpc1lowhighp value Ï2testage years¥gendermalefemalemetastasisyesnolymph node invasionyesnotumor size cm¥ cmtnm stageiiiiiiivkras mutationmutationno mutationgradually downregulated in normal tissue primary crc andliver metastasis crc crlm tissues figure 2d furthermore survival analysis showed that patients with lowermpc1 expression had a worse outcome compared to thepatients with higher mpc1 expression using the tcgacohort figure 2e and ren ji cohort figure 2f additionally among patients with metastasis worse prognosiswas emerged in the mpc1 low cases figure 2g while nosignificant association was observed in patients withoutmetastasis figure 2h mpc1 overexpression impaired crc cells motility bothin vitro and in vivo to evaluate the role of mpc1 on themotility of crc cells the transwell assay was performedfirstly we examined low mpc1 protein expression in humanand high mpc1 protein expression in mouse mc38 crccells by western blot figure 3a mpc1overexpressingstable cell lines were established using a lentivirus carrying the mpc1 gene in lovo and sw480 cells and theoverexpression eï¬ciency was conï¬rmed by immunoblotsfigure 3b mpc1 knockdown in lucmc38 cells wereestablished and veriï¬ed by wb figure 3c mpc1 overexpression exhibited significantly weaker migration and invasion ability than the control cells in both lovo figure 3dand sw480 figure 3e cells following the liver metastasismodel of crc was established by spleen orthotopicallyinjecting mc38 cells transplanting luciferaseexpressingwhich would simulate mc38 metastasis to the liver throughsplenic veinportal vein the results revealed that the mpc1knockdown promoted mc38 cells metastasis to the liverthrough detecting the luminescence intensity monitoredby bioluminescence imaging figure 3f notablythenumber of metastatic liver nodules in the mpc1 silencinggroup wasin the control groupfigure 3g histological examination also proved thatmpc1 knockdown decreased the metastatic potential ofcrc in vivo figure 3gthan thatsmaller decreased mpc1 activated the wntcatenin pathwayby promoting nuclear translocation of catenin to furtherexplore the underlying mechanism of mpc1mediated inhibition of crc metastasis the tcga database was used toperform gsea analysis the results indicated that mpc1was involved in the wntcatenin signaling when set themrna expression median as a cutoï¬ figure 4a anddualluciferase reporter gene assay revealed that mpc1 overexpression obviously inhibited the activity of wntcateninpathway figure 4b which conï¬rmed the result abovewe then tested the distribution changes of catenin innuclear and cytoplasmic which was a crucial step inwntcatenin pathway as shown in figure 4c no significant diï¬erence was emerged in the total amount ofcatenin between mpc1 overexpression cells and the control cells figure 4c however obviously decreased distribution of nuclear catenin was presented in mpc1overexpression cells compared to that in the control cellsas revealed by the stronger gray corresponding to cateninfigures 4c and 4d consistently immunoï¬uorescenceif staining showed that mpc1 overexpression weakenednuclear catenin localization in both lovo and sw480 cellswhen compared to control cells figures 4e and 4f asimilar phenomenon was observed in mouse liver metastasistissues by ihc figure 4g following qpcr analyses displayed some downstream target genes of catenin such asmmp7 ecadherin snail1 and myc obviously increasedin ecadherin and decreased in mmp7 snail1 and mycwere observed after the mpc1 overexpression in both lovoand sw480 cells compared to that in the control cellsfigures 4h and 4i meanwhilethe opposite trendwas observed in mc38 cell after mpc1 knockdownfigure 4j and similar results were observed in mouseliver tissue detected by ihc figures 4k4n takentogether the data above indicated that mpc1 mediatedcrc cell metastasis through the wntcatenin pathway discussionaccumulating evidences have shown that reprogrammedenergy metabolism conduces to the tumor malignant propertiesincluding enhanced crc liver metastatic capacity[] mitochondria as the primary site of energy production regulate the pyruvate metabolism under both physiologic and pathologic conditions the ï¬rst step of the tcacycle is mediated by mpc which transports pyruvate into 0cnoisserpxe cpm evitalergse21510normal crc mcrcnoisserpxe cpm evitalernormalacrccrlmesacesac of immunology researchgse67675pmct26 hmct26ctcgan n p hr time daysgse89393normal crcmcrcbnoisserpxe cpm evitaler egatnecreplavivrus llarevolamroncrcmlrclavivrus llarevoren ji cohortn p hr n time monthsfdlavivrus llarevolavivrus llarevocrc with metastasis n n p hr n time monthsmpc1 higher expressionmpc1 lower expressiongecrc without metastasis n n n p hr time monthshfigure decreased mpc1 enhances tumor metastasis and predicts poor prognosis in crc a b data mining showed mpc1 expressionwas gradually decreased in normal tissue primary crc and liver lesion of metastatic crc mcrc from two independent geo datasetsoneway anova a gse21510 p b gse89393 p c data from gse67675 revealed that mpc1 expressionwas lower in high liver metastasis ct26 cells hmct26 than that in poor liver metastasis ct26 cells pmct26 students ttestp d gradually decreased mpc1 expression was presented in normal tissue primary crc and liver metastasis crccrlm tissue scale bar μm n fishers exact test p e kaplanmeier overall survival os curves in the tcgadataset of crc patients according to the mrna expression of mpc1 the lower quartile value of expression was utilized as a cutoï¬logrank test p f kaplanmeier os curve for the mpc1 expression in the ren ji cohort logrank test p gkaplanmeier os curve for the mpc1 expression in patients with metastasis logrank test p h kaplanmeier os curve forthe mpc1 expression in patients without metastasis logrank test p mitochondrial from cytoplasm in the beginning tcgawas used to analyze the relative genes involved in pyruvatemetabolism which is located in mitochondria the resultsrevealed that mpc1 expression was significantly downregulated in crc tissues meanwhile the geo datasets analysisas well as ihc staining on crc patients tissue and mousemodels conï¬rmed this trend these phenomena may indicate that loss of mpc activity enhanced tumorigenic glucoseutilization by blocking mitochondrial pyruvate uptake andoxidation interestingly in the course of data analysis wefound that the expression of mpc1 was decreased at thestage of intestinal inï¬ammation which was not diï¬erentfrom that in tumor tissue mpc1 has been reported to beinvolved in immune regulation of peripheral t cell homeostasis through metabolic regulation and a decrease inmpc1 was found at the earliest stages of crc hence 0c of immunology researchhumanmouselovosw480lovosw480mpc1gapdhmpc1mpc1gapdhmpc1mpc1gapdhwsthwsovolokrcmtchablovorotcevitnelcpmitnelrotcevitnelcpmitneldlefi rep sllec edavnidlefi rep sllec edavnirotcevitnelcpmitneldsw480rotcevitnelcpmitnelcncpmcncpmdlefi rep sllec detargimdlefi rep sllec detargimccncpmcncpmmc38shnc shmpc1¨¯ sp xufl latotcnhscpmhscnhscpmhsradiancepseccm2srfemc38suledon sisatsatemcnhscpmhsgfigure mpc1 overexpression inhibits the motility of crc cells in vitro and in vivo a mpc1 expression in human and mouse crc celllines examined by western blot gapdh serves as loading control b mpc1 overexpression in lovo and sw480 cells c mpc1 silencing by shrnampc1 in mouse mc38 cells d e transwell assays showed that upregulated mpc1 suppressed the invasion and migration ability of lovod and sw480 e cells quantiï¬cation of invaded and migrated cells was performed for ï¬ve randomly selected ï¬elds values are means ± sd frepresentative bioluminescence photograph of mice spleen implanted with luciferaseexpressing mc38 cells treated with shmpc1 or controlvector total ï¬ux was quantiï¬ed by the ivis system to verify the ability of liver metastasis g representative image of liver metastases andquantiï¬ed by the nodules in mice inoculated with mc38 cells treated with shmpc1 or control vector as well as representative images ofhe staining of the liver metastatic lesions scale bar μm students ttest p p p 0c of immunology researchse erocs tnemhcirnewnt signaling pathwaynes p mpc1 highmpc1 lowoitar pof pot evitalerð½cateninlamin acð½cateningapdhmpc1alovosw480csuelcunlatot icanmalnnetacð½ilovoncmpc1bsw480lovosw480ncmpc1dð½catenindapimergeð½catenindapimergeð½catenincpmcnovolnoisserpxe evitaleregnahc doflelovolianscympmmncmpc1nirehdacehcpmcnwsfsw480lianscympmmncmpc1nirehdaceifigure continuedcmcnhscpmhsmc38glianscympmmnirehdaceshncshmpc1jnoisserpxe evitaleregnahc doflnoisserpxe evitaleregnahc dofl 0c of immunology researchmmp7ecadherinsnail1myccmcnhscpmhscmcnhscpmhscmcnhscpmhscmcnhscpmhsklmnfigure decreased mpc1 activates the wntcatenin pathway by promoting nuclear translocation of catenin a gsea analysis ofmpc1 expression in crc using the tcga dataset nes normalized enrichment score b luciferase reporter gene assay of crc cellstreated with mpc1 overexpression or not c the expression of total catenin and nuclear catenin was detected in control and mpc1overexpression crc cells respectively gapdh and lamin ac were used as the loading control of total and nuclear protein respectivelyd the gray value analysis of nuclear catenin in mpc1overexpression cells and control cells e f mpc1 overexpression could inhibitthe nuclear translocation of catenin in crc cells scale bar μm g ihc staining of catenin in mouse liver metastatic lesionsinoculated with mc38 cells treatment with shmpc1 or control vector scale bar μm h i relative mrna expression level of catenin target genes in crc cells with mpc1 overexpression or control vector j relative mrna expression level of catenin targetgenes in mc38 cells with shmpc1 or control vector kn relative protein expression level of catenin target genes in mouse livertissue detected by ihc scale bar μm students ttest p p we suspect that mpc1 is involved in bowel inï¬ammation totumorigenesis and more studies need to be devised to illustrate this processfollowing in the analysis between the clinical signiï¬cance and mpc1 expression in crc we found that mpc1expression was especially correlated with metastasis inspiredby this we detected the mpc1 expression pattern in normaltissue primary crc and metastasis crc by geo datasetsand patients tissue all results revealed that gradually downregulated mpc1 was in patients with an increase in metastasis ability survival analysis indicated that worse outcome waspresented in patients with lower mpc1 expression especiallyin patients with metastasis additionally function assays veriï¬ed that mpc1 overexpression could attenuate the migration and invasion capacities of crc cells in vitro andmpc1 knockdown could enhance the metastasis capacityin vivo and existing studies have revealed that the mpc1was participated in metastatic dissemination of pgc1αtransduced cholangiocarcinoma through elevating reactiveoxygen species ros production besides mpc inhibitor uk5099 treatment could trigger strong invasive capacitythrough blocking pyruvate translocation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation and trigger aerobic glycolysis these phenomenatogether with our results indicate that mpc1 could act as atumor suppressor through inhibiting tumor metastasis andexisting studies have shown that mpc1 could alter the maintenance and fate of stem cells through regulating cancermetabolism in crc however the relationship betweenthe metabolism and tumor metastasis regulated by mpc1was not being mentioned thus further evidence should bereceived to conï¬rm thissubsequently the underlying mechanism of mpc1 inregulating metastasis was explored for this purpose gseaanalysis was performed the results indicated that mpc1was involved in the wntcatenin signaling the next seriesof experiments also conï¬rmed that mpc1 could mediate thewntcatenin pathway by redistribution of catenin previous reports showed that cytoplasmic catenin phosphorylated in nterminally localized to sites of cellcell contact isassociated with ecadherin and was required for intact cellcell adhesions without any change detected in the levels oftotal catenin [] simultaneously cellcell adhesionbased on cadherin binding with catenin limited wnt signals in addition catenin was reported to interact withusp9x to inhibit the degradation of catenin through thedeubiquitination of catenin in breast cancer a constitutive irs1 and catenin protein interaction activated mycexpression in acute lymphoblastic leukemia cells inhcc catenin was reported to interact with yap1 to leadto rapid tumorigenesis hence it is reasonable to guessthat accumulated cytoplasmic catenin maybe crosstalkwith other genes or involved in other biological processeswhat is more some downstream target genes of cateninsuch as mmp7 ecadherin snail1 and myc were changedin expression as known to us mmp7 is a member of theproteolytic enzyme family which promotes the invasionand metastasis of tumor cells by degrading the basementmembrane and extracellular matrix and previous studies had evidenced for involvement of mmp7 activation incolorectal cancer liver metastases [ ] ecadherin andsnail1 were considered as the epithelialmesenchymal transition emt marker which was involved in metastasis ofmalignant tumor moreover ecadherin was reportedto be involved in cellcell junction to regulate cancer invasionand metastasis [ ] and the gsea analysis also revealedthat mpc1 could aï¬ect the cellcell contacts data notshown as described previously mmp7 could facilitatemorphological transition by cleaving ecadherin thecommunication between the cells is disrupted when ecadherin was shredded leading to destructed cell adhesionand induction of emt followed by increased cell migration inspired by this we assumed that mpc1 could mediatetumor cell motility through aï¬ecting mmp7 activity cellcell 0c of immunology researchcontacts and emt however further studies need to be performed to clarify the detailed underlying mechanisms conclusionsin conclusion we ï¬rstly demonstrated that decreased mpc1was closely correlated with patients metastasis as well as ledto poor outcome moreover mpc1driven nuclear translocation of catenin contributed to crc cell motility thismeans that mpc1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patientsabbreviationscrc colorectal carcinomampc mitochondrial pyruvate carrieremt epithelialmesenchymal transitiongsea gene set enrichment analysisdata availabilitythe data used to support the ï¬ndings of this study are available from the corresponding author upon requestconflicts of interestall authors declare no conï¬icts of interestauthors contributionsyahui wang and guangang tian conceived and designedthe study chunjie xu and kaixia zhou obtained and anized the data guangang tian analyzed the data zhigangzhang and jianren gu contributed reagentsmaterialsanalysis tools xueli zhang and guangang tian wrote themanuscript guangang tian chunjie xu and kaixiazhou contributed equally to this workacknowledgmentsthis work was supported by the national natural sciencefoundation of china no to zhigang zhangand the natural science foundation of shanghai no18zr1436900 to xuelili zhangsupplementary materialssupplementary figure expression analysis of mpc1 innormal ibd and tumor tissues using the gse4183 datasetoneway anova was used to analyze the statistical diï¬erences between ibd adenoma and crc tissues ns no significance students ttest p p p supplementary materialsreferences h brody colorectal cancer nature vol no r l siegel k d miller s a fedewa et al colorectal cancerstatistics ca a cancer for clinicians vol no pp a j rauckhorst and e b taylor mitochondrial pyruvatecarrier function and cancer metabolism current opinion ingenetics development vol pp m g vander heiden l c cantley and c b thompsonunderstanding the warburg eï¬ect the metabolic requirements of cell proliferation science vol no pp s herzig e raemy s montessuit et al identiï¬cation andfunctional expression of the mitochondrial pyruvate carrierscience vol no pp c yang b ko c t hensley et al glutamine oxidationmaintains the tca cycle and cell survival during impairedmitochondrial pyruvate transport molecular cell vol no pp t bender g pena and j c martinou regulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes the embo vol no pp a g ramstead j a wallace sh lee et al mitochondrialpyruvate carrier promotes peripheral t cell homeostasisthrough metabolic regulation of thymic development cellreports vol no pp 2899e6 x zhou z j xiong s m xiao et al overexpression ofmpc1 inhibits the proliferation migration invasion and stemcelllike properties of gastric cancer cells oncotargets andtherapy vol pp d li c wang p ma et al pgc1α promotes cholangiocarcinoma metastasis by upregulating pdha1 and mpc1 expression to reverse the warburg eï¬ect cell death diseasevol no j c schell k a olson l jiang et al a role for the mitochondrial pyruvate carrier as a repressor of the warburg eï¬ectand colon cancer cell growth molecular cell vol no pp y li x li q kan et al mitochondrial pyruvate carrierfunction is negatively linked to warburg phenotype in vitroand malignant features in esophageal squamous cell carcinomas oncotarget vol no pp h zou q chen a zhang et al mpc1 deï¬ciency accelerateslung adenocarcinoma progression through the stat3 pathway cell death disease vol no l wang m xu j qin et al mpc1 a key gene in cancermetabolism is regulated by couptfii in human prostatecancer oncotarget vol no pp y zhong x li d yu et al application of mitochondrialpyruvate carrier blocker uk5099 creates metabolic reprogramand greater stemlike properties in lncap prostate cancer cellsin vitro oncotarget vol no pp x p tang q chen y li et al mitochondrial pyruvatecarrier functions as a tumor suppressor and predicts theprognosis of human renal cell carcinoma laboratory investigation vol no pp g a tian c c zhu x x zhang et al ccbe1 promotesgist developmentthrough enhancing angiogenesis andmediating resistance to imatinib scientiï¬c reports vol no article c xu g tian c jiang et al nptx2 promotes colorectalcancer growth and live
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range of diseases including malignancies and autoimmune disordersIts high eï¬ectivenessprice ratio also won extensive application in ophthalmology On the other hand although MTX has anexcellent pharmacological eï¬cacy MTX associated side eï¬ects in clinical use which vary from patient to patient are nonnegligible ere is no comparatively systematic review on MTX associated side eï¬ects and its risk factors is review aimed toreveal novel clinical approaches of MTX and its adverse eï¬ects in order to provide a reference for ophthalmic scholars in clinicalapplication of MTX IntroductionMethotrexate MTX is an antifolate metabolite that inhibitsDNA synthesis repair and cellular replication It was ï¬rstlyused as one of the essential treatments of pediatric leukemia[ ] According to previous studies MTX has also beenused to treat rheumatoid arthritis RA and psoriasis as antiammatory and immunomodulatory agent [] as MTXcould not only optimize the eï¬cacy of biological diseasemodifying antirheumatic drugs DMARDs [ ] but alsomake the therapeutic goals via lower doses in comparisonwith other conventional synthetic DMARDs [] Figure shows the pathway of folate in DNA synthesis the cellularpathway of MTX and how MTX works inside the cell Whileimmediate and lowdose MTX is used to treat nonmalignantand immunemediated disorders highdose MTX HDMTX more than mgm2week is widely used to treatmalignancies Until now HDMTX with or without radiation therapy is still the backbone of most modern chemotherapy regimens [] as well as the prevention ofsystemiccentral nervous system CNS lymphoma recurrence at a dose of gm2 per week []MTX has also been widely applied in ophthalmic diseases systemically and locally Recently published spay more attention to new clinical applications routes ofadministration and newly discovered side eï¬ects which arefoci of this review Clinical Applications in OphthalmologyAs one of the known corticosteroidssparing agents MTXhas been widely used in the treatment of anterior intermediate posterior or pan uveitis scleritis and ocularmucous membrane pemphigoid [] as well as advancedproliferative diabetic retinopathy [] However followedresearches reveal that MTX works with a significant difference in eï¬ectiveness ratings by anatomic location ofammation [] with treatment success achieved mostcommonly in patients with anterior uveitis and scleritis []In the treatment of noninfectious intraocular ammationoral and intravenous are the most common routes with ausual dose range of mg to mg weekly e typical doseobserved was mgweek [ ] which is in the range oflowdose MTX e median time to achieve the success of 0cJournal of OphthalmologyFigure e cellular pathway of folate and MTX Dietary folate enters the cells through RFC1 as well as MTX In lowdose MTX treatmentMTX inhibits enzymes of the folate pathway Ultimately MTX leads to an increase in intracellular adenosine level which would cause antiammatory eï¬ects RFC1 � reduced folate carrier ABC family � adenosine triphosphatebinding cassette ABC family DHF � dihydrofolate THF � tetrahydrofolate GGH � cglutamyl hydrolase FPG � folylpolyglutamate synthase MTXPG � methotrexate polyglutamate DHFR � dihydrofolate reductase MTHFR � methylene tetrahydrofolate reductase AICAR � aminoimidazole carboxamideribonucleotide ATIC � AICAR transformylasetreatment deï¬ned as control of ammation with theability to taper corticosteroids to mg or less daily rangesfrom months to months for MTX [ ]Intravitreal MTX injection with or without systemicchemotherapy and radiotherapy has already been used totreat primary intraocular lymphoma patients [ ]According to Larkin [] intravitreal MTX injectioncould achieve remission in a proportion of patients withprimary intraocular lymphoma What is particularly noteworthy is that although MTX has a slow rate of onset ofeï¬ect when it was used to treat intraocular lymphoma viaintravitreal injection it prolonged local remission of oculardisease even with an aggressively growing tumor []erefore it has been taken as a relatively ï¬rstline choice forthe treatment of recurrent intraocular lymphoma [] although the treatment for primary intraocular lymphoma islacking solid justiï¬cation because of the limited retrospectiveand prospective case series [] Local treatment via intraocular injection provides a consistent therapeutic MTXconcentration to reduce the systemic MTX associated sideeï¬ects [] erefore intraocular MTX injection is worthtrying especially for unilateral ocular diseases New Approaches of Applications of MTX MTX Used against Epithelial Downgrowth Previousstudies have already demonstrated safety of intravitrealMTX [] It has been used to treat intraocular lymphomaand proliferative vitreoretinopathy because ofits antiproliferative properties [] ere is a novel use of intravitreal MTX for recurrent epithelial downgrowth which wasnot treated by surgical and medical methods Lambert et al[] administered intravitreal MTX to patients with refractory proliferative membrane after cataract surgery whilemembrane peel and endolaser treatment failed e injectionof MTX was administered alone based on previous protocols and the presumed halflife of drugs in vitreous cavityAfter injections totally there was no membrane recurrence is case suggests that intravitreal MTX plays a role intreatment against epithelial downgrowth MTX Used in Conventional erapyResistant DiseasesGenerally the antivascular endothelial growth factor antiVEGF therapy has dramatically improved the prognosis ofneovascular agerelated macular degeneration nAMDHowever there are still some patients who remain refractoryto antiVEGF therapy which is termed as treatmentresistant nAMD As there is evidence that MTX has eï¬ects ininterrupting the angiogenesis cascade at various levels []Kurup oï¬ered intravitreal MTX to patients who wererefractive to standard antiVEGF therapy [] Although itwas an oï¬label use the patients visual acuity improved atfollowup visit while ophthalmic imaging examinationsshowed significantly reduced cystoid macular edema usFolatefolic acidRFC 1Folatefolic acidDHFTHFDHFR5MTHFMTHFRPurine and pyridine synthesis DNA synthesisMTXMTXABC familyFPGGGHMTXFGAICAR5FormylAICARATICAdenosine AdenosineCell membraneCell membrane 0cJournal of Ophthalmologypatients who are refractory to traditional antiVEGF therapymight beneï¬t from intravitreal injection of MTXis approach is not alone Khalil [] had μg01 ml of MTX intravitreal injection once monthlyadministrated to adult Behcets disease BD patientssuï¬ering from BDassociated ocular ammation withposterior segment involvement eir results prove thatintravitreal MTX improves visual acuity reduces posteriorsegment manifestations associated with Behcets disease andallows the reduction of corticosteroids and immunosuppressive drugs [] ese results also supported Taylor andassociates who conducted trials on patients with unilateral uveitis andor cystoid macular edema [] eirclinical trials suggest that intravitreal MTX may help patients with uveitisassociated posterior segment involvementto regain normal anatomical structure and then allowed thereduction of immunosuppressive therapy The Pharmacogenetics of MTXWith molecular sequencing and highthroughput technology large numbers of genetic polymorphisms can now bedetected accurately and rapidly [] Researchers pay moreattention to pharmacogenetics the study of genetic polymorphisms in drugmetabolizing enzymes and the translation of inherited diï¬erences to diï¬erences in drug eï¬ects[] e genes encoding transporting proteins and metabolizing enzymes for MTX are also known to harborfunctionally significant SNPs e SNPs may uence theeï¬cacy of MTX and have been suggested as potential riskfactors for enhanced MTX toxicity even in lowdose regimens based on previous researches []e research of pharmacogenetics of MTX could bedivided into genetic polymorphisms aï¬ecting MTX transport and SNPs thatuence enzymes in the cellularpathway of MTX []Once taken MTX enters the cell through an activetransport which is mediated by the reduced folate carrier RFC1 e loss of RFC1 gene expression might lead toeï¬ects of uptake and intracellular levels of MTX A G80ASNP of RFC1 was proposed [] making a decreasing [] orincreasing [ ] eï¬ect on intracellular level of MTXerefore a significant association between RFC1 SNPs andMTX toxicity should be considered Chango state thatthese SNPs strongly impact the overall MTX associated sideeï¬ects by resulting in altered cellular MTX concentrationbut with no uence on MTX eï¬cacy [] However someresearchers argue that these SNPs have no deï¬nite eï¬ect[] us it remains controversial whether SNPs of RFC1aï¬ect the transport of MTX Moreover Pglycoprotein amembrane transporter that has uences on the dispositionand bioavailability of MTX [] was studied SNPs ofABCB1 including C3435T SNP and C1236T SNP werebelieved to have eï¬ects on the expression of Pglycoprotein[] Gervasini speculate the C1236T SNP of ABCB1aï¬ects the administered doses of MTX and the incidence ofhematological toxicity [] However just like G80A SNPthere are disputes about the uences of these SNPs asdiï¬erent studies had diï¬erent outcomes []Metabolizing enzymes were also being analyzed giventhe critical role of transporters in disposition of MTX and itsactive products as well as the folate metabolism MTXpharmacogenetics mostly focused on the SNPs in theMTHFR gene e present study shows that genetic polymorphisms in the folate metabolic pathway and in MTXtransporters uence the toxicity but not the eï¬cacy of thelowdose MTX treatment in patients with autoimmunediseases [] For example C677T and A1298C are knownin MTHFR gene to result in a lower enzyme activity []Windsor and associates reported that MTHFR A1298C andC677T were associated with MTX related nephrotoxicityand anemia [] ese SNPs might be associated withdecreased activity of methylenetetrahydrofolate reductaseelevated plasma homocysteine levels and altered distribution of folate [] us patients with this genotype weremore vulnerable to potential MTX induced toxicity sincethese reactions above may lead to slower folate metabolismand slower cell repair [] Weisman used univariatelogistic regression to reveal that the MTHFR C677T alsoincreases the occurrence of side eï¬ects in central nervoussystem manifested as headache and lethargy [] HoweverLambrecht argued that MTHFR C667T was not apredictive factor for toxicity [] Berkani found noassociation between A1298C polymorphism and MTXtoxicity [] Interestingly Grabar claimed that thepatients with MTHFR 1298C genotype have a lower risk forMTX toxicity than the carriers of MTHFR 1298A allele []To date the study of pharmacogenetics of MTX continues An increasing number of SNPs have been found to bepossibly associated with the eï¬cacy and toxicity of MTXe newly discovered genotypes include C347G in ATICand ²UTR 28bp repeat and ²UTR 6bp deletion inTYMS which may uence both eï¬cacy and toxicity ofMTX similarly factors that may aï¬ect MTX associatedtoxicity are for example A2756G in MS and A66G in MTRR[ ] e genes and their SNPs that might beassociated with the eï¬ects and side eï¬ects of MTX aresummarized in Table Growing evidences suggest that asingle genetic factor is unlikely to adequately predict theeï¬cacy and toxicity of MTX in polygenic disease such as RAand autoimmune associated ocular disease Given the impactof MTX in several metabolic pathways a complex of multiple risk genotypes examination would help to predict theeï¬cacy of MTX and to identify patients who may haveadverse eï¬ects from MTX administrationTaken together the eï¬cacy and toxicity of MTX mayremain associated with the genetic markers in the patientserefore although this remains a controversial subject it isreasonable to believe that pharmacogenetics may be able topredict who is at risk of MTX associated adverse eï¬ects andmay help in maximizing the beneï¬trisk ratio of MTX The Side Effects of MTXe doselimiting toxicity of MTX mainly includes hepatotoxicity and nephrotoxicity [] but mortality hasoften been reported due to either pneumonitis or secondaryinfections [] 0cJournal of OphthalmologyTable Summary of genes and their SNPs which might have possible clinical eï¬ects towards MTXTransportingproteinsABCfamilyGeneRFC1 []ABCB1 [ ]ABCC1 []ABCC2 []ABCC4 []MetabolizingenzymesMTHFR []ATIC []TYMS [ ]GGH [ ]DHFR [ ]MS [ ]MTRR [ ]SNPsG80AC3435TC1236Trs246240Srs3784862A2412GG1249AG1058AC934AC677TA1298CC347G²UTR 28bp²UTR 6bprepeatdeletionC452TC401TT721AC830TA2756GA66GSome experts divided MTX associated pulmonarycomplications into ammatory infectious and lymphoproliferative [] In the authors opinion all MTX relatedside eï¬ects can be classiï¬ed into these three categoriesaccording to the pharmacological eï¬ects of MTXMajor adverse events for MTX are related to the folateantagonism and primarily aï¬ect highly proliferative tissuessuch as bone marrow and gastrointestinal mucosa []Given the immunosuppression eï¬ect of MTX pancyt iawas one of the most frequent severe toxicities of methotrexate [] Meanwhile the risk of developing an infectiousprocess is increased all along the treatment and the severityof the infected disease would be worsen [ ] includingcommon bacterial infections herpes zoster eruptions andopportunistic infections According to previous studies therisk is larger than that with other disease modifying antirheumatic nonbiological drugs DMARDsSecondly the MTX acts as the hapten [] and is likely toreact directly with nucleophilic groups present in proteins ieto combine with endogenous protein [ ] e proteinadducts thus act as an antigenic signal to direct the eï¬ector armof the immune response [] e provoked immune responsesare most commonly type I immediate hypersensitivity andtype III immune complex reactions [] Hypersensitivitypneumonitis is the most common severe and unpredictablecomplication with a mortality of up to almost []Moreover a few studies have shown that longterm MTXuse can lead to lymphoproliferative disorders LPDs in bothnodal sites and extra nodal sites such as the skin lungsepipharynx thyroid gland nasal cavity spleen and kidneysespecially for patients who are positive for EBV infection[] e reported frequency of EBV positive in MTXassociated LPDs patients is [] Although themechanism of onset is not fully understood it is believedPossible clinical eï¬ectsIncreasingdecreasing intracellular MTX levelAï¬ecting eï¬cacy of MTXAï¬ecting the distribution of MTX and incidence of hematological toxicityAssociation with MTX related toxicityLeading to accumulation of MTX to nephrotoxic levelsAssociation with MTX related gastrointestinal toxicityAssociation with MTX related hepatotoxicityAssociation with MTX related hematological toxicityAï¬ecting the toxicity but not the eï¬cacy by resulting in a lower enzymeactivity association with related nephrotoxicity anemia and neurologicside eï¬ectsAï¬ecting eï¬cacy and toxicity of MTXAï¬ecting eï¬cacy and toxicity of MTXAï¬ecting eï¬cacy of MTXAï¬ecting eï¬cacy of MTXAï¬ecting eï¬cacy of MTXAssociation with MTX associated toxicityAssociation with MTX associated toxicitythat the combination of immunodeï¬ciency and the immunosuppressive eï¬ect of MTX has been implicated in thepathogenesis of MTX associated LPDs e World HealthOrganization WHO has classiï¬ed MTX associated LPDs aslymphoid neoplasms whether iatrogenic or immunodeï¬ciency associated diseases [ ] MTX associated LPDsoften take a spontaneous remission which tends to completemostly within weeks after the discontinuation of MTX[] But there are a few reports showing that the lymphoidneoplasms occur even after stopping using MTX [] e Eï¬ects of Administration Routes Generally the sideeï¬ects of MTX depend on the route of administration Dosedependent [] gastrointestinal side eï¬ects are the mostfrequent side events with orally administered MTX as oraladministration is the most common delivery method[ ] More than of MTX is excreted by therenal system thus MTX associated nephrotoxicity is common among patients taking MTX Fortunately the resolution usually occurs after discontinuation of therapy andsalvage treatment with highdose corticosteroids[]erefore to achieve treatment with less side eï¬ects theappropriate route of administration and dose of MTX arenecessary During the treatment monitoring of patientsgeneral condition mattersAdverse eï¬ects of intravitreal injections of MTX occuronly within the eyeincluding hyperemia keratopathycataract iridocyclitis vitreous hemorrhage retinal detachment maculopathy and endophthalmitis []Splitting doses of MTX rather than intravenous administration is a new attempt to avoid MTX associated sideeï¬ects MTX is split and given twice or thrice in a week toachieve higher bioavailability and better clinical response 0cJournal of Ophthalmology[ ] thus providing us with a novel method of oraladministration of MTX with less adverse eï¬ects Is LowDose MTX Safer Based on clinical cases observation side eï¬ects which can lead to discontinuation ofMTX are rare during the typical ophthalmology treatmentbecause of the lower dose of MTX required [] e application of lowdose MTX regimen has also become one ofthe main therapies of a variety of immunemediated diseasesbecause of its eï¬cacy and an acceptable safety proï¬le asmost lowdose MTX associated toxicity has been describedin case reports and relatively small case series []However although welltolerated and mostly reversibleeven a lowdose regimen of MTX can result in clinicallysignificant toxicity with substantial death rates about according to Kivitys cohort study [] e lowdose MTXassociated severe adverse eï¬ects include major centralnervous system complications [] mucositis pulmonaryinvolvement hepatotoxicity [] and myelosuppression Is MTX Safe to the Pregnant and Fetuses As one of thelipidsoluble and low molecular weight drugs MTX could bereadily transferred across the placental membrane duringpregnancy and adversely aï¬ect the fetus [] In addition MTXmight take longer time for elimination in fetal tissues []Regarding pharmacogenetics mutations caused by MTXlead to severe decrease of the expression of folate and nucleobaseenzymes which are critical for cellular homeostasis [] Inpractice MTX aï¬ected formation of the blastocyst and causeddysmorphic features and neurologic defects in early pregnancyleading to malformations in some cases [] Multiple congenitalabnormalities have been observed after weekly MTX treatmentsat a mg dose during the ï¬rst months of pregnancy [] evenfetal death [] Verberne had reviewed cases of congenitalanomalies after in utero exposure to MTX and proved that somecongenital anomalies such as microcephaly craniosynostosistetralogy of Fallot pulmonary valve atresia limb reductiondefects and syndactyly were truly part of the fetal methotrexatesyndrome [] Administration of MTX in childhood mightalso cause manifestations including visual defect [] andSmithMagenis syndrome [] among patients with speciï¬cmutations us special care should be taken with pregnantpatients and children in particular e Risk Factors of MTX Associated Side Eï¬ects e mostcommon risk facts of MTX induced adverse eï¬ects areadvanced age age years and underlying disease incaused by MTX administration with100 g alcohol concluding renal andor hepatic insuï¬ciency and lung diseaseespecially patients with chronic hepatitis B and diabetesmellitus [] Patients with a history of alcohol intakemight have a greater risk of liver ï¬brosis and hepatotoxicitysumption per week [] Also preexisting hypoalbuminemiaand past use of any of the DMARDs and protonpumpinhibitors have been described in studies to increase theincidence of MTX induced side eï¬ects [ ] Moreovertaking drugs that may interact with MTX at the same timemight also be dangerous these drugs include salicylatescotrimoxazole chloramphenicol sulfonamides cyclosporine and pyrimethamine [] Although no significantprotective eï¬ect of folate supplementation on MTX relatedtoxicity has been found [] the folate deï¬ciency is anotherreason for the side eï¬ects based on clinical cases []Heidari found that MTX administration elevatedkidney ROS levels decreased tissue antioxidant capacityincreased lipid peroxidation and depleted renal glutathionestores eir research data indicate that MTX caused tissuedamage and organ dysfunction through oxidative stresserefore they proposed that patients with preexistingmitochondrial defects might be vulnerable to MTX inducedrenal injury []e use of highdose MTX HDMTX is also the riskfactor of adverse eï¬ects MTX induced liver ï¬brosis is morelikely to become morphologically evident with high cumulative doses possibly largely exceeding to mg[ ] and the side eï¬ects caused by omeprazole use inthe past were found in cancer patients receiving HDMTXtreatment []e distribution of MTX in vivo also plays a role in MTXrelated side eï¬ects As MTX tends to accumulate in theextravascular compartment patients with pleural eï¬usionascites and massive edema should get extra caution due tothe risk of toxicity from reabsorption of extravascular ï¬uid[]Another noteworthy risk factor is UV UV recall phenomenon also known as MTX associated UV reactivationhas been reported [ ] It is reactivity of sunburn areaswithin to days of the treatment with MTX [ ]According to Adams and associates this phenomenon mightbe due to the immune response by uncontrolled sunburninduced ammation released by MTX [] Patients whopreviously suï¬ered sunburns deserve more detailed monitoring when methotrexate is needed Is Folate Supplementation Necessary for OphthalmicPatients To prevent MTX associated side eï¬ectsit iscommon to take folate [as either folic acid FA or folinicacid FLA] in clinic [ ] However there is noconsistent and evidencebased guideline for folate supplementation in ophthalmic patientsFolate and folic acid play significant roles in the de novosynthesis of purines and thymidylate which are required forDNA replication and repair [] Funk and associates founda significant reduction of circulating folate concentrations in of patients receiving MTX treatment [] Patientstreated with highdose MTX HDMTX got routine folatesupplementation to reduce HDMTX associated side eï¬ects[] After a systematic literature review of HDMTXtherapy and folate supplementation Van der Beek et al[] found lower incidence of MTX associated adverseeï¬ects in regimens with higher cumulative doses and earlieradministration of folate supplementation in similar HDMTX dosage studies Folate supplementation in patientswith lowdose methotrexate is also being studied Ortiz et al[] had proved the protectivefolateeï¬ectof 0cJournal of Ophthalmologysupplementation by conducting a Cochrane review including more than patients taking lowdose MTX Untilnow folate supplementation had been proved to prevent andimprove MTX associated eï¬ects including gastrointestinalrespiratory and neurologic side eï¬ects [ ] Mori et alsupported the protective eï¬ect by demonstrating that patients treated with lowdose MTX without folate supplements were significantly associated with the development ofmyelosuppression and pancyt ia []However Arabelovic and associates preliminary studyshowed a significant increase of MTX dose needed []since folic acid fortiï¬cation enriched cereal grain productswere fully implemented in the USA and Canada [] isconveyed a message to us that high dose of folate supplementation might have uence on the eï¬cacy of MTXAlDabagh found that the reduction in eï¬cacy ofMTX cannot be ignored while folate supplementation didmake a significant reduction in associated adverse eï¬ects[] Salim declared the decreasing uence betweenthe antiammatory eï¬ect of MTX and folate supplementation by carrying out a doubleblind clinical trial []Chladek had conducted an labeltwowaycrossover study supporting the opinion above [] Additionally because of the unequal distribution of folic acidand MTX in organs and tissues [] MTX discontinuationis more common for some MTX associated side eï¬ects inophthalmic clinic [] rather than higher dosage of folatesupplementationere are no ophthalmic studies to demonstrate theprotective eï¬ects of folic acid supplementation us although the folate supplementation is widely used amongpatients treated with lowdose MTX [ ] the necessityand standardized dosage of folate supplementation in speciï¬c patients [] as well as the MTXfolate interactionstill warrant further studies DiscussionMethotrexate as one of the alternative pharmacologicalsteroidsparing immunosuppressive agentsis becomingmore and more popular as the preferred treatment in severalautoimmune conditions requiring longterm immunosuppression [] Lowdose MTX has antiammatory andimmunomodulatory properties by increasing levels of intracellular and extracellular adenosine [] which is thefoundation of ophthalmic MTX treatment e standardizedand recommended administration of ophthalmic MTXtreatment is once a week starting with a dose of mg andescalating every to weeks up to mgweek whennecessary [ ] In patients with insuï¬cient response toMTX alone cyclosporin with or without azathioprine wasadded []To avoid side eï¬ects split doses of MTX administrationand folate supplementation are gradually being used inophthalmic clinic Prescription of to mg of folatesupplementation has a significant role in MTX safety []but the higher dosage is less applied even with higher dose ofMTX [] Prophylactic folate supplementation is notnecessary in most patients [] ere is also research toconvey that ml100 g or above dosage of ï¬sh oil is aseï¬ective as folinic acid in therapeutic potential in preventingbone loss during MTX chemotherapy [] For some resistant andor mortal adverse eï¬ects the discontinuation ofMTX will work instantlyWith the increasing longterm use of MTX it is importantto monitor patients blood examination results including bloodroutine and liver and renal functions As pancyt ia can be alate manifestation [] elevation of urea creatinine aminotransferases and albumin as well as electrolytes disturbancesmay result in MTX associated liver and renal side eï¬ects []Plasma MTX level is not a reliable predictor for adverse eventsin MTX therapy [] On the contrary circulating folate levelsand folate polyglutamate distribution change sensitively withMTX exposure and exogenous folate supply [] and could beused as a biomarker of MTX eï¬cacy [] It should be notedthat as erythrocytes have a halflife of approximately daysthe results of blood examinations might reï¬ect both pretreatment and posttreatment status which need to be analyzedcarefully []Numerous studies had been conducted to prove thatMTX could be used as a welltolerated safe and eï¬ectiveï¬rstline treatment Hence the MTX administration shouldnot continue to be stigmatized as a cancer drug or to bediscouraged because of associated adverse eï¬ects Contrarily the indication and the routes of administration areabout to gradually widenConflicts of Intereste authors declare that they have no conï¬icts of interestReferences[] R Q H Kloos R Pieters C van den Bos e eï¬ect ofasparaginase therapy on methotrexate toxicity and eï¬cacy inchildren with acute lymphoblastic leukemia Leukemia Lymphoma vol no pp [] R K Bath N K Brar F A Forouhar and G Y Wu Areview of methotrexateassociated hepatotoxicity Journal ofDigestive Diseases vol no pp [] W Wang H Zhou and L Liu Side eï¬ects of methotrexatetherapy for rheumatoid arthritis a systematic review European Journal of Medicinal Chemistry vol pp [] J Smolen and R Landew´e EULAR recommendations forthe management of rheumatoid arthritis with synthetic andbiological diseasemodifying antirheumatic drugs update Annals of the Rheumatic Diseases vol no pp [] J A Singh K G Saag S L Bridges Americancollege of rheumatology guideline for the treatment ofrheumatoid arthritis Arthritis Rheumatology vol no pp [] M Holdhoï¬ P Ambady A Abdelaziz Highdosemethotrexate with or without rituximab in newly diagnosedprimary CNS lymphoma Neurology vol no pp [] J Peer J M Rowe S Frenkel and E J Dann Testicularlymphoma intraocularvitreoretinal lymphoma and brainlymphoma involvement of three immunoprivileged sites in 0cJournal of Ophthalmologyone patient American Journal of Hematology vol no pp [] S Gangaputra C W Newcomb T L Liesegang et alSystemic immunosuppressive therapy for eye diseases cohort study methotrexate for ocular ammatory diseasesOphthalmology vol no pp [] P W Hardwig J S Pulido J C Erie K H Baratz andH Buettner Intraocular methotrexate in ocular diseasesother than primary central nervous system lymphomaAmerican Journal of Ophthalmology vol no pp [] E Esterberg and N R Acharya Corticosteroidsparingtherapy practice patterns among uveitis specialists Journalof Ophthalmic Inï¬ammation and Infection vol no pp [] K Durrani F R Zakka M Ahmed M MemonS S Siddique and C S Foster Systemic therapy withconventional and novel immunomodulatory agents for ocular ammatory disease Survey of Ophthalmology vol no pp [] S S Gangaputra C W Newcomb M M Joï¬e et alComparison between methotrexate and mycophenolatemofetil monotherapy for the control of noninfectious ocularammatory diseases American Journal of Ophthalmologyvol pp [] V K Ayuso E L van de Winkel A Rothova and J Helenade Boer Relapse rate of uveitis postmethotrexate treatmentin juvenile idiopathic arthritis American Journal of Ophthalmology vol no pp [] S R Rathinam M Babu R undikandy A randomized clinical trial comparing methotrexate and mycophenolate mofetil for noninfectious uveitis Ophthalmologyvol no pp [] A Galor D A Jabs H A Leder Comparison ofantimetabolite drugs as corticosteroidsparing therapy forammation Ophthalmologynoninfectiousvol no pp ocular[] M D de Smet V S Vancs D Kohler D Solomon andC C Chan Intravitreal chemotherapy for the treatment ofrecurrent intraocular lymphoma British Journal of Ophthalmology vol no pp [] E Kim C Kim J Lee and Y Cho A case of primaryintraocular lymphoma treated by intravitreal methotrexateKorean Journal of Ophthalmology vol no pp [] J Smith J T Rosenbaum D J Wilson Role ofintravitreal methotrexate in the management of primarycentral nervous system lymphoma with ocular involvementhistorical image Ophthalmology vol no pp [] CC Chan and D J Wallace Intraocular lymphomaupdate on diagnosis and management Cancer Controlvol no pp [] K L Larkin U S Saboo G M Comer Use ofintravitreal rituximab for treatment of vitreoretinallymphoma British Journal of Ophthalmology vol no pp [] C P Fox E H Phillips J Smith Guidelines for thediagnosis and management of primary central nervoussystem diï¬use large Bcell lymphoma British Journal ofHaematology vol no pp [] A Sadaka R Sisk J Osher O Toygar M Duncan andinfusion forIntravitreal methotrexateC Riemannproliferative vitreoretinopathy Clinical Ophthalmologyvol pp [] N G Lambert DJ Wilson D M Albert andW D Chamberlain Intravitreal methotrexate for recurrentepithelial downgrowth JAMA Ophthalmology vol no p [] A M Joussen F E Kruse HE V¨olcker and B KirchhofTopical application of methotrexate for inhibition of cornealangiogenesis Graefes Archive for Clinical and ExperimentalOphthalmology vol no pp [] S K Kurup C Gee and C M Greven Intravitrealmethotrexate in therapeutically resistant exudative agerelated macular degeneration Acta Ophthalmologica vol no pp e145e146 [] H E M Khalil H A Raafat N A Azab H E Haroun andH A Elgendi e role of intraocular methotrexate in themanagement of uveitis and posterior segment involvementin Behçets disease patients e Egyptian Rheumatologistvol no pp [] S R J Taylor A Banker A Schlaen Intraocularmethotrexate can induce extended remission in some patients in noninfectious uveitis Re
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mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5
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low levels of physical activity pa and high levels of physical inactivity pi are associatedwith higher mortality and cardiovascular diseases higher age is associated with a decreaseof pa only yearolds achieve the pa times recommended by who theaim of this study was to identify levels of and determinants for moderate pa overall pa andpi in a sample of individuals aged � yearsmethodswe analyzed baseline data from an interventionstudy aiming to increase pa and decreasepi by automatically generated feedback letters to objectively measured pa and pi recruitment was multimodal including recontacting participants of previous studies and advertisements in regional public buses and newspapers at baseline participants wore anaccelerometer over a period of consecutive days pa was categorized using cutpointssuggested by freedsoon in light moderate and vigorous physical intensity as well asphysical inactivity potential determinants selfefficacy education were measured byquestionnaires or in a physical examination bmi multiple linear regression models were fitted to identify determinants for pa and piresultsn persons mean age years sd female participated in the studythe weekly amount of overall pa for men was on average minutes sd forwomen on average minutes sd of the women and of the menachieved the who recommendation of minutes moderate paday at baseline the timeof pi during the observation time period of days was on average minutes in men anda1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation kleinke f penndorf p ulbricht s do¨rr mhoffmann w van den berg n levels of anddeterminants for physical activity and physicalinactivity in a group of healthy elderly people ingermany baseline results of the movingstudy one e0237495 101371 pone0237495editor thalia fernandez universidad nacionalautonoma de mexico mexicoreceived november accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237495copyright kleinke this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0237495 august one 0cfunding the study was funded by the federalministry of education and research bmbf as asite project of the german centre forcardiovascular research dzhk funding sign81z7400174 the funders have had no influenceon the conceptualization and conduct of the studyand will not have any role in the data analysis andpublication of the resultscompeting interests the authors have declaredthat no competing interests existlevels of and determinants for physical activity and physical inactivity minutes in women in males age was found to be a significant negative determinantfor overall pa p and for moderate pa p0001 higher education was positivelyassociated with higher levels of overall pa p and moderate pa p in menbmi was a significant negative determinant for overall pa both in men p andwomen p as well as for moderate pa for women p only in women not inmen selfefficacy was to be a significant positive determinant for overall pa p aswell as negatively associated with pi p discussionthe participants of our study showed high levels of pa this is likely due to selection bias inthis convenience sample however also levels of pi are very high and those correspondwith average levels in the german population the determinants for higher pa and lower pidiffered between males and females thus strategies for improving pa and decrease pi arelikely different with respect to sex and should take individual factors eg age bmi intoaccounttrial registration numberdrks00010410 date may introductionphysical activity pa is defined as any bodily movement produced by skeletal muscles thatrequires energy expenditure there is strong evidence that regular pa is a very effectivenonpharmacological and noninvasive healthpromoting method [] a papromoting lifestyle is associated with a reduced risk of mortality and is correlated with improved overallhealth status additionally high levels of pa significantly decreases overall mortality by and cvd mortality by recent reviews showed that regular pa is alsoassociated with a reduced risk of developing dementia in older adults [ ] and is considered as one of the proxies of the concept of cognitive reserve one of the two most significant modifiable risk factors for dementia is pi additionally pa in particular aerobicexercise is positively associated with a less agerelated reduction of anic brain matter international pa guidelines recommend for healthy individuals aged over years at least minutesweek of moderate pa or at least minutesweek of vigorous pa or an equivalent combination of weekly pa in fact there is no difference to recommendations for healthyyounger individuals aged years pa should be performed in uninterrupted time periodsbouts of a duration of at least minutes [ ]data from the united kingdom show that of men and of women aged years reach the recommended level of pa a study from norway showed that of menand of women aged years meet the recommendations in the age group years only of women and of men reach the whorecommendations in the usanational health and nutrition survey nhanes the proportion of people aged over years who achieved the recommended amount of pa was in germany only of men and of women aged years achieve the who recommendations regardingpa selfreported via questionnaire in the age group years this proportion declines to in men and to in women one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivityin europe in of the total population was years or older in germany of the population was years or older in this proportion will furtherincrease to until older age is associated with an higher risk for chronic diseasesmultimorbidity [] prevalences of cvd including coronary heart disease chd andstroke will increaselow pa is one of the leading risk factors for global mortality globally of theadults are insufficiently physically active a reduction of the prevalence of insufficient pais a global target of the who [ ] physical inactivity pi defined as any waking behaviour characterized by an energy expenditure � mets metabolic equivalent of task whilein a sitting or reclining posture sedentary behaviour research network tremblay causes million deaths per year worldwide and in was estimated to beresponsible for million dalys disabilityadjusted life years globally besides lowlevels of pa also pi is a crucial risk factor for mortality [ ] frequently interrupted piis associated with positive effects on health status and a reduced risk for premature death lack of pa and a high level of pi are associated with an array of noncommunicable diseasesncd and eg responsible for approximately of breast and colon cancers of diabetes cases and approximately of ischaemic heart disease prevalencesworld healthanization additionally it seems that physical inactivity is an important preventablefactor for alzheimers dementia older people spent on average to hours a day sedentary which correspondents with of their waking time depending on the exact definition the distrubution of pi acrosseuropean countries ranged from in schweden up to in portugal in germany of men and of women showed a sedentary lifestyle low energy expenditure of theleisuretime expenditure in activities requiring � metabolic equivalents met the amount of pa and pi depends on individual factors such as age bmi gender education social status and selfefficacy [] additionally environmental and policy factors weather conditions and length of the day have an effect on the amount of pa of people[ ]overweight and obesity bmi30 kgm2 have a negative influence on the level of pa andpi in the elderly in a crosssectional study in which subjects were surveyed from member states of the european union it was found that people with a low bmi kgm2and normal bmi kgm2 have low prevalence in pi both genders in contrast peoplewith a bmi above kgm2 showed a more sedentary lifestyle [ ]higher education has a positive influence on pa and pi varo eu study showed thatpeople with primary level education were more sedentary than participants with higher levelsof education greater difference in women further factors that influence the amount of pa and pi are marital status income wellbeing psychosocial variables such as selfefficacy and social and cultural parameters [ ]a report from the who about the prevention of noncommunicablediseases ncd insoutheastern european countries showed that the promotion of pa and reduction of pi arekey aspects in public health efforts promoting physical activity through mass media was a primary goal for immediate action in addition the global strategy on diet physical activityon health and the european charter on counteracting obesity underline the relevance of pa to fight against obesityto develop effective prevention strategies adapted to the elderly detailed information onthe levels of pa and pi and their determinants are necessary in this analysis we assessed thelevels of pa and pi and identified positive and negative determinants for pa and pi in a sample of healthy people aged � years one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitymaterials and methodsthe data for this analysis were retrieved from the baseline assessment of an intervention studywith the goal to increase pa and reduce pi in elderly people with a lowthreshold interventionmovingmotivation oriented intervention study for the elderly in greifswald thisstudy is a twoarm randomized controlled trial consisting of assessment of eligibility baselineexamination randomization intervention only the participants in the intervention groupand followup examinations at and months after baseline the study region was western pomerania a rural area in the northeast of germanystudy participants in the intervention group received two individual feedback letters containing objectively measured pa and pi times based on data from the accelerometer devicecaptured at baseline and months followup measurement feedbackletters were automatically generated in r software version or later lucent technologies murray hill njusaa comprehensive description of the study protocol for the moving study is publishedelsewhere inclusion of the participantsthe study participants had to meet the following inclusion criteria¢ age � years¢ ability to be physically active in daily lifeexclusion criteria were¢ inability to walk independently eg permanent use of a wheelchair¢ simultaneous participation in other studies addressing pa or pi¢ not accessible by telephone or cell phone¢ already fulfilling the who recommendations for pa selfreported for people � yearsat baseline � minutes moderate pa per weekrecruitment was performed in in several ways¢ recontacting participants of a previously performed study ¢ recruitment at venues frequented by older people eg senior sport hours in the publicswimming pool rehearsals of senior choirs events at meeting places for elderly people¢ persons contacted the study centre after reading s about the project in regional newspapers and television advertisements in regional buses and flyers and posters that were distributed in gp practices hospitals and meeting centers for elderly peopleall participants were informed in detail about the study and had to give their writteninformed consentmeasuresall study participants received a baseline examination consisting of the assessment of bloodpressure somatometry data body weight waist and hip circumference as well as sociodemographics sex age education job and partnership status general health status the ssa scaleselfefficacy towards physical exercise was used to asses the participants level of selfefficacy one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitythe result of that scale is a sum in which higher values indicate higher selfefficacy towards pa after that the study participants received an accelerometer device actigraph gt3xbtpensacola fl usa which captures and records pa and pi continuously at a sampling frequencyof hz over a period of seven consecutive days starting at midnight after the baseline examination participants were instructed to wear the accelerometer device for seven days only during daytime on the right hip and to remove it only at bedtime and for waterbased activities egshowering swimming in addition all study participants were asked to document their physicalactivities in a semistandardized protocol for each day of the observation time besides the objective assessment of pa and pi the participants were instructed to answer paperbased questionnaires to assess selfreported pa and pi with regard to the observation period of the accelerometerto asses selfreported pa the international physical activity questionnaire short formipaqsf in german version was used the ipaq consists of seven items addressing intensity and duration of pa in daily life over the last days by selfreport in addition the german physical activity questionnaire for german paq was used to assess type andduration of pa in daily life by selfreport pi was assessed by the measure of older adultssedentary time most questionnaire in the german version body mass index was calculated from measured body height and body weight kgm2 � kgm2 and kgm2 � kgm2after seven days the participants had to bring the accelerometer device and the fulfilledquestionnaires back to the examination centerdata analysisdata assessment and documentation were conducted based on ecrfs in an itsupported documentation system which is characterized by an independent operation synchronization andmonitoring the software is based on the concept of offline clients and each staff member ofthe dzhk had individual login data the paperbased questionnaires were documented using the software cardiff teleform1electric paper lu¨neburg germany all questionnaires and the daily physical activity protocol contained 1d barcodes to ensure anonymization of the study participantsthe actilife software versions to actigraph cop pensacola fl usawas used to download the pa data from the accelerometer the raw data were calculated into10second epochs and saved in raw format as gt3x files a valid measurement day wasdefined as a record of at least hours of total wearing time measurements of at least validdays were required to be included into data analysis to categorize pa intensity we used specific cut points based on freedson pa intensity was divided into sedentary counts light counts moderate counts and vigorous counts pa nonwearing time was defined as having � minutes of continual zero countsrange � minutes between and countsdescriptive statistics were used to describe the population with respect to the levels of paand pi to identify determinants of pa and pi multiple linear regression models were calculated dependent variables for the regression models were overall and moderate pa as wellas pi the effect of the independent variables age bmi education and selfefficacy was examined for all regression models all independent variables were checked for multicollinearityusing a correlation pearson to include education as a determinant in the multiple linearregression models we used dummy variables referring to the gauÃmarkovtheorem we analyzed the residuals and requirements of multiple linear regression like distribution andhomoscedasticity one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitystatistical significance was assumed for pvalues data processing and statistical calculations were performed with ibm spss statistics or later by ibm corp armonk new york usa all statistical analysis were performed based on pseudonymizeddata all identifying data were separated from the project data to the earliest possible timepoint all appointments were ensured facetoface by the study staffethicsthe study was approved by the ethics committee of the university medicine greifswaldethic approval protocol number bb07116 and registered at the german clinical trials register id drks00010410resultsof screended study participants could be included in the analyses fig fig number of recruited participants and participants included in the analysis101371 pone0237495g001 one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable descriptive characteristics of the study sample n characteristicssex womenage yreducation yrbody mass index kgm2 years years years � � currently smoking yesnumber of participants currently having a partnership yeswearing timen number of subjects sd standard deviation101371 pone0237495t001nmean sd or n sd sd participants were included in the analysis thereof women and men the mean age was years sd at the baseline measurement of pa participants had a mean wearing time of the accelerometer of minutes sd per weekwhich corresponds to an average of hoursday table overall there were no significant difference between women and men according tooverall pa mean women minutes mean men minutes per week tdf p men in higher age groups showed lower levels of weekly light pa themean number of minutes of moderate pa increased with higher education in men and inwomen both men and women with higher bmi showed lower levels of moderate and vigorouspa table male participants spent of their waking time in pi female participants olderage was associated with an increasing time of pi in men the level of pi increased with age yr minutes minutes and with higher education yr minutes yr minutes in women the time of pi was largely independent of age yr minutes minutes table n of the men and n of the women achieved the international recommendations for moderate pa � minutes moderate pa the proportion of participantswho fulfilled the recommendations decreased with age table a multiple linear regression was calculated to predict overall pa based on independent variables in table in men lower age p a lower bmi p and higher educationp were found to be significant positive determinants for overall pa higher bmi is anegative determinant for overall pa in women p additionally selfefficacy wasfound to be a significant positive determinant for overall pa p the overall model fitwas r2 for men and r2 for women table for moderate pa higher age p0001 was found to be a significant negative determinantin men in women a higher bmi was a significant negative determinant p for maleparticipants lower education yr was a significant negative determinant p aswell as years of education p table in women better selfefficacy was found to be a significant positive determinant p for pi table one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable mean duration of weekly pa [min] without specific cut points by intensity of pa and overall pamean duration of weekly [min]mean duration of weeklylight pa ci[min] moderate pa cimean duration of weekly[min] vigorous pa mean duration of weekly [min]overall pa cicimalefemalemalefemalemalefemalemalefemaleage yr education yr bmi kgm2 � � currently having apartnership yesyes no total m mean ci confidence interval min minutes101371 pone0237495t002discussionthe results of this analysis show that the levels of light moderate and overall pa in our sampleof older people are high of the male and of the female study participants fulfilledthe age specific whorecommendations for moderate pa for people aged over years� minutes moderate pa or � minutes vigorous pa especially female participants inthe age group years were above average physically active in people in the same age allwomen in this age group n reached the recommendations for moderate pathe study results show that age is a significant negative determinant for moderate and overall pa in men it can be concluded that men become more physically inactive with age this isin contrast to other studies in which women are generally less physically active than their malecounterparts [ ] this finding can also be explained by the fact that women in our sample in particular were physically active to an aboveaverage extenteducation was found to be a significant positive determinant regarding moderate pa inmen and women and overall pa in men these results are consistent with other studies people one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable mean duration of weekly pi [min] proportion of sedentary time of total wake time in minutesmean min of weekly pi cimean proportion of daily pi daytime in malefemalemalecifemaleage yr education yr bmi kgm2 � � currently having a partnership yesyes no total ci confidence interval min minutes101371 pone0237495t003 716s with higher education tend to be more physically active and generally pay more attention totheir personal health [ ]bmi was found to be a significant determinant for moderate pa in women additionallyhigher bmi in men and women was statistically significantly associated with a decreasing levelof overall pa self efficacy was a significant negative determinant for pi only in women thusfemales with higher self efficacy showed lower levels of pi there was no systematic differencebetween the gender self efficacy was the only statistically significant predictor for pi thusmotivation can be seen as an important factor to reduce piour study participants spent most of their waking time in pi men spent and female per day in pi which correspondends to results from other studies examining the sameage group [ ] in industrialized countries high pi values are therefore associated with an enormous burden on health systems table number and proportion of study participants who fulfilled the who recommendations for moderate pa separately for men and womenn of age group of all menn of age group of all womenmalefemaleage yr n n n n total n of of of of of of of of of of n number of subjects fulfillment of who recommendations for moderate pa for people aged and older � minutes moderate pa or � minutes vigorous pa oran equivalent combination per week101371 pone0237495t004 one 101371 pone0237495 august one 0ctable multiple linear regression model to identify determinants for overall pa separately for men and womenlevels of and determinants for physical activity and physical inactivitypredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci yr abitur qualification for the university otherselfefficacy β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t005p������female n adj r2 β ci p����the participants showed a very good adherence in wearing the accelerometer device themean wearing time per day was hours only of participants had to be excluded fromthe analysis because of insufficient wearing time days and 10h per day thus the intervention in our study can be seen as feasible and practicable for this age groupthe public health relevance of pa and pi is high due to the demographic changes maintaining high levels of pa over older ages will become even more important regular pa is positively associated with several physical health outcomes besides that high levels of pa havealso the potential to maintain cognitive health over the long term into higher ages [ ] ingeneral high levels of pa can reduce incidence of dementia and cognitive restrictions significantly norton has shown that changeable risk factors such as pa and pi are responsible for around a third of alzheimers disease worldwide in europe usa and uk physicalinactivity was the strongest risk factor table multiple linear regression model to identify determinants for moderate pa separately for men and womenpredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci yr abitur qualification for the university otherselfefficacy β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t006p�������female n adj r2 β ci p���� one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitytable multiple linear regression model to identify determinants for pi separately for men and womenpredictorsinterceptagebmieducationa yr yrmale n adj r2 β ci yr abitur qualification for the university otherselfefficacy β regression coefficient ci confidence interval��p001�p005a reference high school degree101371 pone0237495t007p��female n adj r2 β ci p����effective and practicable strategies to increase pa and decrease pi are needed especially forthe elderly a report from who for the european region mentions specific suggestions toincrease peoples physical activity eg by promotion of green spaces or cycle paths etc therefore to design scale and implement effective noncommunicable disease preventionprogrammes accurate and valid data on physical activity levels and on sedentary behavior areneeded as well as valid knowledge about significant determinants of both pa and piregular pa is a highly effective healthpromoting method with strong evidence [ ] international recommendations for pa are existing for all age groups [ ] but it stillremains under debate how people should accumulate their recommended time of pa over theweek in recent years the number of interventions targeting pi has increased however there isstill a lack of recommendations for pi in addition it is also not conclusively establishedwhether pi is an independent risk factor for chronic diseases in summary recent literature points out that public health activities should emphasise increasing pa at any intensityespecially in the elderly limitationsthis analysis is based on a convenience sample of participants we used a variety of samplingmethods including the possibility of selfrecruitment some of which have likely increased theproportion of participants with above average pa compared to pa at the population level weobserved high levels of pa particularly among the older age groups and females which indicate some selection biasin general the use of accelerometer devices for objective assessment of pa allows a validand reliable record of pa intensity frequency and duration but data from the accelerometerdevice can potentially differ from the real levels of pa and pi especially in the elderly becauseseveral activities are carried out in standing eg gardening or sedentary positions eg gymnastic on stools which as a consequence can not be assessed reliablyin general the explanatory value for both pa models are acceptable the explanatory valuefor our pi model is low thus research should focus on further environmental and interpersonal factors eg the walkability of neigbourhoods and attractive activities for seniors one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivityconclusionthe number of guidelines and recommendations in pa and pi increases continuously however there are still certain aspects especially of pi which can not described in an accurate wayto our knowledge we still know only little about the independent negative health effects ofsedentary behavior in general there is international consensus regarding recommendationsfor pa but recommendations about pi are still under debatebecause of demographic change and the associated increase of proportion of older peoplethe need of global prevention strategies is high simultaneously knowledge to improve ourunderstanding of pa and pi in the elderly prevalence of dementia and chronic diseases egcvd will probably increase in the next decades therefore the relevance of modifiable riskfactors as preventive measures will rise our analyses confirm that especially individual factors eg age sex has the largest impacton pa the results for pi are less clear thus pi may have other predictors another importantfinding of this study is that pa and pi can be seen as mostly independent factors of activityparticipants with a high level of pa showed also high levels of pi further research should paymore attention to effective predictors for the reduction of pi and should focus more on environmental and interpersonal factors especially in pisupporting informations1 file datasetxlsxauthor contributionsconceptualization fabian kleinke sabina ulbricht marcus do¨rr wolfgang hoffmannneeltje van den bergdata curation neeltje van den bergformal analysis fabian kleinkemethodology wolfgang hoffmann neeltje van den bergproject administration fabian kleinkesoftware peter penndorfsupervision neeltje van den bergwriting original draft fabian kleinkewriting review editing sabina ulbricht marcus do¨rr wolfgang hoffmann neeltje vanden bergreferences australien institute of health and welfare insufficient physical activity [internet] available fromwwwaihwgovaugetmedia44533aa45704447d8d2a4d68d9fa2416insufficient physicalactivitypdfaspxinline true miles l physical activity and health br nutr found nutr bull allender s foster c boxer a occupational and nonoccupational physical activity and the social determinants of physical activity results from the health survey for england j phys act health jan 101123jpah51104 pmid lewis ba napolitano ma buman mp williams dm nigg cr future directions in physical activityintervention research expanding our focus to sedentary behaviors technology and dissemination j one 101371 pone0237495 august one 0clevels of and determinants for physical activity and physical inactivitybehav med [internet] feb available from 101007s10865016 pmid lollgen h lollgen d [risk reduction in cardiovascular diseases by physical activity] internist berl jan ahlskog je geda ye graffradford nr petersen rc physical exercise as a preventive or diseasemodifying treatment of dementia and brain aging mayo clin proc sep 104065mcp20110252 pmid blondell sj hammersleymather r veerman jl does physical activity prevent cognitive decline anddementia a systematic review and metaanalysis of longitudinal studies bmc public health may 101186147124581451
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Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learnings groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center DiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patients life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images []Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined []Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the identityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called BONENAVI version The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed CADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing à convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the networks weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physicians scores Taking a closer lookat the results it can be concluded that networks performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physicians further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patients whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center Diagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatients cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution à pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center DiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patientscategory for example malignant_ and healthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value was assigned for malignant_ prefixes whereas the value for healthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Pythons randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po
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" adenovirus serotype ad5 is a commonly used viral vector for transient delivery of transgenes primarily for vaccination against pathogen and tumor antigens however endemic infections with ad5 produce virus specific neutralizing antibodies nabs that limit transgene delivery and constrain target directed immunity following exposure to ad5 based vaccines indeed clinical trials have revealed the limitations that virus specific nabs impose on the efficacy of ad5 based vaccines in that context the emerging focus on immunological approaches targeting cancer self antigens or neoepitopes underscores the unmet therapeutic need for more efficacious vaccine vectorsmethods here we evaluated the ability of a chimeric adenoviral vector ad5f35 derived from the capsid of ad5 and fiber of the rare adenovirus serotype ad35 to induce immune responses to the tumor associated antigen guanylyl cyclase c gucy2cresults in the absence of pre existing immunity to ad5 gucy2c specific t cell responses and antitumor efficacy induced by ad5f35 were comparable to ad5 in a mouse model of metastatic colorectal cancer furthermore like ad5 ad5f35 vector expressing gucy2c was safe and produced no toxicity in tissues with or without gucy2c expression importantly this chimeric vector resisted neutralization in ad5 immunized mice and by sera collected from patients with colorectal cancer naturally exposed to ad5s these data suggest that ad5f35 based vaccines targeting gucy2c or other tumor or pathogen antigens may produce clinically relevant immune responses in more ¥ patients compared with ad5 based vaccines inhibitor introductiontherapies immune checkpoint have revolutionized cancer treatment and cancer drug development by engaging the immune system to target various cancers1 despite this success many tumors are immunologically cold characterized by a dearth of immunogenic neoepitopes3 and lack of tumor infiltrating lymphocytes4 and remain refractory to checkpoint inhibitors6 one emerging strategy to modify a cold tumor into one responsive to immunotherapy is through combination with cancer vaccines8 the goal of this strategy is to use cancer vaccines to create a pool of tumor reactive t cells with antitumor activity alone andor in combination with checkpoint therapies however this approach is significantly limited by the paucity of effective vaccine platforms to safely deliver tumor specificassociated antigens to elicit beneficial antitumor immunitythe ability of adenovirus serotype ad5 to mediate gene transfer and induce potent immune responses has made it a popular vector for experimental vaccines infectious diseases10 against cancer and indeed there have been more than clinical trials using the ad5 vector with most trials focused on developing cancer treatments10 however on natural infection the host immune system develops neutralizing antibodies nabs to the ad5 capsid limiting viral spread and blocking reinfection because ad5 infections are endemic in many human populations pre existing nabs present in of the worldwide population limit ad5 based vaccine strategies12 these considerations highlight the need for improved vectors for use in vaccines targeting cancer and pathogen associated antigens that can create therapeutic immune responses in the greatest number of patients importantly while the adenovirus capsid is composed of hexon penton and fiber proteins nabs elicited by natural ad5 infection in humans are directed primarily to the ad5 fiber15 suggesting that strategies to flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access circumvent pre existing immunity to this element may improve ad5 based vaccineshere we sought to overcome pre existing ad5 nabs by replacing the ad5 fiber with that of a rare adenovirus serotype ad35 international seroprevalence to improve antitumor immunity in mouse models expressing the gastrointestinal gi cancer antigen guanylyl cyclase c gucy2c preclinical models demonstrated that an ad5 based gucy2c directed vaccine ad5 gucy2c s1 elicited cd8 t cell and antibody responses without autoimmunity17 further ad5 gucy2c s1 vaccination of mice induced long term t cell mediated protection against metastatic colorectal cancer in lung and liver19 moreover those results were recapitulated in a recent first in human phase i clinical trial nct01972737 demonstrating that a humanized version of the vaccine ad5 gucy2c padre safely induced gucy2c specific cd8 t cell responses in patients with colorectal cancer following conventional therapies21 however patients possessing high pre existing titers of nabs against ad5 failed to generate gucy2c specific immunity following ad5 gucy2c padre vaccination21 to overcome ad5 nabs we generated a chimeric ad5 vector possessing the fiber of ad35 ad5f35 with equivalent safety and antitumor activity to ad5 and resistance to ad5 nabs in mice and humans this chimeric vaccine can be translated to patients with gi cancer to safely induce gucy2c specific immunity not only in those patients with low ad5 immunity but also in those with high pre existing ad5 nabsmaterials and methodsadenovirus vectorsadenovirus containing mouse extracellular domain gucy2c1429 with the influenza ha107119 cd4 t cell epitope known as site s1 was described previously ad5 gucy2c s120 here gucy2c s1 was cloned into pshuttle and subcloned into the e1 region of previously generated replication deficient chimeric adenovirus ad5f35 in which the ad5 fiber was replaced by the ad35 fiber22 to generate ad5f35 gucy2c s1 all adenovirus vaccines used in this study were produced in hek293 cells and purified by cesium chloride ultracentrifugation under good laboratory practices by the baylor college of medicine in the cell and gene therapy vector development lab and certified to be negative for replication competent adenovirus mycoplasma and host cell dna contamination in vitro gucy2c expression experiments doseresponse and timecourse were carried out in a549 american type culture collection atcc cells virus was added to the cultures at the indicated doses and culture supernatants were collected at the indicated time points relative gucy2c levels were quantified in supernatants by western blot using μgml ms7 mouse anti gucy2c monoclonal antibody23 and μgml horseradish peroxidase conjugated goat antimouse secondary antibody jackson immunomice and immunizationseight week old male and female balbcj mice were purchased from the jackson laboratory for experiments animal protocols were approved by the thomas jefferson university institutional animal care and use committee protocol for immunizations mice received or vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or ad5f35 gfp control administered as two μl intramuscular injections one in each hind limb using a ml insulin syringequantifying tcell responses by elispotelispot assays were performed using a mouse interferonÎ ifnÎ single color elispot kit cellular technology according to the manufacturers protocol26 briefly well plates were coated with ifnÎ capture antibody overnight at °c the next day plates were washed with phosphate buffered saline pbs and splenocytes from immunized mice were plated at cellswell with no peptide or μgml gucy2c254262 peptide in dimethyl sulfoxide dmso in ctl test medium cellular technology for hours at °c for t cell avidity studies splenocytes were plated at cellswell with decreasing concentrations of gucy2c254262 peptide μgml to pgml normalized to cellswell26 after incubation cells were removed and development reagents were added to detect ifnÎproducing spot forming cells the number of spot forming cells per well was determined using the smartcount and autogate functions of an immunospot s6 universal analyzer cellular technology gucy2c specific responses were calculated by subtracting mean spot counts of dmso wells from peptide stimulated wells26 tumor studiesgucy2c expressing mouse balbc ct26 colorectal cancer cells were used for in vivo tumor studies17 luciferase expressing cells were generated by transduction with lentiviral supernatants produced by 293ft cells invitrogen with plenti4 v5 gw luciferase28 for tumor experiments balbcj mice were immunized with vp of ad5 gucy2c s1 ad5f35 gucy2c s1 or pbs control days before delivering à ct26 cells into tail veins tumor burden was quantified weekly by subcutaneous injection of mg of d luciferin potassium salt gold biotechnologies in pbs followed by an min incubation and imaging with a s exposure using a caliper ivis lumina xr imaging station perkinelmer total radiance photonssecond was measured using living image in vivo imaging software perkinelmerantibody neutralization assayserum samples were obtained previously from patients before ad5 gucy2c padre nct01972737 approved by the thomas jefferson university institutional review board21 neutralizing antibody titers against ad5 and ad5f35 vectors were quantified as immunization with flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0cdescribed21 briefly dilutions of heat inactivated serum samples were added to well tissue culture plates containing a549 cells atcc and infected with vp of gfp expressing ad5 or ad5f35 virus ad5 cmv egfp or ad5f35 cmv egfp respectively baylor vector development lab following a hour incubation at °c egfp fluorescence nm excitation nm emission was quantified using a polarstar optimate plate reader bmg labtech sample fluorescence was normalized to control wells containing cells and virus neutralization or wells containing cells alone neutralization titers were quantified using non linear regression as the serum dilution producing neutralization prism v8 graphpad softwaread5 neutralizing immunity studiesto induce anti ad5 immunity mice were exposed intranasally to ad5 gfp once or twice at a week interval thirty days after the last exposure ad5 nabs were quantified in sera as described above and mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1biodistribution and toxicology studybalbcj mice were immunized intramuscularly with a single dose of vp of ad5f35 gucy2c s1 three doses of vp of ad5f35 gucy2c s1 at day intervals or pbs control animals were monitored for adverse events once daily with additional evaluations on the day of dosing min hour and hours after dosing on days and designated animals were sacrificed and brain salivary glands stomach small intestine colon heart lungs kidneys liver and injection site were harvested and weighed for histopathological analysis by a blinded pathologist pathology evaluation was performed by idexx bioanalytics and detection of viral dna by quantitative pcr qpcr using the previously described assay for the gucy2c transgene19 also spleens were collected for histopathological analysis and detection of viral dna as described above as well as quantification of gucy2c specific t cell responses by ifnÎ elispot as described abovestatistical analysisstatistical analyses were conducted using graphpad prism software v8 statistical significance was considered as follows nsp p p p and p cohort sizes were powered based on prior studies with β02 and α005 for multiple comparisons of survival outcomes significance thresholds were corrected using the bonferroni method to identify vaccine induced t cell responders and non responders a previously described21 modified distribution free resampling approach was employed and positive t cell responses were defined as à compared with dmso and specific spots106 cells to determine the impact of gender and number of vaccinations on responses log transformed vaccine response magnitude was compared in mice of different genders cohorts and treatment regimens for up to three way interactions with stepwise backward variable selection by akaike information criterion using r29 package mass30open accessresultsad5gucy2cs1 and ad5f35gucy2cs1 vectorswhile ad5 seroprevalence worldwide exceeds in some regions ad35 is and associated with lower titers figure 1a12 thus we constructed a chimeric adenovirus ad5f35 composed of ad5 in which the fiber was replaced by the ad35 fiber and evaluated its ability to induce gucy2c specific immunity and resist ad5 specific immunity in humans and mice ad5 gucy2c s1 is a replication deficient human ad5 expressing the mouse gucy2c extracellular domain fused to the i ed restricted cd4 epitope known as site at its c terminus20 to generate ad5f35 gucy2c s1 the ad5 fiber l5 was replaced with the ad35 fiber figure 1b replication deficient ad5 gucy2c s1 and ad5f35 gucy2c s1 generated in hek293 cells produced dose dependent figure 1c and time dependent figure 1d expression of gucy2c s1 protein in a549 human alveolar basal epithelial cells in vitroad5f35gucy2cs1 induces gucy2cspecific antitumor immunityfollowing in vitro validation of gucy2c expression by ad5f35 gucy2c s1 we confirmed its ability to induce gucy2c specific immune responses after vaccination in vivo balbc mice immunized intramuscularly with vp of ad5f35 gucy2c s1 produced lower gucy2c specific cd8 t cell responses figure 2a and no gucy2c specific antibody responses figure 2b compared with ad5 gucy2c s1 importantly ad5 and ad5f35 vaccines produced gucy2c specific cd8 t cells of comparable avidity figure 2c a critical determinant of the antitumor efficacy of gucy2c targeted vaccines26 in contrast gucy2c specific antibody responses have no detectable antitumor activity20 similarly ad5 and ad5f35 vaccines produced comparable s1 specific cd4 t cell responses figure 2dluciferase this model previous studies revealed that ad5 gucy2c vaccines induced protective antitumor cd8 t cell responses in murine models of metastatic colorectal cancer17 thus balbc mice were immunized with ad5 or ad5f35 expressing gucy2c s1 and challenged days later with ct26 colorectal cancer cells expressing gucy2c and firefly specifically emulates secondary prevention of metastatic disease the clinical setting for which the gucy2c vaccine is being developed21 as previously demonstrated ad5 vaccination nearly eliminated metastatic tumor burden figure 3ab delayed disease progression figure 3c and improved survival figure 3d similarly ad5f35 also reduced tumor burden figure 3ab disease progression figure 3c and prolonged survival figure 3d importantly the efficacy of ad5 based and ad5f35 based gucy2c vaccines in flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure construction of ad5f35 gucy2c s1 and antigen expression a reported international seroprevalence of ad5 and ad3512 b the l5 gene encoding the fiber protein from ad5 was replaced with the l5 gene from ad35 producing the chimeric adenoviral vector ad5f35 recombinant ad5f35 gucy2c s1 was produced by inserting mouse gucy2c s1 into the e1 region of e1e3 deleted ad5f35 c and d the human alveolar basal epithelial cell line a549 was transduced in duplicate with ad5f35 gucy2c s1 at a multiplicity of infection moi from to for hours c or at an moi of for and hours d supernatants from infected cells were analyzed for gucy2c s1 protein expression by immunoblot protein expression was quantified by densitometry and plotted relative to uninfected cells error bars indicate mean±sem ad5 adenovirus serotype reducing tumor burden opposing disease progression and promoting survival was identical figure 3adad5f35 resists ad5directed immunity in mice and humansnabs against ad5 correlated with poor gucy2c specific immune responses in patients receiving ad5 gucy2c padre vaccination and prior exposure of mice to ad5 similarly blunted vaccine induced immunity21 ad5f35 based vaccine resistance to pre existing ad5 immunity was quantified in a model of respiratory pre exposure to ad5 the natural route of infection in patients33 followed by vaccination and quantification of gucy2c specific t cell responses control mice not pre exposed to ad5 naive and those that were pre exposed once à or twice à to intranasal ad5 were vaccinated after weeks with intramuscular ad5 or ad5f35 expressing gucy2c s1 and immune responses were quantified weeks later immunogenicity of ad5 gucy2c s1 and ad5f35 gucy2c s1 ad balbc mice n4 micegroup were figure immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and serum and splenocytes were collected days later gucy2c specific cd8 t cell responses were quantified by interferon gamma ifnÎ elispot a and antibodies were quantified by elisa b c gucy2c specific t cell avidity measurements were analyzed by elispot using non linear regression logagonist versus normalized response with comparisons made using the extra sum of squares f test avidity plots depict the regression line solid with cis dashed d s1 specific cd4 t cell responses were measured by ifnÎ elispot t cell responses in a and d were analyzed by one way analysis of variance values in a b and d indicate individual animals and bars in a and d indicate means tcr t cell receptor ad5 adenovirus serotype flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure antitumor efficacy of ad5 gucy2c s1 and ad5f35 gucy2c s1 ad balbc mice n10 micegroup were immunized intramuscularly with control or vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 and challenged days later with a mouse colorectal cancer cell line ct26 expressing gucy2c and luciferase on days and following challenge mice were injected with d luciferin and imaged a to quantify tumor burden day b mice were weighed twice weekly c and monitored for survival d tumor burden b was analyzed by one way analysis of variance and survival comparisons d were analyzed by the mantel cox log rank test in b and d asterisks indicate comparisons of gucy2c vaccines to the control and brackets ] indicate comparisons between ad5 and ad5f35 vaccines ns not significant ad5 adenovirus serotype figure 4a as expected one ad5 pre exposure induced moderate ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses while two pre exposures induced high ad5 nabs online supplementary figure s1 and reduced gucy2c specific t cell responses following ad5 vaccination figure 4b in contrast gucy2c specific t cell responses were reduced only à pre exposure and à pre exposure following ad5f35 vaccination figure 4b importantly ad5f35 produced t cell responses in a substantially greater fraction of the population cohort responses compared with ad5 cohort responses following serial pre exposures to ad5 figure 4cthese observations in mice were recapitulated using sera from patients with colorectal cancer in the ad5 gucy2c padre phase i trial nct0197273721 here nab titers against ad5 and ad5f35 were quantified using an established ad5ad5f35 reporter virus inhibition bioassay in serum samples collected prior to vaccination with ad5 gucy2c padre21 in these patients ad5f35 specific nab titers were substantially lower than ad5 specific titers figure 4d most importantly of patients possessed low ad5 nabs titers figure 4de which closely correlated with a gucy2c specific response rate21 in striking contrast had low ad5f35 nab titers suggesting that the vast majority of patients immunized with ad5f35 based vaccines could produce gucy2c specific responses figure 4e collectively these observations suggest that pre existing viral immunity induced by repeated environmental exposures which neutralizes ad5 delivery platforms may be overcome by the chimeric ad5f35 vector to enhance fractional population vaccine responsessafety biodistribution and toxicity of ad5f35gucy2cs1food and drug administration ind investigational new drug enabling studies quantified the toxicity biodistribution and immunogenicity of ad5f35 gucy2c s1 in balbc mice employing three schemes to examine acute and chronic effects figure 5a cohorts balanced for sex received ad5f35 gucy2c s1 either as a single intramuscular injection or as three intramuscular injections spaced weeks apart monitored daily and sacrificed on day or for analysis as indicated figure 5a there were no signs of acute or chronic toxicity in the in life phase by observation weight changes or survival figure 5bd similarly there were no clinically significant differences in organ weights online supplementary figure s2 or histopathology not shown at necropsy small statistical differences in organ weights were considered clinically insignificant and were unrelated to vaccine exposure dose time online supplementary figure s2 biodistribution quantified by qpcr detected ad5f35 gucy2c s1 at the injection site and in the spleen but not appreciably in other organs after acute and chronic exposures online supplementary figure s3 moreover robust cd8 t cell responses were quantified at day that persisted through day in of mice after a single administration figure 5eg as expected cd8 t cell responses were greater and persisted in more mice at days after three vaccinations figure 5egdiscussionthrough decades of gene therapy trials ad5 has remained a popular vector while high ad5 seroprevalence remains a barrier to universal vaccination33 natural respiratory infection can generate long lived antibodies that neutralize ad5 based vaccines eliminating transgene delivery and potential therapeutic benefit in that context ad5 seroprevalence is across multiple countries12 highlighting an unmet need for alternative vectors here we demonstrate that the chimeric ad5f35 resists pre existing ad5 immunity and induces transgene specific antitumor immunity indeed ad5f35 is less susceptible to neutralization associated with ad5 exposure in mice and humans and generates a substantially higher proportion of vaccine responders in mice pre exposed to ad5 these observations support the suggestion that ad5f35 flickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access figure ad5f35 resists neutralization associated with pre existing anti ad5 immunity in mice and humans ac to generate pre existing immunity to ad5 balbc mice n10 micegroup were exposed intranasally once or twice to vp of ad5 gfp at week intervals four weeks after the final ad5 gfp exposure ad5 exposed and naive mice were immunized intramuscularly with vp of ad5 gucy2c s1 or ad5f35 gucy2c s1 b two weeks after immunization gucy2c specific cd8 t cell responses in each group were quantified by interferon gamma ifnÎ elispot and calculated as the of mean responses in naive mice values indicate individual animals and bars indicate means ad5 and ad5f35 were compared by two way analysis of variance c the fraction of animals producing a detectable gucy2c specific cd8 t cell response filled regions in naive à and à ad5 exposed mice was determined from b d and e sera from patients with colorectal cancer collected prior to ad5gucy2c padre vaccination were tested for the ability to neutralize ad5 and ad5f35 vectors and titers were quantified d analyzed by paired t test the dotted line indicates a titer of the threshold for high neutralizing antibody nab titers21 e while subjects had high nab titers against ad5 only had high titers to ad5f35 vector filled regions binomial test ad5 adenovirus serotype will produce a higher proportion of vaccine responders in patient populationsthe extent to which nabs to the ad5 fiber limit reinfection is controversial in some studies replacing the ad5 fiber with that of another serotype circumvents pre existing ad5 immunity34 in contrast other studies suggest that these chimeric adenoviruses do not evade pre existing ad5 nabs suggesting the hexon as the major target of antibody neutralization35 in contrast to those previous studies which generated pre existing ad5 immunity by intramuscular35 or intravenous administration36 here ad5 immunity was induced by intranasal exposure in mice recapitulating natural infection33 moreover natural human respiratory pre existing ad5 nabs in patients with colorectal cancer uniformly produced by repeated respiratory infections33 similarly were overcome by the ad5f35 vector importantly the quality of antibody responses following adenovirus infection is dependent on the route of exposure indeed respiratory infections elicit fiber specific nabs while intramuscular exposure induce capsid specific nabs15 these qualitative differences in nab responses reflecting varying routes of immunization may contribute to observational discrepancies between laboratories the present studies using relevant animal models confirmed and validated with patient samples support the suggestion that ad5f35 based vaccines should produce clinically relevant immune responses in a substantial proportion of patientsflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen accessfigure safety and immunogenicity of multiple ad5f35 gucy2c s1 administrations ag balbc mice n10 micegroup were immunized intramuscularly with one or three administrations of vp ad5f35 gucy2c s1 or control at week intervals following immunization body weights b female and c male were recorded weekly and mice were monitored for survival d at days and following first immunization mice were euthanized to quantify organ pathology by weight online supplementary figure s2 biodistribution by quantitative pcr online supplementary figure s3 and gucy2c specific cd8 t cell responses by interferon gamma ifnÎ elispot eg g pie charts indicate proportion of responding animals ad5 adenovirus serotype recognizing the pervasive limitations imposed by endemic ad5 immunity in global populations12 there is an emerging interest in alternative serotypes and chimeric constructs as a tractable strategy in vaccine development ad26 ad35 and ad48 vectors have been advanced into phase i clinical trials37 in that regard a comparison of ad5 ad26 ad35 and ad48 immunity among healthy patients revealed that endemic ad35 seropositivity was lowest across global populations12 reinforcing chimeric strategies employed herein similarly the first hexon chimeric adenovirus comprising ad5 and ad48 components was safe and immunogenic in patients39 interestingly ad5 ad35 chimeric vectors more efficiently transduce a variety of human cell types in vitro compared with either parental vector40 these observations underscore the future potential of intelligently designed chimeric adenoviruses strategically constructed to deliver transgenes for replacement therapy or vaccination and targeted precisely to the cellular or disease context40while antitumor efficacy was equivalent cd8 t cell responses were lower and antibody responses were absent for ad5f35 gucy2c s1 compared with ad5 gucy2c s1 however the antitumor efficacy of gucy2c directed immunotherapy is driven primarily by t cell avidity rather than effector t cell quantity26 in that context the functional avidity of gucy2c specific cd8 t cells following ad5 and ad5f35 immunizations were equivalent consistent with their comparable antitumor efficacy quantitative differences in transgene specific immunity between vectors may reflect a variety of factors thus the quantity and persistence of gucy2c s1 transgene following ad5f35 immunization is lower compared with ad519 consistent with prior observations that ad5 transduction efficiency in vivo may be several fold higher than ad5f3541 moreover the ad5 fiber binds to cxadr coxsackievirus and adenovirus receptor42 while the ad35 fiber binds to cd4643 suggesting the two viruses may infect distinct cell typesflickinger jr a0jc et a0al j immunother cancer 20208e001046 101136jitc2020001046 0copen access while checkpoint inhibitors have generated practice shifting results in the clinic and defined immunotherapy as an effective strategy for the treatment of several malignancies they have not been universally successful in that context the dearth of neoepitopes in many cancer types including microsatellite stable colorectal and pancreatic second and third leading causes of cancer mortality respectively makes them insensitive to checkpoint blockade7 indeed examination of neoepitopes presented on the surface of five colorectal cancer specimens revealed a total of three neoepitopes3 thus vaccines targeting cancer associated self antigens have re emerged alone and in combination with checkpoint inhibitors as a strategy to prevent and treat metastases from these cold tumors44 checkpoint inhibitors have become first line therapy in the metastatic setting for some cancers46 while chimeric antigen receptor expressing t cells car t cells are being deployed in patients with metastatic and refractory disease47 in contrast few cancer immunotherapies have been developed for early stage cancer patients with no evidence of disease ned following conventional surgicalradiochemotherapies who are at significant risk of disease recurrence indeed25 of stage ii and of stage iii patients with colorectal cancer recur following surgery and chemotherapy49 while of patients with resectable pancreatic cancer experience recurrence50 vaccines targeting tumor associated antigens such as ad5f35 gucy2c padre may provide safe and effective immunotherapies for the secondary prevention of metastatic disease in patients with ned who are otherwise ineligible to receive checkpoint inhibitors or car t cellsthe present studies suggest that the chimeric adenoviral vector ad5f35 may be preferable to the widely used ad5 vector and warrants further investigation indeed they suggest that ongoing clinical investigations of gucy2c directed immunotherapy in patients with gucy2c expressing cancers including colorectal pancreatic gastric and esophageal could benefit from using the ad5f35 rather than the ad5 vector in that context an upcoming clinical trial will examine the safety immunogenicity and resistance to pre existing immunity of ad5f35 gucy2c padre in patients with gi cancer nct04111172 safe generation of gucy2c targeted immunity in a high proportion of patients will lead to efficacy trials to establish the ability of ad5f35 gucy2c padre to prevent recurrence following standard therapy in patients with gi cancer who represent of all cancer deaths51 and for whom established immunotherapies are ineffectivetwitter adam e snook adamsnookphdacknowledgements the authors thank adrian p gee phd zhuyong mei md deborah lyon and malcolm brenner md phd center for cell and gene therapy baylor college of medicine for assistance in vaccine manufacturingcontributors jcf js bb saw and aes designed the studies jcf js rc el trb jb ec ap jar and jr carried out the studies tz carried out data analysis and statistical analysis in discussion with aes jcf and aes wrote the manuscript and all authors critically reviewed and approved the final version of the manuscriptfunding this work was supported by the national institutes of health nih r01 ca204881 r01 ca206026 and p30 ca56036 the defense congressionally directed medical research program w81xwh17 prcrp ttsa and targeted diagnostic therapeutics to saw aes received a research starter grant in translational medicine and therapeutics from the phrma foundation a degregorio family foundation award and was supported by the defense congressionally directed medical research programs nos w81xwh1710299 w81xw
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"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et
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Neuropilin1 regulated by miR320a participates in the progression of cholangiocarcinoma by serving as a coreceptorthat activates multiple signaling pathways The present study sought to investigate upstream lncRNAs that control theexpression of miR320aneuropilin1 axis and dissect some of the underlying mechanisms Here we report lncRNATTNAS1 titinantisense RNA1 acts as a sponging ceRNA to downregulate miR320a and is highly expressed in humancholangiocarcinoma tissues and cells The expression of the above three molecules is correlated with theclinicopathologic parameters of cholangiocarcinoma patients In this study multiple bioinformatics tools anddatabases were employed to seek potential lncRNAs that have binding sites with miR320a and TTNAS1 wasidentiï¬ed because it exhibited the largest folds of alteration between cholangiocarcinoma and normal bile ductepithelial cells The regulatory role of TTNAS1 on miR320a was further evaluated by luciferase reporter and RNApulldown assays coupled with in situ hybridization and RNA immunoprecipitation analyses which showed that TTNAS1 bound to miR320a through an argonaute2dependent RNA interference pathway in the cytoplasm ofcholangiocarcinoma cells Knockdown and overexpression assays showed that the regulatory effect between TTNAS1and miR320 was in a oneway manner TTNAS1 promoted the proliferation and migration of cholangiocarcinomacells via the miR320a neuropilin1 axis The function of TTNAS1 on tumor growth and its interaction with miR320awere conï¬rmed in animal models Further mechanistic studies revealed that TTAAS1 through downregulating miR320a promoted cell cycle progression epithelialmesenchymal transition and tumor angiogenesis by upregulatingneuropilin1 which cointeracted with the hepatocyte growth factorcMet and transforming growth factor TGFTGF receptor I pathways In the present results demonstrate that lncRNA TTAAS1 is a sponging ceRNAfor miR320a which in turn downregulates neuropilin1 in cholangiocarcinoma cells indicating these three moleculesrepresent potential biomarkers and therapeutic targets in the management of cholangiocarcinomaIntroductionCholangiocarcinoma CCA arises from the epithelialcells facing the lumen of the biliary trees and is the secondmost frequent primary hepatic tumor after hepatocellularCorrespondence Jun Lu lujunsd126com orXueying Sun sunxueyinghrbmueducn1Department of Hepatobiliary Surgery Shandong Provincial Hospital Afï¬liatedto Shandong First Medical University Jinan China2The Hepatosplenic Surgery Center the First Afï¬liated Hospital of HarbinMedical University Harbin ChinaFull list of author information is available at the end of the Edited by A Stephanoucarcinoma globally12 CCA is usually diagnosed atadvanced incurable stages due to the absence of priorrecognizable clinical manifestations coupled with thecurrent unavailability of speciï¬c tumor biomarkers3Despite the latest progress in the development of molecular targeted therapies the prognosis for this devastatingcancer remains grim3 Pemigatinib has recently beenapproved for of CCA patients harboring a fusionor rearrangement of growth factor receptor gene4 andivosidenib has been shown to significantly improve theprogressionfree survival of patients with isocitrate The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Ofï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of dehydrogenase1 mutant advanced CCA in a phase clinical trial5 However CCA is a heterogeneous malignancy and bears a high mutation burden6 thus thepotential druggable genome alterations in a small proportion of CCAs are not ideal therapeutic targets owing tosignaling pathways7the anticipated redundancy ofTherefore there is great urgency in further elucidating themolecular mechanisms and pathways underpinning thisdisease so that the clinical outcome of CCA patients couldbe improvedNeuropilin1 NRP1 is a nontyrosine kinase transmembrane molecule overexpressed in gastrointestinalcancers89 and serves as a coreceptor for several cellularsignaling pathways involved in cancer progression10We have recently demonstrated that human CCA tissuesexpressed higher levels of NRP1 which coactivates thevascular endothelial growth factor VEGF epidermalgrowth factorEGF and hepatocyte growth factorHGFmediated pathways involved in the progression ofCCA14 It is known that microRNAs miRNAs regulatemultiple cellular functions and have emerged as potentialtargetsIn exploring themiRNAmediated mechanisms that lead to the overexpression of NRP1 we have shown that miR320anegatively regulates NRP1 by binding to the ²UTR of itspromoter and is expressed at low levels in CAA tissuesand cells14 MiR320a is regarded as a tumorsuppressivemiRNA16 and inhibits the proliferation and metastasis ofCCA cells in vitro and in vivo through downregulatingNRP114 However its upstream regulatory mechanismsremain unknownin anticancer campaign15Long noncoding RNAs lncRNAs are a group of noncoding RNAs ncRNAs with over nucleotides inlength and comprise of ncRNAs Emerging studiesprovide strong evidence that lncRNAs exert pivotal rolesin regulating gene expression in many diseases17 One ofthe main regulatory functions of lncRNAs is to act ascompeting endogenous RNAs ceRNAs to sponge miRNAs leading to the loss of the ability to degrade silenceor hamper translation of their downstream genes17 ManylncRNAs have been shown to regulate key factorsinvolved in cancer cells18 and some of them representpotential diagnostic markers and therapeutic targets forCCA1920 Therefore we carried out the present study toexplore potential upstream lncRNAs that can regulate themiR320aNRP1 axis in CCAResultsIdentiï¬cation of lncRNA TTNAS1 as a potential targetin CCAThe overexpression of NRP1 in clinical CCA tissueswas conï¬rmed by using immunohistochemistry of tissuemicroarrays Supplementary Fig S1 A panel of CCA celllines expressed different levels of NRP1 where the orderOfï¬cial journal of the Cell Death Differentiation Associationof cell lines with the highest to lowest expression was RBEHCCC9810 QBC939 CC262 and FRH0201 but allexpressed higher levels of NRP1 than normal humanbiliary epithelial HIBEC cells Supplementary Fig S2A BRBE cells expressed the highest levels of NRP1 proteinand mRNA which were and fold higher thanHIBEC cells respectively and expressed the lowest level ofmiR320a which was of that of HIBEC cells FigS2C A negative correlation was found between expression levels of miR320a and NRP1 mRNA Fig S2DLncRNAs that have binding sites with miR320a werescreened by using multiple bioinformatics tools anddatabasestarbasesysueducn DIANATarBasewwwlncrnadb LncBase Experimental v2 lncactdb20omictoolscom and httpbioinfolifehusteducnand potential candidates were selected based on thecriteria of free energy kcalmol and score Supplementary Table S1 We then detected their expressionlevels in RBE and HIBEC cells by quantitative reversetranscription polymerase chain reaction qRTPCR withspeciï¬c primers Supplementary Table S2 Among the candidates TTNAS1 titinantisense RNA1 was shownto have the largest folds of alteration between RBE andHIBEC cells Supplementary Fig S3A B Notably TTNAS1 is a novel lncRNA derived from the opposite strandoftitin TTN gene and has partial sequence complementarity with TTN gene21 LncRNA TTNAS1 hasbeen shown to promote the progression of several cancertypes including esophageal squamous cell carcinomaESCC21 lung adenocarcinoma22 and papillary thyroidcancer23 The expression of TTNAS1 was also detected inall the available CCA cell lines and showed a positivecorrelation with NRP1 but a negative correlation withmiR320a Fig S3CEAssociation of TTNAS1 expression with clinicopathologicparameters of CCA patientstissuesThe qRTPCR analyses revealed that CCA tumor tissuesexpressed significantly higher levels of TTNAS1 Fig 1aand NRP1 mRNA Fig 1b and significantly lower levelsof miR320a Fig 1c compared with adjacent normal bileductIn CCA tissues an inverse correlationbetween expression levels of TTNAS1 and miR320aFig 1d and between miR320a and NRP1 mRNAFig 1e and a positive correlation between TTNAS1 andNRP1 mRNA Fig 1f were found by using Pearsoncorrelation analyses Based on the expression levels ofTTNAS1 we divided CCA cases into the high meanand low mean groups and analyzed the associationbetween TTNAS1 expression and clinicopathologicparameters The results showed that the expression ofTTNAS1 was significantly correlated with tumor differentiation and lymph node metastasis and marginallycorrelated with portal vein invasion while not with gender 0cZhu Cell Death and Disease Page of Fig The expression of lncRNA TTNAS1 miR320a and NRP1 and their correlation in CCA tissues The expression of TTNAS1 a NRP1mRNA b and mature miR320a c in pairs of human CCA tissues and corresponding adjacent normal biliary tissues was detected by qRTPCRn number of samples examined Statistical analyses were performed by a Students t test df The correlation between miR320aTTNAS1NRP1mRNAmiR320a and TTNAS1NRP1 mRNA expression was analyzed with a Pearson testage tumor location or TNM staging Table NamelyCCA patients with poor tumor cell differentiation positivelymph metastasis and portal vein invasion had higherexpression of TTNAS1 Table By using the sameanalyses based on expression levels of NRP1 mRNA andmiR320a we found that both were correlated with tumordifferentiationlymph node metastasis and portal veininvasion Further NRP1 mRNA expression levels alsocorrelated with TNM staging Table TTNAS1 functions as a ceRNA to sponge miR320aFor examining the regulatory effects between TTNAS1and miR320a we ï¬rst showed that transfection of miR320a mimics had little effect on TTNAS1 expression butdepletion of TTNAS1 significantlyincreased theexpression of miR320a in RBE and HCCC9810 cellsSupplementary Fig S4A Bimplying that TTNAS1might negatively regulate miR320 in CCA cells Based onthe putative binding sites between TTNAS1 and miR320a luciferase reporter and RNA pulldown assays wereemployed to examine their direct binding SupplementaryFig S5 The luciferase intensity was decreased by cotransfected miR320a mimics and wildtype TTNAS1reporter vector but not the mutant reporter vector lackingthe miR320a binding site Consistently miR320a wasprecipitated by wildtype TTNAS1 but not TTNAS1mutant and TTNAS1 was pulled down by biotinlabeledwildtype miR320a but not miR320a mutant Fig S5TTNAS1 regulates miR320a in an argonaute2dependentmannerThe above results indicate that miR320a binds tolncRNATTNAS1 without causing TTNAS1 degradation TTNAS1 and miR320a were both located in thecytoplasm of CCA cells as detected by In situ hybridization Fig 2ac suggesting that TTNAS1 may bind tomiR320a through the argonaute2 Ago2dependentRNA interference pathway24 As expected RNA immunoprecipitation RIP assay showed levels of miR320aand TTNAS1 precipitated by an antiAgo2 Ab weremarkedly increasedresulting in a and 3foldenrichment compared with controlIgG respectivelyFig 2d e Meanwhile endogenous TTNAS1 pulldownby the antiAgo2 Ab was speciï¬cally enriched uponectopic overexpression of miR320a Fig 2f These datasuggest that TTNAS1 binds to miR320a in the cytoplasm in an Ago2dependent mannerIn addition the expression of miR320a was downregulated by TTNAS1 overexpression and upregulatedby TTNAS1 knockdown and these effects could beabolished by miR320a mimics and antagomiR320arespectively Supplementary Fig S6A B However nosignificant difference in TTNAS1 expression was detected by transfection of miR320a mimics or antagomiR320a Fig S6C D These results indicate that the regulatory effects between TTNAS1 and miR320a are in aoneway mannerOfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Table Correlations of the expression of TTNAS1 NRP1 mRNA or miR320a with clinicopathological parameters ofCCA patientsParametersTotaln TTNAS1P value NRP1 mRNAP value miR320aP valueLown Highn Lown Highn Lown Highn GenderMaleFemaleAge years¥Tumor differentiationWellmoderatePoorTumor locationIntrahepaticPerihilarTumor size mm¥ mmTNM stagebIIIIIIIVLymph metastasisNegativePositivePortal vein invasionNoYes00042a00182a001823a001543a002709a003307aaindicates a significant differencebAccording to the 8th UICC Union for International Cancer ControlTNM staging system P value was estimated by a Ï2 testCCA cholangiocarcinoma TTNAS1 lncRNA titinantisense RNA1 NRP1 neuropilin10009076a00194a004106aTTNAS1 promotes the proliferation of CCA cellsvia miR320aNRP1We have previously reported that NRP1 depletion andectopic expression of miR320a inhibited the proliferationof CCA cells14 In accord we conï¬rmed that depletion ofNRP1 significantly reduced cell viability while miR320amimics showed a similar effect by downregulating NRP1expression Supplementary Fig S7 We could furthershow that knockdown of TTNAS1 significantly reducedcell viability while antagomiR320a partially restored cellviability Supplementary Fig S8A Mechanistically TTNAS1 knockdown led to a significant downregulation ofNRP1 cyclindependent kinase CDK2 and cyclin E asignificant upregulation of p27 but had little effect on theexpression of cyclin D1 and p21 The above molecules arekey factors involved in cell proliferation and cycle progression25 AntagomiR320a counteracted the effect ofTTNAS1 knockdown Fig S8B Cell cycle distributionassays showed that knockdown of TTNAS1 led to morecells arrested at the G0G1 phase while antagomiR320apartially abolished this effect of TTNAS1 knockdownSupplementary Fig S9On the other hand exogenous overexpression of TTNAS1 increased the viability of FRH0201 cells while miROfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig TTNAS1 regulates the expression of miR320a in the cytoplasm of CCA cells in an Ago2dependent manner a RBE cells weresubjected to in situ hybridizations of miR320a ²DIG tagged probe identiï¬ed with Cy3conjugated Ab in red and TTNAS1 ²DIG tagged probeidentiï¬ed with FITCconjugated Ab in green and stained with DAPI blue Three images from the same cells were merged Scale bar μmb c Total RNA was extracted from nuclear NU and cytoplasmic CY fractions of RBE cells and the expression of TTNAS1 b and miR320a c wasmeasured by qRTPCR and normalized U1 and U6 were used as internal nuclear controls for TTNAS1 and miR320a respectively and GAPDH as aninternal cytoplasmic control d e RBE cells were subjected to RNA immunoprecipitation RIP assays The fold enrichment of miR320a d andTTNAS1 e by an antiAgo2 Ab was normalized to a nonspeciï¬c IgG acting as a negative control f RBE cells transfected with negative control NC ormiR320a mimics were subjected to RIP to measure relative enrichment of TTNAS1 by the antiAgo2 Ab P indicates a significant differencefrom respective controls320a mimics partially abolished this effect Fig S8CTTNAS1 overexpression resulted in the upregulation ofNRP1 cyclin E and CDK2 and downregulation of p27while miR320a mimics could neutralize the effect ofTTNAS1 overexpression Fig S8DTTNAS1 promotes the migration of CCA cells via the miR320aNRP1 axisKnockdown of TTNAS1 significantly reduced theability of RBE cells to migrate while antagomiR320aFig 3ad CCA cellspartially abolished this effectacquire the migratory and invasive properties through acritical process known as epithelialmesenchymal transition EMT26 Therefore we examined the effects ofTTNAS1 knockdown on the expression of decisive facinvolved in the process of EMT27 TTNAS1torsknockdown significantly downregulated the expressionof NRP1 Snail Ncadherin matrix metalloproteinaseMMP2 and MMP9 and upregulated the expression ofEcadherin Fig 3e The results were supported bygelatin zymography assays which showed that TTNAS1knockdown significantly reduced activities of MMP2 andOfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig Knockdown of TTNAS1 inhibits cell migration via regulating miR320a RBE cells were transfected with scrambled shRNA controlshRNATTNAS1 or shRNATTNAS1 antagomiR320a for h Cells were subjected to transwell migration a b and scratch c d assays a Migratedcells were visualized using Giemsa staining Scale bar μm a and µm c b Numbers of migrating cells were counted c Scratch areas wererecorded d Scratch distances were quantiï¬ed at indicated time points e f Cells were immunoblotted for detecting key EMT proteins and the densityof each band was normalized to actin g Cells were subjected to gelatin zymography assays for analyzing the gelatinolytic activity of MMP9 andMMP2 P vs controls and P and P vs shRNATTNAS1MMP9 while antagomiR320a partially counteracted thiseffect Fig 3f On the other hand overexpression ofTTNAS1ofFRH0201 cells while miR320a mimics partially abolishedthis effect Supplementary Fig S10the migratoryincreasedabilityTTNAS1 contributes to the growth of CCA tumors inanimal modelsThe functional role of TTNAS1 was also conï¬rmed inCCA tumors in vivo Subcutaneous RBE tumors wereestablished in mice which were randomly assigned todifferent treatments when tumors reached mm3Tumors treated with shRNATTNAS1 were significantlysmaller ± mm3 than control tumors ± mm3 however cotreatment of antagomiR320aOfï¬cial journal of the Cell Death Differentiation Associationcould partially restore the growth of tumors ± mm3 as measured days after treatment commencement Fig 4a The results of tumor volume correlatedwith the weight of tumors Fig 4b Treatment of shRNATTNAS1 led to TTNAS1 downregulation and miR320aupregulation in tumors harvested days after treatmentsby in situ hybridization and downregulation of NRP1 byimmunohistochemistryofantagomiR320a partially abolished the effects of shRNATTNAS1 on miR320a upregulation and NRP1 downregulation but had little effect on TTNAS1 expressionFig 4c Treatment of shRNATTNAS1 significantlyinhibited cell proliferation in situ Fig 4d e and reducedtumor vasculature while antagomiR320a neutralized theeffects of shRNATTNAS1 Fig 4d f In agreement with4c CotreatmentFig 0cZhu Cell Death and Disease Page of Fig Knockdown of TTNAS1 inhibits the growth and angiogenesis of CCA tumors in vivo Subcutaneous CCA tumors were established inmice by inoculation of RBE cells and received respective treatments as described in Supplementary Information a The growth curve of RBE tumorswas recorded b RBE tumors were resected weighed and photographed at the end of experiments c Two mice were killed from each group toharvest tumors days after treatments and the expression of TTNAS1 and miR320a was examined by in situ hybridization magniï¬cation à Scalebar μm and NRP1 expression by immunohistochemistry Magniï¬cation à Scale bar μm Tumors harvested at the end of experimentsd Illustrated are representative tumor sections immunostained by Abs against Ki67 and CD31 respectively Magniï¬cation à Scale bar μm Insitu cell proliferation index e and tumoral microvessel density f were quantiï¬ed g Tumor tissue homogenates were immunoblotted for detectingthe expression of key proliferation proteins P vs controls P and P vs shRNATTNAS1the in vitro results Fig S8A B immunoblotting analysisof tumor homogenates showed that shRNATTNAS1treatment led to downregulation of NRP1 cyclin E andCDK2 and upregulation of p27 while antagomiR320acounteracted the effects of shRNATTNAS1 Fig 4gOn the other hand by adopting another subcutaneousCCA tumor mouse model with FRH0201 cells whichwere shown to express a lower level of TTNAS1Fig S2A we demonstrated that exogenous overexpression of TTNAS1 promoted tumor growth bypromoting in situ cell proliferation and tumor angiogenesis while miR320a mimics partially abolished theseeffects Supplementary Fig S11TTNAS1 regulates the cMet and TGF pathways via NRP1We have previously demonstrated that NRP1 coactivates the HGFcMet pathway in CCA cells14 Controland shRNATTNAS1transfected RBE cells were incubated with recombinant human HGF protein in the presence or absence of tivantinib a cMet inhibitor and anOfï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Fig TTNAS1 regulates the cMet and TGF pathways via NRP1 RBE and cells were transfected with negative control or shRNATTNAS1 andthen incubated for h in the presence or absence of recombinant HGF protein ngml and tivantinib μgml a or TGF protein ngmland LY2157299 μgml b Cell lysates were immunoblotted to determine the expression of key proteins involved in the above pathways asindicated The density of each band was normalized to actin P and P indicate a significant difference P indicates asignificant difference from negative control cells treated with vehicleanticancer drug used in CCA clinical trial28 TTNAS1knockdown led to downregulation of NRP1 expressionresulting in downregulation of phosphorylated cMetpcMet and sequential downregulation of phosphorylated Akt pAkt and upregulation of p27 Fig 5aIncubation of HGF protein did not affect NRP1 expression but could activate the cMet pathway evidenced byupregulation of pcMet and pAkt and downregulationof p27 and incubation of tivantinib showed the oppositeeffects to HGF ligand Fig 5aNRP1 cointeracts transforming growth factor TGFpathway29 which is crucial for EMT of cancer cells30Therefore we examined the effect of TTNAS1 knockdown on this pathway in CCA cells Control and shRNATTNAS1transfected RBE cells were incubated withrecombinant human TGF protein orand LY2157299 aspeciï¬c TGF receptor TGFR inhibitor31 Incubationof TGF protein or LY2157299 did not affecttheexpression of NRP1 or TGFRI Fig 5b HoweverTGF induced the upregulation while LY2157299expression of pTGFRI TTNAS1reduced theknockdown had little effect on TGFRI expression butsignificantly inhibited its phosphorylation Fig 5b TheOfï¬cial journal of the Cell Death Differentiation Associationactivation of TGF pathway by TGF protein increasedthe sequential expression of pSmad23 and Snail whileLY2157299 and TTNAS1 knockdown demonstratedopposite effects and abolished the activating effects ofTGF protein Fig 5bDiscussioninotherrole wasconï¬rmedLncRNA TTNAS1 was initially reported to participatein the progression and metastasis of ESCC21 Later itsfunctionalcancertypes22233233 Importantly TTNAS1 exerts regulatoryeffects via acting as a ceRNA to sponge different miRNAsin different cancers For instance TTNAS1 regulated themiR133bactinbinding protein fascin homolog axis inESCC cells21 while promoted the migration and EMT oflung adenocarcinoma cells by sponging miR1425p toregulate CDK522 As schematically summarized in Fig we have in the present study found that TTNAS1 servesas a ceRNA to sponge miR320a through complementarybinding sites in an Ago2dependent manner in CCA cellsLncRNAs exhibit different functions depending on theirsubcellular localization This study showed that TTNAS1was mainly localized in the cytoplasm of CCA cells while 0cZhu Cell Death and Disease Page of dependent way and in a oneway manner in CCA cells Inaccord it has been reported that TTNAS1 was locatedmainly in the cytoplasm and acted as a ceRNA spongingmiRNAs in ESCC and papillary thyroid cancer cells2123MiR320a is one of the two most highly downregulatedmiRNAs in clinical CCA tissues and is closely associatedwith the progression and severity of CCA35 MiR320aalso represents a critical suppressor component of theprogression of other cancers163637 We have previouslyreported that miR320a negatively regulated the expression of NRP1 by binding to the ²UTR of NRP1 promoter and inhibited cell proliferation and migration ofCCA cells14NRP1 functions as versatile coreceptors that bind to anumber of growth factors and couple with cognatereceptor tyrosine kinase signaling pathways involved incancer progression111438 In the present study we havefurther demonstrated that NRP1 acts as a coreceptor forthe activation of HGFcMet pathway which induces thephosphorylation of Akt39 a downstream of cMet signaling40 Akt activation leads to the sequential downregulation of p2741 which inactivates the CDK2cyclinE complex resulting in cell cycle arrest41 Howevertivantinib a speciï¬c cMet inhibitor can block NRP1induced activation of the HGFcMet pathway Fig Onthe other hand NRP1 cointeracts with TGF29 leadingto the activation of the TGFTGFRI pathway whichin turn increases the expression of phosphorylated Smad2and Smad3 The latter two combine with Smad4 to form atrimeric SMAD complex that upregulates the expressionof Snail which conveys TGFinduced repression ofEcadherin and stimulation of Ncadherin42 thus promoting EMT of CCA cells However LY2157299 a speciï¬c TGFR inhibitor31 can block NRP1inducedactivation of the TGFTGFRI pathway Fig Asdemonstrated previously14 but not investigated in thisstudy the above signaling pathways may also crosstalkwith each other and contribute to the proliferation andmetastasis of cancer cells43In summary to the best of our knowledge this is theï¬rst study that reports the functional role of TTNAS1 asa sponging ceRNA for miR320a its high expression inCCA tissues and a significant association with clinicopathologic parameters of CCA TTNAS1 displays itsregulatory activity by binding to miR320a through theAgo2dependent RNA interference pathway and in a oneway manner in the cytoplasm of CCA cells Throughdownregulating miR320a TTAAS1 promotes cell cycleprogression EMT and angiogenesis via NRP1 which cointeracts HGFcMet and TGFTGFRI pathways inCCA cells Taken together the present study has unveileda novel axis consisting of TTNAS1miR320aNRP1which may also represent a therapeutic target and biomarkers in the management of CCAFig Schematic diagram of the TTNAS1miR320aNRP1 axiscontributing to the progression of CCA LncRNA TTNAS1 serves asa ceRNA to sponge miR320a through complementary binding sites inan Ago2dependent manner in CCA cells On the other hand miR320a downregulates the expression of NRP1 by binding to its ²UTRAn NRP1 protein molecule is composed of ï¬ve extracellular domainsa1 a2 b1 b2 and c one transmembrane domain and a shortcytosolic tail and acts as a coreceptor for ligands HGF and TGF tostimulate the activation of respective cMet and TGF signalingpathways indicates promotion positive regulation or activation¥ indicates inhibition negative regulation or blockade p indicatesphosphorylation of proteins Ago2 argonaute2 CCAcholangiocarcinoma CDK2 cyclindependent kinase HGFhepatocyte growth factor NRP1 neuropilin1 ORF readingframe TGF transforming growth factor TGFR TGF receptorUTR untranslated regionmiR320a was located in both nuclear and cytoplasmsubcellular compartments It is well established that thematuration of miRNAs occurs in the cytoplasm wherethey execute posttranscriptional gene silencing via anRNAinduced silencing complex pathway34 Intriguinglythe ectopic expression of miR320a reduced the luciferaseactivities of the wildtype TTNAS1 reporter but theexpression of TTNAS1 remained unchanged uponoverexpression of miR320a Moreover endogenousTTNAS1 and miR320a could be pulled down by an antiAgo2 Ab These data suggest that miR320a recognizesand binds with TTNAS1 without triggering the degradation of TTNAS1 which plays a posttranscriptionalregulatory role in downregulating miR320a via an Ago2Ofï¬cial journal of the Cell Death Differentiation Association 0cZhu Cell Death and Disease Page of Materials and methodsClinical CCA tissuesA total of pairs of CCA and matched adjacent normalbile duct tissues were collected at the Department ofHepatobiliary Surgery Shandong Provincial HospitalAmong them pairs have been described previously14while new pairs of tissues were collected between April and September Of the cases were perihilar CCA and were intrahepatic CCA The criteria ofthe included specimens were consistent with our previousstudy14 The current study has been approved by the EthicsCommittee of Shandong Provincial Hospital Jinan Chinaand informed consent was obtained from all subjectsCells antibodies and reagentsHuman CCA celllines HCCC9810 RBE QBC939CC262 and FRH0201 and normal human biliary epithelialHIBEC cells were obtained from the Cell Bank of theChinese Academy of Sciences Shanghai China1444 Cellswere routinely cultured at °C in RPMI1640 mediumsupplemented with vv fetal bovine serum in ahumidiï¬ed atmosphere of CO2 Cell lines were conï¬rmed to be negative for mycoplasma infection by using aPCRbased Universal Mycoplasma Detection kit AmericanType Culture Collection Manassas VA USA Relevantinformation regarding antibodies Abs reagents and kitsare described in detail in Supplementary InformationAnimal experimentsThe experimental protocol has been described previously1444 and approved permit SYXK20020009 by theInstitute Animal Ethics CommitteeImmunodeï¬cientnude BALBc mice H2b were housed in the AnimalResearch Center the First Afï¬liated Hospital of HarbinMedical University China Two sets of experiments weredesigned to examine the effects of TTNAS1 knockdownand overexpression on tumor growth Detailed information for animal experiments is included in SupplementaryInformation Brieï¬y cells were injected subcutaneouslyinto mice and palpable tumors were monitored Around weeks later mice bearing tumors with a volume of¼ mm3 were randomly assigned to different groupsn The TTNAS1 knockdown study had threegroups of animals which received intratumoral injectionsof control shRNATTNAS1 or shRNATTNAS1 antagomiR320a respectively while the TTNAS1 overexpression study comprised three groups of animalswhich received injections of either control TTNAS1 orTTNAS1 miR320a mimics respectively Two micefrom each group were killed days after injection fordetecting gene expression The remaining mice werefurther monitored and euthanized days after treatments commencedOfï¬cial journal of the Cell Death Differentiation AssociationImmunohistochemistry Tissue microarrays Establishment of stable transfectants depleted of NRP1 Assays ofcell viability cell cycle Transwell migration Cell scratchqRTPCR western blot and Gelatin zymography Cellfraction isolation In situ hybridization RNA pulldownand RIP assays Transfection of miR320a mimicsantagomiR320a and TTNAS1 expression vectors Plasmid constructs and luciferase assay In situ Ki67 proliferation index and Assessment of tumor vascularityThe detailed description for these methods is includedin Supplementary Information and has also been described previously1114Statistical analysisGraphPad Prism GraphPad Software San DiegoCA USA was employed for performing statistical analyses Data are expressed as mean values ± standarddeviation Multiple comparisons were made with a oneway analysis of variance ANOVA followed by a Tukeyposthoc test Comparisons between two groups weremade by a ttest Correlations of TTNAS1 NRP1mRNA or miR320a with clinicopathological parameters were estimated by a Ï2 test The relationshipbetween two variables was analyzed by using Pearsonscorrelation coefï¬cient P was considered statistically significantAcknowledgementsThis study was supported in part by the grants from the National Key Researchand Development Program of China 2017YFC1308602 the Supportive Fundby Heilongjiang Provincial Department of Science and TechnologyGX18C010 Natural Scientiï¬c Foundation of Shandong ProvinceZR2019MH089 Natural Scientiï¬c Foundation of Heilongjiang ProvinceH2018028 and LH2019H018 and Research Projects from the Fourth Afï¬liatedHospital of Harbin Medical University HYDSYXH201904 an
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"Immigrant statusfamily relationsACP contemplationACP discussionburial planninga b s t r a c tObjectives To examine how immigrant status and family relationships are associated with advance careplanning ACP engagement and endoflife EOL preference in burial planning among older ChineseAmericans the largest subgroup of Asian AmericansDesign Crosssectional surveySetting Communities in Honolulu HawaiiParticipants Participants were older Chinese Americans aged years and olderMeasures Measures included ACP contemplation ACP discussion and EOL preference in burial planningimmigrant status family cohesion family conï¬ict demographic information and health statusResults Results show that in comparison to foreignborn Chinese Americans USborn Chinese Americanswere more likely to have ACP contemplation [odds ratio OR conï¬dence interval CI ] ACP discussion OR CI and preferences for burial plans at the end of life OR CI Family conï¬ict increased the possibility of having ACP contemplation OR CI ACP discussion OR CI and EOL preference in burial planning OR CI whereas family cohesion was not associated with these study outcomesConclusions and Implications This study suggests that ACP should be adapted to be more culturallyappropriate especially in a time of coronavirus and xenophobia such as framing ACP as a tool to helpfamilies reduce stress while fulï¬lling ï¬lial obligations in order to ensure equitable access to ACP AMDA e The Society for PostAcute and LongTerm Care MedicineAdvance care planning ACP is a process of understanding andcommunicating individuals values goals and preferences regardingendoflife EOL care12 Contemplation of individuals EOL wishes anddiscussions with families can be as important as discussions withphysicians and completion of an advance directive in guiding care34ACP is a social process built on relationships and alleviation ofburden on others a means to prepare for death and a measure toexercise the ethical principle of patient autonomy5 Burial planningcan ensure individuals wishes are executed and relieve the burden ofloved ones to determine what the deceased would have wantedduring the time of grief In this sense burial planning is an importantThis study was supported by a research grant from the Rory Meyers College ofNursing at New York UniversityThe authors declare no conï¬icts of interest Address correspondence to Bei Wu PhD Rory Meyers College of Nursing NewYork University First Avenue Room New York NY USAEmail address beiwunyuedu B Wu101016jjamda20200604015258610 AMDA e The Society for PostAcute and LongTerm Care Medicineelement of ACP6 Therefore it makes sense to examine ACP contemplation ACP discussion with family and EOL preference in burialplanning togetherACP can improve quality of EOL care for individuals including lessinhospital death and increased hospice use7 Despite the beneï¬ts ofACP the participation rate of ACP remains low especially among olderadults of racial and ethnic minorities Studies found that in the UnitedStates Blacks and Hispanics are less likely to have an EOL discussion adurable power of attorney and an advance directive than their Whitecounterparts89 but there is a lack of knowledge on ACP engagementamong Chinese Americans the largest subgroup of Asian Americansand the fastestgrowing minority group in the USA10Compared with nativeborn Chinese AmericansforeignbornChinese Americans may face more cultural and logistical challengesin ACP engagement because of their limited English proï¬ciencygreater cultural burden in discussing death and dying and acceptingindividual autonomy and lack of ACP knowledge1112 In addition theeffectiveness of ACP may rely on the involvement knowledge and 0cY Pei JAMDA xxx 1e4cooperation of family members13 however because of the lack of richand comprehensive measures of family relationships in previousresearch on ACP few studies have examined the extent to whichfamily relationships uence individuals ACP engagement To ï¬ll thisknowledge gap this study aimed to examine how immigrant statusand family relationships are associated with ACP contemplation ACPdiscussion with family and preference in burial planning among olderChinese AmericansMethodsDataData were derived from a survey conducted in Honolulu Hawaiiwhere approximately of the total population is composed ofChinese Americans and of the adult population are immigrants14We used snowball sampling and convenience sampling to identify andrecruit key informants from local Chinese groups social anizationsbusinesses and faithbased agencies based on their capacity ofaccessing Chinese communities and their willingness to assist inrecruiting Chinese older adults in the community We collaboratedwith key community leaders This is a common and effective strategyto recruit respondents from minority populations15 as random sampling is challenging because of the unfeasibility of constructing acompleted sampling frame cultural appropriatenesstime andexpense16 The inclusion criteria for the survey participants includedHonolulu residents aged years and older who selfidentiï¬ed asChinese The detailed recruitment and data collection methods werereported in previous studies17 The participants provided informedconsent prior to the data collection This study was approved by theinstitutional review board at the university with which the secondauthor was afï¬liated A total of participants were recruited fromJanuary to September MeasuresDependent variables ACP engagement and EOL preference in burialplanningACP engagement includes ACP contemplation and ACP discussionACP contemplation and ACP discussion was assessed by asking respondents if they previously had thought about their endoflifecare plan with family and had discussed the plan with familyrespectivelyEOL preference in burial planning was measured by a hypothesizedquestion Respondents were asked whether formulating a burial planwas one of the most important things for them to consider if theywere diagnosed with a terminal illness and only had months to liveamong several other options Other mentioned options includedhaving religious beliefssupport alleviating pain reducing care andï¬nancial burden on family and extending their lifeIndependent variablesImmigrant status was measured by asking respondents whetherthey were US or foreignbornFamily relationships were measured by reliable and valid existingscalesdfamily cohesion and family conï¬ict The index of familycohesion was assessed by asking respondents whether familymembers like to spend free time with each other family membersfeel very close to each other and family togetherness is veryimportant18Family conï¬ict was measured using the 5item Family CulturalConï¬ict scale which assesses cultural and intergenerational conï¬ictperceived by respondents in their family19CovariatesSociodemographic variables included gender age marital statuseducation ï¬nancial strain living arrangement and social activityparticipation Health need factors included selfrated health comorbidity a continuous variable that examines the existence of at least chronic conditions including heart diseases stroke cancer diabeteshypertension high cholesterol thyroid disease arthritis liverrelateddiseases and others disabilities in activities of daily living andpsychological distress Psychological distress was assessed by theKessler Psychological Distress Scale K1020AnalysisFirst we summarized the sample characteristics Then we usedlogistic regression models and calculated odds ratios ORs to testwhether immigrant status family cohesion and family conï¬ict wereassociated with ACP engagement and EOL preferences All the analyses were conducted using Stata version The missing rates for ACP contemplation ACP discussion and EOLpreferences in burial planning were and respectively Toreduce sampling errors and attain more stable analytical results weconducted multiple imputations MIs for each model All thedependent variables were imputed and the imputed values wereretained in the analysis We used imputed data sets as there werehigh levels of missingness on the dependent variables21 For sensitivity analysis a dependent variable was imputed and imputed valueswere deleted for analysis MID The MID method produced ORs thatwere almost identical to those in the model where the imputed valueswere retainedResultsTable summarizes sample characteristics It shows that less thanhalf of the participants had ACP contemplation and ACP discussion Only had EOL preference in burial planning inthe hypothesized situationTable shows ORs with conï¬dence intervals CIs from logisticregressions The USborn Chinese Americans were more likely to haveACP contemplation OR CI ACP discussion OR CI and preference in burial planning OR CI than the foreignborn Higher levels of familyconï¬ict were associated with higher likelihood of ACP contemplationOR CI ACP discussion OR CI and preference in burial planning OR CI whereasfamily cohesion was not signiï¬cantly related to these outcomesDiscussionThis study aimed to examine the roles of immigrant status andfamily relationships in the associations between ACP engagement andgiving EOL preferences to burial planning among older ChineseAmericans The USborn Chinese Americans were more likely to haveACP contemplation and ACP discussion than the foreignborn Thismay be because the foreignborn Chinese Americans have lower socioeconomic status less English proï¬ciency lower levels of acculturation and less knowledge about ACP and the US healthcare systemthan their USborn counterparts111222 In addition these individuallevel differences may be mixed with other systemlevel barrierswithin the US healthcare system to worsen the disparities in ACPengagement23 For example Chinese American immigrants may havea stronger belief that family and society are held in higher regard thanindividuals and attribute a higher value to collectivism of family andsociety rather than patient autonomy in EOL decision making12Moreover because traditional Chinese culture expects children tocarry the role of protecting their parents health safety and generalwellbeing many Chinese children may construe this responsibility as 0cTable Characteristics of the Study Sample of Chinese Americans N ¼ or Mean SDCodingY Pei JAMDA xxx 1e4ACP engagementACP contemplationACP discussionEOL preferences in burial planningUSbornFamily RelationshipsFamily cohesionFamily conï¬ictFemaleAgeMarriedEducationFinancial strainLiving aloneParticipation in social activitiesSelfrated health as excellentgoodNumber of chronic diseaseADL disabilityPsychological distressADL activities of daily living never and dont want tonever but want toreluctant to yes never and dont want tonever but want toreluctant to yes no yes no yes least cohesivee12 most cohesive least cultural conï¬icte10 most cultural conï¬ict male female unmarried married not at alle3 a great deal no yes no yes no yes no help needed needs help least distressfule5 most distressfulmaking every effort to prolong their older parents life which maysometimes be in opposition to their parents own wishes24 Thesepotential factors surrounding older Chinese immigrants may helpexplain this populations lack of engagement in ACP Healthcare providers in turn should pay closer attention to these factors in order tothoroughly evaluate patients EOL wishes It is noted that the USbornChinese Americans were far more likely to have preferences in burialplanning than the foreignborn The ï¬nding is consistent with a previous study in that decisions such as EOL care and funeral and burialpreplanning are impacted by similar factors25 Indeed EOL care decision making and burial planning are integrated processes at theend of life26 and burial plan is included in some advance directivedocuments in practice Future studies on ACP need to consider burialplanningSecond family cohesion was not associated with ACP contemplation ACP discussion and EOL preference in burial planning whereasfamily conï¬ict increased the possibility of ACP contemplation ACPdiscussion and EOL preferences in burial planning The ï¬nding isinconsistent with previous study conducted among White olderadults revealing that the positive family relationship encourageswhereas problematic family relationship hinders ACP engagement27The inconsistency is likely due to the fact that Chinese Americansvalue family in the process of EOL decision making2829 The lack ofassociation between family cohesion and ACP engagement may bebecause older adults with higher levels of family cohesion have tobalance between the potential beneï¬t and harm of ACP engagementOn the hand older Chinese Americans may have positive attitudesabout ACP engagement and believe that ACP engagement is importantand necessary because it allows them to witness their loved onesdeath and dying experience12 On the other hand closeknit familialrelationships may make both older Chinese Americans and theirfamilies feel more uncomfortable to start a conversation on EOL carebecause discussions about death and dying are often considered ataboo in Chinese culture30 In this sense strong family ties may havelimited impact on ACP engagement An explanation for the signiï¬cantrelationship between family conï¬ict and ACP engagement could bethat higher levels of family conï¬ict may indicate a greater need for ACPengagement This is because the members in these families are lesslikely to know about the EOL care preferences of older adults and betrusted in the EOL decision making13 These ï¬ndings suggest thatculture may play an important role in the complex association between family relationships and ACP engagementSeveral limitations of the study deserve mentioning First thecrosssectional data from a small region limit our ability to generalizeï¬ndings to older Chinese Americans living in other parts of the UnitedStates as well as to make causalinferences Second the ACPengagement in our study only included ACP contemplation ACP discussion and preference in burial planning Future studies need toinclude more ACP options such as the completion of living wills oradvance directives and the selection of a durable power of attorneyfor health care to understand more about ACP engagement in ChineseAmerican families Third ACP knowledge is an important confoundingvariable for both immigrant status and ACP engagement Futurestudies on ACP engagement need to consider this variableConclusions and ImplicationsDespite these limitations this study sheds light on how immigrantstatus and family relationships shape ACP engagement among olderChinese AmericansIt is found that immigrant status decreaseswhereas family conï¬ict increases the likelihood of having ACPcontemplation ACP discussion and preference in burial planningTable Factors Associated With Advance Care Planning Engagement Results of Logistic Regression N ¼ USborn ref ¼ foreignbornFamily relationshipsFamily cohesionFamily conï¬ictACP ContemplationOR CI ACP DiscussionOR CI EOL Preferences inBurial Planning OR CI All models adjusted for age gender marital status education ï¬nancial strain living alone social activity participation selfrated health number of chronic disease activitiesof daily living and psychological distress 0cY Pei JAMDA xxx 1e4Health care providers may consider patients immigrant status andfamily relationships to better serve ethnically diverse populationsGiven that cultural factors play an important role in ACP engagementACP should be adapted to be more culturally appropriate amongChinese Americans especially in a time of coronavirus and xenophobia such as framing ACP as a tool to help families reduce stresswhile fulï¬lling ï¬lial obligations in order to ensure equitable access toACPAcknowledgmentWe thank Katherine Wang for her editorial assistance We wouldalso like to thank the research team at the University of Hawaii fortheir data collectionReferences Rietjens JAC Sudore RL Connolly M Deï¬nition and recommendations foradvance care planning An international 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relationship179e184 Tong M Sentell T Insights in public health Challenges investigating healthoutcomes in Chinese Americans using populationbased survey data Hawaii JMed Public Health Ibrahim S Sidani S Strategies to recruit minority persons A systematic reviewJ Immigr Minor Health 20145882e888 Spring M Westermeyer J Halcon L Sampling in difï¬cult to access refugeeand immigrant communities J Nerv Ment Dis 2003191813e819 Zhang W Liu S Zhang K Wu B Neighborhood social cohesion resilience andpsychological wellbeing among Chinese older adults in Hawaii Gerontologist201960229e238 Rivera FI Guarnaccia PJ MulvaneyDay N Family cohesion and its relationship to psychological distress among Latino groups Hisp J Behav Sci 30357e378 Alegria M Vila D Woo M Cultural relevance and equivalence in theNLAAS instrument Integrating etic and emic in the development of crosscultural measures for a psychiatric epidemiology and services study of LatinosInt J Methods Psychiatr Res 200413270e288 Kessler RC Andrews G Colpe LJ Short screening scales to monitor population prevalences and trends in nonspeciï¬c psychological distress PsycholMed 200232959e976 Johnson DR Young R Toward best practices in analyzing datasets withmissing data Comparisons and recommendations J Marriage Fam 926e945 Carr D The social stratiï¬cation of older adults preparations for endoflifehealth care J Health Soc Behav 201253297e312 Shen MJ Prigerson HG Tergas AI Maciejewski PK Impact of immigrant statuson aggressive medical care counter to patients values near death amongadvanced cancer patients J Palliat Med 20192234e40 Bowman K Singer P Chinese seniors perspectives on endoflife decisions SocSci Med 200153455e464 Kelly CM Masters JL DeViney S Endoflife planning activities An integratedprocess Death Stud 201337529e551 Tanaka M Takahashi M Kawashima D Endoflife activities amongin Japan Omega Westport communitydwelling older adults Carr D Moorman SM Boerner K Endoflife planning in a family context Doesrelationship quality affect whether and with whom older adults planJ Gerontol B Psychol Sci Soc Sci 201368586e592 Hinderer KA Lee MC Chinese Americans attitudes toward advance directivesAn assessment of outcomes based on a nursingled intervention Appl Nurs Res20194991e96 YonashiroCho J Cote S Enguidanos S Knowledge about and perceptions ofadvance care planning and communication of ChineseAmerican older adultsJ Am Geriatr Soc 2016641884e1889 Chi HL Cataldo J Ho EY Rehm RS Can we talk about it now Recognizing theoptimal time to initiate endoflife care discussions with older ChineseAmericans and their families J Transcult Nurs 201829532e539 0c"
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"Tumor microenvironment TME plays an important role in malignant tumors Our study aimed toinvestigate the effect of the TME and related genes in osteosarcoma patientsMethods Gene expression profiles and clinical data of osteosarcoma patients were downloaded from the TARGETdataset ESTIMATE algorithm was used to quantify the immune score Then the association between immune scoreand prognosis was studied Afterward a differential analysis was performed based on the high and lowimmunescores to determine TMErelated genes Additionally Cox analyses were performed to construct two prognosticsignatures for overall survival OS and diseasefree survival DFS respectively Two datasets obtained from the GEOdatabase were used to validate signaturesResults Eightyfive patients were included in our research The survival analysis indicated that patients with higherimmune score have a favorable OS and DFS Moreover genes were determined as TMErelated genes Theunsupervised clustering analysis revealed two clusters were significantly related to immune score and T cells CD4memory fraction In addition two signatures were generated based on three and two TMErelated genesrespectively Both two signatures can significantly divide patients into low and highrisk groups and were validatedin two GEO datasets Afterward the risk score and metastatic status were identified as independent prognosticfactors for both OS and DFS and two nomograms were generated The Cindexes of OS nomogram and DFSnomogram were and respectivelyConclusion TME was associated with the prognosis of osteosarcoma patients Prognostic models based on TMErelated genes can effectively predict OS and DFS of osteosarcoma patientsKeywords Tumor microenvironment Osteosarcoma Prognosis Immune features NomogramBackgroundOsteosarcoma is the most common bone tumor especiallyin children and adolescents [] It was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group [] Currently surgery and Correspondence 407404159qqcom4Wenzhou Medical University Wenzhou ChinaFull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years However due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable[] Previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively [] Thereforeit is necessary to The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cHu BMC Cancer Page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsRecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified For example Chen [] reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients Moreover Huang [] discovered that dysregulated circRNAs serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microRNA In addition lncRNA [] microRNA [] and many clinical data [] were also identified asprognostic biomarkers for osteosarcoma patients However osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humansTherefore it is necessary to find accurate biomarkers forosteosarcomaIn recent years researchers have paid more and moreattention to the role of the tumor microenvironmentTME in malignant tumors The function of TME inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] More importantly Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data ESTIMATE an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in [] Based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [] Generally the above research indicatedthat TME can serve as the prognostic biomarker in tumorsand many TMErelated genes were determined as the prognostic genes However the role of TME and TMErelatedgenes in osteosarcoma patients remains unclearIn the present study gene expression data and corresponding clinicopathologic data were obtained from TheTherapeutically Applicable Research to Generate EffectiveTreatments TARGET dataset Then the ESTIMATEalgorithm was performed to quantify the immune score ofosteosarcoma and the TMErelated genes were identifiedby the differential expression analysis Subsequently theprognostic value of TME and TMErelated genes weredetermined by a series of bioinformatics methodsMethodsGene expression datasetsLevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom TARGET dataset ocgcancergovprogramstarget accessed on Oct The correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus After data were extracted from the public domainthe ESTIMATE an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in R software version [] Meanwhile the messenger RNAmRNA expressionprofiles and clinical data ofincludingGSE21257 [] and GSE39055 [] were obtained fromthe Gene Expression Omnibus as external validationcohortstwo cohortsSurvival analysis and correlation analysisAfter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score The KaplanMeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival OS and diseasefreesurvival DFS between high and lowscore cohorts Inaddition the association between clinicopathologic dataand TME score was also studied MannWhitney signedrank test was performed to compare the differences ofimmune score between each clinical group All statisticalanalyses in the present study were performed using Rsoftware Except for the special instructions p value twoside was identified as statistically significantin the present studyDEGexpressed geneDifferentially expressed gene analysisDifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group The limma package in Rsoftware was used to perform the differential analysisand genes with log FC and adjusted pvalue qvalue were identified as DEGs []To further understand the function of DEGs identifiedin the present study Gene Ontology GOincludingbiological processes BP molecular functions MF andcellular componentsCC and Kyoto Encyclopedia ofGenes and Genomes KEGG analysis were performedby clusterProfiler package in R software []Evaluation of association with immune cellsTo further investigate the association between DEGs andimmune cells the CIBERSORT package was used toestimate the relative proportions of types of immunecells [] Meanwhile the ConsensusClusterPlus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping DEGs [] Cumulative distribution function CDF and relative change inarea under the CDF curve were used to determine theoptimal number of clusters k Then MannWhitney 0cHu BMC Cancer Page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters []Survival analysis of DEGsBased on the DEGs the univariate COX analysis was performed to determine the prognostic value of immunerelated genes Then the OSrelated genes were validatedin the GSE21257 dataset while the DFSrelated geneswere validated in the GSE39055 dataset Only genes successfully validated were selected for further analysis Afterward based on the validated genes the multivariate COXanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsThe risk score for each patient was calculated based onthe coefficient from the multivariate COX analysis and thecorresponding gene expression Meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score The survival receiver operating characteristic ROC curve was used to show the discrimination of signatures and the KaplanMeier survivalcurve with the logrank test was generated to show thedifferences of OS and DFS between high and lowriskgroups In addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature The KaplanMeier survivalcurve and survival ROC curve were generated to show thepredictive ability of the signature in the validation cohortDevelopment of a nomogram for osteosarcoma patientsNomogram is a tool to visualize the predictive model andconvenient for clinical practice Therefore we attemptedto develop a nomogram based on the TMErelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients Firstlythe univariateCOX analysis was performed to filter prognostic variableswhich will be further included in the multivariate COXanalysis Secondly based on independent prognostic variables two nomograms were established for predicting theOS and DFS respectively The Cindex was used to assessthe discriminatory performance of the nomogram whichrange from to [] A Cindex of means agreement by chance and a Cindex of represents perfectdiscriminatory performance The higher value of the Cindex the better performance of the nomogram is Furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsResultsImmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females The immunescore of the cohort range from to Tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group The survival analysis indicated thatpatients with higher immune score had a favorable OSand DFS Fig 1a and b After adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both OS and DFSFig 1a and b Inaddition the relationship between immune score and clinical features was also investigated However there was nosignificant relationship between immune score and clinicalvariables Supplementary Figure 1A1CDifferential expression analysisAccording to the median of the immune score patients were divided into highscore n and lowFig Association between immune score and prognosis in osteosarcoma patients a KaplanMeier survival analysis of overall survival for patientswith high vs low immune score b KaplanMeier survival analysis of diseasefree survival for patients with high vs low immune score 0cHu BMC Cancer Page of score group n There were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesFig 2a b and Supplementary Table To furtherunderstand the function of DEGs GO analysisand KEGG analysis were performed The top significant results of GO analysis among three types wereillustrated in Fig 2c Interestingly we can find that theresults of GO analysis are mostly associated with immunity which further verify that the immunerelated DEGsare associated with immune features In addition the results of KEGG also confirmed it Such as PhagosomeAutoimmune thyroid disease Antigen processing andpresentation B cell receptor signaling pathway Intestinal immune network for IgA production Inflammatorybowel disease Primary immunodeficiency Th1 andTh2 cell differentiation Th17 cell differentiation Natural killer cell mediated cytotoxicity and NFkappa Bsignaling pathway Fig 2dconsensusunsupervisedEvaluation of DEGs and immune cellsTo further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns Based on the consensus matrix heat map patientswere clearly divided into two clustersFig 3a In additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined Fig 3bc The immune score betweentwo clusters was significantly different Fig 3d In additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot Fig 3e Interestinglywe can see that the T cells CD4 memory activated ofcluster is significantly higher than cluster Fig 5fPrognostic value of TMErelated genesPrevious studies indicated that TMErelated genes canserve as the prognostic biomarker for tumor patientsFig Differentially expressed genes with the immune score in osteosarcoma patients a Heatmap of significantly differentially expressed genesbased on immune score b The volcano figure to show the upregulated and downregulated genes c GO analysis of differentially expressedgenes d KEGG of differentially expressed genes GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes 0cHu BMC Cancer Page of Fig The immune landscape of the tumor microenvironment ac Unsupervised clustering of all samples based on the overlapping DEGs dComparison of immune score between two clusters e The distribution of types of immune cells in osteosarcoma patients f The comparisonof types of immune cells between clusters DEG Differentially expressed geneHence we performed the univariate COX analysis toidentify prognostic DEGs The results showed that and genes were identified as OS and DFSrelatedDEGs respectively Supplementary Table and Afterward five OSrelated genes were successfully validated inthe GSE21257 data set and five DFSrelated genes were successfully validated in the GSE39055 cohort Furthermoremultivariate COX analysis was performed and two prognostic signatures were generated for predicting the OS andDFS respectively The risk score for predicting the OS wasasrisk score FCGR2B0766 GFAP0702 MPP70387 In addition the risk score for predicting theDFS was as follows risk score CYP2S10574 ICAM3 The AUC values of OSrelated signature were follows 0cHu BMC Cancer Page of and in and 3year respectively Fig 4aand the AUC values of DFSrelated signature were and in and 3year respectively Fig 5aMoreover survival curves showed that patients in the highrisk group had worse OS and DFS compared with the lowrisk patients Figs 4b and 5b Heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients Figs 4ceand 5ce Then both signatures were validated in independent cohorts For OS signature the AUC values ofvalidation cohort were and at and3year Fig 4f For DFS signature the AUC values ofvalidation cohort were and at and3year Fig 5f Additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients Figs 4g and 5gHeat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsFigs 4hj and f 5hjDevelopment of a nomogram for osteosarcoma patientsTo generate a nomogram for clinical use the COX analysiswas performed to select the clinical prognostic variables InFig Establishment and validation of the prognostic model for overall survival based on significant DEGs a Receiver operating characteristiccurves of prognostic signature in the training cohort b The survival curve showed the different overall survival status between high and lowriskpatients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each sample reordered by riskscore e The scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f Receiver operating characteristiccurves of prognostic signature in validation cohort g The survival curve showed the different overall survival status between high and lowriskpatients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reordered by riskscore j The scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0cHu BMC Cancer Page of Fig Establishment and validation of the prognostic model for diseasefree survival based on significant DEGs a Receiver operatingcharacteristic curves of prognostic signature in the training cohort b The survival curve showed the different diseasefree status between highand lowrisk patients c The heat map showed the expression of prognostic genes in the training cohort d The risk curve of each samplereordered by risk score e The scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f Receiver operatingcharacteristic curves of prognostic signature in validation cohort g The survival curve showed the different diseasefree status between high andlowrisk patients h The heat map showed the expression of prognostic genes in the validation cohort i The risk curve of each sample reorderedby risk score j The scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate COX analysis risk score and metastatic status were identified as both OS and DFSrelated variablesFig 6a and e Afterward risk score and metastatic statuswere determined as both independent OS and DFSrelated variables in the multivariate COX analysis Fig 6band f Based on independent variables two nomogramswere established for predicting the OS and DFS in osteosarcoma patients respectively Fig 6c and g The Cindexvalues were and in OS nomogram and DFSnomogram respectively The results of Cindex mean thatboth two nomograms have good discrimination Meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve Fig 6dand h which indicated a good calibrationDiscussionThe relationship between TME and tumor have beenwidely studied in recent years In the present study ESTIMATE algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from TARGET database We confirmed that theTME is significantly associated with the prognosis ofosteosarcoma patientsInadditionfunctional enrichment analyses of TMErelated genes indicated that immunerelated processesincluding OS and DFS 0cHu BMC Cancer Page of Fig Nomograms based on the tumor microenvironment related genes for osteosarcoma patients a Univariate COX analysis of overall survivalrelated variables b Multivariate COX analysis of overall survivalrelated variables c Nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curveS of overall survival nomogram e Univariate COX analysis of diseasefree survivalrelated variables fMultivariate COX analysis of diseasefree survivalrelated variables g Nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curveS of diseasefree survival nomogramknown to contribute to tumor progression More importantly DEGs based on the TME were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the OS and DFS of osteosarcoma patientsrespectivelyIn recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the TME and the ESTIMATE algorithm is oneof the most important quantitative tools for this researchfield Based on the ESTIMATE algorithm the association between the prognosis and TME has been initially 0cHu BMC Cancer Page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ ] However previousstudies indicated that TME scores serve as a differentrole in different tumors For example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ ] However for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] Therefore we can find great heterogeneity among different tumors from the perspectiveof TME For osteosarcoma patients the present studyindicated that patients with higher immune score had abetter OS and DFS Hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionIn our research TMErelated genes were identified by comparing the highscore and lowscore osteosarcoma patients The functional enrichment includingGO and KEGG analyses showed that TMErelated geneswere mainly involved in the immune features such asregulation of leukocyte activation MHC protein complex MHC protein and complex binding More importantly the unsupervised cluster analysis based on DEGswas performed and all patients were divided into twoclusters Immune score and T cell CD4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between DEGs and immune featuresDue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantThe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients Emilie etal []performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma Recently the prognostic signature based onTMErelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients [] Compared with the study performedby Zhang [] we think that our research have someadvantages Firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts Secondlythe outcome of DFS was not reported in the previousstudy As reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]Hence the DFS nomogram can improve the managementof osteosarcoma patients Finally two nomograms incorporated TMErelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsIn our research five genes were incorporated into thefinal prognostic signatures FCGR2B GFAP and MPP7were identified and validated as OSrelated biomarkerswhile CYP2S1 and ICAM3 were DFSrelated biomarkersThe role of these genes in tumor prognosis had beenwidely reported in previous studies [] FCGR2Bhas been confirmed as an immunerelated gene previously [] Although the relationship between FCGR2Band prognosis in sarcoma patients had not been reported the prognostic value of FCGR2B had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] In addition NewM etal [] demonstrated that MPP7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomaCYP2S1 described as Cytochrome P450 Family Subfamily S Member was reported significantly associatedwith colorectal cancer In primary colorectal cancerCYP2S1 was present at a significantly higher level ofintensity compared with normal colon [] More importantly the presence of strong CYP2S1 immunoreactivity was associated with poor prognosis [] The roleof ICAM3 in cancer was also widely reported in published studies and the Akt pathway plays an importantrole in the impact of ICAM3 on tumors YG Kim etal[] reported that ICAM3 can induce the proliferationof cancer cells through the PI3KAkt pathway Additionally JK Park etal showed that the ICAM3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing MMP2 andMMP9 via Akt pathway [] showed that the ICAM3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingMMP2 and MMP9 via Akt pathwayAlthough the role of TME and TMErelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated Firstly the treatmentinformation cannot be obtained from the TARGET database which may influence the prognosis of osteosarcomapatients Secondly two nomograms were generated andshowed good performance in our study However externalvalidation by a large cohort is needed Thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear Finally inthe training cohort and DEGs were identified asOS and DFSrelated DEGs respectively However onlyfive OS and five DFSrelated genes were identified in thevalidation cohort The different age structures smaller 0cHu BMC Cancer Page of sample sizes and the platform covering only part of thegenes may contribute to this resultReceived February Accepted July ConclusionIn conclusion TME plays an important role in osteosarcoma patients and related with the progression of thetumor Moreover TMErelated genes can serve as prognostic biomarkers in osteosarcoma patients Howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studySupplementary informationSupplementary information accompanies this paper at doi101186s12885020072162Additional file Additional file Additional file Additional file AbbreviationsTME Tumor microenvironment DEG Differentially expressed genesOS Overall survival DFS Diseasesfree survival ROC Receiver characteristiccurve ESTIMATE Estimation of STromal and Immune cells in MAlignantTumor tissues using Expression data TARGET Therapeutically ApplicableResearch to Generate Effective Treatments GO Gene Ontology BP Biologicalprocesses MF Molecular functions CC Cellular components KEGG KyotoEncyclopedia of Genes and Genomes CDF Cumulative distribution functionAcknowledgementsNoneAuthors contributionsC H L Y Sq T C L and Yh W conceived of and designed the study C H R Sand C L performed literature search R S L Y and B C generated the figuresand tables L Y Hl R X Y and Jy L analyzed the data C H wrote themanuscript and Sq T and L Y critically reviewed the manuscript L Ysupervised the research All authors have read and approved the manuscriptFundingWe received no external funding for this studyAvailability of data and materialsThe data of this study are from TARGET and GEO databaseEthics approval and consent to participateThe research didnt involve animal experiments and human specimens noethics related issuesConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Joint Surgery the Affiliated Hospital of Qingdao UniversityQingdao China 2Department of Medical Oncology the First Hospital ofChina Medical University Shenyang China 3Department of Nursing Sir RunRun Shaw Hospital Affiliated to Zhejiang University Hangzhou China4Wenzhou Medical University Wenzhou ChinaReferencesJaffe N Bruland OS Bielack S Pediatric and adolescent osteosarcoma vol New York Springer Science Business Media Vander RG Osteosarcoma and its variants Orthopedic Clin North Am Biermann JS Adkins D Benjamin R Brigman B Chow W Conrad EU 3rdFrassica D Frassica FJ Gee S Healey JH Bone cancer J Natl ComprCancer Netw Simpson S Dunning MD de Brot S GrauRoma L Mongan NP Rutland CSComparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics Acta Vet Scand Chen X Cates JM Du YC Jain A Jung SY Li XN Hicks JM Man TKMislocalized cytoplasmic p27 activates PAK1mediated metastasis and is aprognostic factor in osteosarcoma Mol Oncol Huang X Yang W Zhang Z Shao Z Dysregulated circRNAs serve as prognosticand diagnostic markers in osteosarcoma by sponging microRNA to regulatethe downstream signaling pathway J Cell Biochem Liu M Yang P Mao G Deng J Peng G Ning X Yang H Sun H Long noncoding RNA MALAT1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis Int J Surg Xu K Xiong W Zhao S Wang B MicroRNA106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma Oncol Lett Zheng W Huang Y Chen H Wang N Xiao W Liang Y Jiang X Su W WenS Nomogram application to predict overall and cancerspecific survival inosteosarcoma Cancer Manag Res Kahlert C Kalluri R Exosomes in tumor microenvironment influence cancerprogression and metastasis J Mol Med Binnewies M Roberts EW Kersten K Chan V Fearon DF Merad M CoussensLM Gabrilovich DI OstrandRosenberg S Hedrick CC Understanding thetumor immune microenvironment TIME for effective therapy Nat Med Yoshihara K Shahmoradgoli M MartÃnez E Vegesna R Kim H TorresGarcia WTreviño V Shen H Laird PW Levine DA Inferring tumour purity and stromaland immune cell admixture from expression data Nat Commun Yang S Liu T Nan H Wang Y Chen H Zhang X Zhang Y Shen B Qian PXu S Comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma J Cell Physiol Deng Z Wang J Xu B Jin Z Wu G Zeng J Peng M Guo Y Wen Z MiningTCGA database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma Biomed Res Int Zhao K Yang H Kang H Wu A Identification of key genes in thyroid Cancermicroenvironment Med Sci Monit Xu WH Xu Y Wang J Wan FN Wang HK Cao DL Shi GH Qu YYZhang HL Ye DW Prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment AgingAlbany NY Chen B Chen W Jin J Wang X Cao Y He Y Data Mining of PrognosticMicroenvironmentRelated Genes in clear cell renal cell carcinoma a studywith TCGA database Dis Markers Li X Gao Y Xu Z Zhang Z Zheng Y Qi F Identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods Cancer Med Pan XB Lu Y Huang JL Long Y Yao DS Prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma Aging Albany NY Wang H Wu X Chen Y Stromalimmune scorebased gene signature aprognosis stratification tool in gastric Cancer F
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lipases are very versatile enzymes and produced the attention of the several industrial processes lipase can be achieved from several sources animal vegetable and microbiological the uses of microbial lipase market is estimated to be usd million in and it is projected to reach usd million by growing at a cagr of from microbial lipases ec catalyze the hydrolysis of long chain triglycerides the microbial origins of lipase enzymes are logically dynamic and proficient also have an extensive range of industrial uses with the manufacturing of altered molecules the unique lipase triacylglycerol acyl hydrolase enzymes catalyzed the hydrolysis esterification and alcoholysis reactions immobilization has made the use of microbial lipases accomplish its best performance and hence suitable for several reactions and need to enhance aroma to the immobilization processes immobilized enzymes depend on the immobilization technique and the carrier type the choice of the carrier concerns usually the biocompatibility chemical and thermal stability and insolubility under reaction conditions capability of easy rejuvenation and reusability as well as cost proficiency bacillus spp achromobacter spp alcaligenes spp arthrobacter spp pseudomonos spp of bacteria and penicillium spp fusarium spp aspergillus spp of fungi are screened large scale for lipase production lipases as multipurpose biological catalyst has given a favorable vision in meeting the needs for several industries such as biodiesel foods and drinks leather textile detergents pharmaceuticals and medicals this review represents a discussion on microbial sources of lipases immobilization methods increased productivity at market profitability and reduce logistical liability on the environment and userkeywords microbial lipase fatty acids triglycerides protein engineering biosensor food industry candida antarctica lipase b calbintroductionthe serine hydrolases are present in abundantly and known as lipase enzyme which belong to triacylglycerol ester hydrolase family ec they can catalyze the hydrolysis and synthesis of longchain triglycerides to fatty acids diacylglycerol monoacylglycerol and glycerol known as carboxylesterases [ ] besides hydrolysis activity they display interesterification esterification aminolysis and alcoholysis activity which are contributed correspondence pchandrabbaugmailcom food microbiology toxicology department of microbiology school for biomedical and pharmaceutical sciences babasaheb bhimrao ambedkar university a central university lucknow uttar pradesh indiafull list of author information is available at the end of the in wide range industries [ ] lipase synthesizes esters from glycerol and longchain fatty acids in nonaqueous medium the microbial lipases are more valuable comparison to derive from plants or animals due to their variety of catalytic activities available high yield production and simplicity of genetic manipulation absence of seasonal fluctuations regular supply more stability safer and more convenient and the growth rate of microanisms very high in economically media [ ] the bacterial isolates offer higher activities such as neutral or alkaline ph optima and the thermostability associated to yeasts bacterial strains such as pseudomonas alcaligenes p aeruginosa p fragi p fluorescens bj10 bacillus subtilis b nealsonii s2mt and some species of fungi are penicillium expansum trichoderma penicillium chrysogenum the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchandra a0et a0al microb cell fact page of aspergillus niger produces lipases in higher quantities [] the increasing awareness about animal health and quality of animal produce and increasing consumption of enzymemodified cheese emc and enzymemodified dairy ingredients emdi the lipase market has been extensively increased [ ] due to the more benefits of microbial lipases over animal and plant lipases are also motivating the market growth the request for microbial sources is projected to witness significant growth in the near future due to their wide range of food processing applications [ ] the microbial lipase market is projected to dominate due to cleaning agent segment through the forecast period the growth of industrial microbial lipases in the detergents industry is the innovative key factor to replacing harsh chlorine bleach with lipase and reduced the industrial as well as sewage pollution from fresh water [ ] the microbial lipases in the form of powder is projected to dominate the microbial lipase markets due to its stability easy to handle and easier for packaging and its transportation preferred by the consumers [ ] a0these are extensively applicable in several another industries such as dairy food and beverage animal feed cleaning biofuel pharmaceuticals textile cosmetic perfumery flavour industry biocatalytic resolution esters and amino acid derivatives fine chemicals production agrochemicals biosensor and bioremediation [] additionally altering in the dietary patterns have led to augmented the consumption of dairy products in the region increasing in trepidations about superior hygiene in consciousness of personal hygiene contagious diseases and bleaching household industrial surfaces the manufacturers who operate on a global level and the rising in implementation of lipase enzymes drive the demand for microbial lipases in the region [ ]between the and the market scope of lipase is expected to reach million by globally at a cagr of the asiapacific was the largest market for lipase consumption in [ ] and during the forecast period the asiapacific market is estimated to grow at the highest cagr moreover the rising prospects in the developing markets such as india china and brazil are expected to enhance the market scope of lipases over the forecast period novozymes as denmark e i du pont de nemours and company genencor us koninklijke dsm nv netherlands and chr hansen holdings as denmark are the key industries reported for the consumption of lipases at worldwide httpwwwmarke tsand marke tscom due to the specific properties such as enantioselectivity regioselectivity and broad substrate specificity properties the lipase showing more interest between all the enzymes [ ] this present review focused on discussing the sources of microanisms immobilization methods and their potential applications of lipases including commercially availablehistorical inside or outside the cells enzymes are proteins and have ability of catalyzing the various chemical and biochemical reactions they are highly specific natural catalysts to the various types of substrates and operate under insignificant conditions of environmental factor such as temperature pressure ph with high conversion rates [ ] lipase was first discovered in pancreatic juice as an enzyme by claude bernard in which hydrolysed unsolvable oil droplets and transformed them to soluble products after that the productions of lipase have been observed in the bacteria bacillus prodigiosus b pyocyaneus and b fluorescens in and in the current scenario serratia marcescens pseudomonas aeruginosa and pseudomonas fluorescens species of bacteria have been detected for the production of lipases on large scale lipolase was the first commercial recombinant lipase industrialized from the fungus thermomycesl anugiwnosus and expressed in aspergillus oryzae in traditionally lipase has been achieved from the animal pancreas and was made applicable as digestive supplements in the form of crude or in purified grade it has been extensively used as biocatalytic procedures for the synthesis of several novel chemical compounds []definition of a0lipaseslipases ec are known as triacylglycerol acylhydrolase which acts on carboxylic ester bonds is the part of hydrolases family [ ] they do not require any cofactor and belongs to the class of serine hydrolases triglycerides hydrolyzed into diglycerides monoglycerides fatty acids and glycerol by using the lipases naturally fig a01a the carboxylic esters bonds can be hydrolyzed by esterases in addition to lipases [ ]the hydrolysis of ester bonds at the interface catalyzes by lipases between an unsolvable phase of substrate and aqueous phase where the enzymes keep on liquefied under natural conditions fig a0 1b however pseudomonas aeruginosa candida anatarctica b and burkholderia glumae possessed a lid but did not show interfacial activation [ ] esterification transesterification interesterification acidolysis alcoholysis and aminolysis conversion reaction takes place by lipases [ ]the presence of a lid and the interfacial activation are not the suitable criteria for to categorize a true lipase carboxylesterase simply defined that catalyzes the hydrolysis and synthesis of longchain acylglycerols 0cchandra a0et a0al microb cell fact page of fig a hydrolysis of triglyceride converts into glycerol and fatty acid b representation of a molecule of lipase with its featuresproperties and a0characteristics of a0lipasesthe molecular weight of lipases is in the range of a0kda and reported to be monomeric protein the position of the fatty acid in the glycerol backbone chain length of the fatty acid and its degree of unsaturation are the factors and the physical properties of lipases depend on it [ ] the sensory and nutritive values of given triglyceride also affected by these features several lipases catalyze a number of useful reactions such as esterification due to their activeness in anic solvents [ ] lipases displayed ph dependent activities generally at neutral ph or up to ph and lipases are stable chromobacterium viscosum a niger and rhizophus sp produced extracellular lipases are active at acidic ph and p nitroaeducens produced alkaline lipase and active at ph under certain experimental conditions lipases have capability to reversing the reactions which leads to esterification and interesterification in the absence of water [ ] for the expression of lipase activities the cofactors are not necessary but calcium is the divalent cation stimulates the activity [ ] co ni2 hg2 and sn2 inhibited the lipase activities drastically and zn2 mg2 edta and sds inhibited slightly the halflife values determined temperature stability profiles of lipases and lower temperature shows more stability [ ] according to the regionspecificity lipases divided into two groups and revealed with acyl glycerol substrate without display of regiospecificity only fatty acids are discharged from all three positions of glycerols in the first group of lipases [] the fatty acids regiospecifically discharged from the positions of acylglycerols in the second group of lipase triacylglycerol hydrolysed by lipases and constructed 2monoacylglycerol and free fatty acids 3diacylglycerols in a arrhizus r delemar c cylindracea and p aeruginosa the partial stereospecificity have been detected in the hydrolysis of triacylglycerols [] these enzymes may be used to extract optically pure esters and alcohols due to these properties at low water activity using the anic media offers an exceptional prospect over variation of the solvent so varying the properties of the solvents an enzymes specificity may be transformed any solvent may utilize a substantial influence on the catalytic properties of an enzyme due to the possession of soft structures and delicate [ ]kinetic model of a0lipolysisat the substratewater interface lipolysis arises so the michaelismenten model cannot be described it in a homogeneous phase which is effective only for biocatalysis in which enzyme and substrate are soluble [ ] at an interface to describe the kinetics of lipolysis simple models has been proposed and be made up of two consecutive equilibrium [ ] the alterable adsorption of enzyme to the interface ee happens in the first equilibrium phase a single substrate molecule s 0cchandra a0et a0al microb cell fact page of binds by the adsorbed enzyme e in the formation of es complex as a result in the second phase of equilibrium [ ] for the enzymesubstrate complex to the michaelis menten equilibrium this latter equilibrium is equivalent ending with the discharge of the products and renovation of the enzyme in the e form the subsequent catalytic steps take place once the es complex is formed [ ] the adsorbed lipase in the vicinity of substrate concentration at the interface is at the surface concentration instead of volumetric concentration conventional in the atmosphere [ ] the rejuvenated lipase remnant adsorbed to the interface and is only unrestricted after a number of catalytic cycles in this model fig a0the activity of lipase is a utility of interfacial conformation the enzyme can be denatured as well as triggered or neutralized and the interface is a suitable spot for restraining lipolysis the directly interaction of lipase inhibitor with the enzyme and obstructs the activity of lipase on the other hand via the adsorption to the interphase or to the substrate molecules few compounds can postpone the lipolytic reaction []lipase inhibitors are grouped into two categoriesa synthetic lipase inhibitors including phosphonates boronic acids and fats analogues andb natural compounds βlactones and several botanical foodstuffsplant extracts and metabolites chiefly polyphenols saponins as well as peptides and particular nutritive fibers lipases are essential enzymes for lipid absorption so the absorption of fat or obesity controlled by the lipase inhibition β lactones including orlistat are the natural compounds have the ability to inhibit the lipase activity [ ] over of total dietary fats the pancreatic lipase is responsible for the hydrolysis in several countries for the treatment of obesity orlistat is the registered drug fig lipase catalyzed different reactionslipase inhibitors from a0microbial sourcesfrom microanisms several metabolic products have potent pancreatic lipase pl inhibitory activity the several bacterial fungal and other marine species continued search of effective antiobesity agent screened to find new compounds with pl inhibitory activity [ ]lipstatinthe digestive activity of pancreatic lipases controls by the lipstatin is a βlactone molecule which also controls the absorption of fat in the small intestine lipstatin was first isolated from streptomyces toxytricini is a precursor for tetrahydrolipstatin also known as orlistat xenical and alli the only fdaapproved antiobesity medication for longterm use is a very potent inhibitor of pl [ ] lipase inhibitory activity was lost on opening of βlactone ring the catalytic hydrogenation product of lipstatin is crystalline tetrahydrolipstatin and generally known as orlistat is currently on the market as an antiobesity agent [ ]panclicinsstreptomyces sp nr produced panclicins is another class of potent pl inhibitors nformylalanyloxy or nformylglycyloxy substituent are two alkyl chains are found in panclicins too contains blactone structures panclicins a and b are alanine type while panclicins c d and e are glycine type of compounds the inhibitory activity was recognized to the amino acid moiety alaninecontaining compounds being two to three folds weaker than glycinecontaining compounds valilactonevalilactone first isolated from streptomyces albolongus mg147cf2 strain from shaken culture and jar fermentation valilactone potently inhibited hog pl with an ic50 of a0ngml it also influenced inhibitory activity of esterase from hog liver with an ic50 value of a0mgml ebelactonesebelactone a and b are two ebelactones were isolated from the fermentation broth of actinomycetes strain g7gl closely related to streptomyces aburaviensis both a and b revealed pl inhibitory activity with ic50 values of against hog pl are a0 ngml and a0 ngml respectively esterastinesterastin was isolated from actinomycetes streptomyces lavendulae md4c1 strain from the fermentation 0cchandra a0et a0al microb cell fact page of broth competitively esterastin introverted the hog pancreas lipase with ic50 value of a0ngml caulerpenynecaulerpenyne extracted and purified from an extract of caulerpa taxifolia competitively introverted the activity of lipase with ic50 values of a0mm and a0mm using creamed triolein and disseminated 4methylumbelliferyl oleate as substrates individually [ ] the inhibitory activity of caulerpenyne was independent of substrate concentration suggesting direct interaction but dependent on the lipase concentration with the lipase protein slightly than interacting with the substrate oral supervision of corn oil with caulerpenyne to rats demonstrated a reduced and hindered peak plasma triacylglycerol concentration signifying its potential as a lipid absorption inhibitor [ ]vibralactonevibralactone secreted from boreostereum virens microfungi is a scarce fused βlactonetype metabolite covalently but reversibly transforms the active site serine of the enzyme via acylation by the blactone the ic50 of the vibralactone was resolute to be a0mgml [ ]percyquininpercyquinin obtained from the cultures of basidiomycetes stereum complicatum st inhibited pl with an ic50 of a0mm is another βlactone metabolite in one study on βlactone class of compounds the stereochemistry 2s 3s of the βlactone ring was found to impart specificity for the pl while 2r 3r stereochemistry was accountable for inhibition of hmgcoa synthase sources for a0microbial lipasesmicrobial lipases found universal in nature and are commercially substantial due to the low manufacturing cost superior stability and more availability than animal and plant lipases naturally or recombinant microbial lipases are generally used in diverse bioengineering applications a wide diversity of microbial resources provides by nature microbes have more adaptation abilities and inhospitable atmospheres like dead sea antarctica alkaline lakes hot springs volcanic vents and contaminated soils which provides extraordinary potential for the lipases production with specific features [ ] an enormous spinoff with esteem to the enantioselectivity hydrolysis and the formation of carboxyl esters has produced ready availability the marine microfloras have more capabilities for the formation of enzymes and proteins active compounds mostly lipase fashioned extracellularly secretion from fungi and bacteria [ ]in numerous biocatalytic procedures candida antarctica lipase b calb is the most habitually used enzyme and have a more amount of patents candida rugosa lipase crl is another scientifically significant lipase from the yeast which is a mixture of different isoforms and is commercially accessible and this grounding is known as generally recognized as safe gras and used in the food industry pla1s and pla2s from fusarium oxysporum t lanuginosus a niger and trichoderma reesei between the yeast and fungal phospholipases are used in the degumming of vegetable oils and commercialized while mostly used in the food industry are pla1s pla2s and plbs extracted from a oryzae and a niger [ ] due to their high transphosphatidylation and hydrolytic activities plds isolated from actinomycete strains are commercially available and used in several industrialized procedures mostly the bacterial genera for the production of lipases and phospholipases have been reconnoitered are pseudomonas bacillus and streptomyces followed by burkholderia chromobacterium achromobacter alcaligenes and arthrobacter some lipases producing microanisms reveal new sources and applications of industrial enzymes as shown in table a0bacterial lipaseslipase has been detected initially in b prodigiosus and b fluorescens presently serratia marcescens and p fluorescens observed todays best lipase producing bacteria subsequently [] the glycoproteins and lipoproteins are bacterial lipases in most of the bacteria the enzyme production is affected by the certain polysaccharides have been observed [] some bacterial lipases are thermostable and most of the bacterial lipases are reported as constitutive and nonspecific in their substrate specificity [ ] achromobacter sp alcaligenes sp arthrobacter sp pseudomonas sp staphylococcus sp and chromobacterium sp have been exploited for the manufacturing of lipases between the bacteria fungal lipasessince ²s fungal lipases have been studied due to their affluence in thermal and ph stability substrate specificity and activity in anic solvents and downstream processing these lipases have been exploited the contemporary period machinery favors the procedure of batch fermentation and low cost extraction methods so the fungal lipases have assistances over bacteria major filamentous genera of fungi included are rhizopus aspergillus penicillium mucor ashbya geotrichum beauveria humicola rhizomucor fusarium acremonium alternaria eurotrium and ophiostoma for the production of 0cchandra a0et a0al microb cell fact page of table microbial source of a0lipase and a0their industrial applicationmicrobial sourcesfungal species fusarium solani nfccl yarrowia lipolytica aspergillus oryzae rhizomucor javanicus meih rhizomucor miehei geotrichum candidum and c antarctica candida antarctica candida rugosa candida lipolytica penicillium camembertii trichoderma lanuginosus penicillium roquefortii aspergillus niger meyerozyma guilliermondii a niger gzuf36 aspergillus flavus candida antarctica rhizomucor meihei rhizomucor meihei rhizomucor miehei candida tropicalis aspergillus oryzae penicillium abeanum rhizopus nodosus candida rugosa p chrysogenum rhizomucor meihei p chrysogenum thermomyces lanuginose m miehei c parapsilosis m miehei c antarctica m miehei rhizopus arrhizusbacterial species achromobacter sp hegn virgibacillus pantothenticus hegn pseudomonas mendocina acinetobacter radioresistens bacillus sp fh5 staphylococcus pasteuri p fluorescensapplicationsreferenceshalophilic lipase for biodiesel productiondegrades very efficiently hydrophobic and unusual substrates such as nalkanes oils fats and fatty acids as lowcost carbon sourcessaturated fatty acids synthesized faster cheese ripening flavour customized cheesenonhydrogenated solid fatscocoabutter equivalentsthrough biocatalytic processes preparation of chiral intermediates which synthesized the pharmaceutical compounds related to the elimination of bad cholesterol for the treatment of the alzheimers disease oils and fats enriched removal of size lubricants denim finishinghuman milk fat substitutecheese ripening fatty acid productionproduction of glycerolglycolipidssynthesis of saturated triacyl glyceridesproduced a lipase containing detergent lipoprime®production of characteristic flavor of blue cheese in dairy productsfaster cheese ripening flavor customized cheese dough stability and conditioningpromising feed lipase using cheese wheypotential of the enzyme in the synthesis of functional oilsfat stain elimination synthesis of pharmaceuticals polymers biodiesels biosurfactantspitch control in paper and pulp industry polycondensation ring opening polymerization of lactones carbonates in polymeras a biocatalyst in personal care products such as skin and suntan creams bath oils etcsurfactants for baking industry dairy products noodlesoils and fats enriched cocoa butter substitutes synthesis of bioactive moleculesdegradation of crude oil hydrocarbonsuse for docosahexaenoic acid enrichment of tuna oilleather processing and dehairing and fat removalactivated sludge treatment aerobic waste treatmentfood industry waste treatmentsurfactants for baking industry dairy products noodlesfood industry waste treatmentnonhydrogenated solid fatsused as aroma and fragrance in the food beverage and pharmaceutical industrieshydroxamic acids food additivesynthesis of short chain flavour thioester in solvent free mediumproduction of flavour esterstreatment of oily wastewaterdishwashinglaundry removal of fat strainused in detergent industryusing in oil degradationenantioselective transesterification of a racemate rs4methyl1heptyn4en3ol a component of the insecticide s2852 [ ] 0cchandra a0et a0al microb cell fact page of applicationsreferencesthe production of flavour estersused in leather processinginvolved in enantioselective degradation of aopp herbicidescommonly used detergents enhance the removal of oily stains from various types of fabricswaste water treatmentanic solventtolerant lipolytic enzymeanic solventtolerant lipase for biodiesel productionbaking industry for bread makingenhanced stability in methanolbiodegradation of oil and anics determination as chemical oxygen demand cod biodegradation of food wastewater from restaurantsfood processing and oil manufactureapplication in biocatalysisalkaline lipases able to removing fatty stains when used in a washing machinesolvay enzyme products applicable for is a nonionic andor anionic detergent formulationdetergent formulations containing alkaline lipase used in laundry detergent topdegrading of the fatty material in the waste water management table continuedmicrobial sources staphylococcus warneri and s xylosus bacillus sp brevundimonas sp qpt2 micrococcus sp bacillus cereus hss marinobacter lipolyticus haloarcula sp g41 bacillus subtilis geobacillus stearothermophilus pseudomonas aeruginosa hfe733 pseudomonas sp natronococcus sp p alcaligenes m1 pseudomonas plantarii chromobacterium viscosum acinetobacter sp bacillus thermocatenulatus lactobacillus casei lactobacillus paracasei lactobacillus rhamnosus and lactobacillus plantarumused in medical industrycheese industry for improvement of flavor penicillium roquefortii staphylococcus warneri s xylosus pseudomonas cepacia pseudomonas spcheese industry for cheese ripeningproduction of flavour estersbiodiesel fuel productionformation of 15deoxyspergualin in drug industry as antitumor antibiotic and immunosuppressive agentlipases [ ] other species such as candida rugosa candida antarctica t lanuginosus rhizomucor miehei pseudomonas mucor and geotrichum colletotrichum gloesporioides produced ul of lipase are the most productive strain identified from the brazilian savanna soil by using enrichment culture techniques [ ] a niger c rugosa h lanuginosa m miehei r arrhizus r delemar r japonicus r niveus and r oryzae are the principal manufacturers of these commercial lipases []purification of a0lipasesto get consistency of lipase from a large number of bacteria and fungi various novel purification technologies are available generally several steps are contains for the purification of lipases contingent upon the purity estimated for food application the extracellular microbial lipases from the culture broth eliminated by the centrifugation or filtration in the fermentation process and cells are became freed [ ] the ammonium sulphate precipitation ultrafiltration or extraction with anic solvents is concentrated the cellfree culture broth the gel filtration and affinity chromatography like several combination of numerous chromatographic approaches purified about of the using precipitation steps and then ammonium sulphate and ethanol a homogenous product produces is the final step of gel filtration the novel purification machineries such as the i membrane separation procedures ii immuno purification iii hydrophobic interaction chromatography using epoxyactivated spacer arm as a ligand and polyethylene glycol restrained on sepharose iv polyvinyl alcohol polymers as column chromatography stationary phases and v aqueous two phase systems are frequently engaged after these prepurification steps [ ] the enzyme recovery and fold purification outcomes are found acceptable using of hydrophobic interaction chromatography [ ] an 0cchandra a0et a0al microb cell fact page of acid resilient lipase has been filtered from crude profitable arrangements by size exclusion on biogelp100 and ion exchange on monoq from a niger fungi using the chromatography on hydroxyapatite octylsepharose and sephacryl s200 the lipase was purified to homogeneity from r japonicus nr400 substrates for a0lipasea chiral alcohol moiety possesses by the glycerides which is the natural substrate for lipases the lipases were mostly valuable for the resolution or asymmetrization of esters bearing a chiral alcohol moiety was assumed []methods for a0lipase assaydue to the wide substrate specificity of lipases a number of assay protocols are engaged for lipase assay at the lipid water interface the determination of lipase activity is the analytical of free lipase using various physiochemical approaches the determination activities can be carried as with all reactions catalyzed by enzymes and observing the vanishing of the substrate or by the product release for the determining of the hydrolytic activity several methods are presented such as titrimetry spectroscopy photometry fluorimetry and infrared chromatography radio activity interfacial tensiometry turbidimetry conductimetry immunochemistry and microscopy [ ] the triacylglycerol hydrolysis reaction catalyzed by lipases generally can be written asmultifold benefits such as increase in thermal and ionic stability are applicable using immobilized lipases which upturns its proficiency when the enzyme is immobilized it is easier to control reaction parameters like flow rate and substrates convenience [ ] for immobilization include large surface area low cost reusability good chemical mechanical and thermal stability and insolubility the desirable characteristics of solid supports used according to the interface among the enzyme and support the enzyme immobilization approaches can be classified like physical and chemical procedures the interactions among the enzymes and support are by weaker bonds like hydrogen bonds van derwalls exchanges which create these interactions adjustable in the physical method for the interface among the enzyme and support are stronger by covalent bonds the procedure created irrecoverable in chemical methods [ ]physical methodsadsorptionin the physical approaches of immobilization adsorption procedure the enzymes immobilized by van der waals bonds hydrophobic interactions hydrogen bonds and ionic bonds on the surface of the support the enzyme becomes adsorbed bound and the substrates used mostly for this procedure are cation and anion exchange resins activated carbon silica gel alumina triacylglycerols diacylglycerols free fatty acids monoacylglycerols free fatty acids glycerols free fatty acidsthe activity of lipases can be
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Purpose Pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition Encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer LAPC prevents surgical resection This study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationMethods In this phase III trial patients with LAPC were randomised to gemcitabinenab paclitaxel plus Arm A n24 or minus Arm B n13 pamrevlumab Those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria Resection rates progression free and overall survival were evaluatedResults Eighteen patients in Arm A and seven in Arm B completed six cycles of therapy with similar toxicity patterns In Arms A and B carbohydrate antigen response as defined by ¥ decline from baseline occurred in and respectively Sixteen per cent of patients were radiographically downstaged by National Comprehensive Cancer Network criteria in Arm A and in Arm B Positron emission tomography normalised in vs of patients in Arm A vs Arm B respectively and correlated with surgical exploration Eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in Arm A vs Arm B p01193 respectively Postoperative complication rates were not different between armsConclusions Neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with LAPC without added toxicity This combination merits evaluation in a larger patient cohortIntRoduCtIonPancreatic cancer is currently the third leading cause of cancer death in the USA1 and by it will likely become the second leading cause of cancer related death after Key questionsWhat is already known about this subject º Pamrevlumab is anti CTGF1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseaseWhat does this study add º This study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerHow might this impact clinical practice º This study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged OS curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 Surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 However only of patients have disease amenable to upfront curative resection at the time of diagnosis4 Approximately of patients are diagnosed with locally advanced disease5 determined surgically unresectable per National Comprehensive Cancer Network NCCN guidelines6 Patients with locally advanced pancreatic cancer LAPC have a prognosis similar to those with metastatic disease with a historical median overall survival OS of Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access months with recent trials demonstrating median OS of months7 Recent single institution retrospective studies have reported the potential for resection of LAPC with neoadjuvant therapy irrespective of imaging findings with promising results8 However these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation Current prospective trials have documented resection rates of LAPC in the range of to therefore novel approaches are needed to improve patient outcomesThe tumour biology inherent to pancreatic ductal adenocarcinoma PDAC significantly contributes to the poor outcomes seen in this disease Notably PDAC exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor CTGF expression12 CTGF appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition This leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 This stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours Executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticsPamrevlumab is a human monoclonal antibody that targets CTGF Preclinical studies showed that CTGF overexpression is associated with both desmoplasia and gemcitabine resistance in the KPC pancreatic cancer mouse model14 When pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of XIAP an antiapoptotic protein15 When tested in patients with advanced pancreatic cancer Stage IV and locally advanced Stage III treated with gemcitabine and erlotinib in a phase III study n75 pamrevlumab displayed multiple favourable outcomes16We hypothesised that through inhibition of the downstream effects of CTGF overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of PDAC tumours With this in mind this novel phase III randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in LAPC with special emphasis on surgical eligibility and safetyMetHodsstudy designThis was a phase III randomised trial of safety and efficacy in patients with LAPC who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy The randomisation was preplanned and blinded to the investigator The study was approved by individual institutional review boards at nine US institutions and conducted according to the Declaration of Helsinki The trial was registered at clinicaltrials gov as NCT eligibilityKey protocol eligibility requirements included biopsy proven diagnosis of PDAC radiographic staging consistent with locally advanced unresectable disease as defined NCCN guidelines V2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per Response Evaluation Criteria in Solid Tumors RECIST V11 Eastern Cooperative Oncology Group ECOG performance status of or adequate haematological renal and hepatic function no prior therapy for PDAC and no concomitant cancer diagnosis within the past yearsstudy schemaEligible patients were randomised to Arm A or Arm B to receive a total of six treatment cycles weeks of therapy figure Patients in Arm A received pamrevlumab mgkg by intravenous infusion on Days and of each day cycle with an additional dose given on Day in the first cycle Patients in both Arms A and B received gemcitabine mgm2 by intravenous infusion on Days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on Days and of each day cycle Doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care SOC15 Patients remained on therapy for six treatment cycles weeks unless they had disease progression an intolerable adverse event AE or toxicity withdrew consent or were withdrawn at the investigators discretion All patients were followed for drug toxicity until days after the last drug dose Patients undergoing surgery were followed for days following hospital discharge for surgical complications CTGF levels were obtained prior to treatment from all patients Plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug After all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy Postoperative complications including day readmissions and day mortality were notedResponse assessmentPatients were evaluated for response by the following measures carbohydrate antigen CA measured at baseline first day of each cycle and end of treatment EOT RECIST V11 read based on full body CT imaging high resolution dual phase fine cut CT imaging at baseline and every weeks thereafter fluorodeoxyglucose FDG positron emission tomography PET imaging and NCCN V2 resectability criteria at baseline and EOTPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessFigure Patient flow and surgery outcomes In Arm A four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery In Arm A four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive SMA encasement In Arm B one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement SMA superior mesenteric arterysurgical assessmentSubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol PP defined criteria Given that patients included in the trial were determined to be initially unresectable by radiographic imaging and NCCN criteria objective criteria were developed to standardise attempts at surgical resectionPatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma CA level by ¥ at EOT compared with baseline reduction in FDG PET maximum standardised uptake value SUVmax by ¥ at EOT compared with baseline radiological tumour response per RECIST of partial response PR or complete response CR at EOT or met the definition of resectable or borderline resectable per NCCN guidelines Subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on CT scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein PVsplenic vein thrombosis pancreatitis or decline in performance status to a Karnofsky score ¤ or Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes The final decision regarding whether resection was to be performed was made by the treating surgeonendpointsSafety endpoints included serious adverse events SAE during neoadjuvant therapy and surgical complications postresection The efficacy endpoints included surgical eligibility R0 resection R0R1 resection median OS progression free survival PFS and year survival rate All patients were followed and data analysis was stratified by PP population and intention to treat ITT cohortstatistical considerationsThe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the ϲ test Exact CIs for the point estimates as well as the treatment difference were obtained from the SAS PROC FREQ procedure with the EXACT option The two treatment arms were compared using the Cochran Mantel Haentzel test controlling for baseline factors TNM stage ECOG CA PET SUVmax 0c accesssuperior mesenteric artery SMA involvement coeliac abutment and so on as prespecified in the protocol All cause mortality was used in determining OS which was analysed by the Kaplan Meier method Survival status was updated within month before the data cut off date Data from patients who were alive at the cut off date were censored for survival analysis All statistical tests were performed at the significance level of α005 using two sided testsResultsPatient characteristics and dispositionThirty seven patients were randomised to study treatment to Arm A pamrevlumabgemcitabinenab paclitaxel and to Arm B gemcitabinenab paclitaxel alone Patient characteristics at baseline are summarised in table All patients enrolled were unresectable by NCCN criteria patients had tumour arterial involvement SMA encasement ° coeliac abutment Table Patient characteristicsBaseline demographics years years ¥ years Median Male FemaleAge group Sex BMI kgm2 Mean SD Median Min maxECOG Grade Grade TNM stage T3 N0 M0 T3 N1 M0 T4 N0 M0 T4 N1 M0 T4 NX M0Location of the tumour in the pancreas Non resectability per NCCN criterion Head Body Tail Median tumour size mm ° SMA encasement Any coeliac abutment Inferior vena cava invasion or encasement Unreconstructible SMVportal occlusion Aortic invasion and encasementArm AGNPPN24 Arm BGNPN13 TotalN37 to to to · OK as isNot mutually exclusiveBMI body mass index ECOG Eastern Cooperative Oncology Group G gemcitabine n number of subjects NCCN National Comprehensive Cancer Network NP nab paclitaxel P pamrevlumab PV portal vein SMA superior mesenteric artery SMV superior mesenteric veinPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible PVsuperior mesenteric vein SMV occlusion A higher percentage of patients with SMA encasement ° were randomised to Arm A vs Arm B Patient disposition is summarised in figure Twenty four patients in Arm A received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease three patients AEs two patients or physician decision one patient Thirteen patients in Arm B received gemcitabinenab paclitaxel patients completed six treatment cycles Six patients discontinued treatment early due to progressive disease two patients AEs two patients or patientphysician decision two patientssafetySAEs are summarised in table Forty one per cent of patients had a treatment emergent SAE Arm A Arm B No individual toxicity category occurred with frequency except systemic infection patients There was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapyTable Summary of treatment emergent serious adverse eventsSystem organ classpreferred term Ascites Nausea Pancreatitis Vomiting Device occlusion Drug withdrawal syndrome FeverNo of patients with any treatment emergent SAEBlood and lymphatic disorders Haemolytic uremic syndrome LymphadenopathyCardiac disorders Cardiac failure Supraventricular tachycardiaGastrointestinal disorders General disorders and administrative site conditions Hepatobiliary disorders Infections Sepsis Cellulitis Urinary tract infectionInjury poisoning and procedural complications Respiratory thoracic and mediastinal disorders Skin and subcutaneous disorders Cholangitis Hyperbilirubinaemia Craniocerebral injury Pneumonitis Pulmonary embolism RashArm An24n Arm Bn13n Overalln37n · OK as isPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c accessResponse to therapyIn Arm A had ¥ CA decline at EOT response by RECIST PR ¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria During the treatment period the median CA decline was patients were non secretors Seven out of patients had best objective RECIST response CRPR Some patients had exceptional responses defined as normalisation or ¥ decline of CA patients or normalisation PET SUVmax in In Arm B had ¥ CA decline at EOT response by RECIST PR ¥ decline in PET SUVmax and were radiographically downstaged by NCCN criteria Four out of patients had best objective RECIST response CR PR In Arm B of patients had an exceptional CA response and had an exceptional PET response as defined by either ¥ normalized Ca response normalized SUV max andorradiographic downstaging post therapy completion surgical evaluationOverall of the total study patients were eligible for surgical exploration using protocol defined criteria Arm A Arm B p00019 Resection was completed in of the patients Arm A Arm B p01193 Details of the nine resected patients are shown in table In Arm A of the patients were eligible for surgical exploration in the ITT population and of the patients were eligible in the PP population patients who completed six cycles of treatment In Arm A out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined Eight out of patients in Arm A were resected R0 R1 The remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively In Arm B of the patients were eligible for surgical exploration in the ITT population and were eligible in the PP population Of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionPredictors of resectionHigh CA response ¥ decline andor normalisation was contributive to surgical eligibility vs p03 Normalisation versus non normalisation of PET SUVmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 Combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection All nine successful resections were identified by one or both of these criteria Table Summary of resected patientsSitesubject IDTreatmentarmResponse to treatmentNCCNbaselineNCCNend of treatmentResection statusAAAAAAAAB UnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacUnresectablecoeliacUnresectableSMVUnresectableSMAUnresectableSMA SMV coeliacUnresectableSMAUnresectablecoeliacUnresectablecoeliacUnresectableSMA SMVUnresectablecoeliacBorderline resectableUnresectableSMVUnresectableSMAUnresectablecoeliacUnresectableSMAUnresectablecoeliacR0R1R0R0R1R1R1R0R0Protocol defined criteria CA decrease FDG PET SUVmax decrease ¥ RECIST V11 response PR or CR NCCN resectable or borderline resectable criteriaCA carbohydrate antigen CR complete response FDG fluorodeoxyglucose NCCN National Comprehensive Cancer Network PET positron emission tomography PR partial response RECIST Response Evaluation Criteria in Solid Tumors SMA superior mesenteric artery SMV superior mesenteric vein SUVmax maximum standardised uptake valuePicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cConversely radiographic features of response did not correlate with operative potential Neither RECIST response nor radiographic downstaging per NCCN criteria statistically correlated with completed resectionsurgical complicationsPostoperative complications were summarised according to the Clavien Dindo classification posthoc analysis Ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in Arm A Grade II There was one episode of clinically significant pancreatic leak in each arm Grade IIIA no reoperations and no day or day surgical mortality were noted One patient in Arm B had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively Grade IIIB No wound complications or superficial site infections were noted in either group Four out of patients and out of patients in Arm A and B respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalAs of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months PFS was months CI to and months CI to in Arm A and Arm B respectively One year survival and median OS were and months CI accessto in Arm A and and months CI NR in Arm B The median OS for all patients who were eligible for surgical exploration Arm A Arm B vs ineligible Arm A Arm B was months CI NR vs months CI to p00766 The median OS for resected Arm A Arm B vs non resected patients Arm A Arm B was not reached CI NR vs months CI to p00141 figure dIsCussIonThe treatment of LAPC with neoadjuvant therapy remains challenging and there is no established SOC Several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 The combination of more active regimens delivered over an extended period and surgeons comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates A meta analysis of studies using FOLFIRINOX has demonstrated resection rates ranging from to in LAPC17 One of the larger studies including patients with LAPC reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen FOLFIRINOX or gemcitabine based18 Recently a single institution and single arm prospective study of neoadjuvant FOLFIRINOX and losartan with selective use of radiation in patients with LAPC reported an R0 resection rate of Figure Overall survival Resected vs Non resected patientsPicozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0c access However the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease These retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of LAPCthe anti CTGF mechanism of action With respect to gemcitabine based therapy a recent large scale prospective trial of patients with LAPC treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 Furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation More recently the LA PACT trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with LAPC were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigators choice11 Last although FOLFIRINOX has been the most studied induction combination chemotherapy regimen in this population recent randomised data from European patients who received neoadjuvant FOLFIRINOX versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to R0R1 to resection rate vs p0135 or OS vs months p0268Given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates Previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a softening of tumours by endoscopic ultrasound elastography21 This stromal depletion also translated into a decrease of SUV uptake on PET22 In the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsThe protocol specified therapeutic response criteria CA PET SUVmax RECIST and NCCN criteria were used as criteria to determine eligibility for surgical exploration in LAPC This is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards NCCN criteria For example by NCCN conversion alone ie converted from unresectable to borderline resectable only of patients in Arm A would have been eligible for surgical exploration However by protocol criteria of patients in Arm A were eligible for surgical exploration A higher percentage of patients were eligible for surgical exploration by the above criteria in Arm A vs Arm B vs respectivelyOverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size Of the nine subjects that were successfully resected in this trial only one was converted by NCCN criteria to borderline resectable prior to surgical exploration Despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti CTGF mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients This hypothesis needs to be confirmed and patients should be stratified by coeliac andor SMA involvementThe most common predictive factors for eligibility for surgical exploration and resection were CA decline and PET SUV max response which are indicators of tumour response to treatment The combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success Both the ability of CA response and the inability of radiographic response RECIST and NCCN criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials In the MPACT study both CA and PET response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 Recent surgical series of patients with borderline resectable and LAPC have also corroborated their impact in the localised setting25 Correlation of clinical response with plasma levels of endogenous CTGF and pamrevlumab exposure as shown in the prior study by Picozzi et al16 may provide added prognostic and predictive insightWith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel In addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone Pamrevlumab is well tolerated and considered safe compared with the SOC drugs for patients with PDAC These observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in LAPC In addition there were no signals of increased surgical morbidity or wound healing problems with CTGF blockade by pamrevlumab In fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres Similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationFinally while survival data are not yet mature both patients who were eligible for surgery and those that Picozzi a0V et a0al ESMO 20205e000668 101136esmo 2019000668 0cwere ultimately resected had longer PFS and OS highlighting the importance of surgical resection of the tumour Therefore more investigation into newer agents targeting LAPC and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseaseIn conclusion this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in LAPC with prespecified criteria for surgical exploration The use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates Further evaluation of this drug combination in the neoadjuvant treatment setting for LAPC is warranted and a larger phase III trial with resection and survival endpoints is ongoingContributors FibroGen Inc was the study sponsor that designed the study in consultation with the Principal Investigator VP and surgical co investigator FGR All authors except those of the sponsor contributed patients to the study FibroGen was responsible for data collection and analysis All authors reviewed the manuscript and signed off on its accuracyFunding The study was funded by FibroGen Inc San Francisco CAdisclaimer The corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article if accepted to be published in ESMO editions and any other BMJPGL products to exploit all subsidiary rights as set out in our licenceCompeting interests MC MZ SP EK and EC are employees of FibroGen and hold stock andor stock options
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" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the eï¬cacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInï¬ammatory bowel disease IBD is a long life disease with remission and relapse periods IBD arises as a result of inappropriateimmune response to intestinal commensal anisms in individualswith genetic predisposition and consequently causes ammation andintestinal ulcers [] In addition IBD has a complex pathogenesis andmany factors such as dysbiosis oxidative stress and epigenetics thatmay also be involved in disease pathogenesis [] Ulcerative colitisUC and Crohn's diseases CD are known as two main forms of IBDAccordingly these diseases cause intestinal ulcers and some annoyingsymptoms such as diarrhea abdominal pain and rectal bleeding Occasionally the severity of these symptoms is very high which can leadpatients to be hospitalized In this regard therapeutic approaches totreat these diseases mainly focus on prolonging remission and are almost similar however diï¬erential diagnosis can also help to treat thedisease in a more eï¬ective way For example 5ASA which is acommon drug in the treatment of IBD is less eï¬ective on maintainingremission in CD patients On the other hand antibiotic therapy is notrecommended for the treatment UC but it can be eï¬ective on CD patients [][] Diï¬erential diagnosis is a serious challenge because CDand UC have significant similarities in terms of their clinical endoscopic and histological features However there are some diï¬erencesbetween UC and CD which are summarized in Table1 In addition tointestinal complications UC and CD also have significant extraintestinal manifestations For example it was shown that UC is significantly associated with Primary sclerosing cholangitis and CD is alsoassociated with cholelithiasis especially in cases that the ileum is involved [] Furthermore CD can cause ï¬stulas to the urinary systemwhich leads intestinal bacteria to enter the urethra and recurrent urinary tract infections [] Both CD and UC can cause several disorderssuch as arthritis Erythema nodosum pyoderma gangrenosum andanemia which are known as the most important extraintestinal manifestations of IBD [][] The latest statistics showed that the global Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationï¬stulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperï¬cialNONOOccasionallyFrequentlyUCRareFrequentlyNot commonprevalence of IBD currently is on the rise and it is not an exaggerationif we consider it as a global serious health problem [] According to areport published in IBD has the highest prevalence rate inEurope and its prevalence in the newly industrialized countries of AsiaAfrica and South America also appears to be increased over the pastthree decades []Unfortunately the peak of the disease is at the young age of years old [] therefore in addition to the suï¬ering from icts on the patients it also has many negative eï¬ects on societyMoreover many ï¬nancial burdens are annually imposed on countriesfor controlling and treating this chronic disease The invasive diagnosticand therapeutic measures are currently undertaken to diagnose andmanage IBD which are unpleasant for patients as well as having thehigh associated costs Now the gold standard method for diagnosingIBD and monitoring patient status is performing colonoscopy examination and histopathologic evaluation which are invasive measures[] Therefore in recent years many studies have been conducted toï¬nd a suitable laboratory marker with suï¬cient sensitivity and speciï¬city for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the eï¬cacy offecal calprotectin in diagnosing and monitoring patients Althoughsome of these studies reported auspicious results there are still somedoubts on the eï¬ectiveness of fecal calprotectin on diagnosing andmonitoring IBD patients So in this review we addressed the advantages and limitations of fecal calprotectin for the diagnosis andmanagement of IBD The role of fecal calprotectin in diagnosis and management ofIBDThe eï¬cacy of fecal calprotectin as an laboratory marker in various areas of IBD diagnosis and management have been studied including IBD diï¬erentiation from irritable bowel syndrome IBS evaluation of endoscopic activity of the disease evaluation of histologicalactivity of the disease and prediction of disease recurrence andClinica Chimica Acta response to treatment In following after a brief introduction andmentioning the important points regarding laboratory measurement offecal calprotectin we reviewed the most interesting ï¬ndings in all ofthe abovementioned areas Calprotectin A clinically valuable proteinCalprotectin is an antimicrobial protein mainly secreted by neutrophils This protein competes with bacteria over zinc thus kills thebacteria However this is not the only contribution that it has to antimicrobial activity Moreover this protein has many potential clinicalapplications such as the elevated serum levels that have been observedunder various immunological and immunopathological conditionsSerum calprotectin levels rapidly increase in response to bacterial infections in the kidney and heart or during transplant rejection At theearly stages of ammation of the lung serum calprotectin can also beconsidered as a reliable marker besides plasma levels of calprotectinappear to be useful in reï¬ecting disease activity in ammation of thejoints [] In addition it has been demonstrated that serum calprotectin levels are increased in patients with bacterial sepsis so it can beconsidered as a reliable biomarker [] In Neonatal Sepsis the serumlevel of calprotectin increases as well as a sensitivity of and aspeciï¬city of that have been reported for serum calprotectin indiagnosis of Neonatal Sepsis [] It has been recently shown thatserum calprotectin levels increase in patients with aneurysmal subarachnoid hemorrhage and higher levels in the ï¬rst of onset areassociated with a poor prognosis at the ï¬rst three months [] Serumcalprotectin levels also increase in patients with rheumatoid arthritisand even in patients with a moderate to high disease activity who havenormal or low CRP levels so they appear to be more eï¬cient at reï¬ecting disease activity []Some studies have also investigated the eï¬cacy of serum calprotectin in the diagnosis of cancers Correspondingly in one of thesestudies it was shown that serum calprotectin levels significantly increased in patients with laryngeal carcinoma compared with healthyindividuals and those with benign laryngeal pathologies Moreover inthis study a direct relationship was also observed between serum levelsof calprotectin and stage of cancer [] Another study showed that theserum level of calprotectin increased in patients with papillary thyroidcarcinoma but it significantly decreased after operation [] Alsoregarding the eï¬cacy of serum and saliva calprotectin for the diagnosisof IBD impressive results have been reported [][] A study onpatients with IBD both UC and CD have shown that serum calprotectinlevels were directly correlated with fecal calprotectin levels and weremore potent in IBD diagnosis compared to CRP and albumin This studyalso indicated that the combination of serum calprotectin with CRP oralbumin can be helpfulin the prediction of treatment escalationespecially in patients with CD [] However no significant correlationwas observed between serum calprotectin and fecal calprotectin levelsin patients with CD and UC as well as a slight correlation betweenserum calprotectin level and CRP that was observed only in patientswith UC [] Another study showed that the serum level of calprotectin was significantly higher in patients with CD compared to healthyindividuals In addition although a significant correlation was observedwith the clinical activity of the disease no significant correlation wasfound between the level serum calprotectin and endoscopic activity ofthe disease [] The eï¬cacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at °C []Measurement of fecal calprotectin can be done both qualitatively andquantitatively Accordingly in the qualitative measurement monoclonal antibodies are used to detect fecal calprotectin and the positiveresults are characterized by the appearance of colored lines on the testcassette However in the qualitative one only positive or negative results are reported and despite of sensitivity test speciï¬city in theevaluation of disease activity was reported to be only It seems thatthe main application of this test is to diï¬erentiate healthy individualsfrom IBD patients rapidly however some studies have shown that it isnot accurate enough in this case as well [][] Nevertheless asignificant concordance has been reported between home test resultsIBDoc and fecal calprotectin laboratory measurement results whenQuantum Blue calprotectin ELISA kit was used Notably the agreements between results were and depending on the selectedcutoï¬s [] Several commercial kits are also available for fecal calprotectin qualitative test known as rapid calprotectin These tests reportpositive results ranged from to µgg There are also severalcommercial kits that can be used for the quantitative measurement offecal calprotectin These kits are usually designed in terms of the ELISAmethod and some have a measurement range between and µgg Moreover the chemiluminescence immunoassays CLIAmethod can also detect values between and µgg Fluoro enzyme immunoassays FEIA and particle enhanced turbidimetric immunoassays PETIA can also be used for the measurement of fecalcalprotectin In this regard one of the most serious challenges to thelaboratory evaluation of fecal calprotectin is the determination of theupper limit in healthy individuals Among healthy adults there is asignificant agreement on µgg as an upper limit One study suggested values up to µgg in people over years old and up to µgg in children aged between and years old as referenceranges of fecal calprotectin in healthy individuals []Fecal calprotectin levels appear to be higher in healthy infants andchildren under four years old than in adults and further studies areneeded in this regard to determine the acceptable upper limit for diagnosis of pediatric IBD [] Table lists the median levels of fecalcalprotectin in healthy individuals with diï¬erent ages reported in somestudies According to these reports age can aï¬ect fecal calprotectinlevels Fecal calprotectin and IBD diagnosisOnly a small percentage of patients complaining of abdominal painand diarrhea have IBD In many cases IBS as a functional gastrointestinal disorder is known as the cause of such clinical symptomsPatients with IBS have normal colonoscopy results while IBD patientsindicate abnormal colonoscopy results and have intestinal ulcersUnfortunately the significant prevalence of IBS and the overlap between clinical symptoms and IBD can increase the colonoscopy rateTherefore a noninvasive diagnostic marker can be very helpful in thisregard Notably the ï¬rst evidence of the eï¬cacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the ï¬rst and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow conï¬rmed and complemented the ï¬ndings of this study In another study published in AUC values of CI were reported for fecal calprotectin in thediagnosis of colorectal ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a speciï¬city of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a speciï¬city of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a speciï¬city of at a cutoï¬ of μgg were observed for fecal calprotectin in the diagnosis of IBD however oursample size was and the majority of patients were in the active phaseof the disease []In another study conducted on patients with ulcerative colitis asensitivity of and a speciï¬city of at cutoï¬ of μgg havebeen reported in this regard [] In one study it was shown that fecalcalprotectin in cutoï¬ of μgg is able to distinguish patients withIBD from patients without IBD patients with diseases other than IBDpatients with IBS and healthy persons with sensitivity and speciï¬city [] Caviglia et al in their study reported a sensitivity of and a speciï¬city of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a speciï¬city of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy ï¬ndings in patients with Crohn's disease Asensitivity of and a speciï¬city of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE ï¬ndings anddiagnosis of Crohn's disease [] In another study lower sensitivityand speciï¬city rates sensitivity speciï¬city were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the eï¬cacy of fecal calprotectin in predicting wirelesscapsule endoscopy ï¬ndings a sensitivity of and a speciï¬city ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren yearsChildren yearsAdultsOver years Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ammation in Crohn's disease [] Given these ï¬ndings it seems that fecal calprotectin has no ideal sensitivity and speciï¬city for the diagnosis of IBD where the small intestine is involvedBesides there are some preanalytical limitations which are explainedin the next sections Therefore optimistically speaking fecal calprotectin measurement can eliminate the need for colonoscopy Howeverin a metaanalysis performed to evaluate the eï¬cacy of fecal calprotectin and some other ammatory markers to diï¬erentiate betweenIBD and IBS the probability of IBD was less than at fecal calprotectin values lower than µgg or CRP values lower than mgdL[] Therefore it seems that fecal calprotectin can be helpful at leastin ruling out the possibility of IBD in patients with IBSlike symptoms aswell as reducing the rate of colonoscopy Moreover it should be notedthat although a systematic review has reported pooled sensitivity andspeciï¬city above for fecal calprotectin to diï¬erentiate between IBDand IBS it emphasized more on the possibility of falsepositive resultsin low cutoï¬ points [] Hence performing extensive studies indiï¬erent countries on the healthy population and the IBD patient is beneeded to determine a suitable cutoï¬ with maximum sensitivity andspeciï¬city and minimum falsepositive resultsTable summarizes the results of various clinical investigationsregarding fecal calprotectin utility in the diï¬erential diagnosis of IBDfrom IBS and Table4 summarizes some metaanalysis results in thisregard As shown in Table the most important limitation of the majority of clinical studies conducted to date is the small sample size Alarge global study may be helpful in providing a more precise evaluation of fecal calprotectin clinical value in discrimination between IBDand nonIBD diseases Fecal calprotectin and endoscopic and histologic activity evaluationUndoubtedly one of the most serious challenges in the managementof IBD is evaluating the endoscopic and histologic activities of thedisease Nowadays colonoscopy and histopathologic examinations arethe routine tools for the assessment of mucosal healing in patients withIBD As noted earlier several scoring systems have been devised toscore disease activity based on the ï¬ndings of colonoscopy and histopathologic examinations In recent years many promising results havebeen reported regarding the correlation between these scores and fecalcalprotectin levels In addition many studies have been performed inthe last decade all of which cannot be reviewed in this article The ï¬rstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the ï¬rst studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting ï¬ndings indicate that fecal calprotectin can beconsidered as a biomarker in the evaluation of endoscopic activity andClinica Chimica Acta Table4summarized results of some metaanalysis regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDSample sizePooled SensitivityPooled Speciï¬cityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and speciï¬city of fecal calprotectin in predicting endoscopic activity of Crohn's disease [] Correspondinglythe researchers used the Crohn's Disease Endoscopic Index of SeverityCDEIS scoring system in their study to evaluate the endoscopic activity of Crohn's disease As a result they found that there was a significant correlation between the endoscopic activity of the disease andthe level of fecal calprotectin Besides the ï¬ndings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and speciï¬city In another study CDEIS and Mayo Disease Activity IndexMDAI were used to evaluate the endoscopic activity of Crohn's diseaseand ulcerative colitis respectively According to the results of thatstudy on IBD patients there was a significant correlation between fecalcalprotectin levels and disease endoscopic activity [] Another studyshowed that fecal calprotectin is more strongly correlated with theendoscopic activity of the disease in ulcerative colitis compared to theRachmilewitz clinical activity index In addition in this study theoverall accuracy of fecal calprotectin for endoscopically active diseaseidentiï¬cation was obtained as []Some studies have also shown the superiority of fecal calprotectinover traditional ammatory markers like CRP Besides one studyfound that fecal calprotectin was more strongly correlated with theSimple Endoscopic Score for Crohn's disease SESCD compared to theCRP and even Crohn's disease activity index CDAI [] The modiï¬edBaron Index was also used in another study to evaluate the endoscopicactivity of ulcerative colitis As a result it was shown that calprotectinis more strongly correlated with the endoscopic activity of ulcerativecolitis compared to CRP and clinical activity of the disease [] In thisregard similar results were also observed in our recent study in whichthe Ulcerative Colitis Endoscopic Index of Severity UCEIS and SESCDwere used [] Therefore fecal calprotectin appears to be superior totraditional ammatory markers in the prediction of IBD endoscopicactivity The high values of sensitivity and speciï¬city that were mentioned earlier have raised the hope that using fecal calprotectin canreduce colonoscopy rate for patients monitoring However severalrecent studies have reported some significantly lower values Accordingly in a recent study in which Mayo Endoscopic Score [MES] wasused to evaluate the endoscopic activity of ulcerative colitis aTable Summary of the results of some studies regarding the utility of fecal calprotectin in discrimination between patients with IBD and without IBDNumber of IBD patientsAge groupLocationCut oï¬SensitivitySpeciï¬city CD and UC CD and UC CD and UC and unclassiï¬ed68CD and UC CD and UC and unclassiï¬ed CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSreï¬dbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoeï¬cientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland Modiï¬edCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a speciï¬city of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand speciï¬city of fecal calprotectin at a cutoï¬ of µgg for diï¬erentiating MES ¤ in patients with ulcerative colitis were and respectively [] Overall as presented in Table several studiesperformed in diï¬erent countries reported the correlation between fecalcalprotectin and IBD endoscopic activity Although some of these studies reported a strong correlation some others reported a relativelyweak correlation As noted earlier there are significant diï¬erencesbetween the reports on the sensitivity and speciï¬city of fecal calprotectin to predict the endoscopic activity of IBD Undoubtedly a widerange of factors from sample size and the inclusionexclusion criteriato preanalysis variables and indexes used to evaluate the endoscopicactivity may also contribute to these diï¬erences However fecal calprotectin does not appear to be a very reliable marker for the predictionof IBD endoscopic activity so currently it seems a bit optimistic toconsider fecal calprotectin as a reliable alternative for colonoscopy Inthis regard further studies are still needed However under some certain circumstances such as pregnancy or pandemics the use of fecalcalprotectin to evaluate IBD endoscopic activity can be helpfulPregnant patients with IBD have serious limitations for colonoscopyexamination and it has been recommended that colonoscopy should beonly performed in the second trimester of pregnancy and where there isa strong indication [] Therefore noninvasive markers such as fecalcalprotectin can be helpful during pregnancy In one study physicianglobal assessment [PGA] which is a clinical symptombased criterionwas used to evaluate IBD activity and subsequently the associationbetween fecal calprotectin and this criterion was investigated in pregnant women with IBD The results of this study showed a significantcorrelation between fecal calprotectin and PGA levels at prepregnancyduring pregnancy and postpartum stages [] In another study asignificant association was reported between fecal calprotectin levelsand clinical activity of IBD in pregnant women Moreover it was shownthat stool calprotectin at a cutoï¬ of mgkg had a sensitivity between and as well as a speciï¬city between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also conï¬rmed the conclusions obtained from these studies [] According tothese results it seems that fecal calprotectin is not aï¬ected by physiological changes during pregnancy however it is significantly correlatedwith IBD clinical activity during pregnancy Therefore from the viewpoint of relatively acceptable sensitivity and speciï¬city in predictingthe endoscopic activity of IBD fecal calprotectin may be considered as anoninvasive biomarker for the evaluation of IBD endoscopic activity inpregnant women In addition under pandemic conditions fecal calprotectin can be very helpful Following the COVID19 pandemicwhich began in late and is still ongoing healthcare systems indiï¬erent countries were forced to impose significant limitations oncolonoscopy Therefore noninvasive IBD management and fecal calprotectin as a noninvasive laboratory marker have become moreimportant than before The combination between disease clinical activity and fecal calprotectin has been recommended as a noninvasiveapproach that can help in making decisions on treatment duringCOVID19 pandemic [] Therefore it seems that fecal calprotectincan be considered as an alternative for colonoscopy used for IBD endoscopic activity evaluation during pandemic Fecal calprotectin appears to be associated with IBD histologic activity as well Given thediï¬culty in the evaluation of the histologic activity of Crohn's disease[] some studies have been focused on the ulcerative colitis andmany scoring systems have been devised so far Correspondingly thesesystems score the disease's histologic activity based on histologic observationsTherefore for this purpose a biopsy of the intestinal tissue is required which can be prepared by colonoscopy and then sent to thelaboratory In this regard one of these histologic scoring systems isRoberts score that was used in one of our recent studies where weobserved a significant correlation between the level of fecal calprotectinand the histologic activity of ulcerative colitis which was calculatedbased on the Roberts scoring system [] Theede et al also used themodiï¬ed Harpaz Index and performed some interesting studies in thisregard In one of their studies fecal calprotectin was found to be significantly associated with the histologic activity of the ulcerative colitisand it was shown that it could predict histological mucosal healingAUC CI95 Sensitivity Speciï¬city andCutoï¬ mgkg [] In another study on patients with endoscopically inactive ulcerative colitis Mayo endoscopic score the researchers showed that patients with ulcerative colitis who were inendoscopic remission but had histologically active disease had higherlevels of fecal calprotectin compared to patients with no histologicallyactive disease versus mgkg P Also despite thehigh speciï¬city the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes
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"Immune checkpoint inhibitors ICIs can induce immunerelated adverse events irAEs includingthyroid dysfunction There are only a few reports on Graves disease induced by ICIs We report a case of newonsetGraves disease after the initiation of nivolumab therapy in a patient receiving gastric cancer treatmentCase presentation The patient was a 66yearold Japanese man who was administered nivolumab mg every weeks as a thirdline therapy for stage IVb gastric cancer His thyroid function was normal before the initiation ofnivolumab therapy However he developed thyrotoxicosis before the third administration of nivolumab Elevatedbilateral and diffuse uptake of radioactive tracer was observed in the 99mTcpertechnetate scintigraphyFurthermore the thyroidstimulating hormone receptor antibody TRAb and thyroidstimulating antibody TSAbtest results which were negative before the first administration of nivolumab were positive after starting thetherapy The patient was diagnosed with Graves disease and the treatment with methimazole and potassiumiodide restored thyroid functionConclusions This is the first complete report of a case of newonset Graves disease after starting nivolumabtherapy confirmed by diffusely increased thyroid uptake in scintigraphy and the positive conversion of antibodiesagainst thyroidstimulating hormone receptor It is important to perform thyroid scintigraphy and ultrasonographyto accurately diagnose and treat ICIinduced thyrotoxicosis because there are various cases in which Graves diseaseis developed with negative and positive TRAb titresKeywords Graves disease Nivolumab Thyrotoxicosis Immune checkpoint inhibitor 99mTcpertechnetatescintigraphy Thyroidstimulating hormone receptor antibodyBackgroundImmune checkpoint inhibitors ICIs such as cytotoxicTlymphocyteassociated protein CTLA4 programmed cell death protein1 PD1 and programmeddeath ligand PDL1 inhibitors have been widely usedas a standard cancer treatment during recent yearsimmunerelatedHowever occasionallycauseICIs Correspondence yhiroshinmsacjp1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo JapanFull list of author information is available at the end of the adverse events irAEs which affect different anssuch as the lung gastrointestinal tract liver nervous system skin and endocrine glands The endocrine irAEsinclude hypophysitis thyroid dysfunction adrenal insufficiency and type diabetes While endocrine irAEs dueto CTLA4 inhibitors such as ipilimumab and tremelimumab mainly include pituitary dysfunction those dueto PD1 inhibitors such as nivolumab and pembrolizumab are mainly related to thyroid dysfunction []The PD1 inhibitorinduced thyroid dysfunction oftenincludes hypothyroidism rather than hyperthyroidism [ ] Thyrotoxicosis following ICI therapy is caused The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYamada BMC Endocrine Disorders Page of spontaneouslyrecover withmostly by thyroiditis syndrome which has been reportedsubsequenttohypothyroidism in many cases[ ] HoweverGraves disease induced by ICI treatment has not beenextensively explored Here we present a case of Gravesdisease shortly after the initiation of nivolumab therapyfor gastric cancerthecancerCase presentationA 66yearold man was diagnosed with stage IVbT4bN0M1 human epidermal growth factor receptor HER2positive gastricat Nippon MedicalSchool Chiba Hokusoh Hospital one and a half yearsbefore the onset of thyrotoxicosis After diagnosis hewas not referred for surgery because of liver metastasiswith a portal tumour thrombus rather the patient received cycles of first line chemotherapy with a combination of tegafurgimeraciloteracil S1 cisplatin andtrastuzumab However the patient presented with progressive disease assessed based on the computed tomography CT and oesophagogastroduodenoscopy OGDevaluations following the first line therapy Hence he received a second line chemotherapy with paclitaxel andramucirumab After cycles of the second line chemotherapy although there was a reduction in tumour sizeafter cycles the patient presented with progressivedisease as assessed by CT At this stage nivolumab mg every weeks was started The patient had anormal thyroid function before the first administrationHowever TSH suppression was observed before the second administration and thyrotoxicosis occurred beforethe third administration of the drug hence nivolumabtherapy was discontinued and the patient was referred toour departmentThe patient had complained of fatigue and shortnessof breath during exertion His height was cm bodyweight was kg heart rate was beats per minute and blood pressure was mmHg There wasno evidence of Graves orbitopathy or pretibial myxedema He and his family members had no history ofandTgAbantibody ngmL Thyroidthyroid diseases Thethyroidstimulating hormoneTSH free triiodothyronine FT3 and free thyroxineFT4 levels were μIUmL pgmL and ngdL respectively Table The titres of thyroidstimulating hormone receptor antibody TRAb andthyroidstimulating antibody TSAb were positive IUL and respectively whereas those of antithyroglobulinantithyroidperoxidase antibody TPOAb were negative IULrespectively The thyroglobulin Tgand IULlevel wasultrasonographyshowed slight goitre Fig 1a and rich blood flow in theparenchyma Fig 1b 99mTcpertechnetate scintigraphywhich was performed on the first consultation day of thepatient at our department showed elevated bilateraland diffuse uptake of the radioactive tracer Fig Wemeasured antithyroid autoantibodiesin preservedserum samples The titres of TRAb and TSAb werenegative before the first administration of nivolumabwhereas they were positive IUL and respectively before the second administration Thus we diagnosed his thyrotoxicosis as newonset Graves diseaseafter the initiation of nivolumab therapy The humanleukocyte antigen HLA typing of the patient showedthe following allelic variants A24022601 B510154 C01021502 DRB104051501 DQA1010203 DQB104010602 DPA10202 and DPB10501We treated the patient with methimazole MMI at adose of mgday and potassium iodide KI at a doseof mgday One month after the initiation of the therapy when the FT3 and FT4 levels of the patient werenormal we discontinued KI Gradually we reduced thedosage of MMI and the continued administration tillthe death of the patient of MMI at a dose of mg everyalternate day stabilised his thyroid function Fig Furthermore as nivolumab was found to be ineffectivebased on the CT and OGD evaluations the patient received irinotecan therapy However after cycles ofchemotherapy the patient was diagnosed with brain metastasis by magnetic resonance imaging MRIforTable TSH FT3 FT4 and Tg levels and TRAb and TSAb titres in our patientTSH μIUmLFT3 pgmLFT4 ngdLTRAb IULTSAb Tg ngmLDay of nivolumab administration NA NANA NANANANANADay first administration of nivolumab Day second administration of nivolumabThe normal range of the thyroid parameters is as follows TSH μIUmL FT3 pgmL FT4 ngdL TRAb IUL TSAb and Tg ngmL 0cYamada BMC Endocrine Disorders Page of Fig Thyroid ultrasonography of the patient a Slight swelling in isthmus b Rich blood flow in parenchymawhich he received gamma knife and steroid therapyThe patient died months after his first visit to ourdepartmentDiscussion and conclusionsWe present a case of newonset Graves disease after theinitiation of nivolumab therapy in a patient receivinggastric cancer treatment Thyrotoxicosisinduced byICIs is mainly a form of destructive thyroiditis Threecases of newonset Graves disease during nivolumabtherapy other than the present case have been reported[] Table Iadarola [] reported a case ofGraves diseaselike hyperthyroidism after the second administration of nivolumab in a patient with left lungIn this case 99mTcpertechnetate scintigcarcinomaraphy in the patient with T3toxicosis showed diffusethyroid uptake of the radionuclide suggesting Gravesdiseaselike hyperthyroidism whereas the TRAb testswere consistently negative Thyroid ultrasonographyshowed a multinodular goitre with a normoechoic pattern and normal vascularity ofthe parenchyma []Brancatella [] reported a case similar to that ofIadarola [] with diffuse thyroid uptake and negative TRAb titre In this case ultrasonography showed anenlargement of the thyroid with a hypoechoic patternand mild hypervascularity Kurihara [] reported acase of simultaneous development of Graves disease andtype diabetes mellitus during nivolumab therapy InFig 99mTcpertechnetate scintigraphy showing elevated bilateral and diffuse uptake of the radioactive tracer 0cYamada BMC Endocrine Disorders Page of Fig Clinical course of the patient MMI methimazole KI potassium iodide Day first administration of nivolumab Day secondadministration of nivolumabTable Comparison of case reports on newonset Graves disease during nivolumab therapyStudyTSHμIUmL FT3pgmLFT4ngdLTRAb IULBeforeNAIadarola []AfterNegativeTSAb BeforeNANAAfterNANANAUSNormalHypervascularNormalRAIU99mTcuptakeHigh99mTcHighRAIUNAHLANANADRB104Brancatella []NANegativeKurihara []NAPositive NAYamada presentcaseUS ultrasonography RAIU radioactive iodine uptake Before before the initiation of nivolumab therapy After after the initiation of nivolumab therapy at theonset of the thyrotoxicosisNegative NegativeHypervascular PositivePositiveHigh99mTcDPB105 0cYamada BMC Endocrine Disorders Page of this case thyrotoxicosis was detected after the sixth administration of nivolumab with positive TRAb titreHowever ultrasonography showed no enlargement ofthe thyroid and a normal vascularisation pattern Thispatient was clinically diagnosed as mild Graves diseaseand treated with MMI [] Unlike these cases our caseis important in terms of confirmation of both positiveTRAb titre and diffuse thyroid uptake in scintigraphyMoreover titres of TRAb and TSAb were convertedfrom negative to positive after starting nivolumab therapy It seems reasonable to presume that Graves diseasewas induced by nivolumab although there is a possibilityof coincidence Furthermore our patient had HLADPB10501 which has been reported to be associatedwith Japanese Graves disease [ ] Although the involvement of HLA cannot be argued based only on asingle case accumulating similar cases might help clarifythe mechanism of development of rare ICIinducedGraves diseaseGraves disease induced by ICIs other than nivolumabhas been rarely reported Azmat [] reported aipilimumabinduced thyrotoxicosis caused bycase ofGraves disease Gan et al[] reported a case oftremelimumabinduced Graves hyperthyroidism Yajima [] reported a case of Graves disease induced bypembrolizumab a PD1 inhibitor In this case TRAbwas positive after the fifth administration of pembrolizumab and thyroid ultrasonography showed a mild increase in the intrathyroidal blood flow A thyroidscintigraphy was not performed because of the iodinetreatment [] The cases of nivolumabinduced Gravesdisease with negative TRAb titre suggest that performingthyroid scintigraphy and ultrasonography can help to accurately diagnose and treat ICIinduced thyrotoxicosisA relationship between thyroid antibodies and PD1inhibitorinduced thyroid dysfunction has not been explained Kimbara [] suggested that patients withpreexisting TgAb and an elevated TSH level at baselineare at a higher risk of thyroid dysfunction induced bynivolumab Osorio [] reported an association between positive thyroid antibodies antithyroglobulin orantimicrosomal antibodies and thyroid dysfunction induced by ICIs In the studies on newonset Graves disease during nivolumab therapy it is interesting to notein two caucasian patients with Graves diseasethatTRAb was negative [ ] Furthermore TRAb waspositive in two Japanese patients including our patient[] Table However additional evidence is requiredto reveal the role of TRAb in the pathogenesis of ICIinduced hyperthyroidismA limitation of our case was radioactive iodine uptakeRAIU was not performed Because imaging with99mTcpertechnetate reflects both blood flow and uptakevia the symporter and does not assess anificationmalignant nodules may appear hyperfunctioning in pertechnetate imaging but hypofunctioning in 123IimagingIn our study tumours were not detected although a partof 99mTc uptake was strongerIn conclusion we reported a case of Graves diseaseshortly after the initiation of nivolumab therapy for gastric cancer Our case presented a typical Graves diseasewith both positive TRAb titre and diffuse thyroid uptakein scintigraphy Moreover our case is valuable in termsof confirming the conversion of TRAb and TSAb fromnegative to positive titres after starting the therapy It isimportant to perform thyroid scintigraphy and ultrasonography because there are cases of nivolumabinducedGraves disease with negative TRAb titre as previouslyreported To revealthe pathogenesis of ICIinducedGraves disease it is necessary to study additional casesof similar natureAbbreviationsCT Computed tomography CTLA4 Cytotoxic Tlymphocyteassociated protein FT3 Free triiodothyronine FT4 Free thyroxine HER2 Humanepidermal growth factor receptor HLA Human leukocyte antigenICI Immune checkpoint inhibitor IrAE Immunerelated adverse eventKI Potassium iodide MMI Methimazole MRI Magnetic resonance imagingOGD Oesophagogastroduodenoscopy PD1 Programmed cell deathprotein1 PDL1 Programmed death ligand RAIU Radioactive iodineuptake Tg Thyroglobulin TgAb Antithyroglobulin antibody TPOAb Antithyroidperoxidase antibody TRAb TSH receptor antibody TSAb Thyroidstimulating antibody TSH Thyroidstimulating hormoneAcknowledgmentsNot applicableAuthors contributionsHY FO and NE interpreted the data drafted the manuscript andparticipated in the endocrinological treatment of the patient HS revised themanuscript TO and SF participated in the gastroenterological treatment ofthe patient All authors have read and approved the final version of themanuscript for publicationFundingNot applicableAvailability of data and materialsThe data that support the findings of this study are stored in NipponMedical School Chiba Hokusoh Hospital Inzai Chiba and available from thecorresponding author on reasonable requestEthics approval and consent to participateThis case report was approved by the ethics committee of Nippon MedicalSchool Chiba Hokusoh HospitalConsent for publicationWritten informed consent was obtained from the patients next of kin forpublication of this case report and any accompanying images A copy of thewritten consent is available for review by the editor of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Endocrinology Diabetes and Metabolism Graduate Schoolof Medicine Nippon Medical School Sendagi Bunkyoku Tokyo Japan 2Department of Gastroenterology Nippon Medical SchoolChiba Hokusoh Hospital Kamagari Inzai Chiba Japan 0cYamada BMC Endocrine Disorders Page of Received April Accepted August ReferencesGonzalezRodriguez E RodriguezAbreu D Spanish Group for CancerImmunoBiotherapy GETICA Immune checkpoint inhibitors review andmanagement of endocrine adverse events Oncologist Michot JM Bigenwald C Champiat S Collins M Carbonnel F PostelVinay S Immunerelated adverse events with immune checkpoint blockade acomprehensive review Eur J Cancer Bertrand A Kostine M Barnetche T Truchetet ME Schaeverbeke T Immunerelated adverse events associated with antiCTLA4 antibodies systematicreview and metaanalysis BMC Med Faje A Immunotherapy and hypophysitis clinical presentation treatmentand biologic insights Pituitary Topalian SL Hodi FS Brahmer JR Gettinger SN Smith DC McDermott DF Safety activity and immune correlates of antiPD1 antibody in cancerN Engl J Med Robert C Schachter J Long GV Arance A Grob JJ Mortier L et alPembrolizumab versus ipilimumab in advanced melanoma N Engl J MedOrlov S Salari F Kashat L Walfish PG Induction of painless thyroiditis inpatients receiving programmed death receptor immunotherapy formetastatic malignancies J Clin Endocrinol Metab Kimbara S Fujiwara Y Iwama S Ohashi K Kuchiba A Arima H et alAssociation of antithyroglobulin antibodies with the development ofthyroid dysfunction induced by nivolumab Cancer Sci Iadarola C Croce L Quaquarini E Teragni C Pinto S Bernardo A et alNivolumab induced thyroid dysfunction Unusual clinical presentation andchallenging diagnosis Front Endocrinol Lausanne Brancatella A Viola N Brogioni S Montanelli L Sardella C Vitti P et alGraves' disease induced by immune checkpoint inhibitors a case reportand review of the literature Eur Thyroid J Kurihara S Oikawa Y Nakajima R Satomura A Tanaka R Kagamu H et alSimultaneous development of Graves' disease and type diabetes duringantiprogrammed cell death1 therapy a case report J Diabetes Investig Dong RP Kimura A Okubo R Shinagawa H Tamai H Nishimura Y et alHLAA and DPB1 loci confer susceptibility to graves disease Hum Immunol Ueda S Oryoji D Yamamoto K Noh JY Okamura K Noda M et alIdentification of independent susceptible and protective HLA alleles inJapanese autoimmune thyroid disease and their epistasis J Clin EndocrinolMetab Azmat U Liebner D JoehlinPrice A Agrawal A Nabhan F Treatment ofipilimumab induced graves disease in a patient with metastatic melanomaCase Rep Endocrinol Gan EH Mitchell AL Plummer R Pearce S Perros P Tremelimumab inducedgraves hyperthyroidism Eur Thyroid J Yajima K Akise Y A case report of Graves' disease induced by the antihuman programmed cell death1 monoclonal antibody pembrolizumab ina bladder cancer patient Case Rep Endocrinol Osorio JC Ni A Chaft JE Pollina R Kasler MK Stephens D Antibodymediated thyroid dysfunction during Tcell checkpoint blockade in patientswith nonsmallcell lung cancer Ann Oncol Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"25 Thus our results of a 2-year DFS rate of 80% and OS rate of 96% appear favorable by comparison. However it is prudent to be cautious because we lost 20 of 81 patients from the survival analysis because of consent withdrawal and a direct comparison of outcomes data among trials cannot account for differences in study populations eligibility and staging criteria and provisions for data collection and analysis. The spectrum of protein levels for ERCC1 and RRM1 significant correlation of levels between both molecules and distribution of patients into the 4 gene expression categories in the current study is consistent with previous experience.91213161826 However the current analysis method for biomarker evaluation (ie antibody-based assessment of in situ protein levels) is not suitable for general clinical implementation for several reasons. First ERCC1 has multiple isoforms that cannot be specifically distinguished by the available reagents and only 1 isoform appears to be involved in platinum-induced DNA damage repair.27 Second the monoclonal antibody 8F1 which is consistently used for ERCC1 protein expression analysis detects a second and unrelated protein that shares a common epitope with ERCC1.2830 This observation may account for the highly batch-dependent performance of this antibody1827 which may explain the significantly lower ERCC1 values in the current study compared with prior results.16 Third protein levels for RRM1 in particular and to a lesser degree for ERCC1 appear to be influenced by the specimen processing and handling procedures used at collection sites.26 Finally although the method for immunofluorescence-based quantitative detection of both molecules performs well if all specimens to be analyzed are processed simultaneously there is considerable interassay variability if specimens need to be processed individually over an extended period of time as required for real-time patient decision-making.18 However it is important to note that the biochemical biophysical and cell biological evidence for ERCC1 and RRM1 as predictive molecules for platinum and gemcitabine efficacy remains undisputed.51012273132 A small number of recent clinical trials have used ERCC1 prospectively for therapeutic decision-making. These include 2 randomized phase 3 trials in patients with advanced-stage NSCLC (1 published [NCT00499109]18 and the other terminated and unpublished [NCT00801736]) and 2 adjuvant trials 1 of which was a terminated and not yet published phase 2 trial [TAilored Post-Surgical Therapy in Early Stage NSCLC (TASTE) NCT00775385] and the other an ongoing phase 3 trial [International TAilored Chemotherapy Adjuvant trial (ITACA); EudraCT 2008-001764-36]. Results from the first trial (NCT00499109) demonstrated no improvement in patient survival; however the authors raised the possibility of a false-negative result because of an inexplicably divergent survival in an internal control group.18 The second trial (NCT00801736) and third trial (NCT00775385) were terminated early after the discovery of ERCC1 isoforms27 and specificity problems with the 8F1 antibody.2830 The fourth trial is using ERCC1 and tumor thymidylate synthase mRNA expression levels for treatment assignment compared with a cisplatin-based control treatment with OS as the primary endpoint and a planned accrual of 700 patients. Results from these trials will help to further delineate the feasibility and technical issues mentioned above. The results of the current study demonstrated the feasibility of our biomarker-based decision algorithm in a multiinstitutional cooperative group environment for patients with surgically resected NSCLC. We identified that the current practice of evaluation and treatment for these patients may present an obstacle to rapid molecular-based decision-making. Although encouraging efficacy data emerged from this trial bioassays that specifically measure platinum-induced DNA damage repair must be developed before further clinical trials are launched that seek to tailor the use of these agents. "
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"Oral cancer is one of the most common noncommunicable diseases worldwide This paper presentsan evaluation of the trends and geographical distributions of oral cancers in the Saudi Arabian populationMethods Data from Saudi Cancer Registry reports were used in this analysis which assessed the period between and All cancer cases are recorded in these reports as well as the age gender region and histologicalcancer sites for each patient Agestandardised and agespecific incidence rates were calculated in these reportsFor the purposes of this paper only cancers of the lips tongue and mouth were considered oral cancersResults Between and the Saudi Cancer Registry identified cancer cases in total Of these were oral cancer The mean agestandardised rate of oral cancer for the study period was per peoplefor females it was and for males it was The incidence of oral cancer varied by region with Jazan displayingthe highest agestandardised rate and Hail displaying the lowest A positive correlation was observed between oralcancer incidence and ageConclusion The overall trend of the agestandardised rate for both sexes remained constant from to However the oral cancer incidence in Saudi Arabia varies by region Studying this variation in more detail will helpto guide awareness programmes in the regions that are most in needKeywords Cancer epidemiology Cancer prevention and control Oral neoplasmBackgroundCancer is an intractable global health problem and theleading cause of death in the developed world in the developing worldit is the secondleading cause [] In the most recent year for which information fromthe International Agency for Research on Cancer IARCis available approximately million new cancer caseswere diagnosed and million people died from cancerworldwide [] In this same year new cases oflip and oral cavity cancers were reported representing of all cancer casesA review of the global prevalence of oral cancer revealsa wide variation in distribution among countries []Correspondence bmalshehrinuedusaDepartment of Clinical Laboratory Faculty of applied Medical SciencesNajran University PO Box Najran Kingdom of Saudi ArabiaTwothirds of the estimated incidence of oral cancer occurred in developing countries with up to of allnew oral cancer cases in Sri Lanka India Pakistan andBangladesh [] Converselyin France which has thehighest rate of oral cancer incidence in the EuropeanUnion only oral cancer cases were reported in representing just of all cancer cases [] Inthe USA the American Cancer Society estimated that in approximately people were diagnosed withoral cavity or oropharyngeal cancer and will dieof these cancers [] In Arab countries the prevalence oforal cancer is concentrated between western and southeast Asia [] While this type of cancer is relatively uncommon across Arab gulf countries Saudi Arabia andYemen are notable exceptions [] No studies have beenpublished discussing the epidemiological parameters andgeographic distribution of oral cancer cases or any of its The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cAlshehri World Journal of Surgical Oncology Page of subtypes in the Saudi Arabian population Thereforethis study analysed and discussed oral cancer trends inthe Saudi population by using the most recent dataavailableAccording to the International Classification of Diseases 10th revision ICD10 oral cancer is classifiedinto six sites mucosal lip ICD10 C00 tongue ICD C02 gum ICD10 C03 mouth floor ICD10C04 palate ICD10 C05 and mouth ICD10 C06However examining trends in oral cancer incidencerates that include all oral sites can be misleading Thedata analysed in this study only include cancers of thelip tongue and mouth ICD10C00C06 which formthe majority of oral cancers moreover they have severalrisk factors in common and share a similar biology []Thus those accounting for a minority of oral cancercases were excludedMaterials and methodsDataThis retrospective descriptive epidemiological study analysed oral cancer cases in a Saudi population that hadbeen diagnosed from January through December The study used a method of analysis similar tothat used by Alshehri [] Their analyses incorporated male and female data on lip tongue and mouthICD10C00C06 cancer cases to evaluate disease patterns in the Saudi population Data for the present studywere obtained from the Saudi Cancer Registry SCR apopulationbased registry established in by theMinistry of Health in Saudi Arabia This data can onlybe obtained from the reports published by the SCRSince the SCR has been publishing reports oncancer in Saudi Arabia with the primary objective of defining populationbased cancer incidences The presentstudy was conducted using these reports to derive a descriptive epidemiology of oral cancer in Saudi Arabia InSCR reports agestandardised ASR and agespecificAIR incidence rates were calculated with a focus ongenderspecific and regional differencesThe analysis included cases recorded in the SCR filesfrom January to December totalling cancer cases overall approximately of which wereoral cancerData analysisThe GraphPad Prism6 software was used to analyse thedata Descriptive analyses of epidemiological data wereconducted by calculating the mean of the percentagesand ASR stratified by age sex region and year of diagnosis The ASR was calculated in the SCR reports byadjusting all Saudi regions populations mathematicallyto have the same age structure On the other hand theAIR was calculated by summation of the number ofcancer cases occurring during the year in a regionspopulation among specific age and sex groups dividedby the midyear population of these age and sex groupsUsing these two standardised rates is important because age is a basic element of the risk of developingcancer globally [] Using summary measure tools suchas the ASR and AIR which represent the schedule ofagespecific rates in different regions and across timewill give us a more representative picture of the characteristics in question and enable comparisons of cancerincidences between several populations of Saudi regionsthat differ with respect to ageResultsIncrease in the number of oral cancer casesThe total number of cancer cases identified by the SCRfrom to was with males and females Of this total cases were oral cancer The number of registeredoral cancer cases increased gradually from MF in to a peak of MF in however only cases were reported in MF Table Table Number of oral cancer cases in Saudi Arabia for theperiod from to YearNumber of female casesNumber of male casesTotal 0cAlshehri World Journal of Surgical Oncology Page of The percentage of cases representing oral cancers was for females and for males Fig in These percentages decreased to for females and for males in Fig The percentage curve fororal cancer out of all cancer types for males and femalescorrelated with increases and decreases over the studyperiod apart from the years and Fig ASR of oral cancer fluctuated over the study periodBetween and the ASR per male casesfluctuated in it was trending downwards to alow of in and peaking at in beforedropping again to in Fig The female ASRper increased from in to a peak of in decreasing again to in Fig For bothsexes ASR curves like oral cancer percentages correlateto increases and decreases over the study period apartfrom the years and Fig and generally remained constant from to ASR of oral cancer varies by regionThe ASR data for oral cancer cases of all persons demonstrated a wide variation across Saudi regions TheASR means per people for the period from to ranged from in Hail to in Jazan with anational average of per Fig The Jazan region had the highest male ASR mean at followed by the Najran and Tabuk regions at each Fig Conversely Qassim Baha Hail and theNorthern province reported the lowest ASR averages at and per respectively Fig Male and female ASR data were generally equivalentin terms of region rankings with the Jazan region posting the highest overall ASR of as an average valueof both genders followed by the Makkah region at and the Najran region at Fig Similarly the HailBaha Qassim and Madinah regions posted the lowestASR averages at and respectively FigAIR of oral cancer increases with ageThe AIR data from to showed a positive correlation between oral cancer incidence and age withmost cancer cases occurring in the older age groups Figure shows the AIR of oral cancer increasing noticeablywith age up until age More than of cases werediagnosed after the age of Some AIR differences were found between the sexesacross age groups From ages to rates of oral cancer were higher in females than in males however thistrend had reversed to favour males in the 75andoverage group The overall AIR per showed onlyslight differences between the sexes at for femalesand for males Fig DiscussionA review of oral cancer data in Saudi Arabia for theperiod from to showed an overall increasingtrend in the numbers of oral cancer patients Despitethis rise ASR data trends for oral cancer remained constant from to Fig This curve stabilised inthe face of a substantial Saudi Arabian population increase from million in to million in [] Many accumulative factors could be contributed tothis stability First the significant increased access tohealth services in Saudi regions has contributed to thedissemination of oral health awareness and early diagnosis of some cases of metaplasia that were discovered before they could develop into cancerous tumours SecondFig Consistency in percentage curves for oral cancer out of all cancer types from to The percentage curve for oral cancer out of allcancer types for males and females are correlated with overall increases and decreases over the period from to with the exception ofyears and 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer fluctuated over the study period Between and the male ASR was per in and dropped to in The female ASR fluctuated between and per the increased level of public health in the Kingdom isusually linked to an increase in the economic level of thecountry and individuals may have contributed to thisconstancy as many infectious factors such as virusesand fungi have been linked to oral cancers Third SaudiArabia is a majority Islamic country wherein many oralcancer risk factors such as alcohol consumption andcigarette smoking are forbidden by Islamic law Islamiclaw may thus mediate the lower number of oral cancercases in Saudi Arabia compared to the rest of the world[] Thus based on the IARC data for eight ofthe nine world regions whose ASR of oral cancer isabove the global rate [ ] were located in nonMuslimcountries [] with Melanesian regions having the highest rate [] In contrast most of the regions locatedwithin Muslim countries were ranked below the globalASR [] Further investigation of this aspect could therefore be valuable to cancer prevention effortsThe ASR data revealed that more females than maleswere diagnosed with oral cancer in Saudi Arabia at for men and for women This finding is in contrastwith global data showing that men are more likely to develop oral cancer than women [] In the most recent year for which IARC information is available theglobal ASR of oral cancer was for men and forwomen [] While these rates do not match the globalFig Agespecific incidence rates AIR of oral cancer increases with age The total AIR of oral cancer increased noticeably with up until patientswere and over More than of the cases were diagnosed after the age of 0cAlshehri World Journal of Surgical Oncology Page of Fig Agestandardised incidence rates ASR of oral cancer varies by region in Saudi Arabia For all persons the ASR means per peoplefor the period from to ranged from in the Hail region to in the Jazan regionsex distribution oral cancer in Saudi Arabia has a relatively low overall ASR when compared to the globalaverage as discussed aboveResults also revealed consistency in the ASR oral cancer curve for both sexes Fig potentially due to thepresence of common risk factors for oral cancer in malesand females This finding could be used as a startingthreshold for studying the risk factors of oral cancer inthe Saudi population through studying the common factors between the sexesAs with many other types of cancer the present studyfound a correlation between the occurrence of oral cancer and age with of cases diagnosed after the age of years In the USA the average age at diagnosis of oralcancer is years and twothirds of individuals with thisdisease are over the age of [] Ageing is accompaniedby increased susceptibility to cancercausing geneticmaturations due to accumulated exposures to environmental and behavioural risk factors Avoiding these riskfactors could greatly reduce the role that ageing plays incancerThis study found a wide variation in the incidence oforal cancers among Saudi regions Such differencescould indicate that regional environmental factors andlifestyle habits affect oral cancer incidence The resultsreviewed above found that the Jazan region possessedthe highest ASR of people with oral cancer In contrastthe Northern province presented the lowest ASR Severalstudies have focused on investigating why the Jazan region has such a high incidence of oral cancer []Ibrahim and others focused on the association ofcertain eating habits and lifestyle behaviours with the development of oral cancer especially the abuse ofshamma a form of smokeless tobacco and the chewingof khat Catha edulis leaves These substances havebeen classified as carcinogens especially in relation tooral cancer Studies by these researchers found that consuming shamma increased the odds of developing oralcancer 29fold suggesting a strong link between oralcancer and diet and lifestyle choicesAccording to the above poor dietary habits related totobacco use and its derivatives are one of the main reasons for the high incidence of oral cancer in some citiesand not others Other factors such as variations in thegenetic background of the Saudi regions citizens cannotbe excluded especially because most of the populationin the Kingdoms regions is tribal so consanguineousmarriages are highly common Thus genomic sequencing can provide information on genetic variants thatmay be present in citizens of these regions and that maybe linked with increased or decreased rates of oral cancer development Populationbased genetic testing issuggestedConclusionDespite the presence of yeartoyear changes in the incidence of oral cancer in the Saudi population there wasoverall no noticeable change in the incidence of oralcancer in the Saudi Arabian population for the periodbetween and In contrast to some international findings females were somewhat more likelythan males to be diagnosed with oral cancer in SaudiArabia The positive correlation between ageing and theincidence of oral cancer for both males and femalesdemonstrates that oral cancer is mainly a disease of theelderly both in Saudi Arabia and across the globe Thewide variation in the incidence rates among Saudi regions raises an important research question concerning 0cAlshehri World Journal of Surgical Oncology Page of World Bank World Development Indicators Washington DC WorldBank Access online via httpwwwirieduarpublicaciones_irianuariocd_anuario_2014Economia4bpdf Albar MA Islamic teachings and cancer prevention J Family CommunityMed Ibrahim EM Satti MB Al Idrissi HY Higazi MM Magbool GM Al QA Oralcancer in Saudi Arabia the role of alqat and alshammah Cancer DetectPrev Alsanosy RM Mahfouz MS Gaffar AM Khat chewing among students ofhigher education in Jazan region Saudi Arabia prevalence pattern andrelated factors Biomed Res Int Quadri MFA Alharbi F Bajonaid AMS Moafa IHY Al Sharwani A AlamirAHA Oral squamous cell carcinoma and associated risk factors in JazanSaudi Arabia a hospital based case control study Asian Pacific J CancerPrev Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspotential causes that need to be investigated furtherThe knowledge produced by this study must be translated into interventions by performing indepth analysesof regional differences This will contribute to the effortsof preventing oral cancer in Saudi ArabiaAbbreviationsAIR Agestandardised incidence rates ASR Agestandardised specific ratesIARC International Agency for Research on Cancer SCR Saudi CancerRegistry ICD10 International Classification of Diseases 10th revisionAcknowledgementsI express my thanks and gratitude to the Saudi Ministry of Health forproviding me with the Saudi Cancer Registry reportsAuthors contributionsI certify that I have participated sufficiently in the intellectual contentconception and design of this work analysis and interpretation of the dataas well as the writing of the manuscript to take public responsibility for itand have agreed to have my name listed as a contributor The author readand approved the final manuscriptFundingThis research received no specific grant from any funding agency in thepublic commercial or notforprofit sectorsAvailability of data and materialsThe data that support the findings of this study are available from SaudiMinistry of Health but restrictions apply to the availability of these datawhich were used under authorization for the current studyEthics approval and consent to participateThis study was approved by the Research Ethics Committee at NajranUniversity The ethical document reference No ETConsent for publicationA secondary data analysis was conducted in this retrospective study byusing a published dataCompeting interestsThe author declares that he is the only author for this work No other authorcontributed to this work He is also in agreement with the content of themanuscript He declares no conflict of interestReceived June Accepted August ReferencesJemal A Bray F Center MM Ferlay J Ward E Forman D Global cancerstatistics CA Cancer J Clin Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin Warnakulasuriya S GloWarnakulasuriya S Global epidemiology of oral andoropharyngeal cancer Oral Oncology httpsdoi101016joraloncology200806002bal epidemiology of oral andoropharyngeal cancer Oral Oncol Rick A Afsaneh B Cancer Facts Figures American Cancer Society Access online via httpswwwcancercontentdamcancerresearchcancerfactsandstatisticsannualcancerfactsandfigures2017cancerfactsandfigures2017pdfAlJaber A AlNasser L ElMetwally A Epidemiology of oral cancer in Arabcountries Saudi Med J Ariyawardana A Johnson NW Trends of lip oral cavity and oropharyngealcancers in Australia overall good news but with rising rates inthe oropharynx BMC Cancer Alshehri B Descriptive epidemiological analysis of thyroid cancer in the Saudipopulation Asian Pacific J Cancer Prev Armitage P Doll R The age distribution of cancer and a multistage theoryof carcinogenesis Br J Cancer 0c"
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cancer is still one of the most prevalent and highmortality diseases summing more than million deaths in This has motivated researchers to study the application of machine learningbased solutionsfor cancer detection to accelerate its diagnosis and help its prevention Among several approaches one is toautomatically classify tumor samples through their gene expression analysisMethodsstomach breast and lung To do so we have adopted a previously described methodology with which we comparethe performance of different autoencoders AEs used as a deep neural network weight initialization technique Ourexperiments consist in assessing two different approaches when training the classification model fixing theweights after pretraining the AEs or allowing finetuning of the entire network and two different strategies forembedding the AEs into the classification network namely by only importing the encoding layers or by inserting thecomplete AE We then study how varying the number of layers in the first strategy the AEs latent vector dimensionand the imputation technique in the data preprocessing step impacts the networks overall classification performanceFinally with the goal of assessing how well does this pipeline generalize we apply the same methodology to twoadditional datasets that include features extracted from images of malaria thin blood smears and breast masses cellnuclei We also discard the possibility of overfitting by using heldout test sets in the images datasetsResults The methodology attained good overall results for both RNASeq and image extracted data Weoutperformed the established baseline for all the considered datasets achieving an average F1 score of Continued on next pageCorrespondence mafaldafferreirafeuppt1Faculty of Engineering University of Porto Rua Dr Roberto Frias sn Porto Portugal2INESC TEC Institute for Systems and Computer Engineering Technology andScience Porto Portugal The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes weremade The images or other third party material in this are included in the s Creative Commons licence unlessindicated otherwise in a credit line to the material If material is not included in the s Creative Commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The CreativeCommons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Continued from previous pageand and an MCC of and for the RNASeq when detecting thyroid cancer the Malaria and theWisconsin Breast Cancer data respectivelyConclusions We observed that the approach of finetuning the weights of the top layers imported from the AEreached higher results for all the presented experiences and all the considered datasets We outperformed all theprevious reported results when comparing to the established baselinesKeywords Cancer Classification Deep learning Autoencoders Gene expression analysisBackgroundCancer is a label for a group of diseases that is characterized by abnormal and continuous cell growth with thepotential to spread through its surrounding tissues andother body parts [] During cancer was the secondleading cause of death globally accountable for milliondeaths where around were in developing countries[] Throughout the years and given the evolution of techniques technology and treatments in medicine cancersurvival rates have been improving [] However there arestill some types that have survival rates of under suchas pancreatic esophagus and liver cancers Its prevalencemakes it more crucial to correctly and accurately classify such diseases For tackling this need many researchgroups have been trying to help on accelerating cancerdiagnosis by experimenting and studying the applicationof machine learning algorithms to this problem []When automatically classifying tumor samples oneapproach is to analyze the samples derived molecularinformation which is its gene expression signatures Geneexpression is the phenotypic manifestation of a gene enes by the processes of genetic transcription and translation [] By studying it this gene map can help to betterunderstand cancers molecular basis which can have adirect influence on this diseases life cycle prognosis diagnosis and treatment There are two main cancer genomicsprojects The Cancer Genome Atlas TCGA [] andThe International Cancer Genome Consortium ICGC[] that aim to translate gene expression systematizing thousands of samples across different types of cancersWith this elevated number of features each representing a particular gene one may find genomewide geneexpression assays datasets in these projects However thistype of data presents some challenges because of alow number of samples an unbalanced class distribution with few examples of healthy samples and a high potential of underlying noise and errors due toeventual technical and biological covariates [] This difficulty in gathering data accurately is underlying for everydataset creation The equipment used to collect the datahas intrinsic errors associated mechanical of acquisitionand others hence the dataset will reflect these errorsSeveral authors have chosen the previously mentionedapproach of analyzing the gene expression of tumor samples Many of the developed methodologies in this scopeuse straightforward supervised training especially whenusing deep neural networks DNNs relying on theirdepth to produce the best results Gao [] proposedDeepCC a supervised deep cancer subtype classificationframework based on deep learning of functional spectra quantifying activities of biological pathways robust tomissing data The authors conducted two studies eachwith a different cancer detection colorectal and breastcancer data The authors claimed that the describedmethod achieved overall higher sensitivity specificity andaccuracy compared with other classical machine learningmethods widely used for this kind of task namely randomforests support vector machine SVM gradient boostingmachine and multinomial logistic regression algorithmswith an accuracy higher than Sun [] proposed Genome Deep Learning GDLa methodology aiming to study the relationship betweengenomic variations and traits based on DNNs Thisstudy analyzed over six thousand samples of Whole ExonSequencing WES mutations files from different cancer types from TCGA and nearly two thousand healthyWES samples from the one thousand genomes projectsThe main goal of GDL was to distinguish cancerous fromhealthy samples The authors built models to identify each type of cancer separately a totalspecific modelable to detect healthy and cancerous samples and a mixedmodel to distinguish between all types of cancerbasedon GDL All the experiments were evaluated througha three performance metrics accuracy sensitivityand specificity and b Receiver Operating Characteristic curves with the respective Area Under the CurveROCAUC This methodology achieved a mean accuracy of on the specific models on mixturemodels and on total specific models for canceridentificationIn [] Kim compared the performances of a neural network a linear SVM a radial basisfunctionkernel SVM a knearest neighbors and arandom forest when identifying types of cancers and 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of healthy tissues The classifiers were trained with RNAseq and scRNAseq data from TCGA where they selectedup to the most significant genes expressed for eachof the cancer variations To determine the optimal number of genes for each classifiers binary classification taskthe methods mentioned above were trained with different sizes of gene expression datasets from to genes When learning with genes the neural network the linear SVM and the radial basis functionkernelSVM models achieved their best performance with a witha Matthews Correlation Coefficient MCC of and respectively The knearest neighbors and random forest models achieved an MCC of and accordingly when using genes Furthermore theauthors identified classes with an accuracy of over and achieved a mean MCC of and a mean accuracy of with the neural network classifierHowever many DNNs besides the known challenges regarding their training setting [] have a highertendency to overfit which one can detect when applying the same architecture to unseen data or to a heldouttest Thus our motivation focuses on exploring unsupervised pretraining methods based on a lowerdimensionallatent representation with the usage of an autoencoderAE This approach is grounded in the hypothesis thata there is unessential information in high dimensionality datasets and b the acquisition and processing errorspotentially present in the dataset are discarded contributing to a lower probability of overfitting [] Furthermorepretraining AEs and using the learned weights as priorsof the supervised classification task not just improves themodel initialization but also often leads to better generalization and performance [] This may be one of thereasons why AEs are found to be the most predominantstrategy when analyzing RNASeq data []To support our motivation and choices we presentsome works that include unsupervised training in theirmethodologies In [] the authors designed a solution by combining a Multilayer Perceptron and StackedDenoising Autoencoder MLPSAE aiming to predicthow good genetic variants can be a factor in gene expression changes This model is composed of layers inputtwo hidden layers from the AEs and output and trained itto minimize the chosen loss function the Mean SquaredError MSE The authors started by training the AEs witha stochastic gradient descent algorithm to later use themon the multilayer perceptron training phase as weight initialization crossvalidation was used to select the bestmodel The performance of the chosen model was compared with the Lasso and Random Forest methods andevaluated on predicting gene expression values for a different dataset The authors concluded that their approach outperformed both the Lasso and Random Forest algorithms with an MSE of versus and respectively and was able to capture the change ingene expression quantificationThe authors in [] described a study of four different methods of unsupervised feature learning PrincipalComponent Analysis PCA Kernel Principal Component Analysis KPCA Denoising AE DAE and StackedDenoising AE combined with distinct sampling methods when tackling a classification task The authorsfocused on assessing how influential the input nodes areon the reconstructed data of the AEs output when feeding these combinations to a shallow artificial networktrained to distinguish papillary thyroid carcinoma fromhealthy samples The authors highlighted two differentresults in their 5fold cross validation experiment thecombination of a SMOTE [] with Tomek links and aKPCA was the one with the best overall performancewith a mean F1 score of while the usage of a DAEachieved a mean F1 score of In [] presented a stacked sparse autoencoder SSAEsemisupervised deep learning pipeline applied to cancer detection using RNASeq data By employing layerwise pretraining and a sparsity penalty this approachhelps to capture more significant information from theknown high dimensionality of RNASeq datasets usingthe filtered information to the sequent classification taskThe SSAE model was tested on three different TCGARNASeq datasets corresponding to lung stomach andbreast cancers with healthy and cancerous samplesand compared it to four others classification methodsan SVM a Random Forest a neural network supervisedlearning only and a vanilla AE The authors performed5fold cross validation and evaluated the models performance through four metrics accuracy precision recalland F1 score The results show that the semisuperviseddeep learning approach achieved superior performanceover the other considered methods with an average F1score of across the three used datasetsThe authors in [] developed a methodology for detecting papillary thyroid carcinoma They analyzed how theusage of AEs as a weight initialization method affectedthe performance of a DNN Six types of AEs were considered Basic AE Denoising AE Sparse AE DenoisingSparse AE Deep AE and Deep Sparse Denoising AEBefore being integrated into the classifier architecture allAEs were trained to minimize the reconstruction errorSubsequently they were used to initialize the weights ofthe first layers of classification neural network meaningthat the AE layers become the top layers of the wholeclassification architecture using two different strategieswhen importing the weights just the encoding layersand all the pretrained AE Moreover in the training phase the authors studied two different approacheswhen building the classifier a fixing the weights ofthe AE and b allowing subsequent finetuning of all 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of the networks weights The authors used stratified 5foldcrossvalidation and evaluated the model through distinct metrics Loss Accuracy Precision Recall and F1score The authors reported that the overall best resultwas achieved through a combination of Denoising AEfollowed by its complete import into the classification network and by allowing subsequent finetuning throughsupervised training yielding an F1 score of in which the main goalIn [] the authors present a transfer learning methodologyis to explore whetherleveraging the information extracted from a large RNASeq data repository with multiple cancer types leadsto extract important latent features that can help complex and specific prediction tasks such as identifyingbreast cancer neoplasia The authors used the TCGAPanCancer dataset which is composed of approximately RNASeq gene expression examples of distincttumor types This data was split into two sets breast cancer and nonbreast cancer data The nonbreast data isfirstly used to train the three selected architectures forthis study a sparse AE a deep sparse AE and a deepsparse denoising AE models Then the breast data isused to finetune the resulting AEs After pretrainingthese models the authors aim to predict the breast tumorintrinsicsubtypes which is given by the PAM50 subtype information included in the clinical data included inthe PanCancer data The extracted features from the AEbased architectures are then fed as input to three differentmachine learning classifiers namely Logistic RegressionSupport Vector Machine and a shallow Neural NetworkTo assess the deep AEs performance as feature extractionmethods the authors compared them to other classical feature extraction methods combining them with theclassification algorithms previously mentioned ANOVAMutual Information ChiSquared and PCA A 10foldcross validation was performed and all the combinationswere compared through the accuracy metric The resultsshowed the deep sparse denoising AE performs best whenusing the AE extracted features where the combinationwith a shallow neural network leads to the best overall of ±In [] Ferreira used the same methodologydescribed in [] to discriminate different types of cancerinstead of distinguishing cancerous samples from healthyones In this case they aimed to identify thyroid skin andstomach cancer correctly Given that a Denoising AE wasthe AE that lead to the best results in previous studiesthe authors chose to single it out instead of the original The rest of the experiments remained the same strategies for importing the pretrained AE into the top layersof the classifier two approaches when training the classifier to detect different types of cancer same evaluation ofthe obtained results Although in a different domain thebest outcome was reached with a combination of the samestrategy and the same approach in the previous work []with an F1 score of when identifying thyroid cancerMethodsWe extend the previously described work in [] byassembling three different types of experiments dividedinto two main parts where we use three different AEsand five types of cancer samples In the first one we analyze the performance of a deep neural network DNNusing the same pipeline to identify different types of cancer In the second part we choose one of the used AEsto assess how the variance of its latent vector dimension impacts the essential information capture and therefore possibly influencing the classifiers performance and different data imputation strategies can influence theoverall performance in the classification task Moreoverwe study if the network architecture is correlated withits overall performance and how the model reacts whentraining with a different data type dataset We built thispipeline in Python using the Numpy [] and Pandas []packages for the data preprocessing step the Keras deeplearning library [] running on top of TensorFlow andthe ScikitLearn [] package to train and evaluate themodels and the Matplotlib [] library for visualizationAdditionally we used an NVIDIA GeForce RTX TiGPU on a Ubuntu operating systemThis section is anized as follows The data subsection describes the used data and its inherent preprocessing Autoencoders subsection overviews the AEs considered to this study Methodology subsection outlinesthe pipeline for each of the referred experiments Evaluation subsection details how we evaluate the results toprovide statistical evidence Finally Baseline subsectionpresents the established baseline results for all the useddatasetsThe dataIn our experiments we use two different types of datawhich are described in the subsections that followRNASeq dataWe used five different RNASeq datasets from The Cancer Genomes Atlas TCGA [] each representing a typeTable Five instances of the thyroid RNASeq dataset we have usedTPTEP1 AKR1C6PUBE2Q2P2 HMGB1P1 LOC155060 ZZZ3 NANANANANANANANANANA NA NA NA NA NAThe first line the header contains the genes names and the column valuesrepresent its expression samplewise except for the first column which is thesample ID NA stands for missing value for a particular gene and sample 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of of cancer thyroid skin stomach breast and lung Onecan find a sample of the described data in Table The datasets were downloaded from the cBioPortal []which gathers cancerrelated data from different projectsincluding TCGA To train DNNs we need as many data aswe can get Ergo our first criterion was to choose cancertypes that had the highest number of examples Additionally we decided to gice priority to cancer types withhigh mortality and high incidence rates We use the samethyroid skin and stomach datasets presented in []alongside the lung and breast datasets The data filteringprocess in the cBioPortal comprised searching with thekeywords PanCancer sorting the obtained results fromhighest to lowest RNASeq examples and finally selectingthe thyroid skin stomach breast and lung datasetsAll five datasets are composed of approximately thousand features Each column feature in these datasetsrepresents a specific gene and the cell values for each column are the expression of that gene in a particular sampleAll the RNASeq data were normalized according to thedistribution based on all samples The expression distribution of a gene is estimated by calculating the mean andvariance of all samples with expression values and discarding zeros and nonnumeric values such as NA Nullor NaN which are substituted by NA [] With the fivedatasets we gathered examples of thyroid cancer of skin cancer of stomach cancer of breast cancer and of lung cancer We would like to emphasizethat this dataset is only a toy dataset since the data doesnot fairly reflect the immense difficulty associated withidentifying cancer in a real scenarioThe preprocessing pipeline was executed for each RNASeq dataset separately Firstly we removed the columnsthat had only one value throughout all samples Whena value is constant for all the examples there is noentropic value with no value variation one cannot inferany information In total and columns were removed on the thyroid skin stomach breast and lung datasets respectively By default weattributed the remaining missing values represented byNA in the dataset as observable in Table with the meanvalue of the column where the missing value is [] Further normalization was not applied in the data Finally weadded the Label column to link the instances to their typeof cancer when training the classifierSince we aim to distinguish several cancer variations wetest all cancers against each other assigning the positivevalue one to the class of interest and zero to the remainingones When detecting thyroid cancer all thyroid examplesare labeled as one and the skin stomach breast and lunginstances as zero and henceforwardAfter processing all the datasets it is improbable thatthe preprocessing phase removed the same columns in allof them To guarantee the same features describe all thesamples we intersect all the datasets and use the resultas our final dataset Also given that the breast cancerdatasets had almost the double of instances we applydownsampling and randomly select breast cancerexamples to keep the final dataset as evenly distributedfor all the cancers as possible In the end the resultingdataset has approximately instances and more than thousand genesData of features extracted from imagesWe use two datasets of two different diseases composedof features extracted from images malaria and breast cancer Since we aim to evaluate how well this methodologygeneralizes by using distinct types of data we are nowable to gather evidence supporting this premiseThe malaria dataset was created by the FraunhoferAICOS institution through the MalariaScope project[] Their main goal is to develop lowcost solutions thatcan provide fast reliable and accurate results on detecting such disease particularly in developing countries In[] the authors thoroughly describe the feature extraction process from thin blood smear images exclusivelyacquired with smartphones The resulting dataset is composed of samples and features These featureswere normalized between [ ] via scaling and groupedinto three main groups geometry color and textureFrom all the examples approximately contain malariaparasites Due to the high unbalance between Malariaand NonMalaria labels we performed downsampling onthe NonMalaria class where we randomly selected examples We decided to choose instead of dueto a wide variety of nonparasite artifacts Once the samples were selected and similarly to the preprocessing stepof the RNASeq data we verify if there are features withconstant values and remove them if that is the case Ourworking malaria dataset has instances negativeand positive and feature columnsThe Wisconsin Breast Cancer dataset [] from the UCIMachine Learning Repository is composed of examples and features These features are computed from afine needle aspirate digitized image of a breast mass anddescribe the cell nuclei characteristics present in thoseimages such as texture area concavity and symmetryFrom the examples approximately are benignsamples and are malign ones No under or oversampling techniques were applied since we do not find it to beneeded As performed in the malaria data we checked ifthere were columns with constant values for which therewere not The data was used as is with the proportionsand characteristics described aboveAutoencodersAn autoencoder AE [] is an unsupervised featurelearning neural network that aims to copy its input based 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of on a lower dimensional representation This type of architecture is able to extract features by reducing the dimension of its hidden layer [] which helps the AE to focuson capturing the essential features that best represent thedataLet the encoding and decoding functions of the AE be fand g parameterized on θe and θd respectively where θ θeª θd L being the loss function and J the cost function tobe minimized When learning the AE aims to find value θthatargminθJθ X LX gθdfθe Xpenalizing the reconstruction of the input given by ËX fθe X the more distinct ËX is the bigger the appliedgθdpenalty When training an AE we use Mean Squared ErrorMSE as the loss function and the Rectified Linear Unitsactivation function ReLU [] for all its layers Currentlyusing ReLU as activation is the default recommendationwhen training neural networks [] Similarly using MSEas the loss function is a fairly common practice present inthe literature when training AEs [ ]We use the AEs as a weight initialization technique[] since evidence supports that using unsupervised pretraining guides the learning towards basins of attractionof minima that support better generalization from thetraining dataset [] Thus we pretrained them beforeimporting the encoding part or all their layers to theclassification neural networkBasic autoencoder AEThe simplest AE has only one hidden layer This type ofAE learns through the optimization cost function presented in Eq With the combination of linear activationsReLU and the MSE loss function these AEs behave similarly to the Principle Component Analysis PCA method when trained with an MSE an AE learns the principalsubspace of the training data consequentially []Denoising autoencoder DAEA Denoising AE DAE [] aims not just to reproduce theinput but also to keep its information intact to undo theeffect of an intentional corruption process applied to theoriginal data Its cost function can be described byFig Overall pipeline of our experiments This figure illustrates the chosen metodology for our work Firstly we pretrain the autoencoders AEsbefore embedding them to the top layers of the classification network fullfilling either Strategy import only the encoding layers from the AE orStrategy import the complete AE Each of the full assembled architectures is then trained to detect one of the cancer types in the input dataThe training process can follow two different approaches regarding the imported weights of the AEs A fixing them or B allowing subsequentfinetune I represents the input layer E the encoding layerËI the output layer of the AE at the classification region of the network D represents thefully connected layer and O the output of the classifer 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of θfθe ËXJθ X LX gθdargminwhere ËX is a copy of the input X intentionally corruptedby a sort of noise [] To simulate a form of BernoulliNoise [] we apply a Dropout layer immediately after theinput layer where of the connections are randomly cutSparse autoencoderSimilarly to a DAE a Sparse AE SAE learning processalso has two main goals minimizing the reconstruction error when aiming to copy the input data and applying a sparsity penatly represented by 01 to theparameters involved in the encoding partJθ X LX gθdfθe X λ · 01θeargminθAlthough it also tries to reproduce X an SAE canaddress unique statistical features of the dataset it hasbeen trained on [ ] To deliver that sparsity elementwe use an L1 penalty with a λ of MethodologyWe have adopted the methodology described in []which was also used in [] Our experiments consist ofan analysis of the performance of a DNN trained to classify different cancer types studying how three differentfactors may impact the network performance The top layers where we use three different AEs asweight initialization The dimension of the latent vector of the AEs thatmeans the encoding layer size The imputation technique to replace missing datawhen preprocessing the datasetsBesides the top layers imported from the AE the classification part of the full architecture is composed of aBatch Normalization layer [] followed by two FullyConnected layers with a ReLU [] activation Since weaim to detect one type of cancer at the time the last layer the predictive one is a single neuron layer with aSigmoid nonlinearity [] This activation considers thatif the probability of the classification is lower than the sample is classified as negative that is not having thedisease otherwise the sample is classified as positiveTo assess the following experiments we decided to onlyuse the AE that achieved the best results in the firstexperiments For points and we try three different dimensions and For the data imputationstudy we use three strategies replacing the data witha the mean column value used as default a constantvalue in this case zero and b with the most frequentvalueFurthermore we want to study if when using Strategy importing the complete AE into the classification network the model yields better results just because it hasone more layer and therefore more parameters to trainTo observe if the classifier is better only by being deeperwe pretrained the AE and at the embedding step forStrategy we add a decoder layer with all its weightsrandomized guaranteeing that there are no discrepanciesconcerning the networks topological complexity for bothstrategiesFinally we want to assess how the pipeline behaveswhen dealing with different data types besides RNAseq entries Hence we apply the same methodologyto the image extracted features datasets described inThe data section to assess if the model can adapt andgeneralize well to these data characteristicsFor all these we follow the same pipeline see Fig Foreach experience we start by pretraining a different AE tominimize the reconstruction error before importing theminto the top of the classification architecture When doingso we choose one of the two strategies considered for thisstudy add just the encoding layers or add all thepretrained AE After the embedding of the AE to the toplayers we consider two different approaches in the training process A fixing the imported weights of the AElayers and B by allowing them to be finetuned duringthe model training for the classification taskWith the complete architectures AE as the top part ofthe classification network assembled we train each oneto distinguish¢ The RNASeq input data as one of cancers namely¢ The malaria input data as Malaria or NonMalaria¢ The breast masses input data as Malign or Benignthyroid skin stomach breast and lungEvaluationWe use stratified 10fold crossvalidation to ensure andprovide statistical evidence The AEs are trained during epochs and the classifier during with a batchsize of The classification model is trained with thebinary crossentropy loss function [] and with an Adamoptimizer [] Furthermore we assess the overall performance of the model in the training and validation setsby analyzing five more metrics Accuracy Matthews Correlation Coefficient MCC [] Precision Recall and F1score and provide the Receiving Operator Curve with therespective Area Under the Curve ROCAUC and thePrecisionRecall CurveFurthermore to study how the model generalizes tounseen data during the training phase we evaluate theperformance of the best architecture combination on aheldout test set for the Malaria and the Wisconsin BreastCancer datasets For both and separately we use a ratio ofone third to create two new splits Therefore 0cFalc£o Ferreira BMC Medical Informatics and Decision Making 20Suppl Page of Table Baseline results for cancer detection using a Fully Connected Neural Network the classification architecture without the AEas top layersThyroidSkinStom
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neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing antiammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these ï¬nd ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the beneï¬t of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as insilico drug re purposing in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to ï¬nd out a new se ries of nep inhibitors to the best of our knowledge this is the ï¬rst study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of Ëa prior to docking and simulation studies the biological unit of protein was prepared using protein preparation wizard in schrodinger suite during the process of protein preparation the protein was subjected to import and reï¬ne review and modify and minimize processes in protein preparation wizard missing side chains and residues were ï¬lled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond Ëa were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force ï¬eld and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workï¬ow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force ï¬eld in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding aï¬nities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer ï¬le of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force ï¬eld to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme proï¬le the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coeï¬cient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of ï¬ve were calculated induced ï¬t docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedï¬t docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifdsp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which reï¬nement of all residues within Ëa of the ligands pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedï¬t protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula prime_energy glide score ifd score glide_ecoul molecular dynamics md simulation the ï¬exibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede ï¬ned spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol Ëa balanced at k tem perature and bar pressure via npt ensemble in the ï¬nal step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme proï¬le further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workï¬ow was used and the cubic box of speciï¬c dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the ï¬nal torsional reï¬nement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were ï¬ltered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identiï¬ed for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this ï¬nding indicates that the selected drugs may act as nep inhibitors induced ï¬t docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced ï¬t docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the ï¬exible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications ï¬rst it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd conï¬rmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme proï¬le was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of ï¬ve given in table all the selected drugs obey the lip inski rule of ï¬ve molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited ï¬exibility which is insuï¬cient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and lig ï¬t prot for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be Ëa and Ëa respectively the rmsd values were found to be in the acceptable range Ëa but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are Ëa and Ëa for protein and ligand respectively for complex the rmsd values were found to be Ëa for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of Ëa for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signiï¬ 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of ï¬ve sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is antiammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding aï¬nity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are ï¬rst it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drugs activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identiï¬ed based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0
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inanic arsenic ias is the chemical form of as commonlyfound in drinking water atsdr and in some foodscubadda chronic exposure to ias has been associatedwith risk of skin bladder lung and liver cancers iarc aswell as with other diseases naujokas including diabetes maull cardiovascular abhyankar moon saquib states respiratorysanchez and neurological diseases caito andaschner parvez humans and most other mammalian species have developed a mechanism for detoxifying iasthat involves the sequential conversion of ias to monomethylasmas and mas to dimethylas dmas thomas both methylation steps are catalyzed by orthologs of a singleenzyme arsenic oxidation state methyltransferase as3mtlin the methylation of ias by as3mt not onlyaddress correspondence to miroslav st½blo department of nutritionuniversity of north carolina at chapel hill chapel hill nc usa telephone email styblomeduncedu or beverly hkoller department of genetics university of north carolina at chapel hillchapel hill nc usa telephone emailbkolleremailuncedusupplemental material is available online 101289ehp6943this document was reviewed by the center for computational toxicology andexposure oï¬ce of research and development us environmental protectionagency and approved for publication approval does not signify that thecontents reï¬ect the views of the agency nor does mention of trade names orcommercial products constitute endorsement or recommendation for usethe authors declare they have no actual or potential competing ï¬nancialinterestspublished august received february revised july accepted july note to readers with disabilities ehp strives to ensure that all content is accessible to all readers however some ï¬gures and supplementalmaterial published in ehp s may not conform to standards due tothe complexity of the information being presented if you need assistanceaccessing content please contact ehponlineniehsnihgov our staï¬will work with you to assess and meet your accessibility needs within working dayspromotes wholebody clearance of as drobn¡ hughes but also produces methylated intermediates that contain highly reactive and toxic trivalent as asiii watanabe andhirano masiii and dmasiii unlike their pentavalent counterparts masvand dmasv exceed ias in potency as cytotoxinsgenotoxins and enzyme inhibitors thomas hencemasiii and dmasiii may be critical determinants of toxic and carcinogenic eï¬ects associated with chronic exposure to ias alteredcapacity to methylate ias has been linked to an increased risk ofdiseases associated with ias exposure ahsan pierce vahter in most mammals the gene encoding as3mt is present asa single copy ¼ kb from the gene encoding bloc1 relatedcomplex subunit borcs7 to date the methylation of ias isthe only known function of as3mt however recent studies haveidentiï¬ed the as3mtborcs7 locus as conferring risk for schizophrenia duarte compared with healthy individualsexpression of borcs7 and of the humanspeciï¬c splicing variantof as3mt as3mtd2d3 has been found to be consistently higher inthe brains of patients with schizophrenia li notablyexpression levels of as3mt and borcs7 were found to be weaklycorrelated li suggesting that the two genes may sharetranscriptional regulatory elements although published datashowed that ias exposure can aï¬ect as3mt expression in micest½blo the role of ias exposure in the expression ofborcs7 or as3mtd2d3 which is thought to lack ias methylationactivity has never been studiedlaboratory studies of the mechanistic basis of iasassociateddiseases have been hindered by substantial diï¬erences between laboratory animals and humans in their capacity to metabolize anddetoxify ias rats unlike humans sequester significant portions ofingested ias in erythrocytes in the form of dmasiii lu mice the species most commonly used in mechanistic studies diï¬erfrom humans displaying very high rates of as methylation thisspecies diï¬erence is associated with faster rates of urinary clearanceof methylated metabolites and the predominance of dmas as themain urinary metabolite of ias in mice vahter interspeciesdiï¬erences in as metabolism may contribute to diï¬cultiesenvironmental health perspectives august 0cencountered replicating some of the eï¬ects of ias exposure reportedin humans including the carcinogenic eï¬ects in laboratory micethus producing an ias methylation phenotype in mice that resembles the phenotype found in humans could create a better animalmodel to study the role of ias metabolism in adverse health eï¬ectsof chronic ias exposure to generate such a model we have humanized the entire borcs7as3mt locus in 129s6 mice by syntenicreplacement in this process the segment of mouse dna carryingthese two genes was removed and replaced with the syntenic regionof human dna in mouse embryonic stem es cells using homologous recombination this ensured that the junctions between thehuman and mouse dna are known to the base pair level mice weregenerated from the es cells with the expectation that they wouldexpress the human as3mt and borcs7 proteinsthe present study describes in detail the creation of thehumanized mouse strain expression of human as3mt andborcs7 in tissues of the humanized mice and metabolism ofias in these mice after a single dose and during a subchronic exposure to ias in drinking watermethodsrationale for humanizing the borcs7as3mt locuswe chose to excise the 61kb segment of mouse dna carrying theas3mt and borc7 genes and replaced it with the correspondingpromoter of as3mt abuts59kb segment of human dna the untranslated region of borc7 and the weak correlation inthe the expression pattern of the genes suggests possible shared transcriptional regulatory elements li in addition the current human transcript databases for example the university ofcalifornia santa cruz genome browser ucsc lists a putative readthrough borcs7as3mt transcript lacking the ï¬nalexon of borcs7 and the initial exon of as3mt this suggests thepossibility that at least some as3mt activity may be linked to transcription driven by the borcs7 promoter thus the inclusion ofborcs7 in the humanized region increases the likelihood that theelements directing the tissue species diï¬erences in the expressionof as3mt are included in the humanized regionassembly of borcs7as3mt displacerthe borcs7as3mt displacer construct was assembled using astandard recombineering approach figure the mouse arms ofhomology were derived from the 129s7ab22 bmq bac librarysource bioscience plc nottingham uk the short homologyarm was derived from bac bmq455l14 and the long arm ofhomology from bac bmq345l20 the segment of humangenomic dna containing the borcs7 and as3mt loci wasderived from the human tile path bac rp11753c18 bacpacresources center childrens hospital oakland research instituteoakland ca the single nucleotide polymorphisms snps previously associated with interindividual diï¬erences in ias metabolism apata or schizophrenia risk li which were included in the this borcs7as3mt segment aredescribed in table s1 a dna segment bp extendingfrom chr1946683032 to were deleted from the mousegenome and replaced with a 59261bp segment of human dnaextending from chr10102845563 to the haplotypeof the as3mt gene carried in the displacer construct is 1a wood the resistance marker gene used for selection of escells in which the displacer construct underwent genomic integration consisted of a phosphoglycerate kinase pgkneo cassetteï¬anked by mutant loxp sites the marker gene can be excised leaving only a nonfunctional lox site in its placeall polymerase chain reactions pcrs carried out during thedisplacer assembly used taq polymerase bio basic with an initial denaturation temperature of °c for s followed by cycles of denaturation for s at °c annealing for s at a°c and extension at °c with an extension time of min per bp of product length all redet recombination reactionswere carried out using a commercially available kit gene bridgescat no k001 all predesigned primers used during the construction of the displacer vector were from sigmaaldrich and arelisted in table s2 in descriptions of speciï¬c assays each of theseprimers is listed by the corresponding number as stated above the short arm of the displacer construct wasderived from the bmq455l15 bac library this was accomplished by ï¬rst replacing the borcs7as3mt region of the bac withan ampicillin resistance marker by redet recombination theampicillin resistance marker was ampliï¬ed from the pbluescriptii sk cloning vector stratagene primers and the homology arms referred to as the red arm primers and and the ds arm primers and respectively were generated by pcr ampliï¬cation ofsegments of the bmq455l15 bac the homology arms werefused to the ampicillin resistance gene by overlap pcrabhuman locuscyp17a1borcs7as3mtmouse locuscyp17a1borcs7as3mt kb deletedcnnm2cnnm2cborcs7 as3mt displacerddisplaced mouse locuscyp17a1floxedneofloxedneoborcs7as3mtcnnm2homologousrecombinationborcs7as3mtcnnm2 kb insertedfigure scheme for humanization of the mouse borcs7as3mt locus a human borcs7as3mt locus showing the relative positions of the borcs7 andas3mt genes as well as the closest ï¬anking genes b mouse borcs7as3mt locus with ï¬anking genes c schematic of the borcs7as3mt displacer construct d humanized locus prior to cremediated marker excision the names of human genes are capitalizedenvironmental health perspectives august 0cwas used to insert the human borcs7as3mt segment and neomycin resistance cassette by redet recombination into thebmq354l20 bac carrying the short arm and ampicillin genesimultaneously displacing the ampicillin gene this reactionyielded the ï¬nal borcs7as3mt displacer vector which wasdigested with noti to release the bac backbone before transfection into es cellsgeneration of mouse es cellsthe parental es cell line phnx43 used in these studies was generated as follows male and female 129s6svevtac mice taconicbioscience were mated females were checked every morning forcopulatory plugs indicative of successful mating three days laterfemales were euthanized by lethal carbon dioxide co2 exposurefollowed by physical euthanasia and the 35d embryos blastocysts were collected by ï¬ushing the uterus with co2independentmedium gibco cat no supplemented with bovine serum albumin sigmaaldrich cat no a3675 individualembryos were collected from the lavage ï¬uid with a pipette andplaced in 2cm2 wells that had been seeded h earlier withprimary mouse embryo ï¬broblasts mefs gibco cat nogsc6005m blastocysts and mefs were cultured in gibcoknockout¢ ko medium cat no supplementedwith esqualiï¬ed fetal bovine serum fbs gibco cat no and 2mm lglutamine gibco cat no after d in culture when the inner cell mass of the embryosreached ¼ mm in diameter the embryos were removed and disrupted with a pipette and placed in a new well seeded with mefsthis process of serial expansion of a single inner cell mass wascontinued until a single inner cell mass was expanded to at least colonies of cells cells were then further expanded by disruptionand exposure to trypsin for min at °c after expansionand cryopreservation in dimethyl sulfoxide sigmaaldrichcat no d2660 a sample of the cell line was subjected to karyotype analysis in karyologic inc rtp nc the cell line used inthis study had a xy normal male mouse karyotypeexpression of human borcs7as3mt in mouse es cellssinglecell suspensions of the parental es cell were prepared bytreatment with trypsin as described above in a volume of ml cells were electroporated in the presence of lgof dna the apparatus used was a btx electro cell manipulator with cuvette btx cheshire analytical the settingsused were v lf r8 ohms after electroporation the cells were distributed onto seven 100mm tissue cultureplates corning cat no in gibco ko medium supplemented with esqualiï¬ed fbs gibco cat no and mm lglutamine gibco cat no after hselection was initiated with the addition of geneticin¢ ï¬nal concentration mgml gibco cat no after d individual neomycin resistant colonies became visible cells from¼ colonies were pooled and used for rna isolation to conï¬rmthat the human genes were expressed see details below once thiswas established individual colonies from the remaining plateswere transferred individually by pipette to a 96cell plate each colony was trypsinized and a portion used for pcr analysis to identify those in which the dna had integrated by homologousrecombination as described below the remaining cells wereallowed to grow and cultures carrying a targeted allele as determined by the assay detailed below were transferred sequentially to and 60cm2 tissue culture plates at this point some cellswere cryopreserved while a portion of each was used for karyotypeanalysis and to conï¬rm the expression of human as3mt by probebased droplet digital pcr ddpcr using commercially availablethe displacer short arm together with the inserted ampicillingene was subcloned from the modiï¬ed bmq455l15 bac into aplasmid vector by redet recombination the plasmid vectorwas prepared by ligation of four pcr products the vector backbone carrying a cole1 origin of replication and a spectinomycingene was derived by ampliï¬cation of the pfloxxerx plasmid previously assembled in kollers laboratory see table s3 primers and the bmq455l15 bac was used as atemplate for the other three pcrs us arm primers arm primers and andand short ds arm primers and all pcr productswere gel puriï¬ed on a agarose gel using a commerciallyavailable kit qiagen cat no digested with bbsi nebcat no r3539s and mlui neb cat no r0198s and columnpuriï¬ed qiagen cat no fifty nanograms of each pcrproduct was combined and ligated neb cat no m0202s in a20ll reaction for h at °c the ligation reaction was heatinactivated at °c for min and then ll of the reaction wastransformed into chemically competent e coli dh5afcellszymo research cat no t3002 according to the manufacturers directions and plated on luria broth agar plates supplemented with spectinomycin at lgml the resulting plasmidvector was digested with mlui and used for redet recombination with the modiï¬ed bmq455l15 bac the resulting plasmidwas in turn digested with bbsi to release the displacer short armand ampicillin resistance gene ï¬anked by the us and dshomology arms this bbsi restriction fragment was introducedinto the bmq345l20 bac by redrecombination to create abac vector in which the ampicillin resistance marker wasï¬anked by the displacer arms of homologythe segment of the human borcs7as3mt locus included inthe displacer vector was prepared for excision from the rp11753c18 bac in two steps in the ï¬rst step a neomycin resistancegene was inserted into the bac upstream of the borcs7 gene toaccomplish this the neo gene ï¬anked by mutant loxp sites wasligated to homology arms referred to as blackred and purplerespectively the neomycin resistance gene was excised from thevector psï¬ijt15neojtz17sï¬i previously assembled in kollerslaboratory see table s4 by digestion with sï¬i neb cat nor0123s the blackred homology arm was ampliï¬ed primers and using the bmq455l15 bac as a templateand the purple homology arm was ampliï¬ed primers and using the rp11753c18 bac as a template all fragments were gel puriï¬ed as described above and the blackred andpurple homology arm pcrs were digested with bgli neb catno r0143s and then column puriï¬ed as described above theloxpï¬anked neomycin resistance gene was ligated to the homology arms as described above in a 20ll reaction containing a totalof lg of dna with the three fragments at an equimolar ratiotwo microliters of the ligation reaction was used for a redetreaction with the rp11753c18 bac in the second step an ampicillin gene was inserted downstream of the as3mt gene in therp11753c18 bac carrying the neomycin resistance marker toaccomplish this homology arms referred to as bluegreen andyellow respectively were added to the ampicillin resistance genethe bmq455l14 bac was used as the template for the bluegreen pcr primers and pbluescript ii sk was used as the template for the ampicillinyellow pcr primers and the two pcr products were gel puriï¬edand then combined in an overlap pcr primers and the resulting pcr product was gel puriï¬ed and ngwas used for a redet reaction with the rp11735c18 bac carrying the neomycin resistance cassetteapproximately ng of the modiï¬ed rp11753c18 bacwas digested with mlui in a 20ll digest and ll of the digestenvironmental health perspectives august 0cprimers applied biosystems hs00960526 here a 20ll reaction mixture was pipetted into a dg8¢ cartridge along with llof droplet generation oil for probes bio rad and loaded into theqx200 droplet generator bio rad according to the manufacturers instructions after droplet generation the droplets weretransferred to an eppendorf twintec® 96well plate and placed in ac100 touch thermal cycler bio rad using the manufacturersrecommended cycling conditions after cycling the plate was readin the qx200 droplet reader bio rad quantasoft¢ softwareversion a portion of each of the es colonies ¼ cells was disruptedby incubation for min at °c in lysis buï¬er consisting of trisedta [ mm trisma base thermo fisher scientiï¬c cat nobp152 mm ethylenediaminetetraacetic acid edta thermofisher scientiï¬c cat no volvol triton¢ x100millipore sigma cat no and mgml proteinase kroche cat no ] lysates from all colonies werescreened for homologous recombination by pcr using primersscreen f and see table s4 with gxl polymerase takarabio according to the manufacturers suggested protocol the pcrprogram used was °c initial denaturation for s followed by cycles with s denaturation at °c s annealing at °c minat °c and a ï¬nal 10min extension at °c pcr products wereanalyzed by agarose gel electrophoresis agarose bio basiccat no d0012 in trisacetateedta running buï¬er the sizeof the pcr product was determined by comparison with 2logladder neb cat no n3200ltwentyseven clones with homologous recombination wereidentiï¬ed and were subjected to further analysis because of thishigh recombination frequency it was not necessary to use clustered regularly interspaced short palindromic repeats and crisprassociated protein 9crisprcas9 to stimulate recombinationevents rna was prepared from pools of the cells and examinedfor the expression of the human genes rna was also preparedfrom a number of the individual clones after expansion brieï¬ycells were lysed directly in the culture dish and homogenized byrepeated pipetting rna was isolated using rna bee or stat60teltest according to the manufacturers instructions rna yieldand quality were assessed using a nanodrop thermofisher one to two micrograms total rna was reverse transcribedwith the high capacity reverse transcription kit appliedbiosystems expression of the human as3mt was examined byqualitative pcr qpcr on a c100 touch thermal cycler biorad using commercially available primers applied biosystemshs00960526generation of mice expressing human borcs7as3mtfor injection into blastocysts the es cells carrying the humanborcs7as3mt locus were again trypsinized to generate singlecell suspensions collection of recipient blastocysts blastocystinjection and transfer to pseudopregnant dams was carried out bythe unc chapel hill animal models core following proceduresdescribed by longenecker and kulkarni and by koller to maintain the mutation on the 129s6svevtac background the chimeric mice were bred to 129s6svevtac micetaconic bioscience the resulting f1 mice were identiï¬edby taq as wildtype wt homozygotes for the mouse as3mtborcs7 locus wtwt or heterozygous wths or homozygoushshs for the human as3mtborcs7 locus the primers usedwere common and endo for the endogenous locus and common anddisplaced for the displaced locus see table s2 the pcr assayswere carried out using taq polymerase bio basic with an initialdenaturation temperature of °c for s followed by cycles ofdenaturation for s at °c annealing for s at a °c andextension at °c for s with a ï¬nal extension of min the f2generationconsisting of hshs homozygotes hswt heterozygotes and wtwt homozygoteswas produced by mating of f1hswt heterozygotes the hshs hswt and wtwt oï¬springfrom the f2 generation were housed together one litter per cagecunder controlled conditions with 12h lightdark cycle at ± and ± relative humidity the parental mice and mice in thef1 and f2 generations were fed purina labdiet 5v5r and drankdeionized water ad libitumall proceduresinvolving mice were approved by theuniversity of north carolina institutional animal care and usecommittee 0ecollection of tissues for mrna expression analysistissues for mrna expression analysis were collected from f2hswt mice both males and females measurement of rnaexpression in hswt mice which were heterozygous for thehuman and mouse gene allowed direct comparison of expressionin the same tissuerna sample minimizing the eï¬ects of physiological variation between animals or quality of sample whichcould result in subtle diï¬erences in rna quality and cdnaformationmice were euthanized by exposure to co2 followed by physical euthanasia the following tissues were collected whole brainparts of brain cortices pons medulla hippocampus cerebellumpituitary spinal cord adrenals liver spleen kidney duodenumjejunum colon stomach uterus ovaries testes heart and skinfrom back of neck neuronal and glial cells were also dissectedbrain parts and spinal cord were collected from male mice at dof age neuronal cells were isolated from embryonic day e17embryos and glial cells from postnatal day p1 pups all othertissues were collected from to 12wkold mice three mice wereused for each tissue blood was collected from lethally anesthetized mice via cardiac puncture with an edtacoated syringe justprior to thoracotomy to isolate mononuclear cells from blood ml of whole blood were layered onto ml of histopaque® sigma and centrifuged at room temperature for min with nobrakes at g the upper layer was discarded and the interfacecontaining the mononuclear cells was transferred to a 15ml conical tube the cells were then washed twice with phosphatebuï¬ered saline and then lysed with rna beeneuronal cell isolation and culture brains were harvestedfrom eight e17 embryos and placed in a petri dish containinghankss balanced salt solution hbss gibco meninges wereremoved cortices dissected and placed in a new petri dish containing hbss cortices were transferred to a 15ml conical tube containing ml hbss and centrifuged at g for min at °cthe tissue was digested with ml tryple express gibco catno and dnase i roche cat no for min at °c the digested cortices were centrifuged at gfor min at °c the tryple dnase i was aspirated and thecortices were washed for min with ml fbs to inactivate thetrypsin the tissue was centrifuged again and resuspended in complete dulbeccos modiï¬ed eagle medium dmem gibco with fbs vwr glutamax gibco with penicillinstreptomycingibco and 2mercaptoethanol sigma the cortices were titurated times with a 10ml pipette and then times with aï¬amedtip pasteur pipette the cells were then passed through a100lm cell strainer into a clean conical tube cells were then pelleted by centrifugation at g for min the cell pellet wasresuspended in ml complete dmem and plated in mm culture dishes coated with lgml poly dlysine sigma cells per dish after h 1lm cytosine bdarabinofuranosidearac sigma cat no c6645 was added using a mediachange without exposing the cells to air the cells were harvestedfor rna isolation on day environmental health perspectives august 0cmixed glial cell culture brains were harvested from six p1pups and placed in a dish containing hbss meninges wereremoved cortices dissected and placed in a conical tube containing cold dmem cortices were centrifuged at g for min at°c and media replaced with ll cold dmem the corticeswere titurated times with a ï¬amedtip pasteur pipette coated inserum the cells were then passed through a 100lm cell strainerinto a fresh conical tube cells were pelleted by centrifugation at g for min the cell pellet was resuspended in ml complete dmem and ml of the suspension was plated on three100mm culture plates the medium was changed h later andthen daily the cells were harvested for rna on day isolation of adrenal cortex and medulla to obtain samplesenriched for each of these two distinct functional regions of thegland the adrenal cortex was removed from medulla of male andfemale adrenal gland by microdissection enrichment was quantitated by qpcr analysis of genes known to be expressed in onlyone of these two regions chgb in the medulla and cyp11b1 inthe cortex the human protein atlas using appliedbiosystem taqman probes mm00483287 and mm01204952respectively rna was isolated from cells and tissues using themethod described for es cells however the tissues were ï¬rst homogenized in stat60 in a desktop homogenizer fastprep mp bio using ceramic beadsanalysis of as3mt as3mtd2d3 borcs7 as3mt andborcs7 expression in collected tissues and cellsboth ddpcr and qpcr were used to detect and quantify as3mtas3mtd2d3 borcs7 as3mt and borcs7 mrna the speciï¬cmethod is indicated in each ï¬gure legend ddpcr is minimallyinï¬uenced by diï¬erences in the eï¬ciency of the mouse and humanprimer sets quan and therefore provides a means ofdirect comparison of expression between the endogenous mouselocus and the humanized locus the distribution of the as3mttranscript in the tissues was compared with as3mt protein distribution described by the human protein atlas for the human tissues sybr green qpcr was used to compare rna expression inwhole brain cortex hippocampus pituitary cerebellum pons medulla spinal cord glial cells neurons adrenals ovaries testesheart kidney liver colon jejunum spleen and blood ddpcr wasused to assess rna expression in es cells whole brain liver adrenals spinal cord ovaries testes spleen and heart for all assays lg total rna was reverse transcribed with the high capacityreverse transcription kit applied biosystemsfor sybr green qpcrthe relative expression of fulllength as3mtthe d2d3 variant and the borcs7as3mtfusion transcript were assessed on a quantstudio6 flex¢ realtime pcr system applied biosystems sybr green reactions were run using itaq universal sybr green supermixbio rad nm of each primer and ngll of cdnaprimers used for quantiï¬cation of as3mt isoforms were d2d3fand d2d3r for the d2d3 isoform and fullf and fullr for thefulllength isoform see table s2 for analysis of borcs7as3mt readthrough transcripts expression in mouse tissues lgrna was reverse transcribed and sybr green quantitative pcrwas run using primers gaacagtcatcggatctacagga3and and itaq sybrgreen universal supermix bioradgaacagtcatcggatctacagga3for ddpcr absolute concentration of as3mt as3mt borcs7and borcs7 was acquired using the ddpcr supermix forprobes no dutp bio rad and taqman gene expressionassays as3mt mm00491075_m1 as3mt hs00960526_g1borcs7 mm01205060_m1 and borcs7 hs00376014_m1all from applied biosystems the ddpcr analysis was carriedout as described aboveevaluation of the metabolism of a single dose of iasthe metabolism of ias was examined in to 22wkold hshsmale n and female n f2 oï¬spring and their wtwtmale n and female n littermates the mice weregiven a single dose of ias sodium arsenite pure sigmaaldrich in deionized water diw by gavage lg askg ofbody weight immediately after dosing each mouse was placedin a metabolic cage mousecage and urine and feces were collected in 24h intervals for d during these d all mice drankdiw ad libitum the mice were fasted during the ï¬rst h buthad free access to puriï¬ed laboratory diet envigo teklad catno ain93g during the second and the third days our published data showed that ias content in this type of diet rangesfrom to ¼ lg askg douillet huang murko the 24h urine and feces samples werefrozen in dry ice and stored at c 0eevaluation of the metabolism of ias during subchronicexposureafter collection of urine and feces following the singledoseadministration the hshs and wtwt mice were again cagedtogether one litter per cage and maintained on the puriï¬ed dietand diw for wk to allow for clearance of as from the bodywhich was conï¬rmed by measuring total as tas levels inurine see the results section for details both wt andhshs mice now to 27wkold were then exposed to iassodium arsenite pure sigmaaldrich in drinking water lg asl for wk spot urine samples ¼ to llwere collected weekly body weights were recorded before andafter the exposure after wk all mice were sacriï¬ced by cervical dislocation without anesthesia and tissues were collectedincluding the liver kidneys pancreas spleen heart lung adrenals kidneys bladder visceral fat calf muscle testes or ovaries and cecum and colon all tissues were ï¬ashfrozen in dryice and stored at c 0eanalysis of as species in urine feces and tissuesspeciation analysis of as was carried out in 24h urines and fecescollected after the single dose of ias and in spot urine samplescollected during subchronic ias exposure the speciation analysis was also performed in livers and kidneys collected after subchronic exposure at sacriï¬ce ten percent homogenates of liverand kidney were prepared in icecold diw wtvol usingwheaton potterelvehjemstyle tissue grinders with a ptfepestle and wheaton overhead stirrer apparatus dwk lifesciences feces were snap frozen in liquid nitrogen and pulverized to powder using a steel mortar and pestle placed in dry icethe powder was mixed with 2n ultrapure phosphoric acid thermo fisher and digested in a mars5 microwave cemcorp for h at °c this method eliminates the biologicalmatrix but does not alter as speciation currier the digestates were neutralized by sodium hydroxide sigmaaldrich to ph the urines tissue homogenates and neutralized digestates were treated with lcysteine sigmaaldrich at room temperature for h to reduce pentavalent asspecies to their trivalent counterparts prior to the analysiscurrier the cysteinetreated samples were analyzed by hydridegeneration atomic absorption spectrometrycoupled with a cryotrap hgctaas as previously describedcurrier hern¡ndezzavala this analysis determined the concentrations of ias mas and dmas tasconcentration in urine feces and tissues was calculated as thesum of ias mas and dmas for assessment of the eï¬ciencyof ias metabolism after a single oral dose the amount of tasenvironmental health perspectives august 0cwas expressed as the percentage of the dose by comparing theamount of elemental as administered as ias to each mousewith the amount of tas in 24h urine and feces samples collected from that mousethe instrumental limits of detection lod for ias mas anddmas using this method are and pg as respectivelyhern¡ndezzavala an imputed value of was usedfor measurements below the lod hgctaas is also capableof detecting and quantifying trimethylarsine oxide tmasohern¡ndezzavala a product of ias metabolism byrat as3mt waters however because tmaso israrely detected in human urine and because tmaso was not aproduct of ias methylation by recombinant human as3mt inour published study ding we did not includetmaso analysis in this studystatistical analysisoneway analysis of variance with studentnewmankeuls ortukeys multiple comparison posttest was used to assess diï¬erences in gene expression and in the concentrations and proportions of as species among wtwt and hshs mice students ttest was applied for comparison of mouse and human geneexpression in a single tissue of hswt mice and for comparisonof as species concentrations or proportions between male andfemale mice and between hshs and wtwt mice of the samesex the statistical analyses were performed using prism software graphpad software diï¬erences with p were considered statistically significantresultsexpression of borcs7 and as3mt in hswt miceexpression of as3mt and borcs7 were directly compared withthose of endogenous mouse genes in tissues collected from f2male hswt mice that carried one copy of the mouse locus andone copy of the human locus figure 2a using both conventionalqpcr and when appropriate ddpcr expression of humanas3mt and borcs7 was easily detected in all tissues expressingthe corresponding mouse genes although the level of expressionof the human and mouse genes diï¬ered figure 2bc notable inthis analysis was
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Genomic characterization of malignant progressionin neoplastic pancreatic cystsMicha«l No«et alIntraductal papillary mucinous neoplasms IPMNs and mucinous cystic neoplasms MCNsare noninvasive neoplasms that are often observed in association with invasive pancreaticcancers but their origins and evolutionary relationships are poorly understood In this studywe analyze samples from IPMNs MCNs and small associated invasive carcinomas from patients using whole exome or targeted sequencing Using evolutionary analyses weestablish that both IPMNs and MCNs are direct precursors to pancreatic cancer Mutations inSMAD4 and TGFBR2 are frequently restricted to invasive carcinoma while RNF43 alterationsare largely in noninvasive lesions Genomic analyses suggest an average window of overthree years between the development of highgrade dysplasia and pancreatic cancer Takentogether these data establish noninvasive IPMNs and MCNs as origins of invasive pancreatic canceridentifying potential drivers of invasion highlighting the complex clonaldynamics prior to malignant transformation and providing opportunities for early detectionand interventionA list of authors and their afï¬liations appears at the end of the paperNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178Pancreatic cancer is a deadly disease with a dismal prognosisthat is predicted to soon be the second leading cause ofcancer death in the United States1 However like otherepithelial malignancies pancreatic cancer arises from noninvasiveprecancerous lesions that are curable if detected and treated earlyenough Although the majority of pancreatic cancers are believedto originate in microscopic precancerous lesions pancreaticintraepithelial neoplasia or PanIN a significant minority arise inassociation with larger cystic neoplasms that can be detectedusing currently available imaging technologies2 These neoplasmswhich includeintraductal papillary mucinous neoplasmsIPMNs and mucinous cystic neoplasms MCNs are frequentlydiagnosed incidentally on abdominalidentifying acohort of atrisk patients with an important opportunity forprevention of invasive pancreatic cancer2 However preventionmust be balanced with potential overtreatment of lowrisk lesionsas pancreatic resection carries significant morbidity and evenoccasional mortality3 There is a critical need to understand themolecular alterations that are associated with the development ofinvasive cancer as these represent potential biomarkers to identify cysts at high risk for progression to carcinoma and thusrequiring clinical interventionimagingAlthough genomic analyses have been performed on hundredsof invasive pancreatic cancers relatively few noninvasive neoplasms have been analyzed comprehensively Whole exome andtargeted sequencing of small cohorts of IPMNs and MCNs haverevealed driver genes characteristic of each type of cystic neoplasm4 while targeted analyses in larger cohorts have conï¬rmed the prevalence of speciï¬c driver gene mutations thatcorrelate with grade of dysplasia or histological subtype7 Thesestudies have conï¬rmed that hotspot mutations in the oncogenesKRAS and GNAS occur in lowgrade lesions while mutations inother driver genesincluding CDKN2A TP53 RNF43 andSMAD4 occur with increasing prevalence in lesions with highgrade dysplasia or associated invasive carcinoma8 Targeted nextgeneration sequencing has been used to analyze pancreatic drivergenes in different regions of IPMNs revealing a surprising degreeof intratumoral genetic heterogeneity even with respect to wellcharacterized driver gene mutations9 However the aboveanalyses were based on studies of either single regions from eachneoplasm or a limited number of genes from multiple regionsand did not provide an analysis of the evolutionary relationshipbetween different regions of pancreatic cysts and associatedcancers These limitations highlight the need for comprehensivegenomic analysis of these cysts and associated invasive cancers tounderstand the molecular alterations that underlie the transitionto invasive carcinomaIn this study we perform whole exome sequencing of IPMNsand MCNs and their associated invasive carcinomas Importantlywe focus our study on small invasive carcinomas cm inorder to more precisely analyze the genetic alterations that occurat malignanttransformation in pancreatic tumorigenesis Inaddition in a subset of our samples we perform deep targetednext generation sequencing on a larger set of additional tissuesamples in order to assess mutated loci through entire neoplasmsincluding areas of lowgrade dysplasia highgrade dysplasia andinvasive carcinoma These analyses reveal important features ofpancreatic tumorigenesisincluding evolutionary relationshipsbetween different regions within cystic neoplasms as well asmolecular alterations that may drive the transition from a noninvasive precursor lesion to invasive cancerResultsOverall approach In order to dissect the molecular relationshipsbetween noninvasive dysplastic lesions and invasive pancreaticcancers we performed whole exome sequencing of neoplastictissue samples from patients with small invasive carcinomas cm associated with neoplastic pancreatic cysts including patients with IPMNs and patients with MCNs Supplementary Data Whole exome sequencing was performed onone sample from the noninvasive component with highgradedysplasia and one sample from the invasive cancer in each caseand for three cases an additional noninvasive sample with lowgrade dysplasia was also analyzed by whole exome sequencingMatched normalsamples were analyzed by whole exomesequencing in each case to exclude germline variants and toidentify somatic mutations Whole exome sequencing was performed with an average total coverage of distinct coverageof generating TB of sequencing data SupplementaryData In addition to whole exome analyses we performed targetednext generation sequencing of microdissected tissue samplesfrom seven of the above cases six IPMNs and one MCN Forthese targeted analyses we performed laser capture microdissection to isolate neoplastic cells from every available tissue block ofthe noninvasive cyst and cancer specimens Separate sampleswere microdissected based on grade of dysplasia cell morphollocation This resulted in ogy architecture and spatialadditional samples per case The targeted panel analyses includedall mutated loci identiï¬ed in the whole exome sequencing of theseseven cases as well as the entire coding regions of wellcharacterized pancreatic driver genes Supplementary Data The targeted sequencing had an average coverage of distinct coverage of Supplementary Data We developed an integrated mutation calling pipeline torigorously assess mutations in all sample types in our analyses inorder to conï¬dently identify even subclonal alterations in sampleswith low neoplastic purity see Methods Fig Supplementary Data In addition we utilized both on target and off targetreads to examine focal copy number changes as well as loss ofheterozygosity throughout the genome Fig SupplementaryData and From our whole exome sequencing analyses weidentiï¬ed an average of somatic mutations in samples fromnoninvasive components range and an average of somatic mutations in invasive carcinoma samples rangeFig 1a Supplementary Data An average of somatic mutations were shared between the noninvasive andinvasive components while somatic mutations were unique tosamples from noninvasive components and somatic mutationswere unique to samples from invasive cancer Fig 1a We alsoidentiï¬ed an average of ï¬ve shared copy number alterationsbetween noninvasive and invasive components as well as anaverage of one copy number alteration unique to samples frominvasive cancer A similar mean proportion of somatic mutationsand copy number alterations were unique to invasive samples for somatic mutations for copy number alterationsAnalysis of our combined whole exome and targeted sequencing data provided multiple insights into IPMN and MCNtumorigenesis In every analyzed case there were multiple sharedmutations between the noninvasive and invasive componentsThese included both driver and passenger mutations indicatingthat they shared a common phylogenetic ancestor Fig 1a b Inaddition accumulation of unique mutations in both noninvasiveand invasive components demonstrated independent evolutionafter the divergence of the subclone that gave rise to the invasiveFig Supplementary Figs S1S18 Analysis ofcanceradditional adjacent lowgrade or highgrade samples from thesame lesions revealed a subset of shared mutations suggestingthat these dysplastic lesions preceded the development of theinvasive carcinoma Fig Supplementary Figs S1 S2 S3 S5and S16 Evolutionary analyses showed a branched phylogeny inNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178asnoitatuMCancerization onlyDuctal cancer onlyMucinous cancer onlySharedIPMN onlyMCN onlyPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMPTMbKRASTP53CDKN2ASMAD4RNF43GLI3GNASMUC16PTPRTTGFBR2ATMCNTN5PIK3CAALKAPCRYR2STK11CTNNB1ERBB4EGFRSF3B1NOTCH1KEAP1ERBB2MYCRETTSC2PrecursorSharedCancerFig Somatic mutations identiï¬ed in matched noninvasive and invasive cancer samples a In each patient sample multiple mutations were sharedbetween the noninvasive and invasive cancer samples gray In addition some mutations were limited to the noninvasive bluegreen while others werelimited to the cancer redpink Darker colors indicate alterations that were likely restricted to one component but where sequencing coverage in thesecond component was limited The proportions of shared and distinct mutations varied between different lesions b Somatic mutations in the mostfrequently mutated genes are categorized as shared between noninvasive and cancer gray limited to noninvasive blue or limited to cancer redMutations in some genes such as KRAS were always shared while others were enriched in samples from noninvasive RNF43 or cancer SMAD4MutationsMTP1MTP2MTP3MTP4MTP5MTP6MTP7MTP8MTP9MTP11MTP13MTP14MTP18MTP19MTP23MTP24MTP26MTP30chr1chr2chr3chr4chr5chr6chr7chr8chr9chr10chr11chr12chr13chr14chr15chr16chr17chr18chr19chr20chr21chr22Copy number log2SamplesLG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationFig Copy number alterations identiï¬ed in matched noninvasive and invasive cancer samples Chromosomal gains red and losses blue are shownfor each chromosome in each patient with noninvasive samples on the left and cancer samples on the rightNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP19HEbefore LCMafter LCMMTP8KRASG12DKRASG12RKRASQ61H1225380275_TGKRASQ61H1225380275_TAKRASG12VTMEM56RWDD3c5662TC [SP]KCNA5A451AC17orf98T142MDUSP27T652TADAMTS12P429LCOL12A1R933HSCAF8G961VTMEM246A372AMCM10E613KPNLIPRP1T465TC13orf35L33LSMAD6L179LGRIN2AS397SAP1G1T798TTP53E171FCHO1c22471GC [SP]LRFN1S632SLTBP4P243LLOHchr17001ABRF645SALPK1R873RLOHchrX275DELchr92182377CDKN2ABSND861NSUPT6HI104TPLEKHF1D258DLOHchr221716LTBRR425GACOT11R306LOR6N1Y120YENPP5Y341YITGB3C562BTKP116TSLC1A7G465DFGF3R104QZBED4P1100LSIM1R192CPHC1S627CMS4A1T41TLOHchr712725SH2D5R199WRNPEPL1G413VGAL3ST2P85PJADE2N352SHHLA1A97VTMEM141Q61QBPTFR296HKLHL22R603RGALEQ261LANKS4BQ368RKRI1S326SOR8I2C240CLRTM2G319VLOHchr97123LAMB3R887LSPTAN1I1484FTP53R175HTissue sourceLG IPMNHG IPMNMUCINOUS CANCERDUCTAL CANCERCANCERIZATIONSequencingTargeted SeqWhole Exome SeqDriver MutationHotspotInactivating_OtherDELLOH µm µmMPT19 LG IPMN µm µm µmMPT19 HG IPMN µm_________ µm µm µmMPT19 ductal cancer µm µm µmMPT19 cancerization T2 T1 µm µmMPT8 LG IPMN µm µm µmMPT8 HG IPMN µm__ µm µm µmMPT8 mucinous cancer T1 T2 TTC39AN83ILRRC8DI741MOBSCNR2837QRGS7A195AOR14A2A156ACCDC13S534SEGFLAMG553VITGA1V27LBDP1N622NKIAA0319V824FRIMS1R211QPRDM13A303VIFNGR1M1PHACTR2A348PGPR85F208FGNED83HHTR7T240MGRM5T946KKRASG12DUNC13CN497SBNIP2D58DCCDC33D354NCLDN9P187PNOTUMR308CPIEZO2R2407QC19orf24G52AMUC16P9201QCCNE1R85WGNASR201HPHF21BA116TTNS1R894QNDUFAF6L320ISLC15A1E26KCLUHR433PRR23AA151TBRD3R313HUSP34R3136HRHOAK27ESIM1A654TNRG1A540VVRK3S129NCCDC166A84TST3GAL4V165MTSPAN17P63PMAD1L1S327SC17orf47E142GCHERPL47ITRPC5E418DZFP37R18WGNASD464NMED12E1497KBAG2T93TRNF43R371CACNG2V255MMAGEB18A63VBRINP3A748APDE1BE401EQRICH2R160RRNF43P370fsINTS1G527VMYOM3G152SPRRT3P407STGFBR2R553HPDZD2I810IFAM120BR646CEPHB4V682MNOBOXR302CCEP164R897QMAB21L1T171MZCCHC14T80MCD68L295LGAS2L2S728LRUNDC3AE49EPHBI122VDNMT1N109NZNF865A721ACASS4S376SKCNJ6R322SLC5A4L390PLRRC8CR355HNPR1R439HPPP1CBV263LBIRC6E4424VIL1Q468QMOGAT3c2359GAUSP20A675TMEX3BS256YMUC13T30AZBTB10R456QSLC26A3V54IC9orf84E8DPPP1R36R303CPLEKHA6Q171SSH3P639PDSCAML1G1252DSLCO1C1R639KENDOVA2VCOL9A1A680TMC2RF228LBPIFB2A427SLOHchr812546DELCDKN2ARNF43P660fsRNF43C471fsRNF43E39fsRNF43M1_G4delRNF43S321RNF43P231LFig Somatic mutations identiï¬ed in MTP19 and MTP8 in targeted and whole exome sequencing We show mutations identiï¬ed in each sampleincluding lowgrade IPMN light blue highgrade IPMN dark blue ductal cancer red mucinous cancer pink and cancerization purple The type ofsequencing analysis targeted or whole exome performed for each sample is indicated in a track on the bottom Representative images of neoplastic tissuestained by hematoxylin and eosin as well as isolated regions before and after laser capture microdissection are shown for MTP19 and MTP8NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178MTP2MTP8MTP3MTP19MTP1MTP5MTP24LG IPMNHG IPMNLG MCNHG MCNDuctal cancerMucinous cancerCancerizationMutationsFig Evolutionary reconstruction of samples analyzed by whole exome and targeted sequencing In all cases noninvasive samples bluegreenprecede invasive samples redpink in the evolutionary history In MTP5 different invasive cancer samples are placed in different regions of thephylogeny highlighting multiple independent invasion events in this lesion In MTP19 a sample of cancerization purple has descended from invasivecancer sampleseach case with multiple clonally related but distinct dysplasticsamples from each neoplasm Fig Importantly removal ofdriver gene mutations from these analyses did not significantlyalter the resulting phylogenies indicating that the evolutionaryrelationships are supported by mutations in addition to driveralterations which might be shared by chance Thus the inferredevolutionary relationships between IPMNMCNs and cancersamples are robust as the probability of sharing a nonhotspotmutation due to chance alone is vanishingly small in million while the mean number of shared point mutations was the vast majority of which did not occur in hotspotsAnother potentialexplanation forshared mutationsthis sample impurityinnoninvasive and invasive samples is the presence of a smallnumber of cancer cells contamination in IPMNMCN samplesOur detailed pathological characterization and macro or microdissection minimized the risk ofInaddition variant allele frequencies VAFs of shared mutationsin IPMNMCN and cancer samples can also help to evaluate thelikelihood of such contamination In mutations shared betweenthe IPMNMCN and cancer the mean VAF in the noninvasivesamples was with only of shared mutations having aVAF below in the noninvasive samples These high VAFsindicate that the shared mutations were not the result of a smallnumber of cancer cells contaminating the noninvasive samplesOverall these data provide evolutionary evidence that IPMNs andMCNs were precursors to invasive pancreatic cancer with lowgrade regions usually preceding highgrade regions and ultimatelyresulting in invasive carcinomaDriver genes of IPMNMCN tumorigenesis Through wholeexome and targeted sequencing analyses of IPMNsMCNs andassociated invasive carcinomas we conï¬rmed the high prevalenceof mutations in previously identiï¬ed pancreatic driver genesincluding mutations of KRAS of cases GNAS CDKN2A TP53 SMAD4 and TGFBR2 Fig 1b Somatic mutations were also identiï¬ed in RNF43 which has been previously highlighted for its role as a driver inmucinproducing pancreatic cysts4 Somatic mutations wereobserved at low prevalence in key positions in the PI3K PIK3CATSC2 and WNT APC CTNNA2 CTNNB1 signaling pathwaysas well as in STK11 Fig 1b Supplementary Data Alteration ofthese genes and pathways has been previously reported in afraction of IPMNs471314 The two MCNs analyzed were similarto the IPMNs in the cohort with hotspot mutations in KRAShomozygous deletion of CDKN2A and inactivating mutations inRNF43 among others but as expected these MCNs did not haveGNAS alterations Supplementary Figs S3 S76In addition to driver genes previously reported in IPMNs ourdata provide an opportunity to discover novel drivers of IPMNtumorigenesis We identiï¬ed somatic mutations in the DNAdamage response gene ATM in of lesions including onenonsense mutation Fig 1b Supplementary Data In additionwe identiï¬ed alterations in Hedgehog pathway member GLI3 in of cases Fig 1b Supplementary Data We alsoidentiï¬ed somatic mutations in a previously described hotspot inSF3B1 which encodes a protein critical for RNA splicing Fig 1bSupplementary Data Ampliï¬cations of the wellcharacterizeddriver genes ERBB2 and MYC were each observed in a single caseand have not been previously reported in IPMNs SupplementaryData Other altered genes with a previously unknown role inIPMN tumorigenesis include MUC16 four cases PTPRT fourcases and CNTN5 three cases Fig 1b Intriguingly in onecase an STK11 mutation was found in combination with bialleliclosstheATM lossand cancerspeciï¬c biallelic KEAP1NATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178combination ofreported in lung cancers Supplementary Fig S315these three mutations has previously beenAlthough KRAS mutations occur in the majority of IPMNs andare thought to initiate tumorigenesis in these lesions two IPMNslacked mutations in this gene One case contained a hotspotmutation in codon of GNAS another potential initiator ofIPMN tumorigenesis as well as alterations in TP53 and RNF43Supplementary Fig S15 The other case lacked mutations in anyof the frequently altered pancreatic driver genes but containedhotspot mutations in both CTNNB1 S45P and SF3B1 H662QSupplementary Fig S10 These cases highlight alternativepathways of initiation and progression in IPMNs lacking KRASmutationsOrder of genetic alterations in IPMNMCN tumorigenesis Ourmultiregion sequencing approach of IPMNsMCNs and associated invasive carcinomas provided insights into the order ofspeciï¬c genetic alterations in pancreatic tumorigenesis In ofthe cases at least one somatic mutation in the initiating drivergenes KRAS and GNAS was shared between the noninvasivecomponent and associated invasive cancer with the remainingcase lacking mutations in these genes Somatic mutations in TP53and CDKN2A were also shared in the noninvasive componentand associated invasive cancer in the majority of cases In contrast SMAD4 had alterations conï¬ned to the invasive carcinomain three cases and was shared between noninvasive and invasivesamples in four cases Fig 1b The majority of SMAD4 alterations in all sample types were biallelic including in noninvasive samples and in invasive samples SupplementaryData Alteration of TGFBR2 which functions in the samesignaling pathway as SMAD4 was also restricted to the cancer inone case Fig 1b The other genes with mutations restricted tothe invasive cancer CDKN2A CNTN5 PIK3CA KEAP1 andRET only had this pattern in a single sample Fig 1bOur study also identiï¬ed driver mutations in subclones ofnoninvasive neoplasms that diverged from and were not presentin invasive cancer These included hotspots mutations in wellcharacterized oncogenes and inactivating mutations in tumorsuppressor geneseg PIK3CA pE545K CTNNB1 pS45FSMAD4 pE33fs and multiple inactivating RNF43 mutations inpatient MTP3 Supplementary Fig S3 Mutations in RNF43were a particularly striking ï¬nding in these cases as somenoninvasive components contained several different RNF43mutations each limited to a small number of sections and noneinvolving the invasive cancer Fig Supplementary Fig S3 Inaddition to heterogeneity in RNF43 in early lesions we alsoidentiï¬ed two cases with multiple mutationsin KRAS inprecursor lesions of which only one was present in the invasivecancer For example in MTP19 KRAS pG12V was present in themajority of IPMN samples as well as all the invasive cancersamples but there were an additional four other KRAS mutationsall occurring in hotspot positions that were present in a smallnumber of sectionsin lowgrade IPMN samples Fig Intriguingly these three lowgrade IPMN regions shared nomutations with the invasive cancertheyrepresented genetically independent clonessuggesting thatNotably while there were often many differences in thesomatic point mutations identiï¬ed in the matched noninvasiveand invasive samples the copy number proï¬les were quite similarbetween IPMNMCNs and invasive cancers Fig Supplementary Data and the proportion of copy number alterationsunique to cancer samples was similar to that observed forsomatic mutations While homozygous deletion of some genesoccurred in the invasive cancer but notthe noninvasivecomponent such as CDKN2A in MTP8 Fig analyses ofchromosomal gains and losses through assessment of allelicimbalance revealed that an average of of the genome wassimilar in copy number in matched noninvasive and invasivesamples Fig Supplementary Data Insights into pancreatic neoplasia revealed by sequencing Thesamples analyzed by targeted sequencing were characterizedmorphologically and meticulously isolated using laser capturemicrodissection Even with this process we identiï¬ed samples intwo cases that were characterized morphologically as IPMNs butthrough genomic and evolutionary analyses were determined tobe identical to or descendants of the associated invasive cancersFor example in MTP19 some of the samples originally identiï¬edmorphologically as noninvasive IPMN and T1 sharedall the mutations present in the invasive cancer sample T2 andcontained additional mutations suggesting that these samplesdescended from the cancer Figs Evolutionary analysessuggested that some of the morphologically identiï¬ed IPMNsamples in this case as well as MTP1 actually representedintraductal spread of invasive carcinoma also referred to ascancerization of the ducts In these cases after invading thestroma the carcinoma invaded back into and colonized the cystsuch that it was morphologically indistinguishable from IPMNwith highgrade dysplasiaIn one case MTP5 we also identiï¬ed an interesting pattern ofmultifocal invasion of the carcinoma In this case we analyzedï¬ve different samples from invasive cancerthree samples wereisolated from a mucinous carcinoma and two samples wereisolated from a ductal carcinoma Based on evolutionary analysesof the patterns of shared and distinct mutations in the cancersand IPMNs we conclude thatthere were multiple separateinvasion events in this lesion as represented by the mutationsshared between the invasive cancers and noninvasive componentsas well as those that were unique to the speciï¬c invasive cancersFig Supplementary Fig S5As our study represents the largest cohort of comprehensivelysequenced IPMNsMCNs we also analyzed mutational signaturesin our dataset Intriguingly our data contrast somewhat with themutational signatures previously reported in pancreatic ductaladenocarcinoma PDAC1617 Like PDAC the most prominentmutational signature was associated with age Signature 1Awhich was identiï¬ed in almost every case SupplementaryFig S19 However we also identiï¬ed signatures associated withAPOBEC enzymes four cases smoking three cases andmismatch repair deï¬ciency cases Although smoking isconsidered a risk factor for pancreatic cancer until now themutational signature associated with smoking has not beenreported in pancreatic neoplasia18Evolutionary timeline of highgrade IPMN to PDAC To estimate the time between the development of highgrade IPMN andPDAC we evaluated Bayesian hierarchical models for the numberof acquired mutations under a range of possible mutation ratesThese models estimate the time interval between a founder cell ofa PDAC and the ancestral precursor cell in the associated highgrade IPMN assuming that mutation rates and cell division timesare constantseeMethods We performed this analysis on the paired WES datafrom of our cases Supplementary Fig S20 We excludedMTP19 because our evolutionary analyses demonstrated intraductal spread of invasive carcinoma and as such we lacked WESdata from an IPMN sample in this case In the analyzed casesthe average median time to progression from IPMN to PDAC was years but the models showed a bimodal distribution Thismedian time was nearly years for patients but nearly yearsthis period of developmentthroughoutNATURE COMMUNICATIONS 101038s41467020179178 wwwnaturecomnaturecommunications 0cNATURE COMMUNICATIONS 101038s41467020179178for patients with more than acquired mutations highlightingpotential variability in progression time between patients Forexample in patient MTP1 most models suggested an average of years between the development of the IPMN and the PDAC CI years In contrast for patient MTP2 with additional mutations acquired in the PDACthe transitionappears to have been slower with an average estimate of years CI years from the Bayesian models Overall theseanalyses suggest that for most patients there is a significantwindow of time between development of highgrade dysplasiaand pancreatic cancer providing an opportunity for surveillanceand interventionDiscussionThis study representsthe largest dataset of whole exomesequencing of IPMNs and MCNs to date Importantly our dataestablished that both IPMNs and MCNs are direct precursors ofinvasive pancreatic cancer Fig This conclusion has beenpreviously suggested by the morphological relationship betweenthe noninvasive neoplasms and invasive cancer on traditionalhistologic sections as well as shared driver gene mutations intargeted sequencing studies679 However the presence ofmany shared driver and passenger mutations clearly demonstrated the common origin of IPMNsMCNs and invasive pancreatic cancers in our study and evolutionary analyses revealedthat dysplastic lesions precede invasive cancers Evolutionaryanalyses suggested that highgrade noninvasive lesions occur over years before invasive carcinoma providing a window ofopportunity for early detection and interventionIn this study we identiï¬ed somatic mutations in driver genesthat had not been previously implicated in IPMNsMCNs Forexample we identiï¬ed alterations in the DNA damage responsegene ATM in of the analyzed cases Germline mutations inATM have been recently reported in patients that developedIPMNs highlighting the potential importance of this gene inIPMN risk19 In addition mucinous colloid carcinomas aresignificantly more common than typical ductal carcinomas inpatients with germline ATM mutations further highlighting thelink between mutations in this gene and IPMNs20 Although theATM gene is large potentially increasing the likelihood of passenger or artifactual mutations even larger genes such as TTNhad a lower mutation prevalence suggesting that at least some ofthe alterations identiï¬ed in ATM are likely to be bona ï¬desomatic mutations Somatic mutations in GLI3 which encodes acomponent of the Hedgehog signaling pathway were identiï¬ed in of cases Somatic mutations in GLI3 were recently reportedin a distinct morphological variant of pancreatic carcinomaundifferentiated carcinoma with osteoclastlike giant cells aswell as at a low prevalence in sporadic PDAC suggesting that theimportance of GLI3 mutations and its signaling pathway inpancreatic tumorigenesis may extend beyond IPMNsMCNs21The hotspot mutations in SF3B1 which encodes a protein criticalfor RNA splicing are also potential drivers in the IPMN pathwayHowever somatic mutations in this gene have been reported in avariety of other neoplasms including hematologic malignanciesand uveal melanoma24We highlight somatic alteration of the SMAD4 pathway as aputative driver of progression to invasive cancer as mutations inSMAD4 or TGFBR2 occurred only in invasive cancer samples in of the cases analyzed SMAD4 was the only gene with cancerspeciï¬c mutations in more than one case highlighting thepotentially unique role this gene plays in pancreatic carcinogenesis This role has been previously suggested by next generationsequencing of highgrade PanINs showing an absence of SMAD4mutations in precancerous lesions as well as cancerspeciï¬cSMAD4 mutationsreported in a paired PanINcarcinomaanalyses2728 Loss of SMAD4 expression limited to invasivecarcinomas has been reported in MCN and IPMNassociatedinvasive cancers and targeted sequencing of a small number ofIPMNs and matched cancers identiï¬ed a single case with aSMAD4 mutation occurring only in the cancer72829 In our datathere were also four cases where mutations in SMAD4 wereshared between noninvasive and invasive cancer samples and twowhere SMAD4 mutations were limited to the noninvasive component Although our wholeexome approach could not detect alltypes of SMAD4 alterations such as rearrangements or epigeneticchanges the majority of SMAD4 mutations observed in bothnoninvasive and invasive components affected both alleles Takentogether this suggests that the role of SMAD4 mutations may notbe universal and may depend on other factors including cellintrinsic such as somatic mutations in other driver genes andcell extrinsic such as stromal and immune microenvironmentmechanismsAlthough some of our cases had SMAD4 mutations limited tothe invasive cancer most of the IPMNMCNassociated cancerslacked driver gene alterations that were associated with invasivedisease suggesting that malignant progression is not universallydriven by point mutations Previous studies have speciï¬callydemonstrated the importance of copy number alterations andchromosomal rearrangements in pancreatic tumorigenesis30 Wedid not identify large differences in the copy number proï¬lesbetween noninvasive components and associated invasive cancers suggesting that global genomic instability may be importantas an early feature of tumorigenesis but is not likely to drivemalignant transformation in many casesOur study also revealed prevalent genetic heterogeneity indriver gene mutations in early lesions demonstrating morecomplex processes than previously suggested by traditional lineartumorigenesis models Similar to our recently reported polyclonalorigin of IPMNs12 we identiï¬ed multiple independent clonesinitiated by distinct KRAS mutations in two cases in the currentstudy In addition our study identiï¬ed multiple distinct inactivating mutations in RNF43 limited to unique tumor subclones apattern previously observed by our group and not shared by othergenes in our whole exome sequencing analyses1231 In most casesRNF43 mutations were enriched in noninvasive components andabsent from the associated invasive cancers More generally weobserved multiple instances of clear driver mutations includinghotspot mutations in oncogenes as well as inactivating mutationsin tumor suppressor genes that were limited to the noninvasivecomponents and not present in the associated invasive cancerThus these mutations occur and clonally expand in the IPMN orMCN but are not present in the subclone that subsequentlyinvades Such independent evolution of premalignant lesions hasbeen observed in other an sites and does not diminish theconclusion of our evolutionary analyses that IPMNsMCNs aretheseprecursors ofobservations suggest unique selective processes at different timepoints in tumorigenesis such that mutations selected in theprecancerous lesion are not selected for or are even selectedagainst in the invasive cancerinvasive pancreatic cancer32 RatherIn addition to these observations about clonal evolution innoninvasive lesions our data also provide genetic evidence formultiple underappreciated processes in pancreatic neoplasiaFirst we provide genetic evidence for intraductal spread ofinvasive carci
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" the popularization of health and medical informatics yields huge amounts of data extracting clinicalevents on a temporal course is the foundation of enabling advanced applications and research it is a structure ofpresenting information in chronological order manual extraction would be extremely challenging due to thequantity and complexity of the recordsmethods we present an recurrent neural network based architecture which is able to automatically extractclinical event expressions along with each events temporal information the system is built upon the attentionbased and recursive neural networks and introduce a piecewise representation we divide the input sentences intothree pieces to better utilize the information in the sentences incorporates semantic information by utilizing wordrepresentations obtained from bioasq and wikipediaresults the system is evaluated on the thyme corpus a set of manually annotated clinical records from mayoclinic in order to further verify the effectiveness of the system the system is also evaluated on the timebank_dense corpus the experiments demonstrate that the system outperforms the current stateoftheart models thesystem also supports domain adaptation ie the system may be used in brain cancer data while its model istrained in colon cancer data our system extracts temporal expressions event expressions and link them according to actuallyoccurring sequence which may structure the key information from complicated unstructured clinical recordsfurthermore we demonstrate that combining the piecewise representation method with attention mechanism cancapture more complete features the system is flexible and can be extended to handle other document typeskeywords clinical text mining event extraction temporal extraction relation extraction piecewise representationattention mechanism precision medicine is an emerging approach for diseasetreatment and prevention it becomes the whole worldbiomedicine domain the research hot spot which needsthe support of biomedical methods eg data mining it correspondence clixjtueducn zhijing li and chen li contributed equally to this work1school of computer science and technology xian jiaotong universityxian shaanxi china2shaanxi province key laboratory of satellite and terrestrial network techrd xian jiaotong university xian shaanxi chinaassociates with key information extracted from clinicalrecords eg symptoms over a disease course the associations are often statistically concluded from the evidencecollected from the clinical records the medical bigdata mostly exists in an unstructured form eg textwhich could store useful information very well aligningbiomedical events in clinical data along the events actually occurring time is a meaningful and efficient way ofstructuring such complex data the result may assistauxiliary diagnosistreatment scheme determinationepidemic prediction and side effect discovery etc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cli bmc medical informatics and decision making page of many works have been devoted in the study of application in the medical era however large data analysis ofmedicaltreatment needs to map the correspondingmedical events in the clinical records the medical eventswith temporal information are very useful in medical erathese efforts will become the foundation of understanding disease facilitating the analysis of large medical dataas well for example the clinical record in fig may bepresented in a structured manner as the occurring eventsalong with temporal information it is easier for understanding the events and corresponding time point for example using the time point april as a referencethe entity bleeding is before the time point and the entitybolus chemotherapy and nausea is after the time pointin such case the actual events and their occurring consequence becomes clear at a glancein this paper we present a novel system which is builtupon deep neural networks to automatically extractevent expressions and their related temporal expressionsfrom clinical records the system has been evaluated onthe temporal histories of your medical event thyme corpus a corpus developed by a number of professionals according to characteristics of the corpusclinical data contains very long sentences that willundoubtedlythe difficulty of processingtherefore we do not simply use neural networks wewant to make full use of the contextualinformationour proposed method anically combines piecewiserepresentation and attention mechanism by a recurrentneural network rnn and achieves the stateoftheartperformance the results show improvements in automatic extraction of clinical event expressions along witheach events temporal informationincreaserelated workextracting clinical events along with temporal information is a complicated task and the existing systems oftenaccommodate several independent components each ofwhich retrieves different parts eg events and time andassemble them together each component may use a setof hand crafted rules or be based on a pretrained mlmodel velupillai develop the blulab systeminclude the cleartk support vector machine and conditional random fields classification approach and get thefirst place in semeval2015 task clinical tempeval macavaney present the system guir includeconditional random fields and decision tree ensemblesusing lexical syntactic semantic distributional and rulebased features guir receive the best score insemeval2017 task clinical tempeval in the way oftemporal expressions extraction tourille use aneural network based approach and achieve goodperformance for both event and relation extraction insemeval2017 task clinical tempeval lin propose a recurrent neural network with multiplesemantically heterogeneous embeddings within a selftraining framework for clinical temporal relation extraction task they achieve good results for both in andcrossdomainafter event and temporal expression extractionassigning each event with the right temporal expressioninvolves more complicated process some systemsmatch event with temporal expression by a set of syntactic rules crafted by experts wang use syntacticrulebased method for automatic pathway relation information extraction from biomedicalliterature these methods are fast but not flexible enough somefig the example of the medical information extraction the texts marked by the underscores ___ are the temporal expressions the textsmarked by the dash lines _ _ _ are the event expressions 0cli bmc medical informatics and decision making page of existing methods for medical relation information extraction are based on machine learning ml models[]conditional random field crf and supper vectormachine svm are often used in the task of relation extraction lu liu propose an svm model toextract the relations between the potential named entitypairs finkel propose a crfbased informationextraction system to determine relationships deeplearning revives the popularity of neural networkswhich can learn effective relation features from the givensentences withoutengineeringsocher is the first work that employs an rnnmodel to classify relation one early work proposed byluo is based on a recurrent neural network and ableto classify relations from clinical notes compared withthe rule based methods these methods are more flexibleneural network based methods take less time by quicklyscreening out the most unlikely candidate entitypairstherefore some approaches attempt to combine bothhence we think rnn is a good choice and we adopt thernn systemcomplicated featurein recent years attention mechanism has been widelyused in various tasks of nlp based on indepth learning li propose a model that combines abidirectional long shortterm memory network with amultiattention mechanism forrelation extractionzhou propose the attentionbased bidirectional long shortterm memory networksattblstmfor relation classification and the modelresults outperforms most of the existing methods withonly word vectors on the semeval2010 relation classification task so in this paper we also introduce theattention mechanismmethodologythe system consists of three components the first component extracts temporal expressions temporal expressions enable events to be chronologically annotatedthe second component identifies the relevant medicalevents event expressions any situation relevant to thepatients clinical timeline the third component detectsthe relations between the events and the temporalexpressionsannotating clinical records are very expensive frequently only a data of disease specific type is availablebesides the regular mlbased extraction we introducedomain adaption to allow the system to be able to extract the information from one type of disease eg braincancer while it is trained on another type eg coloncancerwe show the pipeline of our system in fig thesystem is built upon an annotating pipeline adoptingthemanagementunstructuredinformationfig the dataprocessing pipeline of the system we first extracttemporal expressions and event expressions respectively frommedical records then extract the relations between themarchitecture uima framework the preprocessingincludestokenization partofspeech tagging andlemmatization which used the stanford corenlp toolkit in both time and event extractions spans oftime and event expressions are represented by the offsets in texts the automatic annotations of event expressions temporal expressions and their relations arebased on three rnn models utilizing lexical syntacticand semantic features [] at the core of deeplearning techniques for nlp lies the vector basedword representation which maps words to an ndimensional space for the choice of the word embeddings corpus we do some research work only entities time and event expressions in the clinical records can be found in the wikipedia in comparisonthe bioasq corpus which is full of biomedical information contains more than of the entities weuse word embeddings from the european projectbioasq obtained by using word2vec on pubmed abstracts [ ] and include the vectorsof distinct words each word is representedas a 200dimensional vector if a word could not befound in the bioasq corpus the embedding is generated from wikipedia by word2vec mikolov as a complement [ ]thestateinternalof rnn can demonstratedynamic timing behavior the hidden state vector can be computed by the following formulaht ¼ f wht þ uxtðþin this formula xt is the input ht is the hidden stateu and w are the weight coefficients f is the nonlinearfunction such as tanh or relu 0cli bmc medical informatics and decision making page of extraction of temporal expressions and event expressionsindependent models are trained for the extractions oftemporal expressions and event expressions figure shows the infrastructure of the system there are twoforms of temporal expressions one is numeric temporalexpressions eg etc and the other is casualtemporal expressions eg day weeks during aperiod etc firstly we generalize all the numeric temporal expressions into for example both and become which can be easily recognizedby the regular expression the regular expression is useddue to the characteristics of the data the numeric temporal expressions are not well recognized by rnn if wedo not use it secondly the casual temporal expressionsare recognized by a rnn model the casual temporalexpressions in the training set are tagged to representthe tokens position in a particular expression there are four tags in our proposed method includingb i o and e which state that the token is at thebeginning on the inside on the outside or at the end ofthe entity respectivelyin this section we propose the system arnn whichis based on a recurrent neural network combining theattention mechanism we need to predict the tokensposition tag of each word before we can train the arnnmodel to predict the type of each temporal expressionwe treat each temporal expression as an entity and eachentity is treated as a unit input we use the average valueof all the word embeddings of an entity in the nextprocess the network in the fig shows the flow chartof arnn network to predict the type of the entitythere is an example sentence from the corpus we willget a ct enterography to rule out crohns disease inthis case the given entity is enterography and thecontext words are we will get a ct to rule outcrohn disease in order to better apply the context information we employ the attention mechanism to learnthe weighted score of each context word related to thegiven entitythe higher weight the higher semantic is bound upwith the given entity 01 00αi exp vti u vhtuv is the parameter that has to be learned from fig we can see that st is the state vector that integrates theother context words information with the given entity attime t st can be computed asst ¼xihαihiwe combine st and h3 to obtain h0sent the given entity which can repre ¼ h3 þ sth0in this process the prediction of entitys type will bepredicted from the given input entity vector the methodof using regular expression are also used to match themissing temporal expressions eg similar to temporal expressions extraction the eventexpressions extraction is built on another rnn unliketime expressions event expressions are all single wordsthere is no need to sign each tokens position we usethe softmax classifier to predict the label y² of the temporal and event expressions the state vector h0 is usedas input and y² could be computed by 10 11py ¼ softmax w h0 ¼ arg maxypyyeventtime relation erer extraction is the most important task in this paperin this section we propose the novel system aprnnwhich is based on a recurrent neural network combiningthe attention mechanism and the piecewise representation the eventtime relation are regarded as a classification problem it is divided into four categories based onsome wellknown communities such as semeval orbionlp the event time relation associates the identifiedevent expressions and temporal expressions and indeedindicates the what and when of a medical event inclinical records the four types are before after beforeoverlap and overlapthe shortestsyntactic path used includespiecewise representationthe dependency parsing of each sentence has beenobtained by utilizing stanford corenlp toolkit thepreviously trained word embeddings which representeach word by a 200dimension word vector and theshortest syntactic paths are fed into the rnn modelof er extraction the word embeddings and shortestsyntactic path are used as features the informationofthewords the poss and the length we add all thesevectors as the entity feature after adding up we stillget a 200dimensional vector for each entity if theentity include severaltemporal expressions we add the vectors of each token and get theaverage vector as the entity vector the whole is divided into sentences as input units we extractall entity pairs based on the annotations given a sentence x x1 x2 ¦ xt the words are projected intoa sequence of word vectors denoted by e1 e2 etwhere t is the number of words in this part wewould like to introduce the piecewise representationtokens eg 0cli bmc medical informatics and decision making page of fig architecture of the rnn model for the extraction of temporal expressions and event expressions we propose the attentionbased rnnmodel to do the entity extraction on the left side of the figure is the details of the attention mechanism the right part of the figure is thernn modelinformation as shown in fig in other wordsthe input sentence is divided intothree parts according to the entity pair we call thisprocess piecewise representation the purpose ofpiecewise representation is to better use of the contextthe examplesentence is divided into three parts according to theentity pair will enterography the whole sentence iswe will get a ct enterography to ruleout crohnsdisease the first part is the sequence before the entity pair we the second part is the sequence between the entity pair get a ct and the third partis the sequence after the entity pair to rule¦ thereare reasons for segmenting the sentence the firstone is that in many cases some studies may choosethe sentence between the entity pair as input insteadof using the whole sentence nevertheless thiscan miss some information and some of them maybe useful only several words cannot supply enoughcontextfeatures segmented sentences can be used to extract the effectiveinformation forextractingrepresentsinformation to the greatest extent of each sequenceand avoid the absence of contextualinformationanother reason comes from the network and our corpus in the corpus the longest sentence contains words however the average length of all sentenceshas words with the rnn structure since the information of a sentence is learned word by word thefeature vector produced at the end of the sentenceactuallysentence althoughrnn has the memory in learning process but thememory time is not long accumulation by recurrentconnectionslongterm informationquickly and the feature vector atthesentence is hard to carry the information of earlysteps in model training there are many longdistancesentences more than words in the training dataso the piecewise representation can help the systembetter use the information of the sentence the syntactic analysis and pos information of the examplesentence are also shown in the fig the end ofentirethetendsto fet 0cli bmc medical informatics and decision making page of fig the flow of our proposed model aprnn on the left side of the figure is the details of the attention mechanism the right part of thefigure is the rnn model which is divided into three partsmodelthe er model contained three parts the first component the feature layer the second component thehidden layer catch the information of word sequenceand produces wordlevel features representations andthen merges wordlevel features into a sentencelevelfeature vector by selecting the most valuable featureinformation among allfeatures weshow the whole process in the fig in this part weproposeattention mechanism to obtain thethe wordleveltherepresentation of the sentence not all the words inthe context describe the er relation each word inthe context has different effects on the given entitypair therefore we introduce the attention mechanism to learn the weighted score of each context wordrelated to the entity pair for the first part the attention mechanism is used to screen the most useful information we use the bilinear operator to computethe attention weight αi for each vector h1 and h2 toreflect how the information relevant to the first entity 0cli bmc medical informatics and decision making page of in the entity pair the current state h3 the calculation method of αi and st is the same as the description in the extraction of temporal expressions andevent expressions sectionwe combine st and h3 to obtain h0 which can represent the sequence before the entity pair for the secondpart we choose the state of the last entity in the sequence ht to represent the whole sequence for thethird part the same method is used as the first part wealso use the same attention mechanism to obtain therepresentation oft the singlesentencelevel feature vector need to be obtained to represent the entire sentence for the relation classificationwe introduce the maxpooling approach as in cnnmodels to obtain the single sentencelevel feature vector the maxpooling is formulated as followsthe sequence h0htf gm ¼ maxtnext the sentencelevel feature vector m is passed tothe output layer furthermore the output layer has classes we use the softmax classifier to predict the labely² from a set of labels y from the sentence the statevector m is used as input therefore y² could be computed bypy ¼ softmax wmð ¼ arg maxypyyþin addition there are two settings in er extraction inorder to better compare our approach the first is basedon our proposed method the second is only utilize thernn network without any piecewise representation orattention mechanism as shows in fig experiment and resultsdatathe major medical data are the data of medical institutions diagnosis and treatment collection of massiveclinical data and laboratory data produced every day atall levels of hospitala golden annotated corpus marked up with temporalexpressions events and relationship between them isneeded to allow us to evaluate by our methods thethyme corpus which has been used since isone of the suitable corpora consisting of clinical andpathological notes of patients with colon cancer andbrain cancer from mayo clinic unlike other datasetsthe events in this dataset are all single words which arevery suitable for our system the notes are manually annotated by the thyme project thymehealthnlpusing an extension of isotimeml for the annotation oftemporal expressions events and temporal relations of the corpus is used for training is forfig the flow of the comparison system this is a commonrnn modeldevelopment and is for testing the developmentset is used for optimizing learning parameters thencombine it with the training set to build the system usedfor reporting results table shows the distribution ofthe thyme corpus the colon cancer data are used astraining data and are tested on both colon cancer andbrain cancer data to demonstrate its effectiveness withtable the distribution of the thyme corpus in this table weshow the different types of data in the corpusdatacolon cancertrain dev testbrain cancertrain testdocumenttemporal expressions event expressions er 0cli bmc medical informatics and decision making page of or without domain adaptation and this can reflect thatour approach is not limited to a particular field inevaluation all methods have access to the same set oftraining and testing datatable the temporal expressions extraction results on braincancer we utilize different methods to do the task the resultsare shown in part the result of previously best system isshown in part resultsthe method has been evaluated on both colon cancerdata and brain cancer data to demonstrate the effectiveness with or without domain adaptation in order to dobetter research several methods are used to de the entityextractionsix methods of temporal expression extraction arulebased method a system based on crf a system based on general rnn without any attention mechanism or context rnn a system based on rnnwith easy attention mechanism but without any contextwords rnnatt our proposed method a rnn system with attention mechanism and context words asystem combines the crf and rnn network all the results are compared part in tables and for therulebased methods firstly we find all the prepositionsaccording to our experience and experimental statisticswe extract five tokens behind their own prepositionsthrough careful observation of data we found thatmany time expressions always show up behind a preposition we then judge whether those five words are relatedto time expressions we define a time dictionary to listthe words which we think can be a part of the time exlike month week day hour maypressionsmonday morning once and so on next we contrastthe five tokens with time dictionary and findwhether it can represent a date or a precise time finallywe extract all the continuous tokens that we thoughtmay relate to the time expressions if there is a definite before those tokens extract it as well there existsome expressions do not after a preposition and onlycontain one word and most of them have the same prefix like pre post peri so we use this prefix rule tofind the remain expressions the major feature we usedfor training the crf and svm classifier is simple lexicaltable the temporal expressions extraction results on coloncancer the part shows the results of six different methodsthat we used to do the temporal expressions extraction thepart shows the result of the previously best systempart methodrulebasedcrfrnnrnnattarnnpart crfarnnblulab run prf1part methodrulebasedcrfrnnrnnattarnncrfarnnpart guirprf1features word embeddings partofspeech tag numericlower case blulab run andtype capital typeguir system are the previously best system mentionedin the section two these results are shown in tables and part in both tables and rule based methods achievethe lowest result the recalls are relatively better thanthe precisions due to the welldefined dictionary theerror analysis shows that some pre post and periare considered as time expressions while they should notbe meanwhile the rulebased method often mistakestwo independent expressions as one if they are adjacentin table the rnn systems performances are lowerthan blulab run 3a cleartk svm pipeline usingmainly simple lexical features along with informationfrom rulebased systems the rulebased information iseffective but it has limitations it can extract rules according to the characteristics of data we do not addany rules to the rnn system the observation on theerror analysis shows that without any attention mechanism and context words rnn is not very effective forsimilar combinations of numbers and letters eg h days etc because the form of the corresponding wordvectors are generated randomly and the time series contains a large number of the above type so the modelcannot learn characteristics of time series so it cannotbe correctly extracted after adding the attention mechanism and context words the arnn system achieve therelatively good results because of the good results ofcrf we combine the crf with the arnn and achievethe best result from table we can see that the rnnoutperforms the guir system which is the current bestsystem it is an ensemble of crf and decision tree withlexical syntactic semantic distributional and rulebasedfeatures the guir system can not extract the previously unseen or atypical date formats very well it is obvious that their rules are not comprehensive enoughthis problem also exists in rnn system however whenadding the attention mechanismit can extract more 0cli bmc medical informatics and decision making page of new and otherwise unknown formats the arnn andcrfarnn system achieve the best results in this partwe have two test data sets one is colon cancer anotherone is brain caner we trained all the models on thesame training data and test them on two different testdata sets except for the different test data the parameters are exactly the same the experimental results provethat our model is effective on other test data setsmeanwhilefive methods of event extraction amethod based on svm a system based on crf asystem based on general rnn without any attentionmechanism or context rnn a system based onrnn with easy attention mechanism but without anycontext words rnnatt our proposed method arnn system with attention mechanism and contextwords all the results are evaluated part in tables and for event extraction the svm and crf modelobtain the relatively good results in colon data and perform poorly in brain colon data compared to the bestsystem limsi however rnn achieves preferably results in the two sets of test data even higher than thebest system limsi as shown in both tables and when adding the attention mechanism and contextwords the results are improvedas for the er extraction which is the key point of thepaper first we compare our proposed model with thefollowing methods a general rnn system withoutany attention mechanism or piecewise representationwe use the sentence between the entity pair as the inputrnn a general rnn system without any attentionmechanism or piecewise representation we use thewhole sentence as the input rnnwhole we can seethe results of rnnwhole is better than the results ofrnn it means that the sentence length can affect theperformance of the system therefore we use the sentence between the entity pair as the input for other system a general rnn system with attention mechanismbut without piecewise representation rnnatt ageneral rnn system without attention mechanism butwith piecewise representation rnnpie our proposed system aprnn but only use the word embeddings trained from wikipedia aprnnwiki ourtable the event extraction results on colon cancer different methods are utilized by us all the results are shown inpar1 the part shows the result of the previously best systemf1methodsvmpart prcrfrnnrnnattpart arnnblulab run table the event extraction results on brain cancer we adopt methods to do the task the results can be compared in part the result of the best system is shown in part part part methodsvmcrfrnnrnnattarnnlimsiprf1proposed system aprnn but only use the word embeddings trained from bioasq aprnnbioasq our proposed system which is based on a recurrentneural network combining the attention mechanism andthe piecewise representation all these results are evaluated part in tables and except for model and the word embeddings for other models are from bothwikipedia and bioasq from the results we can seethat both attention mechanism and piecewise representation are useful they can improve the results to someextent we can directly compare the value of attentionin two groups of experiments result and result result and result the result and result result and result can directly demonstrate the performance with and without segmentation the difference between model and is that model is missing thepiecewise representation and the difference betweenmodel and is without or with the attention mechanism the result has been improved with the piecewiserepresentation the experiment and are aboutlooking at the impact of word embeddings the result and result show that different word embeddings canlead to different results after combining the two corpuswikipedia and bioasq the results increase slightlytable the er classification results on colon cancer part shows the results of the relevant methods we used the otherrelated works which achieved the very good results are shownin part part part methodrnnrnnwholernnattrnnpieaprnnwikiaprnnbioasqaprnnblulab run svmattblstmprf1 0cli bmc medical informatics and decision making page of table the er classification results on brain cancer the resultsof our proposed methods are shown in part part shows theresults of other relatedpart part methodrnnrnn wholernnattrnnpieaprnnwikiaprnnbioasqaprnnlimsisvmattblstmprf1aprnn different factors that may affect the resultsare verified from experimental results eg piecewise representation attention mechanism word embeddings allthese factors are utilized to make better use of contextual informationwe compare our work with other related work thelimsi system which achieves the best score on the ertask in semeval2017 task li rao and zhang proposed the litway which is a system that has adopteda hybrid approach that uses the libsvm classifier with arulebased method for relation extraction theyachieve the best score in the seedev task of bionlpst thus we use their approach as a benchmark forour system the bilstmattention networks proposedby zhou were chosen as another benchmarking model attblstm which outperforms most of theexisting methods they designed a bidirectional attention mechanism to extract wordlevel features from thesentence the features for the attentionbased model include word vector
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the molecular heterogeneity of renal cell carcinoma rcc complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge this study investigates the role of thelncrna cdkn2bas1 and mir1413p interactions in the progression and metastasis of kidney cancer human renalcancer cell lines achn and caki1 normal rptec cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study an overexpression of cdkn2bas1 was observed in rcc compared to normal samplesin tcga and our inhouse sfvamc tissue cohorts reciprocally we observed reduced expression of mir141 in rcccompared to normal in the same cohorts cdkn2bas1 shares regulatory mir141 binding sites with ccnd1 andccnd2 genes direct interactions of cdkn2bas1mir141cyclin d1d2 were conï¬rmed by rna immunoprecipitationand luciferase reporter assays indicating that cdkn2bas1mir141cyclin d1d2 acts as a cerna network in rccfunctionally attenuation of cdkn2bas1 andor overexpression of mir141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model furtheroverexpression of cdkn2bas1 is positively correlated with poor overall survival of rcc patients expression of mir141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal rptec cells induced procancerous characteristics cdkn2bas1 attenuation or mir141 overexpression decreased ccnd1ccnd2 expressionresulting in reduced rac1ppxn that are involved in migration invasion and epithelialmesenchymal transition thisstudy for the ï¬rst time deciphered the role of cdkn2bas1mir141cyclin d axis in rcc and highlights this networkas a promising therapeutic target for the regulation of emt driven metastasis in rccintroductionrenal cell carcinoma rcc is one of the most commoncancers in the usa accounting for nearly deathsand new cases in surgery is the ï¬rst line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of morecorrespondence rajvir dahiya rdahiyaucsfedu1department of urology veterans affairs medical center san francisco anduniversity of california san francisco san francisco ca usa2department of surgery university of miami miller school of medicine miamifl usafull list of author information is available at the end of the these authors contributed equally pritha dasgupta priyanka kulkarniedited by e candithan however in approximately of localizedrcc cases recurrence occurs with distant metastasis2the obstinate nature of rcc to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence lack of sensitivity to both chemotherapytherapeuticoptions difï¬cult3 it is therefore of utmost importanceto improve our understanding of rcc pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive rcc6and immunotherapy makesemerging lines of evidence suggest that cancer aggressiveness is associated with epithelialmesenchymal transition emt7 it is a wellorchestrated process involved in the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40ofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8emt changes in cellular morphology and migratoryproperties are governed by numerous factors9 increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like ncadherbronectinvimentin and matrix metalloproteinasemmps and decreased expression of epithelial markerslike ecadherin αecatenin claudin etc10 are common emt phenomena often the progression of cancerthrough emt is significantly induced by the interaction ofcyclind with its binding partner cdk4 which act astranscriptional regulators controlling cell proliferationand migration14 it is well known that cyclind regulates the ratelimiting step in cell cycle progression fromg1 to s phase accumulating evidence also suggest thatabnormal cyclindcdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodreports show that noncoding rnas are involved in thefactors involved in emt18 micrornasregulation ofmirnas a naturally occurring class of small noncodingrna molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mrna degradation20 whereas long noncoding rnaslncrnas with more than nucleotides can also actas regulators for tumorsuppressive mirnas in differentcancers21 recently a class oflncrnas have beencategorized as competing endogenous rna cernawhich involves crosstalk among lncrnas mrnas andtheir shared mirnas thus a novel regulatory mechanismis hypothesized suggesting that lncrnas and mrnascommunicate with each other by competing for commonmirna response elements24in this context we describe the novel role of lncrnacdkn2bas1 and mir1413p mir141 in the regulation of cyclind to govern the metastatic progression ofrcc to our knowledge this is the ï¬rst report to directlydemonstrate that cdkn2bas1mir141 interaction is acrucial component in rcc progression and metastasisthrough the cyclindrac paxillin pathwaymaterials and methodscell lines and cell culturethe normal rptec atcc number crl4031 andrenalcancer achn atcc number crl1611and caki1 atcc number htb46 cell lines were purchased from the atcc manassas va these humanderived celllines were authenticated by dna shorttandem repeat analysis by atcc cell line experimentswere performed within months of their procurementresuscitation achn cells were cultured in mem mediaofï¬cial of the cell death differentiation associationcaki1 cells in and mccoy 5a medium and rptec cellsin dmemf12 medium atcc® ¢ all mediawere supplemented with fbs and 1x antibioticspenicillin and streptomycin cell lines were maintainedat °c and humidiï¬ed atmosphere of co2mirnasirna transfectionsto induce overexpression or knockdown cells were transiently transfected with either mirvana mirna mimic nmoll or antimir mirna inhibitor nmollthermo fisher scientiï¬c and nmoll of sirna sigmaaldrich using lipofectamine rnai max thermo fisherscientiï¬c according to the manufacturers protocol toverify transfection efï¬ciency mirvana mirna mimicnegative control mirna inhibitor control and sirnacontrol were used respectively in each transfection experiment at the same concentration all transfection experimentswere carried out for hclinical specimensformaldehydeï¬xedparafï¬nembedded ffpe tissue specimens from patients undergoing radical nephrectomy wereobtained from the san francisco veterans affairs medicalcenter sfvamc written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines irb approval no allpatient samples were pathologically conï¬rmed for clear cellrcc ccrcc and slides were reviewed by a boardcertiï¬edpathologist for the identiï¬cation of tumor foci and adjacentnormal tissue apart from sfvamc cohort tcgakirctcgakich tcgakirp icgc and geo cohorts forrcc from online databases were also used to check theexpression levelsrnamirna extraction and quantitative realtime pcrqrtpcrtotal rna was extracted from microdissected ffpetissues and cell lines using mirneasy ffpe and mirneasymini kits qiagen respectively in accordance to manufacturers instructions mature mirna and mrnas wereassayed by qrtpcr using quantstudio flexreal timepcr system applied biosystem using fast sybr®green master mixtaqman universal pcr master mixprobes and primers applied biosystems inc foster cityca usa following manufacturers protocol humangapdh and rnu48 were used as endogenous controlsand relative expression of rnamirna were calculatedusing comparative ct threshold cycle primer sequencesare provided in supplementary table t1dna methylation analysis insilico in cell lines and 5azacdr treatmentdna hypermethylation of the mir141 promoter innormal and rcc samples was ï¬rst conï¬rmed in the 0cdasgupta cell death and disease page of tcga database using wanderer software27 in order toconï¬rm the methylation status of the mir141 promoterin rcc cell lines we extracted dna from achn andcaki1 using dneasy tissue kit qiagen sodium bisulphite modiï¬cation was done using ez dna methylationgold kit zymo research orange ca usa followingthe manufacturers protocol bisulï¬tetreated dna wasanalyzed by methylationspeciï¬c quantitative polymerasechain reaction msqpcr with primer pairs speciï¬c formethylated and unmethylated regions of the mir141promoter msqpcr was performed as described earlier28 for each sample the percent of methylation wascalculated by the difference of ct in methylated samplectm and ct in unmethylated sample ctu the primers sequences are mentioned in supplementary table achn and caki1 cells were treated daily with μmoll5azadeoxycytidine 5azacdrfor h29 and total rna was isolated using a mirneasy minikit qiagen to check mir141 expressionsigmaaldrichcell viability clonability migratory invasion andapoptosis assayscell viability was measured at and h using acelltiter aqueous solution cell proliferation assay kitpromega madison wi following the manufacturersinstructions for colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed plates were then stained with giemsafollowed by crystal violet and colonies were countedculture inserts of 8µm pore size transwell costar wereused for migration and invasion assay inserts were coatedwith matrigel bd biosciences µgwell for invasionbrieï¬y h posttransfection cells were counted andplaced on inserts at à cellsml for migration and à for invasion in serumfree medium and wereallowed to migrateinvade for h at °c cellsmigrated or invaded through the pores were ï¬xed stainedwith crystal violet crystal violet was solubilizedwith methanol and quantiï¬ed at nm by a kineticmicroplate reader spectra max molecular devicesfacs analysis for apoptosis was done h posttransfection using annexin vfitc and 7aad kit beckmanin accordance with the manufacturerscoulter incinstructions cold pbs washed cells were resuspended in1x binding buffer and stained with annexin vfitc7aad viability dye after min of incubation at roomtemperature in the dark stained cells were analyzed usingbd facsverse bd pharmingendualluciferase reporter assaythe wild type wt and offtarget ot luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingofï¬cial of the cell death differentiation associationsites and corresponding nontarget mutant sites into thepmirglo reporter vector luciferase constructs µgwere cotransfected into achn and caki1 cells along with nmoll mir141 mimic or controlmir using transfection reagent jetprime polyplustransfection illkirchfrance luciferase activities were measured using thedualluciferase assay promega madison wi h posttransfection relative luciferase activity was calculated bynormalizing fireï¬y luciferase to renilla luminescencerna immunoprecipitation rip assaysimultaneous binding of mir141 to lncrna andmrna was conï¬rmed by rip assay an imprint rip kitwas used following the manufacturers protocol sigmaaldrich st louis mo usa igg control and ago2antibodies were used forimmunoprecipitation theimmunoprecipitated rna fraction was reverse transcribed to cdna using high capacity cdna reversetranscription kit thermo fisher fold enrichment oflncrna and mrna to ago2 with respect to igg wascalculated using quantitative rtpcrwestern blot and immunoï¬uorescence analysistotal protein extraction was performed as describedpreviously18 proteins were then separated by nupage bistris protein gels invitrogen and subsequentlytransferred onto nitrocellulose membraneresulting blots were blocked using odyssey blockingbuffer licor and subsequently probed with primaryand secondary antibodies blots were scanned using anodyssey infrared imaging system scan and quantiï¬cationwas carried out with the licor odyssey® scanner andsoftware licor biosciences the primary antibodiesused are listed in supplementary table for immunoï¬uorescencetransfected achn andcaki1 cells were ï¬xed in paraformaldehyde for minfollowed by blocking 1x pbs5 normal goat serum triton x100 for h at room temperature cellswere then incubated overnight in fold diluted primary antibody at °c cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoledapi for min cells were then mounted on a slideusing prolong gold antifade reagent images were captured using zeiss microscope model axio imagerd2transientlyrenal cancer xenograftswe studied the antitumorigenic effects of mir141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 male nude mice weekold n charles river lab were subcutaneously injected with à caki1 cells oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups ï¬ve in 0cdasgupta cell death and disease page of each synthetic mirna mir141 mimicmircon of μg was complexed with μl siportamine transfection reagent ambion in μl pbs and deliveredintratumorally in 3day intervals tumor volume wascalculated according to the formula x2 y2 where x yx width y length experiments were terminated days after the last treatment day tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups all animal care was in accordance withinstitutional guidelines iacuc approval no statistical analysisall quantiï¬ed data represents an average of at leastthree independent experiments or as indicated statisticalanalyses were performed using graphpad prism andmedcalc error bars represent ± standard error meansem the mannwhitney u test was used to assessthe difference between mirna expressions in tumornormal adjacent tissue significant differences betweenthe groups were determined using the students ttest alltests were performed either one tailed or two tailed andresults were considered statistically significant at p ¤ receiver operating curves roc were performed toevaluate the potential of mir141 to differentiate betweenmalignant and nonmalignant samples using medcalcsoftware showing area under curve auc and conï¬dence interval kaplanmeier analyses for overall survival with respectto mir141 methylation levels intcgakirc cohort were generated using softwareezrhttpsdoi101038bmt2012244 tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsresultslncrna cdkn2bas1 is oncogenic and is a direct target ofmir141initially we found cdkn2bas1 is an oncogeniclncrna in rcc based on tcga fig 1a icgc andgeo databases fig s1 tcga cohort also revealed thathigh cdkn2bas1 expression increases from lower gradeand stage to higher grade and stage fig 1b moreoverhigher expression is significantly p correlated tooverall survival fig 1c in agreement with these datacohorts significantly higher cdkn2bas1 expression wasalso seen in rcc cell lines achn caki1 as compared tonormal rptec fig 1d and sfvamc cohort fig 1epatient and tumor characteristics are summarized insupplementary table t3 roc analysis shows an auc of p ci fig 1f suggesting the diagnostic potential of cdkn2bas1 to discriminate between normal and tumor tissues we usedcomputational algorithms and identiï¬ed putative mir141binding sites in the cdkn2bas1 sequence fig 1g toofï¬cial of the cell death differentiation associationexamine potential mir141cdkn2bas1 interactionexperimentally we performed luciferase reporter assayboth achn and caki1 cells cotransfected with mir141and cdkn2bas1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmir141 directly interacts and regulates cdkn2bas1fig 1h thus all these data suggest that clinicallyimportant cdkn2bas1 is an oncogenic lncrna in rccand is a novel target of mir141cdkn2bas1 inhibition by sirna suppressestumorigenicity in rcctransient transfection of achn and caki1 cells withcdkn2bas1 sirnas for h showed significant reduction in cdkn2bas1 expression fig 2a cdkn2bas1knockdown in both cell lines significantly inhibited cellproliferation figs 2b s2a and clonogenic survivalfigs 2c s2b with a significant increase in apoptosis fig2d e decreased cell migrationinvasion figs 2f s2c dwith simultaneous changes in emt markers such as anincrease in epithelial markers αecatenin and claudinand decrease in mesenchymal markers vimentin andï¬bronectin were also observed figs 2g s3expression of mir141 and its clinical importance in renalcarcinomaand samples lowersince our results conï¬rmed cdkn2bas1 as a directtarget of mir141 we examined mir141 status andclinical importance in rcc expression of mir141 wassignificantly downregulated in rcc cell lines fig 3aand in tumor samples fig 3be compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors fig 3ce patientand tumor characteristics are summarized in supplementary table t3 roc analysis showed an auc of p ci fig 3f suggesting thatmir141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsepigenetic regulation of the mir141 locuswe identiï¬ed a genomic site rich in cpg island locatedupstream of the mir141 in chromosome 12p13 in thetcga cohort we observed hypermethylation of mir141promoter in tumor tissues as compared to normalfigs 3g s4a which is significantly associated with poorpatient survival fig 3h similarly rcc cell lines achnand caki1 also showed hypermethylation compared tonormal rptec cells fig 3i further we treated achnand caki1 cell lines with demethylating agent 5azacdrand observed decrease in methylation fig s4b withconcomitant increase in mir141 expression fig 3jindicating possible epigenetic regulation a significantdecrease in the expression of methylation regulatory 0cdasgupta cell death and disease page of fig lncrna cdkn2bas1 is oncogenic in renal cancer and is a direct target of mir141 a expression levels of cdkn2bas1 among kircnormal tumor kich normal tumor and kirp normal tumor patient samples in tcga cohort using wanderersoftware pvalue calculated by mannwhitney twotailed test b expression of cdkn2bas1 in tcgakirc cohort among different grades normal grade grade and stages normal stage iii and stage iiiiv c overall survival in tcgakirc cohort as performedby kaplanmeier analysis using ualcan software d relative expression levels of lncrna cdkn2bas1 in rcc cell lines achn and caki1 e expressioncdkn2bas1 in matched pairs of rcc tissue samples from sfvamc cohort pvalue calculated by mannwhitney twotailed test f receiver operatingcurve roc analysis on sfvamc cohort showing ability of lncrna cdkn2bas1 to differentiate between malignant and nonmalignant samples sfvamccohort g predicted binding sites of mir141 in cdkn2bas1 sequence h luciferase assays showing decreased reporter activity after cotransfection witheither wildtype wt offtarget ot cdkn2bas1 or luciferase control constructs ev with mirconmir141 in achn and caki1 cellsgenes such as dnmtl dnmt3a and dnmt3b werealso noted after 5azacdr treatment compared to control dmso in both achn and caki1 cell lines31mir141 overexpression phenocopies functional effectsobtained with cdkn2bas1 inhibition in vitro andsuppresses tumorigenicity and in vivowe sought to determine if cdkn2bas1 causes itsantitumorigenic effects through mir141 we checkedthe effect of mir141 overexpression in rcc cellstransient transfection of mir141 mimic in achn andcaki1 cells for h led to over expression of mir141compared to control mircon fig 4a also overexpression of mir141 significantly reduced cdkn2bas1 expression fig 4b indicating a reciprocal correlation between mir141 and cdkn2bas1 a significant decrease in cell proliferation over time fig 4cand marked decreasefig 4din clonogenicityofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of fig knockdown of lncrna cdkn2bas1 reduces tumorigenicity in renal cancer a relative expression of cdkn2bas1 in achn and caki1 cellstransfected with cdkn2bas1 sirnas b cell proliferation assessed by mts assay after knockdown of cdkn2bas1 in both cell lines with sirna2 cgraphical representation showing knockdown of cdkn2bas1 with sirna2 significantly decreased colony formation in achn and caki1 cellsd e achn and caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of cdkn2bas1f reduced migration and invasion in cdkn2bas1 sirna transfected cells compared to control treatment g changes in emt related proteins in bothachn and caki1after knockdown of cdkn2bas1compared to controls were also observed further westudied the therapeutic potential of mir141 in amouse xenograft model a significant decrease intumor growth was observed by intratumoral delivery ofmir141 mimic compared to control over the course ofexperiment average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received mir141 mimic fig 4e in additionmir141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both rcc celllines compared to controlfig 5a this proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyadpribose polymerase parp an increase in bax and adecrease in bcl2 at protein levels fig 5b a significantdecrease in migration fig 5c and invasion fig 5dwas also observed in both rcc cell lines with mir141overexpression we also examined emt markers aschange in migration and invasion are directly associated with emt our results showed an increase inepithelial markers αecatenin and claudin with concomitant decrease in mesenchymal markers ï¬bronectinand vimentin at both protein fig 5e and mrnaofï¬cial of the cell death differentiation associationfig s5 levels taken together overexpression of mir phenocopies the functional effects of cdkn2bas1 inhibition in vitro and tumor growth suppressioneffects in vivolike cdkn2bas1aremir141cdkn2bas1 interaction negatively regulatescyclind and its downstream effectors in rcccyclind1cyclind2alsooncogenic in rcc fig s6 and are direct targets of mir as discussed earlier lncrnas can act as cernas tocarry out their regulatory functions32 we observedthat cdkn2bas1 shared regulatory mir141 bindingsites with cyclind1cyclind2 fig 6a and therebysponges mir141 allowing cyclind1d2 to be expressedin tumors to determine potential mir141cdkn2bas1cyclind interaction experimentally we performedrip assay both achn and caki1 cells over expressingmir141 revealed significant enrichment of cyclind1cyclind2 and cdkn2bas1 with ago2 as compared toigg control fig 6b moreover decreased luciferaseactivity also conï¬rmed direct binding of mir141 tocyclind in mir141overexpressing achn andcaki1 cells compared to controls fig 6c we also found 0cdasgupta cell death and disease page of fig expression clinical signiï¬cance and epigenetic regulation of mir141 in renal cancer a mir141 expression levels in achn caki1 andrptec cells b expression levels of mir141 in kirctcga cohort normal and tumor c expression levels of mir141 in normal n nonmetastatic n metastatic n kirc patient samples in tcga cohort d expression levels of mir141 in kirctcga cohort amongdifferent grades normal grade grade and stages normal stage iii and stage iiiiv e relative mir expression in rcc tissue vs matched adjacent normal regions n among different grades normal grade grade andin different stages normal stage iii stage iiiiv as assessed by qrtpcr sfvamc cohort f receiver operating curve roc analysisshowing ability of mir141 to differentiate between malignant and nonmalignant samples g methylation for kirc patient samples in tcgakirccohort for probe cg02624246 h overall survival with tcgakirc methylation as performed by kaplanmeier analysis i methylation status of mir141promoter in rcc cell lines compared to nonmalignant rptec as assessed by msqpcr j expression of mir141 in 5azacdr treated and untreatedachn and caki1 cell lines results are representative of three independent experiments p value calculated by student t test bar mean ± standarderror mean semthat overexpression of mir141 or inhibition of cdkn2bas1 significantly decreased cyclind1d2 expression atboth the mrna fig 6d e and protein levels figs 6fhs8a b this effect was significantly attenuated by mir inhibitor fig s7 indicating that cyclind expressionis dependent on the interaction between mir141 andcdkn2bas1 we further observed a decrease in rac1 asmall gtpase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mrna levelsfigs s3 s5 and protein levels figs 6g ij s8cf inboth mir141 overexpressed and cdkn2bas1 inhibitedrcc cell lines which in turn are involved in regulatingcellular migrationinvasion cumulatively these resultsindicate that suppression of cdkn2bas1 by mir141inhibits renal cell proliferation invasion and migration byinhibiting cyclind rac1 and phosphorylation of paxillinofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of fig mir141 overexpression mimics the knockdown effect of lncrna cdkn2bas1 in vitro and reduces tumorigenicity in vivo a relativeexpression of mir141 in achn and caki1 cells transfected with mir141 mimic and control b significant decrease in cdkn2bas1 expressioncompared to control in both cell lines overexpressed with mir141 c rcc cell proliferation after transfecting with mir141 mimic and control asassessed by mts assay d colony formation and its graphical representation in mir141 overexpressing achn and caki1 cells compared to controlse pictures of excised tumors are taken at the termination of experiment day graph represents tumor volume after intratumoral injection ofcontrol or mir141 mimic into established tumors injection was started at day and was followed for days each mouse in both groups mirconand mir1413pmimic received a total of eleven injections intermittently data represent the mean of each group and error bars are sem results arerepresentative of three independent experiments p value calculated by student t test bar mean ± standard error mean semattenuation of mir141 exerts tumorigenic attributes innormal rptec cellswe next determined whether attenuation of mir141induces tumorigenic characteristics in normal rpteccells by targeting cdkn2bas1 and cyclind transienttransfection of mir141 inhibitor indeed showed a significant decrease in mir141 expression fig 7a and anincrease in cdkn2bas1 expression fig 7b along withother procancerous phenotypes such as increased cellproliferation fig 7c colony formation fig 7d migration and invasion fig 7e as compared to controlsadditionally a significant increase in cyclind1 cyclind2 rac1 and paxillin pxn expressions were observed inmir141 inhibited rptec cells fig 7f a noticeableincrease in prometastatic ï¬bronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαecatenin genes were also observed in mir141 inhibited rptec cells compared to controls fig 7gdiscussionprior studies have shown the regulatory role of noncoding rnas in tumorigenesis especially in the emtofï¬cial of the cell death differentiation associationpathway leading to cancer aggressiveness cdkn2bas1also known as anril is located at chromosome 9p21cdkn2bas1 is reported to be upregulated in tumortissues and function as an oncogenic lncrna in pancreatic ovarian and laryngeal squamous cell carcinoma36 human mir141 is located at chromosome12p1331 and is transcribed from a mir200 familyclusterinterestingly expression of mir141 is controversial since it exhibits either oncogenic39 or tumorsuppressive roles42 in speciï¬c types of cancer theprime goal of the present study was to understand therole of cdkn2bas1mir141 interactions in regulatingrcc progression and metastasisin this study we identify cdkn2bas1 to be a crucialoncogenic lncrna that plays an important role in renalcarcinogenesis cdkn2bas1significantly overexpressed in rcc and the expression increases fromlower to higher grades and stages lncrna cdkn2bas1directly interacted with mir141 as it was found to be anovel target of mir141 in contrast to cdkn2bas1 weobserved significant attenuation of mir141 expression inrcc cell lines and tumor samples compared to normalis 0cdasgupta cell death and disease page of fig ectopic mir141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a apoptosis assessed by ï¬owcytometric analysis of annexinvfitc 7aadstained achn and caki1 cells transfected with mirconmir141mimic graph represents totalapoptosis early late b immunoblots showing apoptotic proteins in mirconmir141mimic treated achn and caki1 cells with gapdh asendogenous control c migration assay and d invasion assay as seen in pictures and graphical representation of both achn and caki1 cells after mir overexpression compared to control e immunoblot assay showing emt related proteins in mirconmir141mimic treated achn andcaki1 cells with βactin as endogenous control graphs are average of three independent experiments p value calculated by student t testbar mean ± standard error mean semcell line or matched normal samples as it is known thatextensive dna hypermethylation of cpg islands is highlycorrelated to activation of cancerspeciï¬c genes45 wechecked the methylation status of mir141 in normal andrcc tissues interestingly insilico analysis showed thepresence of cpg island in the promoter region of mir and we also found hypermethylation of mir141 intcga samples as compared to normal this hypermethylation is also found to be significantly associatedwith poor survival of patients similar results were alsoobserved in rcc cell lines compared to a normal rpteccell line functionally inhibition of cdkn2bas1 andoroverexpression of mir141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in rcc in vitro in vivodata show suppression of tumor growth by mir141overexpression conversely attenuation of mir141 innormal rptec cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionfrom a clinical point of view noncoding rnas signatures are powerful tools for early cancer diagnosisofï¬cial of the cell death differentiation associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 our results revealed thathigher expression of cdkn2bas1 is positively correlatedwith poor overall survival probability of rcc patientsindicating its prognostic capability cdkn2bas1 canalso discriminate normal from tumor samples showing itsdiagnostic potential similarly mir141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be a diagnostic biomarker for rcc collectivelythese resultshighlight the biomarker potential of cdkn2bas1mir expression in rcc although it needs to be conï¬rmedin a larger independent sample cohortinterestingly we found tha
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"rapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Elizabeth Ann L Enninga2 Fabrice Lucien Matteoni3 Jacob J Orme Heather Dale4 Edwin Burgstaler4 Susan M Harrington3 Matthew K Ball4 Aaron S Mansfield1 Sean S Park5 Mathew S Block1 Svetomir N Markovic1 Yiyi Yan1 Haidong Dong3 Roxana S Dronca6 Jeffrey L Winters4To cite Orme a0JJ Enninga a0EAL Lucien Matteoni a0F et a0al Therapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Journal for ImmunoTherapy of Cancer 20208e001113 101136jitc2020001113 º Additional material is published online only To view please visit the journal online http dx jitc Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Division of Medical Oncology Mayo Clinic Rochester Minnesota USA2Department of Obstetrics and Gynecology Mayo Clinic Rochester Minnesota USA3Department of Urology Mayo Clinic Rochester Minnesota USA4Department of Laboratory Medicine and Pathology Mayo Clinic Rochester Minnesota USA5Department of Radiation Oncology Mayo Clinic Rochester Minnesota USA6Department of Hematology and Oncology Mayo Clinic Florida Jacksonville Florida USACorrespondence toDr Jacob J Orme orme jacob mayo eduBackground Trans acting programmed death ligand PD L1 derives from malignant cells in three known forms High levels of secreted splice variant PD L1 sPD L1 ADAM10ADAM17 shed sPD L1 and PD L1 positive extracellular vesicles evPD L1 each predict poor prognosis and limited response to PD L1 checkpoint inhibitors in cancer To our knowledge no clinical intervention has reduced any of these circulating forms of extracellular PD L1 Here we explore therapeutic plasma exchange TPE as a treatment to reduce circulating extracellular PD L1Results In patients with melanoma sPD L1 levels above ngmL predicted inferior overall survival In patients undergoing TPE for non malignant indications each TPE session removed a mean sPD L1 and evPD L1 detectable in plasma TPE also reduced total and ADAM10 positive extracellular vesiclesConclusion Here we report the first known clinical intervention to remove either sPD L1 or evPD L1 from plasma in vivo TPE reduces plasma sPD L1 and evPD L1 in vivo and may have a role in treatment with immunotherapy TPE may also prove useful in patients with other extracellular vesicle related conditionsINTRODUCTIONOvercoming initial or acquired resistance to programmed death ligand PD L1 immune checkpoint inhibitors is a major area of unmet need for many cancers1 Although the full scope of mechanisms of resistance to these therapies has yet to be determined different forms of tumor derived extracellular PD L1 have been linked to resistance in multiple clinical studies2in Malignant cells produce trans acting extracellular PD L1 three distinct forms First tumor cells transcribe and secrete soluble PD L1 sPD L1 splice variants4 Second enzymes ADAM10 and ADAM17 shed sPD L1 ectodomain directly from the tumor cell surface6 Both forms of sPD L1 carry known homodimerization domains and can be detected by ELISA sPD L1 can outcompete PD L1 inhibitors kill CD8 T cells and limit the ability of healthy peripheral blood mononuclear cells to kill tumor cells in vitro6 In a third form tumors generate extracellular vesicles EVs bearing surface PD L18 PD L1 positive EVs evPD L1 exhibit similar properties to sPD L1 in systemic circulation9 Each type of trans acting extracellular PD L1 correlates with poor survival in multiple clinical trials online supplementary table While broad spectrum pharmacological inhibitors and genetic manipulation have been shown to reduce release of these forms of PD L1 in culture or animal models none are suitable for clinical use To date we know of no reported clinical intervention that safely and reliably eliminates any of these forms of immunosuppressive systemic extracellular PD L1Therapeutic plasma exchange TPE is a procedure in which blood is passed through an apheresis machine separating plasma from cellular components Removed plasma is discarded and replaced with either colloid eg albumin or crystalloid and colloid solutions Unlike dialysis which removes small ions by diffusion TPE removes plasma restricted substances like antibodies that are too large for rapid diffusion On average each TPE session removes approximately of large non cellular plasma restricted intravascular components10It is unknown whether sPD L1 approximately kDa or PD L1 positive EVs nm are plasma restricted non diffusing and unbound If so we hypothesized that TPE could efficiently remove these substances from patient blood Such an intervention could if effective improve response to immunotherapyOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Figure Soluble programmed death ligand PD L1 suppresses antitumor immunity and predicts overall survival in patients with melanoma A A model of three known tumor derived extracellular PD L1 forms evPD L1 ADAM10ADAM17 cleaved soluble PD L1 sPD L1 ectodomain and secreted splice variant sPD L1that downregulate antitumor immunity and prevent response to PDL1 inhibition B A Kaplan Meier plot shows significantly worse overall survival for patients with melanoma exhibiting high ¥ ngmL versus low ngmL plasma sPD L1 levels p0005 C Patients with melanoma exhibited a higher mean plasma sPD L1 level ngmL in comparison to healthy controls ngmL p0001RESULTSsPDL1 levels predict overall survival in patients with melanomaEach form of extracellular PD L1 acts in trans as a systemic immunosuppressant through PD1 signaling figure 1A online supplementary table To confirm the clinical impact of plasma sPD L1 we measured sPD L1 levels in a retrospective cohort of patients with melanoma Exploratory analysis of overall survival OS determined a working cut off value of sPD L1 ¥ ngmL and baseline characteristics at the time of entry into study were similar online supplementary table Patients with high plasma sPD L1 levels experienced inferior median OS compared with patients with low plasma sPD L1 levels figure 1B vs months p0005 In comparison to healthy age matched controls patients with melanoma exhibited higher mean plasma sPD L1 figure 1C ngmL vs ngmL p0001 In a multivariate Cox proportional hazards analysis high sPD L1 prior to treatment predicted worse survival HR CI to p0025 when accounting for advanced age not significant sex not significant late stage p0002 and high serum LDH p001 online supplementary table TPE significantly reduces plasma sPDL1 levelsWe hypothesized that TPE may remove extracellular PD L1 in its various forms figure 2A To address this question we prospectively enrolled patients undergoing planned TPE figure 2B Twenty eight patients met inclusion criteria of which provided informed consent Baseline patient characteristics are in table One patient was excluded for biotin containing supplement use as biotin interferes with the established sPD L1 detection assay The remaining patients underwent plasma exchange and sample collection before and after the procedure as described Discarded plasma samples from the TPE device waste bag for each session were also collected sPD L1 was measured in each sample and most patients undergoing TPE exhibited sPD L1 levels above the clinically relevant ngmL cut off from the retrospective melanoma studyMost patients undergoing TPE did not have an active cancer diagnosis Baseline sPD L1 levels in all patients were compared with matched normal controls and patients with melanoma online supplementary fig and some patients exhibited sPD L1 above the clinically significant cut off level determined in the retrospective melanoma cohort Patients with high baseline sPD L1 levels were significantly more anemic than patients with lower baseline sPD L1 even when controlling for the higher number of female subjects in the high sPD L1 group female only mean Hgb vs p004 male only mean Hgb vs p003 Groups were otherwise similar TPE significantly reduced plasma sPD L1 levels in patients receiving albumin only ie no Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Therapeutic plasma exchange TPE significantly reduces plasma soluble programmed death ligand sPD L1 levels A A model of the TPE procedure in which patient plasma is separated and replaced to extract non cellular substances confined to the plasma B A diagram of the present study in which patients undergo plasma exchange C All plasma levels of sPD L1 immediately prior to pre and after post TPE using albumin replacement fluid are plotted TPE significantly reduced sPD L1 levels in patient plasma by Wilcoxon signed rank test p00001 D In a typical timeline patient sPD L1 levels are reduced by each successive session of TPE gray bars See also table a0 online supplementary figures FFP replacement fluid figure 2C p00001 Removed sPD L1 was detected in matching plasma samples from the TPE procedure waste bag Each TPE session removed a mean of detectable plasma sPD L1 mean regeneration of sPD L1 between sessions was table TPE sessions were usually separated by daysincluding sessions A representative individual patient treatment course showing sPD L1 reduction over four successive TPE sessions is also shown figure 2D All individual patient TPE courses involving donated human blood products eg fresh frozen plasma or FFP are shown in online supplementary fig Pre TPE and post TPE sPD L1 levels for all sessions are also shown online supplementary fig TPE significantly reduced plasma sPD L1 even when sessions requiring donated FFP were included p00001FFP is sometimes given during TPE for patients with increased risk of bleeding We observed that some patients receiving FFP with low baseline sPD L1 experienced rapid increases in sPD L1 levels after TPE presumably passively acquired from donor plasma as this was not observed in patients receiving albumin replacement alone sPD L1 was not detected in the discarded plasma from the procedure for these patients We observed a mild association between post FFP infusion rises in sPD L1 levels and the blood type of the recipient mainly in patients with O type blood Individuals with group O blood are universal recipients of FFP products and universal donors of cellular products due to a lack of ABO group antigens and the presence of preformed anti A and anti B antibodies respectively Recipients of FFP usually receive a mixture of compatible plasma from multiple donors To determine whether blood type in FFP donors is associated with FFP sPD L1 content we measured sPD L1 by ELISA in plasma from multiple FFP donors online supplementary fig O negative plasma donors showed higher sPD L1 levels than donors with most other blood typesTPE efficiently reduces plasma EV levels in vivoWe postulated that TPE may remove PD L1 positive EVs evPD L1 from patient blood To address this question we measured total EV levels and evPD L1 in each sample by flow cytometry We also determined the impact of TPE on platelet derived CD61 positive EVs one of the most abundant EV Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Table Patient baseline characteristicsCharacteristicHigh sPDL1 n17Low sPDL1 n7StatisticStarting sPD L1Age yearsGender FActive cancer YesImmunotherapyNoneAtezolizumabPembrolizumabPlasma exchange indicationCNS demyelination myelitis MS NMO myelopathyImmune encephalitisMyasthenia gravisParaneoplastic syndrome encephalitis neuropathy pemphigusParaproteinemia Waldenström cryoglobulinemia kappa gammopathySusac syndromeTransplant rejection heart kidneyPre TPE white cell countPre TPE hemoglobinPre TPE creatinine to to to to to to to to to to F1223619 p0001F122035 p0558X2070 p0404 X20 p0967 X2288 p0237 X2288 p0315 F122078 p0385F122860 p0008F122382 p0063Patients undergoing therapeutic plasma exchange TPE are compared by starting soluble programmed death ligand sPD L1 level above or below survival cut off established in patients with melanoma ngmL For categorical variables n is given For continuous variables mean quartiles is givenKruskal Wallis PearsonCNS central nervous system MS multiple sclerosis NMO neuromyelitis opticasubpopulations in blood CD61 is a platelet marker and ADAM10 positive low density EVs ADAM10 has been implicated in exosome loading and pathogenesis11TPE significantly reduced total plasma particle concentration figure 3A average per exchange p00001 TPE sessions requiring FFP or other human blood product were excluded from analysis leaving session pairs PD L1 positive evPD L1 and ADAM10 positive EVs were Table Soluble programmed death ligand sPD L1 reduction and regeneration per exchange Reduction per exchangen44Mean SDMedian min max Regeneration between exchangesMean SDMedian min maxRegeneration per cycle pgmLMean SDMedian min max n44 38k 154kFor each exchange not requiring FFP percent sPD L1 reduction and regeneration between each exchange is calculated n44FFP fresh frozen plasmasignificantly reduced by TPE figure 3BC p0028 and p00001 respectively and were detected in waste plasma data not shown Each TPE session using albumin based replacement fluid with pre TPE levels above one million removed a mean of detectable PD L1 positive EVs from patients online supplementary table Platelet derived CD61 positive EVs while abundant were not significantly reduced by plasma exchange figure 3DIndividual patient courses showing total plasma PD L1 positive ADAM10 positive and CD61 positive EV levels before and after each TPE session are shown in online supplementary fig with exemplary nanoflow plots in online supplementary fig Three successive TPE sessions consistently depleted total PD L1 positive and ADAM10 positive but not CD61 positive EVs These trends were less pronounced when sessions in which patients received donor FFP were included online supplementary fig In normal control FFP donors blood type did not correlate with plasma EV concentrations online supplementary fig DISCUSSIONExtracellular PD L1in the form of splice variant sPD L1 ADAM10ADAM17 cleaved sPD L1 ectodomain or evPD L1 positive EVs evPD L1mediates resistance to PD L1 inhibitors4 These forms are resistant to clinically tested Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Plasma exchange efficiently reduces total programmed death ligand PD L1 positive and ADAM10 positive extracellular vesicle EV levels in vivo Plasma levels of total EVs immediately prior to pre and after post therapeutic plasma exchange TPE are plotted TPE significantly reduced A total p00001 B PD L1 positive p0028 and C ADAM10 positive p00001 but not D CD61 positive EVs p094 by Wilcoxon signed rank test See also online supplementary figures and online supplementary table combinations of chemotherapy and immunotherapies in vitro and in animal models and are associated with poor prognosis in many cancer types In the present study we found that TPE reliably reduces sPD L1 and evPD L1 This reduction was most pronounced over approximately three consecutive single plasma volume treatment sessions Given the dramatic reduction in sPD L1 and evPD L1 TPE may provide a novel approach to combating these mechanisms of resistanceWhile promising the present study was limited to patients receiving TPE mainly for non oncological indications over a short time horizon One patient in the study had melanoma receiving pembrolizumab and exhibited high pre TPE evPD L1 that was reduced on treatment Another patient had a uterine neuroendocrine tumor receiving atezolizumab and exhibited high pre TPE sPD L1 that was reduced on treatment The purpose of TPE in all cases however was to blunt paraneoplastic autoimmunity or treat some other coexisting autoimmune disordernot the underlying malignancy Neither of these patients experienced improvement in their autoimmunity after TPE The source of sPD L1 and evPD L1 in these cases is uncertain as no assay currently differentiates tumor derived and non tumor derived PD L1 We observed that these forms of immunosuppressive extracellular PD L1 exist naturally although at lower levels in healthy subjects than in patients with cancer suggesting a potentially beneficial immunoregulatory role While we observed some regeneration for both sPD L1 and evPD L1 between TPE sessions it is unknown to what degree malignant cells may regenerate and maintain extracellular PD L1 homeostasis Relatedly it is uncertain how other plasma substances removed by TPE may affect response to immunotherapy or more broadly cancer immunity overall Nor is it known at what level sPD L1 andor PD L1 positive EV removal would become clinically relevant These facets will be tested in future studiesImmunotherapy resistance is widespread and costly In most instances PD L1 inhibitors such as pembrolizumab nivolumab atezolizumab durvalumab and avelumab are used in situations in which less than half of tumors are expected to respond Of patients that benefit many do not experience a sustained durable response These treatments represent a major investment the cost of PD L1 checkpoint blockade commonly reaches several hundred thousand dollars over the course of therapyTo our knowledge this is the first report of an intervention to achieve consistent rapid reduction in either sPD L1 or PD L1 positive EVs in a clinical setting TPE is safe and commonly prescribed Thus preimmunotherapy TPE may combat immunotherapy resistance In light of the heavy investment that anti PD L1 therapy entails the added cost of TPE in selected patients may be practical14 While the durability of extracellular PD L1 reduction in malignancy will be explored in future studies the present study suggests that this approach warrants further investigationOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Beyond evPD L1 this is also to our knowledge the first known intervention to reliably deplete EVs in a clinical setting EVs have been implicated in oncogenesis and metastasis through miRNA carriage and direct protein signaling independent of PD L115 Beyond cancer EVs have also been implicated in autoimmunity17 agingneurodegeneration18 infection19 obesity20 and heart disease21 The selective removal of ADAM10 positive ie likely immune derived versus CD61 positive ie likely platelet derived EVs in this study suggests flexible selective EV depletion may be both possible and expedient in other indications The present study is only a proof of concept and additional exploratory studies in these areas are necessaryIn summary TPE reduces extracellular forms of PD L1 associated with PD L1 checkpoint inhibitor resistance Future studies will explore the potential role of TPE in improving cancer immunotherapyMETHODSRetrospective melanoma outcomes study designIn a retrospective analysis baseline blood samples from patients with melanoma prior to treatment in one of three clinical trials by the North Central Cancer Treatment Group N057e22 N077523 and N087924 between and were tested for sPD L1 of patients were diagnosed with cutaneous melanoma and none received immunotherapy treatments Blood from healthy volunteers undergoing blood donation at Mayo Clinic was also testedProspective TPE study designIn an investigator initiated label single center observational study adults undergoing TPE were approached from December through March In consenting subjects samples of whole blood immediately prior to TPE and on completion of the procedure were collected in ACD vacutainers BD In each case the first mL of blood was discarded to avoid contamination after which an mL sample was obtained in sequence Plasma was isolated by centrifugation A postprocedure blood sample was obtained after completion of the procedure In addition matching samples from discarded plasma from the procedure waste bag were collected Samples were obtained from up to four consecutive procedures for each patient If a patient underwent fewer than four TPE procedures samples were obtained from as many procedures as possiblePatients included were adults able to give consent and undergoing TPE for a variety of hematological neurological and renal diseases as indicated by published guidelines from the American Society for Apheresis ASFA or according to the medical judgment of the referring physicians25 Patients taking biotin supplements were excluded from the study due to biotin interference with the sPD L1 ELISA assay Procedures were performed using centrifugation based cell separators either the Fenwal Amicus Fresenius KABI USA LLC Lake Zurich Illinois USA or the Spectra Optia Terumo BCT Lakewood Colorado USA For each patient a single plasma volume was exchanged using either peripheral intravenous preferred or central lines for vascular access For this study due to the possibility of sPD L1 or PD L1 positive EVs present in donor plasma only TPE sessions using no donor plasma ie fresh frozen plasma FFP in the replacement fluid were included in calculations Anticoagulation consisted of either mL of acid citrate dextrose solution A ACD A or mL of ACD A with units of unfractionated heparin Anticoagulant to blood ratios were when ACD A was used and when ACD Aheparin was used Patients did not receive routine electrolyte replacement but mL of calcium gluconate was administered by slow intravenous push for signs and symptoms of hypocalcemia related to the ACD A anticoagulant in one patientELISAELISA was performed as previously published26 Both secreted splice variant and shed sPD L1 are reliably detected by this ELISA In brief paired mouse IgG2 monoclonal antibody clones H1A and B11 against extracellular human PD L1 were utilized in a capture detection plate assay using biotinylation and HRP streptavidin detection This assay is specific for sPD L1 and does not exhibit cross reactivity to other B7 H homologues nor to evPD L1 Concentrations were determined by optical density measurements along a known standard curve of recombinant human PD L1 ELISAs were performed by team members who were blinded to the identity of the samplesFlow cytometryFlow cytometry for EVs was performed as previously published27 In brief plasma samples were centrifuged twice at 2000g to deplete platelets Resultant platelet free plasma were analyzed using an A60 Micro Plus Nanoscale Flow Cytometer Apogee FlowSystems gating for mid intensity light angle light scatter and markers of interest Anti PD L1 Genentech atezolizumab ADAM10 RD Systems clone and CD61 BioLegend clone VI PL2 antibodies were conjugated to fluorophores Life Technologies Alexa647 PE phycoerythrin and Alexa488 and titrated prior to use Nanoscale flow cytometer calibration was performed using a standard reference bead mix as previously published Flow cytometry was performed by team members blinded to the identity of the samplesStatistical analysisAll statistical analyses were performed using R Statistical Software R Foundation Retrospective progression free survival was analyzed using Kaplan Meier and Cox proportional hazards modeling Optimal cut off values for sPD L1 levels were determined using the greyzoneSurv package for R Wilcoxon signed rank test was used to compare paired pre TPE and post TPE patient sample sPD L1 and EV levels as indicated Baseline clinical characteristics for the study were compared by Kruskal Wallis test for continuous variables and Pearsons Ï2 test for discrete variables as indicated Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0cOtherwise groups were compared by unpaired two sided Students t test Figures comprising box plots show quartile values and individual data points Mean values and CI are indicated in corresponding online supplementary figures and tables P was considered statistically significant In figures p values are denoted with with and with Twitter Jacob J Orme JakeOrmeMDPhDAcknowledgements Statistical guidance was provided generously by Nathan Foster of the Mayo Clinic Center for Clinical and Translational Science Some illustrations were created using Servier Medical Art templates which are licensed under a Creative Commons Attribution Unported License https smart servier com Additional illustrations were provided by Mayo Clinic Media Services The authors thank Daniel Summerfield MD MS for use of his likeness in Fig 2AContributors JO originated hypotheses designed the study oversaw experiments performed analyses and wrote the article EALE performed retrospective melanoma cohort analysis FL M performed nanoflow cytometry HD and EB oversaw and performed TPE study enrollment sample collectionprocessing and blinding SMH performed ELISAs MB AM SP MB SNM YY HD RD and JLW helped develop hypotheses provided clinical samples and reagents and contributed support and oversightFunding R21 5R21CA19787802 Role of Bim and soluble B7 H1 in monitoring T cell responses to anti PD1 therapy in melanoma HD and RD L30 CA23154101 Soluble B7H1 as a PD1 Checkpoint Remote Control in Cancer JJO U10 CA180790 EE K12 CA090628 YY Richard M Schulze Family Foundation HD and RDCompeting interests Intellectual property has been filed addressing discoveries disclosed in this manuscript The authors report no other relevant conflicts of interestPatient consent for publication ObtainedEthics approval All research protocols involving human subjects were approved by Mayo Clinics Institutional Review Board and all human subjects gave written informed consentProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available in a public access repository All data will be available for download at the Science Framework at https osf io qtskd access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See http creativecommons licenses by nc ORCID iDJacob J a0Orme http orcid REFERENCES O'Donnell JS Long GV Scolyer RA et a0al Resistance to PD1PDL1 checkpoint inhibition Cancer Treat Rev Ando K Hamada K Watanabe M et a0al Plasma levels of soluble PD L1 correlate with tumor regression in patients with lung and gastric cancer treated with immune checkpoint inhibitors Anticancer Res Fan Y Che X Qu J et a0al Exosomal PD L1 retains immunosuppressive activity and is associated with gastric cancer prognosis Ann Surg Oncol Zhou J Mahoney KM Giobbie Hurder A et a0al Soluble PD L1 as a biomarker in malignant melanoma treated with checkpoint blockade Cancer Immunol Res access Mahoney KMet a0al A secreted PD L1 splice variant that covalently dimerizes and mediates immunosuppression Cancer Immunol Immunother Orme JJ Jazieh KA Xie T et a0al ADAM10 and ADAM17 cleave PD L1 to mediate PD L1 inhibitor resistance OncoImmunology Romero Y Wise R Zolkiewska A Proteolytic processing of PD L1 by ADAM proteases in breast cancer cells Cancer Immunol Immunother Chen G Huang AC Zhang W et a0al Exosomal PD L1 contributes to immunosuppression and is associated with anti PD1 response Nature Poggio M Hu T Pai C C et a0al Suppression of exosomal PD L1 induces systemic anti tumor immunity and memory Cell Derksen RH Schuurman HJ Meyling FH et a0al The efficacy of plasma exchange in the removal of plasma components J Lab Clin Med Berckmans RJ Nieuwland R Böing AN et a0al Cell Derived microparticles circulate in healthy humans and support low grade thrombin generation Thromb Haemost Crescitelli R Lässer C Jang SC et a0al Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation J Extracell Vesicles Kowal J Arras G Colombo M et a0al Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes Proc Natl Acad Sci U S A 2016113E968 Winters JL Brown D Hazard E et a0al Cost minimization analysis of the direct costs of tpe and IVIg in the treatment of Guillain Barré syndrome BMC Health Serv Res Lee JC Zhao J T Gundara J et a0al Papillary thyroid cancer derived exosomes contain miRNA 146b and miRNA222 J Surg Res Yang J Wei F Schafer C et a0al Detection of tumor cell specific mRNA and protein in exosome like microvesicles from blood and saliva PLoS One 20149e110641 Nakao R Hasegawa H Ochiai K et a0al Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response PLoS One 20116e26163 Thompson AGet a0al Extracellular vesicles in neurodegenerative disease pathogenesis to biomarkers Nature Reviews Neurology Nature Publishing Group Marcilla A Martin Jaular L Trelis M et a0al Extracellular vesicles in parasitic diseases J Extracell Vesicles Huang Doran I Zhang CY Vidal Puig A Extracellular Vesicles Novel Mediators of Cell Communication In Metabolic Disease Trends in Endocrinology and Metabolism Elsevier Inc Boulanger CM Loyer X Rautou PE et a0al Extracellular vesicles in coronary artery disease Nature Reviews Cardiology Nature Publishing Group Kottschade LA Suman VJ Amatruda T et a0al A phase II trial of nab paclitaxel ABI007 and carboplatin in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group Study N057E1 Cancer Kottschade LA Suman VJ Perez DG et a0al A randomized phase study of temozolomide and bevacizumab or nab paclitaxel carboplatin and bevacizumab in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group study N0775 Cancer McWilliams RR Allred JB Slostad JA et a0al NCCTG N0879 Alliance A randomized phase cooperative group trial of carboplatin paclitaxel and bevacizumab ± everolimus for metastatic melanoma Cancer Padmanabhan A Connelly Smith L Aqui N et a0al Guidelines on the Use of Therapeutic Apheresis in Clinical Practice Evidence Based Approach from the Writing Committee of the American Society for Apheresis The Eighth Special Issue J Clin Apher Frigola X Inman BA Lohse CM et a0al Identification of a soluble form of B7 H1 that retains immunosuppressive activity and is associated with aggressive renal cell carcinoma Clin Cancer Res Gomes J Lucien F Cooper TT et a0al Analytical considerations in nanoscale flow cytometry of extracellular vesicles to achieve data linearity Thromb Haemost Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c"
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Neurologic Manifestations of Systemic Disease D Lapides Section EditorNeurologic Manifestationsof Systemic Disease SleepDisordersEric M Davis MD1Chintan Ramani MBBS1Mark Quigg MD MSc2Address1Division of Pulmonary and Critical Care Department of Medicine University ofVirginia Charlottesville VA USAEmail emd9bvirginiaedu2Department of Neurology University of Virginia Charlottesville VA USA Springer ScienceBusiness Media LLC part of Springer Nature This is part of the Topical Collection on Neurologic Manifestations of Systemic DiseaseKeywords Sleep disorders I Sleep manifestations of systemic diseases I Sleep impacts on health I Sleep apnea IInsomniaAbstractPurpose of review Sleep is intimately involved in overall health and wellbeing We provide acomprehensive report on the interplay between systemic diseases and sleep to optimizethe outcomes of systemic disordersRecent findings Spanning the categories of endocrinologic disorders metabolictoxicdisturbances renal cardiovascular pulmonary gastrointestinal infectious diseases autoimmune disorders malignancy and critical illness the review highlights the prevalentcoexisting pathology of sleep across the spectrum of systemic disorders Although it is rarethat treating a sleep symptom can cure disease attention to sleep may improve quality oflife and may mitigate or improve the underlying disorder Recent controversies inassessing the cardiovascular relationship with sleep have called into question some ofthe benefits of treating comorbid sleep disorders thereby highlighting the need for anongoing rigorous investigation into how sleep interplays with systemic diseasesSummary Systemic diseases often have sleep manifestations and this report will help theclinician identify key risk factors linking sleep disorders to systemic diseases so as tooptimize the overall care of the patient 0c Page of IntroductionCurr Treat Options Neurol All Earths species maintain a solar 24h cycle of rest andactivity and disrupting the cycle affects adaptation andhomeostasis Sleeps quotidian normalness meansthat analogous to fish not knowing about water until itis dry sleep is not commonly thought about until it isdisruptedFor example about of the adult populationcomplain of transient insomnia and about experience chronic insomnia that disrupts daytime function[] Patients with chronic insomnia experience less workproductivity more absenteeism more accidents andmore hospitalizations leading to direct treatment costsof approximately 60B annually [] Considering thepotential widespread reach of comorbid sleep disordersevaluating sleep in the neurological patient is importantThis review will introduce the accepted anizationof sleep disorders review important features in historytaking and evaluation and survey the systemic diseasesthat have important comorbidities with particular sleepdisordersGeneral considerationsClassification of sleep disordersAn abridged listing of sleep disorders from the American Academy of SleepMedicine Table provides an overview of the current classification []Insomnia is a chronic dissatisfaction with sleep duration and quality that isassociated with daytime dysfunction Although pharmacologic treatment isoften pursued for chronic insomnia management outcomes are often betteraddressing underlying factors with the early use of cognitivebehavioral therapyfor insomnia CBTi []Sleeprelated breathing disorders involve dysfunction of the respiratory systemduring sleep usually resulting in daytime hypersomnia Obstructive sleepapnea OSA central sleep apnea CSA and respiratory effort related arousalsare classified under this category Treatment options including continuouspositive airway pressure CPAP positional therapy mandibular advancementdevices healthy weight loss and even a novel cranial nerve stimulator whichprotrudes the tongue forward during sleep [4cid129cid129]Central hypersomnias are defined as a primary dysregulation of sleep resultingfrom dysfunction of the central nervous system that causes daytimehypersomnia Often treatment addresses the underlying cause and may includeuse of strategic napping and wakepromoting medicationsCircadian disorders consist of various lesions or external disruptions of thecircadian timing system that desynchronize the brains clock from the externalsolar lightdark cycle resulting in hypersomnia or insomnia in a clockdependent fashion Treatment of circadian rhythm disorders involves adjustinglife around the patients desired sleep time or augmenting factors that entrainthe bodys clockParasomnias represent disorders of faulty inhibition of waking behaviors thatarise inappropriately during sleep and are divided into those that occur duringnonREM sleep REM sleep or state transitions REM sleep behavior disorder is aparasomnia characterized by loss of muscle atonia during REM sleep thatusually occurs in patients with neurodegenerative disorders It is often treatedeffectively addressing other sleep disturbances and treating with clonazepam ormelatonin [] 0cCurr Treat Options Neurol Page of Table Abridged classification of the AASM sleep disordersInsomniaChronic insomnia disorderShortterm insomnia disorderExcessive time in bedShort sleeperSleeprelated breathing disordersObstructive sleep apneaCentral sleep apneaSleeprelated hypoventilation disordersSleeprelated hypoxemia disordersCentral disorders of hypersomnolenceNarcolepsy types and Idiopathic hypersomniaKleineLevin syndromeHypersomnia due to medical disorder medication substance psychiatric disorderInsufficient sleep syndromeCircadian rhythm sleepwake disordersDelayedAdvancedIrregularNon hShift workJet lagParasomniasNREM relatedArousal disordersConfusional arousalsSleepwalkingSleep terrorsSleeprelated eating disorderREM relatedREM sleep behavior disorderRecurrent isolated sleep paralysisNightmare disorderOtherExploding head syndromeSleeprelated hallucinationsEnuresisSleep talkingSleeprelated movement disorders 0cCurr Treat Options Neurol Page of Table ContinuedRestless legs syndromePeriodic limb movement disorderLeg crampsBruxismRhythmic movement disorderBenign sleep myoclonus of infancyPropriospinal myoclonus at sleep onsetNormal variantsSleep historySleeprelated movement disorders consist of fragmentary often repetitive bodymovements that can disrupt sleep or sometimes worse disturb the sleep of bedpartners Periodic limb movement disorder PLMD and restless legs syndromeRLS both fall under this category and are treated with repletion of iron storesand consideration of dopaminergic agonists []A sleep history helps a patient disclose sleep findings and helps the physiciananize it into categories of hypersomnia sleep habits and scheduling sleepcharacteristics environmental issues and sleep interrupters Table The Epworth Sleepiness Scale quantifies the degree of hypersomnia []Most adults require h of daily sleep [] and prefer it anized into eithera monophasic nocturnal schedule or in a biphasic pattern augmented with anafternoon siesta The sleep pattern characterizes the presence and severity ofsleeponset insomnia sleep maintenance insomnia or terminal insomnia insomnia distributed within the last half of the sleep period Catchup sleep aphenomenon of prolonged sleep on a free day is a classic sign of sleepdeprivation Habitual earlyphase advances morning larks latephase delays night owls or a chaotic irregular schedule can be a sign of circadiandisorders One also must inquire about common sleep disruptors including legmovements snoring witnessed apneas and environmental factorsDiagnostic testing modalitiesSleep diaryPolysomnographyThe sleep diary often available through standardized forms or evenwebsites or smartphone apps consists of weeks of selfreported sleeptimesThe overnight polysomnography PSG is the goldstandard measurementof sleep architecture respiratory disorders such as OSA and parasomniasIn the case of OSA the unattended home sleep study has had an 0cCurr Treat Options Neurol Page of Table A categorical sleep historyHypersomniaEpworth Sleepiness Scale Considering the last weeks how likely would you fall asleep while doing each task not at all points slight moderate severe Normal ¤ pointsSitting and readingWatching TVSitting inactive in public lecture church ¦Car passenger for an hourLying down to rest in the afternoonSitting conversationSitting quietly alone after lunchDriving stopped in trafficSchedulesleep timeWorkday bedtime and out of bedtimeWeekday bedtime and out of bedtimeWhat is your estimated sleep latency If min what are you doing in bed before you fall asleepHow often do you awaken at night and whyDo you need an alarm clock to awaken in the morningHow many days of the week do you nap and for how longEnvironmentDo you have a bedroomDo you have a bedpartner TV Mobile phone or other electronicsWhat are you doing right before bedtimeHow much caffeine coffeeteasoda popenergy drinks and alcohol do you consume and when is the latest intakeInterruptersDo you have leg pain or restlessnessDo you have chronic pain that prevents or interrupts sleepDo you have daytime hallucinations or dreams severe or lucid nightmares sleep paralysis or cataplexyDo you snore or have witnessed apneasMultiple sleep latency testincreasing role as a diagnostic testing alternative to the traditional inlabPSG Concerns of other sleep disorders or those that may be presentcomorbidly with probable OSA require inlab PSG that can measure sleeparchitecture and sleepassociated movementsThe multiple sleep latency test MSLT consists of a series of daytime napsfrom which sleep onset is calculated The test in combination with PSGperformed the night before is the gold standard in measuringhypersomnia especially in the evaluation of narcolepsy 0c Page of ActigraphyPersonal devicesCurr Treat Options Neurol Wrist actigraphy provides measurements of longterm patterns of rest andactivity as proxies for sleep and wakefulness Such patterns can help tocorroborate histories of sleep duration and timingPopular smartphones and other ambulatory devices with physiologicalmonitoring capabilities may transform the evaluation of sleep However arecent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries orstandard PSG [] The overall conclusion is that at the beginning of wearable devices are not ready for reliable quantification of sleep acrossindividuals Although serial recordings confined to a single individual mayhold some value these measurements have yet to be validatedSleep comorbidities with systemic diseasesEndocrine disordersThyroid diseaseConsidering the various sleep disorders and diagnostic tools afforded by a goodsleep history and sleep testing understanding the relationship between sleepdisorders and systemic diseases has farreaching implications in optimizing thecare of the patient The following sections will address sleep manifestations ofvarious neurological disorders arising from systemic disease based on an systemAlmost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep choking hypersomnia or fatigue [] OSA is presentin approximately [] A unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airways soft tissuesand dilator muscles even though myxedema can be absent [] Larger goiters canalso cause OSA by external compression of the airway []On the other side of the thyroid spectrum hyperthyroidism is most closelyassociated with insomnia occurring in of patients [] Arousaldisordersspecifically sleep walkingalso occur especially in the setting ofthyrotoxicosis [] proposed to arise from frequent arousals and impairmentof attaining slowwave sleep as the direct result of thyroid hormoneBeyond the treatment of the specific sleep disorder sleep problems usuallyremit following appropriate treatment of the underlying thyroid disorder []Type diabetes mellitusSleep disorders affect high proportions of those with type diabetes mellitusDM surveys of patients with DM compared with those of controls show a 0cCurr Treat Options Neurol Page of nearly 2fold propensity for insomnia fourfold higher use of sedativehypnoticsand a 10fold higher rate of hypersomnolence [] OSA is highly prevalent inDM and many are undiagnosed [] Contributors to a multifactorial series ofsleep disruptors include periodic limb movements and restless legs syndromeRLS diabetic neuropathy and fluctuations in blood glucose []DM presents an excellent model by which to demonstrate the reciprocaleffects of sleep disruption on the primary disease First sleep disturbances affectthe regulation of the neuroendocrine control of appetite Sleep deprivationpromotes overeating through hyperactivity of orexin system [] and activatesthe hypothalamicpituitaryadrenal system to increase cortisol secretionresulting in impaired glucose tolerance [ ] These multiple mechanismssupport clinical observations that untreated OSA may be reason for the ineffective treatment of DM and that accordingly treatment with CPAP leads toimprovements in glycemic control in some patients []Sex hormones and gender affect the distribution and susceptibility to a varietyof sleep disorders Men on the basis of relative airway collapsibility haveapproximately a twofold increased risk of OSA compared with women in males and in females [] A potential side effect in thetreatment of hypoandrogenism is the facilitation of OSA given the impacttestosterone has on upper airway collapsibility []Testosterone levels may affect the propensity for chronic insomnia Menwith hypoandrogenism demonstrate reduced sleep efficiency increased nighttime awakenings and reduced deep sleep compared with the normaltestosteronelevel controls although it is not clear whether these features improve with testosterone therapy [] Women experience higher rates of chronicinsomnia risk ratio of for women versus men which becomes even morepronounced in the elderly [] Despite sleeping longer overall sleep quality isoften lower in women than men []The distribution of sleep disorders in women varies with reproductivelifespan Younger women are more susceptible to restless legs syndromeRLS mainly on the basis of mensesassociated irondeficiency During pregnancy women are at significantly increased risk for the development of RLSwith an overall prevalence exceeding of all pregnant patients [] Treatment of RLS in pregnancy involves iron supplementation with a goal ferritinlevel mcgl Often oral iron repletion is adequate although there arereports of intravenous iron therapy in severe cases of pregnancyrelated RLSand irondeficiency [] Pregnancy is also associated with an increased prevalence of OSA up to of pregnant patients during the third trimester whichis associated with increased risks of complications including gestational hypertension gestational DM and preeclampsia []Although not a particular systemic neurological disease pharmacological effectson sleep form an important aspect of neurological sleep medicine since manymedications that are used by neurologists may affect sleep Table showscommon medications that provoke insomnia hypersomnolence respiratorysuppression parasomnias and RLSperiodic limb movement disorderSex hormonesMedications 0c Page of Curr Treat Options Neurol Table Medication classes and specific examples that can cause sleep disturbancesInsomniaCentral nervous system stimulants methylphenidate amphetamines modafinilCaffeineAntidepressantsSelective serotonergic reuptake inhibitors fluoxetine sertralineSelective norepinephrine reuptake inhibitors venlafaxine duloxetineSecondary tricyclic antidepressants desipramine nortriptylineCardiovascularBeta2 agonists albuterolVasopressors epinephrine dopamineCorticosteroidsSympathetic amines phentermineHypersomniaBenzodiazepines alprazolam diazepamNonbenzodiazepine receptor agonists zolpidem eszopicloneOpioidsH1 antihistamines diphenhydramineAntiepileptic agents phenytoin levetiracetamAntidepressantsSelective serotonergic reuptake inhibitors paroxetine sertralineTertiary tricyclic antidepressants amitriptylineTypical and atypical antipsychotics haloperidol olanzapineDopaminergic agonists ropinirole carbidopalevodopaAnticholinergic medicationsCentrally acting α agonists clonidine dexmedetomidineRespiratory suppressionOpioids oxycodone morphineBenzodiazepines diazepam clonazepamAlcoholPhenobarbitalParasomniasAntidepressants clomipramine fluoxetine citalopramNonbenzodiazepine receptor agonists zolpidemCaffeineAlcohol withdrawalRestless legs syndrome and periodic limb movementsSelective serotonergic reuptake inhibitors fluoxetine mirtazapineAntipsychotics haloperidol risperidoneTricyclic antidepressants amitriptyline clomipramine 0cCurr Treat Options Neurol Page of Renal diseaseInfectious diseasesSleep disturbances are highly prevalent in patients with chronic kidney diseaseCKD spanning the broad spectrum of sleep disorders including hypersomniainsomnia sleeprelated breathing and RLSThe prevalence of OSA in CKD ranges from to rates that are notexplained solely by overlapping comorbidities common to both OSA and CKD[] The cooccurrence of both CKD and OSA is associated with increasedcardiovascular events and allcause mortality [] Usually OSA develops inpatients with CKD independent of underlying renal dysfunction but someevidence shows that CKD can cause or exacerbate OSA and central sleep apneaProposed mechanisms for this causal relationship include uremic neuropathyaltered chemosensitivity and hypervolemia [] Accordingly renal replacement therapy and fluid removal [] may improve obstructive or central sleepapnea Conversely treatment of sleep apnea with PAP may improve renalfunction in those with borderline renal impairment []RLS is a common and debilitating symptom in patients with CKD occurringin up to of patients on hemodialysis compared with that in approximately of the general population [] Although RLS symptoms generally follow acircadian rhythmicity with increased symptoms occurring at night RLS symptoms can occur during the long periods of daytime inactivity during hemodialysis [] Treatment is primarily focused on ensuring adequate iron stores thenconsidering medical therapy as per routine care of RLSSleep disorders and infectious diseases have few specific associations In general acute infection is associated with mild encephalopathy that masquerades ashypersomnolence and fatigue Proinflammatory cytokines are implicated inthe development of these constitutional symptoms Some infections howeverdirectly affect regulatory centers of the sleepwake systemEncephalitis lethargica is a historical pandemic cause of hypersomnolence ofrenewed interest since this review is being written in the middle of the COVID pandemic Also known as Von Economos encephalitis it occurred inassociation with the Spanish flu pandemic of [] An estimated millionwere affected worldwide The most common subtype the somnolentophthalmoplegic form developed after flulike symptoms of fever and malaiseand consisted of subsequent ophthalmoplegia accompanied by long periods ofhypersomnia Despite the appearance of deep sleep patients could be easilyawoken and sometimes maintained memories of activities that had transpiredaround them while asleep This state of acute akinetic psuedosomnulencecould be followed by the development of chronic postencephaliticparkinsonismThe pandemic associated with the severe acute respiratory syndrome coronavirus SARSCoV2 ie COVID19 occurring during the writing of thisreview features evolving literature The first reports centered on respiratorysymptoms Although the involvement of the nervous system now appearsprevalent [] sleep disorders have yet to be specifically reported Howeverthe psychological responses to social distancing change in schedules and otherfeatures of an active pandemic have caused a wave of anxiety and depressionwhich in turn have been associated with poor sleep quality For example a 0c Page of Curr Treat Options Neurol survey of Chinese health care workers showed prevalences of depressionat anxiety at and insomnia at []Postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy In certainvaccinations in Europe for the H1N1 pandemic caused narcolepsy at a risk of in pediatric patients [] Fortunately the risk of postvaccinationnarcolepsy appeared confined to specific vaccine formulations The incidenthowever has led to ongoing research in the immunological etiology ofnarcolepsyAfrican trypanosomiasis or sleeping sickness remains important in the developing world It is a parasitic infection spread by the tsetse fly that is endemic insubSaharan Africa The first symptoms include fever headaches and lymphadenopathy Once the parasite enters the central nervous system disorderedfragmented sleep ensues often with inversion of the circadian sleepwake cycleThe World Health anization outlines treatment with a regimen of antiparasitic medications once symptoms have started []Nonalcoholic fatty liver disease NAFLD consists of idiopathic hepatic steatosiswith a prevalence of to of the general population with increasedfrequency in individuals with obesity or DM [] Given these coassociationsOSA is common Untreated OSA may exacerbate liver injury because of oxidative stress and systemic inflammation [] and is a risk in conversion fromNAFLD to liver fibrosis [] Trials with CPAP have shown inconsistent resultsin markers of liver injury following treatment of OSA []The symptoms of gastroesophageal reflux disease GERD worsen during sleepparticularly if sleep occurs soon after a meal [] The lower esophageal sphincter that normally prevents reflux may be compromised by the increase inthoracic pressure in the setting of the upper airway obstruction [] Patientswith symptoms of GERD should be screened for OSA and conversely interruption of sleep in absence of OSA may improve with treatment with a protonpump inhibitor PPI [] or by simply elevating the head of the bedInflammatory bowel disease IBD has bilateral interactions with sleep []Given the relationship between sleep deprivationfragmentation on cytokineregulation and immune dysfunction it is hypothesized that poor sleep qualityworsens overall symptoms of IBD [ ] Additionally the proinflammatorystate disrupts the circadian rhythm [] Subjective and objective measurementsof sleep quality and timing should be considered in patients with IBD particularly in those who have frequent inflammatory flares despite otherwise adequate management An algorithmic approach to sleep assessment in IBD patients has been proposed by Canakis et al []Systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders with a prevalence of sleep disturbancesof greater than [] The mechanisms of sleep disturbances as well as thereciprocal relationship in the contribution of poor sleep to worse autoimmunestatus are thought to be similar to those described above with IBD [ ] Thespecific sleep disorders prevalent in this group are OSA and periodic limbGastrointestinal systemAutoimmune disorders 0cCurr Treat Options Neurol Page of Pulmonarymovement disorder PLMD both with greater than prevalence [ ]As seen above hypersomnolence and activitylimiting fatigue arise from specificsleep disorders pain and medication side effects well as the primary effects ofthe primary proinflammatory status [ ] Often treating the underlyingautoimmune disorder improves associated fatigue However if sleepiness persists then evaluating for a comorbid sleep disorder such as obstructive sleepapnea is indicatedOne syndrome with possible autoimmune origins is chronic fatigue syndromeSleep disturbances insomnia and unrefreshing sleep are common symptoms yetpatients rarely report relief despite appropriate identification and treatment ofcomorbid sleep disorders [] Cognitivebehavioral therapy CBT and gradedexercise therapy are commonly pursued treatment approaches []Obstructive lung diseases most commonly asthma chronic obstructive pulmonary disease COPD and less common disorders such as cystic fibrosisCF or bronchiolitis obliterans may affect nocturnal ventilation OSA andCOPD often overlap given shared body habitus and other mutual risk factorsestimates of comorbid OSA and COPD range from to [] Patients withsevere COPD treated with nocturnal noninvasive ventilation NIPPV a moreadvanced form of positive airway pressure experience an absolute risk reduction of of the risk of hospital readmission or death at months compared with those treated with standard care and without NIPPV [64cid129]Insomnia is another common complaint among patients with COPD Circadian bronchial constriction may cause nocturnal wheezing dyspnea or othersymptoms of asthma prompting the patient to awaken [] In addition thehyperadrenergic response to beta agonist inhalers used in treatment for acutedyspnea impairs sleep onset see Table The growing success in treatments for CF patients means that sleep disordersarising from their intrinsic obstructive lung disease are now coming to theattention of caregivers Many factors contribute to sleep disruption includingchronic cough frequent infections abdominal discomfort reflux frequentstools medication side effects and psychological disease [] In addition tosleep disruption patients with CF are susceptible to hypoventilation thatworsens with disease progression Use of NIPPV in highrisk patients withhypercapnia has been shown to improve physiologic parameters and at timescan positively impact symptoms particularly in patients who have severedisease while awaiting lung transplant []Restrictive lung diseases defined by a reduced total lung capacity includethose with parenchymal damage such as idiopathic fibrosis hypersensitivitypneumonitis or other interstitial pneumonias Alternatively lung parenchymais normal in restrictive diseases such as obesity hypoventilation syndromehemidiaphragm paresis or neuromuscular disorders muscular dystrophiesamyotrophic lateral sclerosis Restrictive lung disease patients as seen abovewith obstructive disease patients are susceptible to nocturnal hypoventilationsubsequent CO2 retention and compensatory sleep fragmentation Use ofNIPPV in patients with severe restrictive lung disease spanning obesityhypoventilation syndrome to muscular dystrophies and ALS has had positiveimpacts on survival and quality of life [ ] 0c Page of CardiacCurr Treat Options Neurol Over of patients with congestive heart failure CHF have comorbid OSAmainly on the basis of mutual risk factors of DM hypertension obesity andolder age [ ] In addition insomnia in those with CHF may arise from avariety of factors including diuretic medications and subsequent nocturiapositional heart failure symptoms increased adrenergic status or psychosocialfactors [] Treatments addressing comorbid OSA and insomnia improve sleepquality but demonstrate mixed results in terms of longterm cardiovascularoutcomes [ ]Patients with acute myocardial infarction AMI experience both acute andchronic sleep disorders Due to the circadian variability of adrenergic hormonesand cardiac and systemic vasculature [] the timings of AMI sudden cardiacdeath and arrhythmia occur with increased frequency at night [] Cardiacischemia may present a series of nocturnal symptoms including paroxysmaldyspnea chest pain agitation or insomnia Surviving patients are at risk forchronic sleep disorders such as insomnia and sleepdisordered breathing withor without the cooccurrence of anxiety or depression []Retrospective longitudinal data demonstrate that those with OSA and whoare adherent with CPAP experience improved cardiovascular morbidity andmortality over nonadherent patients [] However these findings have notbeen clearly supported by prospective randomized trials The Sleep ApneacardioVascular Endpoints Trial SAVE Trial has called into question the causallink between the treatment of OSA and cardiovascular outcomes With a meanfollowup of years those randomized to PAP experienced no significantimprovements in study endpoints of death from cardiovascular causes AMIstroke and hospitalization for unstable angina CHF or transient ischemicattack compared with controls [78cid129cid129] Because of possible insufficient CPAPuse and because of the lack of main indications for CPAP treatment such assevere sleepiness interpretation of the findings of this large trial remainscontroversial In practice these authors often pursue CPAP treatment for patients with OSA and cardiovascular risk factors even in the absence of sleepiness at least for a trial period to assess adherence to treatment and to determineif there are subjective and objective improvements to sleep qualityWith a prevalence range of OSA is common in patients with atrialfibrillation and other arrhythmias [] Accordingly the Sleep Heart HealthStudy showed a two to fivefold higher risk of arrhythmia in patients with severeOSA compared with that in controls [] Retrospective series show that inpatients with atrial fibrillation and untreated OSA the risk of atrial fibrillationrecurrence following cardioversion is compared with in patients whoare adherent to CPAP [] However a prospective randomized control trialcalled retrospective findings into question [] Similar in design to the SAVETrial patients with atrial fibrillation were randomized to CPAP versus usualtherapy from a cohort in which sleepiness was specifically excluded This smalltrial total assessed the primary outcome of time to arrhythmia recurrenceBoth arms had recurrence rates of Although the trial showed that CPAPitself provides no specific benefit to those with atrial fibrillation the outcomesfor treatment of those with both disorders remain unclearAlthough the above studies centered on associations between cardiac diseaseand OSA patients with CHF AMI and atrial fibrillation experience high rates of 0cCurr Treat Options Neurol Page of CancerCritical illnesscentral sleep apnea CSA as well exceeding in patients with mild symptomatic CHF as an example [] CheyneStokes respiration a cyclical form ofCSA results when circulatory impairment perturbs the normal responsivenessin respiratory control resulting in alterations in the loop gain in modulatingchanges in carbon dioxide and oxygen levels in the bloodstream [] analogous to overly aggressive adjustments to a thermostat in response to changingtemperature The presence of CSA has been considered a marker of increasedmortality in patients with CHF although aims to resolve the treatment of CSAwith CPAP or more advanced modalities have not clearly demonstrated animprovement in cardiovascular outcomes []Estimates of the prevalence of sleep disturbances across cancer patients range widelyfrom to [ ] Insomnia is the most common disorder with prevalencelevels ranging from to [ ] Patients with cancer who undergo PSGhave shorter total sleep times longer times in bed low sleep efficiency andproportionately less deep sleep than controls [] Insomnia in patients with canceris driven by a multitude of factors including preexisting socioeconomic andpsychiatric disorders fatigue age RLS pain and medication effects [ ]Treatment follows that for the general population Although sedativehypnoticsare most commonly prescribed no evidence exists for specific pharmacologicinterventions for sleep disturbances in this population [] Cognitivebehavioraltherapy is currently the recommended firstline treatment for chronic insomnia[] Because the rarity of trained psychologists makes finding a provider difficult insome circumstances the electronic delivery of cognitivebehavioral therapy hasbeen sought as an alternative to facetoface therapy [ ]The bilateral interactions between sleep and critical illness form a rapidlychanging area of investigation which is made particularly challenging giventhe difficulties in measuring sleep in critically ill patients [ ] Lack ofsleepor its encephalopathic analogmay affect outcomes in critical illnessesFor example a lack of scorable REM sleep correlates with longer ventilatorweaning time compared with controls with intact REM [] Failure rates onnoninvasive ventilation are impacted by sleep continuity [] Delirium acommon neurobehavioral syndrome seen in upwards of of patients inthe ICU [ ] is associated with significantly worse outcomes i
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increasing relevancy of geospatial technologies such as geographic information system GIS inthe public health domain particularly for the infectious disease surveillance and modelling strategies Traditionally thedisease mapping tasks have faced many challenges authors rarely documented the evidence that were used to createmap before evolution of GIS many errors aroused in mapping tasks which were expanded extremely at global scalesand there were no ï¬delity assessment of maps which resulted in inaccurate precision This study on infectious diseasesgeosurveillance is divided into four broad sections with emphasis on handling geographical and temporal issues to help inpublic health decisionmaking and planning policies geospatial mapping of diseases using its spatial and temporalinformation to understand their behaviour across geography the citizens involvement as volunteers in giving healthand disease data to assess the critical situation for diseases spread and prevention in neighbourhood effect scientiï¬canalysis of healthrelated behaviour using mathematical epidemiological and geostatistical approaches with capacitybuilding program To illustrate each theme recent case studies are cited and case studies are performed on COVID19 todemonstrate selected modelsKeywords Geospatial technology 01 Citizen Science 01 Public health 01 COVID19 01 Mathematical epidemiologyIntroductionThe public health sectors increasing demand for mappinganalytics and visualization had started a date back in thelast years which has resulted in a growing informationage technology for communicable disease surveillance andepidemiology Baker Bos and Blobel Friede Friede Khan Reeder Yu and Edberg This continuous publichealth burden with advances in information technology Sameer SaransameeriirsgovinPriyanka SinghPriyankaiirsgovinVishal KumarVishalkumariirsgovinPrakash ChauhanprakashiirsgovinIndian Institute of Remote Sensing Indian Space Researchanisation Kalidas Road Dehradun Indiacombined with spatial data led to the development ofvarious tools and systems that provides visualization ofdisease data in space and time Dredger Kothari Robertson and Nelson Schriml et alThe ï¬rst integral deï¬nition of public health was given byWinslow as science and art of preventing diseaseprolonging life and promoting health through the anized efforts and informed choices of society anizations public and private communities and individualsThe American Public Health Association APHA mentioned public health as a practice of preventing the spreadof disease and an aim of promoting good health from smallcommunities to across the world Turnock Advances in information technology and spatial features resultedin geospatialtechnology which is acute for mappingsurveillance predicting outbreaks detecting clustering andanalysing spread patterns of infectious diseases with epidemic or pandemic potential in communities and acrossterritories AvRuskin Carpenter Castronovo Dominkovics Gao 0c Heymann and Brilliant Hills Klompas Reis Geospatial technology has provided visualization and analytical tools topublic health professionals and decision makers to executediseases control programs in affected andor suspectedregions and make analysis and predictions possible thatwas once technologically out of reachGeospatial technology includes geographical information systems GIS global positioning systems GPS andsatellitebased technologies such as remote sensing RSGIS is known for geographic data capture input updatemanipulation transformation analysis query modellingand visualization of all forms of geographically referencedinformation through the set of computer programs BonhamCarter GPS provides positioning navigationand timing PNT services by capturing data from satellitesand providing it to users Eldredge and RS isan earth observation instrumentthat delivers regionalinformation on climatic factors and landscape featuresTherefore GPS and RS provide regional and spatialinformation while GIS provides geospatial data integrationas well as accurate geospatial analysis in realtime mannerZhen Geospatial Technology and InfectiousDisease SurveillanceInfectious diseases mostly adapts antimicrobial andmobility features later formed in a shape of pandemic andor epidemic Chen Cheng Lee andNishiura which forced public health authorities tounderstand not only the diseases virulence but also itsdemographic and environmentalfactors that helps inmaking spread patterns though space and time domainCroner For example the global spread of highlypathogenic avian uenza HPAI H5N1 in withno effective vaccines led to concern among public healthdecision makers in spite of many international programsRappole and Huba´lek The reason behind theirconcern was they were lacking of disease surveillance toolin its initial stage which caused inaccessibility to populations atrisk and faced difï¬culties in implementingimmunization strategies at a global scale Kitler Stoto However the impact of environmentaland demographic factors also plays a major role as this caninform about the interaction between hosts and pathogensand patterns of spread in space and timeThe GIS provides dynamic maps to understand geographical distribution of diseases for analysis on frequencyof cases disease mapping spatial cluster of diseases disease association with environmentalfactors networkanalysis etc With such a visualization and analyticalJournal of the Indian Society of Remote Sensingforservice frameworkcapabilities GIS technology is holding a widespreadgrowth in public health Ahmad 2011a b Booman Hanaï¬Bojd Kolivras Martin Nykiforuk and Flaman Abdul Rasam Zhang Zhen Theseamless integration of GIS with realtime infectious diseaserelated diverse datasets through webbased mappingto the development of geospatial dashboardleadsgeospatialinfectious diseasesurveillance Dent Gao Yun Theinfectious diseaserelated data mightinclude diseasesurveillance data activeconï¬rmed cases and health system data hospital visits emergency services availabilitynursedoctor availabilityICUbed availability Many source geospatialstandards of GeospatialConsortium OGC are used as a Web Map ServiceWMS Web Coverage Service WCS Web ProcessingService WPS Web Feature Service WFS etc Bulatovic´ Gao to visualize accesspublish and manipulate geospatial resources Also manyother popular industrial geospatial standards are developedby ESRI Google Yahoo and MapInfo Granell to fetch locationbased data and provide infectiousdisease surveillance dashboard to monitor and control thegeographically spread of disease Zhang TheGeocoded Really Simple Syndication GeoRSS taggedXML ï¬les from GeoRSS services can also be used toprovide geocoded infectious disease news from socialmedia platform Tolentino KassHout andAlhinnawi 2013a b Kodong Historical ContextThe mapping of infectious diseases using geospatial andinformation technology to beneï¬t public health is not a newway of tracking the diseases Ahmad Cui Hirsch Hornsby Matthew May Mujica Nicholson and Mather Noble Perl and Moalem Williams The historical disease mapping has faced manychallenges authors rarely documented the evidencethat were used to create map after mapping had beenimplemented before the beginning of geographical information systems many errors arouse which were expandedextremely at global scales and there were no ï¬delityassessment of maps which resulted in inaccurate precisionBut nowadays wide range of geospatial applications areavailable in public health community with a possibilities ofvisualization analysis detection of clusters formed andcalculate diseaserelated metrics such as incidence andprevalence rate Beck Clarke Hay Jacquez Kleinschmidt Lawson and 0cJournal of the Indian Society of Remote SensingLeimich Moore and Carpenter Robinson Wilkinson The earliest mapping for visualisation ofthe linkbetween disease and place was done in on plagueepidemic in Italy Dent During cholera outbreak in the study of physician John Snow had made a novelcontribution in history of public health and epidemiologyby using cartography applications and geographic visualization in ï¬ghting cholera After years the maps wereidentiï¬ed as a communication tool in understanding andtracking of infectious diseases such as the uenzapandemic yellow fever and cholera Since then revolutionof webbased tools started in applied health geographyBoulos The trend of infectious disease mappingcould be seen from review of the Health GIS literature which demonstrated that research papers out of were focused on infectious disease mapping Lyseen Covid19The ongoing pandemic outbreak targeting humans respiratory system was recently discovered in December by the name of Coronavirus Disease Covid19 WorldHealth anization from a cluster of patients with acuterespiratory distress syndrome in Wuhan Hubei ProvinceChina Huang Lu 2020a b and spreadglobally by March This pathogenic disease is structurally related to the Coronavirus CoV which belongs tofamily Coronaviridae and the order Nidovirales Thisfamily is classiï¬ed into four generaAlphacoronavirusBetacoronavirus Gammacoronavirus and Deltacoronavirus on the basis of their phylogenetic and genomicanalysis The species of Alphacoronavirus and Betacoronavirus infect mammals causes respiratory illness inhumans and gastroenteritis in animals while species ofGammacoronaviruses and Deltacoronaviruses infect birdsbut some of them can also infect mammals Woo et alfrom Betacoronavirus The two virusgenusSevere Acute Respiratory Syndrome SARSCoVor Middle East Respiratory Syndrome MERSCoVhadearlier demonstrated that coronaviruses can cause signiï¬cant public threat Ge The COVID19 is categorizedby World Healthanization WHO on the basis of genomic sequencinganalysis ofrespiratory tract samples which isobtained from total of nine patients Huang Lu 2020a b COVID19 has started behaving like theonceinacentury pandemic by affecting healthy adults aswell as elderly people with some health issues and byinfecting others at an exponential rate of increase thanSARS or MERSinto BetacoronavirusspecieslowerGeospatial TechnologyDuring occurrence of diseases geospatial technologies andservices could help in representing the spatiotemporalinformation and in analysing the dynamic spread of diseases As mentioned by Boulos geospatial technologies and services which performs in real time mannerare tremendously relevantto create a spatial healthinformation infrastructure In this section a review onmany geospatial technologies with enabled IT services iscarried out to understand and analyse the spread and outbreak of disease with a case study on COVID19 pandemicCitizen Scienceissues and concernsThe expansion of Citizen Science from biodiversity andecological domain Haklay MillerRushing to public health community across spatial extentsmade an urgent need to study its different forms Crowl The indepth report of EU describes taxonomyof Citizen Science in three levels European Commission described in Roy Wiggins andCrowston and Haklay Roy categorized Citizen Science by participants number and oftheir spread local and mass and thoroughnesstime and resource investment or King described for the people with the people or by the people about Citizen Science activities Wiggins andCrownston classiï¬ed Citizen Science projects inconservation managing natural resources action addressing localinvestigation answering scientiï¬c questions and education providingknowledge to citizens Haklay classiï¬ed CitizenScience into four levels based on participants engagement level is crowdsourcing in which citizens withless or no knowledge on activity perform as sensors tocomplete computing tasks level is distributed intelligence where citizens are being trained with skills forinterpretation of collected data level is participatoryscience in which citizens decide about research questionsand types of data to be collected and level is extremewhere citizens are fully involved in deï¬ning researchstrategies data collection data interpretation and performing scientiï¬c analysis Apparentlythe concept ofCitizen Science is rare in public health domain but some ofits contribution seen in some studies which not only helpsin predicting disease risks but also in combating theinfectious diseases CurtisRobles Palmer Smolinski Wilson Another approach similar to Citizen Science is popularepidemiology in which experts and laypersons jointly 0ccollect environmental data responsible for particular healthconsequences Brown or street science as a process in which general public communities actively engagedin deï¬ning problems framing of research questions anddecisionmaking activities about research design CorburnCrowdsourceVGI Mobile AppsDespite technological and computational developments inGeoWeb many web technologies such as jQuery andAJAX mapping APIs like Google and GPS devicesresulted in a new revolution of neogeography Turner where mapping is done by crowd and can bereached by anyone from general public members groupSuch revolution brought a trend of Volunteered GeographicInformation VGI which is ï¬rst coined and explained byGoodchild 2007a According to Goodchild 2007b VGIhighlighted the human capabilities in collecting geospatialinformation by using ï¬ve senses and then integrating withexternal sensors of mobile devices like GPS accelerometer camera digital compass and microphone gives valuable datasets which can neither be retrieved from satelliteimagery nor collected with any GPS receivers Anothersuccessful term in geospatial mapping using mobile technology is crowdsourcing Heipke HudsonSmith which was coined by Howe thatinvolves the collection of geospatial information or mapping of any particular activity by an undeï¬ned crowd ornetwork of people Both terms VGI and crowdsourcingslightly differ but they are usually recognized as a synonyms or even as a combined term crowdsourcing geographic information Sui Over the lastdecade VGIoriented source mobileareEpiCollect Aanensen for ecology and epidemiology NoiseTube Maisonneuve httpnoisetubenet and Noise Battle GarciaMartı´ for noise monitoring Skywatch Windoo httpwindoochfor weather monitoring Mappiness httpwwwmappinessukfor behavioural analysis MacKerron andMourato appsThe source mechanism for data collection usingAndroid devices can be performed by Data KitODK suite107 https datakit which is composed of ODK Collect and ODK Aggregate ODK Collecthttps datakitusecollect provides a customizable framework for geospatial data collection and ODKAggregate is a web application that runs on ApacheTomcat server httptomcatapache to store collecteddata through a synchronization with a databaseforexample PostgreSQL Brunette As suchsuites performance can be seen in various activities likeagricultural monitoring Krosing and Roybal Journal of the Indian Society of Remote Sensingmonitoring of deforestation and school attendance documentation of war crimes and health programs Anokwa Digital Contact TracingNowadays COVID19 has become the greatest threat forpublic health in last years and due to such pandemicvarious levels of lockdown are issued across the world tobreak its chain of infection transmission However this isthe ï¬rst approach to invade the contagion but once itwould be lifted this pandemic would start in a new wayand might reach its highest peak by infecting more andmore population Ferguson Thereforetocombat with such a global pandemic threat anotherapproach is discovered by a group of researchers known asdigital contact tracingSmartphonebased contact tracing is known as a digitalcontact tracing which presents a sustainable solution tolimit the transmission of infectious disease by tracing theirpotential transmission routes in a population howeversuch an app presents signiï¬cant concerns regarding privacy The digital contact tracing works on the principle ofcrowdsource data by measuring the proximity to aninfectious person In previous diseases risk surveillancethe contact tracing apps were used to pool location timestamped data to determine the exposures to risk of infectionsSacks Such data are highly personal and leadmany privacy concerns Smith but they werenot always accurate to infer the exposure risks due to noisydata Farrahi Therefore various smartphoneapps are developed in COVID19 pandemic in which someapps use location for proximity and some of them are notusing location services of mobile device subject to theprivacypreserving natureCOVID19 Contact TracingIn order to illuminate the epidemiology of COVID19 andto characterize its severity Lipsitch there is anurgent need of digital platform that captures realtimeaccurate information on COVID19 patients diseasesdiagnosis treatment and clinical reports and whom theyget interacted at which place to detect clusters and generatealerts Such information may help in understanding riskfactors of infection and in predicting the next generation ofinfectious persons FitzGerald Addressing thisunprecedented challenge many mobile apps have beendeveloped and are being used at large scale and some ofthem are as follows 0cJournal of the Indian Society of Remote Sensing¢ COVID Symptom Study COVID Symptom TrackerThis mobile app is developed in collaboration of ZoeGlobal Ltd a digital health care company and a groupof academic scientists from Massachusetts GeneralHospital and Kings College London which waslaunched in UK on March and becameavailable after days in USA This app enquires aboutage location and other diseases risks and also a selfreporting function is enabled which is associated withCOVID19 infection and exposure Drew This app retrieve updates on healthcare workersexperiences who are on COVID19 duty their stressand anxiety and use of personal protective equipmentPPE kits are being surveyed through this app toobserve intensity of health care workers Drew et alappimplemented¢ Aarogya SetuThis mobile app is launched on April by Government of India to aware general publicon COVID19 symptoms government advisory measures online consultation facility and dynamics ofdisease Thiscrowdsourcingapproach by which general public members enter theirdetails for selfassessment and this assessment is thenused to trace the infectious contacts or agents as adigital contact tracing concept This app uses locationservices to geolocate the users and Bluetooth tomaintain the log of contacts when one userdevicecomes in contact with another userdevice and as suchdigital contact tracing activity helps in identifying thecluster of diseases and communities which are at risk ofinfection The Aarogya Setu app was downloadedby million users within days of its launchUpadhyay and by using apps crowdsourcedata the Indian government detected approx positive casesinformed probable users ofbeing at risk and identiï¬ed potential clusters TheTimes of IndiaNumerous digital contact tracing apps are in use indifferent parts of worldTrackCOVID Yasaka TraceTogether Bay WeTrace De Carli and Google and Apples recently announcedjoint initiative Li and Guo COVID19 Data Visualization and ExploratoryData AnalysisWith early experiences of epidemics such as SARSCoV Boulos and the MERSCoVGikonyo and other seasonal ï¬us online realtime or nearrealtime mapping of diseases occurrencesusing geospatial technologies and web applications havealways been used as a pivotal webbased tools in trackinghealth threats and combating infectious diseases Thissection described a range of mapping dashboards based ongeospatialtechnologies for tracking and unfolding thecoronavirus disease around the world Some of the globaland national geospatial initiatives with an aim to supplyinformation faster than diseases are as summarized inTable Infectious Diseases ModellingThe intention of infectious diseases surveillance is to detectepidemics in their early stages so that the countermeasurescould be taken for preventing its wide spread Suchsurveillance tasks require many epidemiological and statistical methods with geospatial features in investigatingepidemics preferably from localized areas The reason forpreferring the local areas for investigation is because epidemics generally emerged in small areas and then spreadwidely if they are not controlled However some methodsrequire rigorous conventions in their underlying modelsand are too problematical to be applied on small areasThereforefordetecting diseases prevalence with case studies on smalldatasets which would be more useful for public healthactivitiessection discussessimple methodsthisClusteringClustering deals with the study of spatialtemporal patternsof the spread of communicable diseases and identiï¬cationof other diseaserelated aspects allied with heterogeneousgeographical distribution which might be helpful in elucidating the diseases spread mechanism Such study andanalysis on spacetime patterns is a kind of diseasesurveillance which involves detecting the outbreak clustersof active cases monitoring of localisation and isolation ofinfectious agents and relative risks assessment of affectedsites at early stage Clements Cromley Kulldorff This study on geographical clustering ofinfectious diseases with temporal features helps in makingstrategies that dynamically update on emergence source ofdisease outbreak to help epidemiologists and decisionmakers for identiï¬cation of spread and risk zones Thusclustering helps to enable timely prevention and containment measures and timely resource allocation to mitigatethe diffusion of diseasesBased on spacetime surveillance of diseases spacetime scan statistic Kulldorff is one of the clusterdetection tools which is widely used in geographicalsurveillance of diseases during epidemic andor pandemicThe spacetime scan statistic comes with two versionsprospective and retrospective Desjardins 0cTable Summary table for geospatial dashboards for COVID19Project nameDatasetsScopePurposeJournal of the Indian Society of Remote SensingWHO Coronavirus Disease COVID19WHOs ofï¬cial dataDashboard Dong httpscovid19whointGlobal Visualization of ofï¬cial daily counts ofconï¬rmed cases and deaths related toCOVID19 with time stamps using EsriArcGIS Online serviceExploratory data analysis using 3D graphto perform countrywise analysis usingpopulation conï¬rmed cases cumulativecases deaths and cumulative deathsProvides daily aggregate case and deathcount in CSVJohns Hopkins University COVID19Aggregated data from WHO EuropeanGlobal Dashboard for visualizing realtimeDashboard Dong httpscoronavirusjhuedumaphtmlCentre for Disease Prevention andControl ECDC WorldoMeters BNONews US CDC 1Point3Arces COVIDTracking Project and DXYmapping of COVID19 with graphs onconï¬rmed and daily casesCritical trend analysis on new cases perday mortality and fatality analysis inpopulation timeline of outbreak etcISROs BHUVAN COVID19 GeospatialSolution httpsbhuvanapp3nrscgovincoronacorona_dashboarddashboarddashboardphptypecitizenCOVID19 data Source MoHFWIndiaTime series visualization of activerecovered and deceased cases fromMarch to till dateGraphical analysis on spread trend ofCOVID19 daywise and statewiseCOVID19INDIA httpswwwCM Health M handles Press Trust ofIndiaVisualization of cumulative and dailycovid19indiaIndia state press bulletins PBI and ANIreportsnumbers of conï¬rmed active recovereddeceased and tested statewise casesthrough maps and graphsProvides daily COVID19 cases in stateand district and cases reassigned to statesthrough APIMapmyIndia COVID19 httpsmapsCOVID19 data Source MoHFWIndiaProvides API on corona dashboards tomapmyindiacomcoronadistricts_containment_zonecontainment_zone_gradientHunger Relief Centres Source MyGovHunger Night Shelter Source MyGovNDMAvisualize cases at district and state levelhotspots treatment centres testing labsquarantine centres containment zoneslockdown issues hunger relief hunterand night sheltersMonthly climate explorer for COVID19httpscdsclimatecopernicuseuappsc3sappc3smonthlyclimatecovid19explorerMonthly COVID19 cases Source JHUGlobal Visualization of COVID19 fatalities withCSSEAtmospheric compositionPM10 and NO2Source CAMS EAC4Meteorological datahumidity hPaand surface air temperature on hourly andmonthly average rate Source ERA5reanalysisclimatic and atmospheric variations onmonthly basisExploratory analysis on correlation ofpollutants and speciï¬c humidity withCOVID19 deathsExperimental COVID19 and GlobalCOVID19 cases Source JHU CSSEGlobal Visualize earthquake as a cause of increaseSeismic Risk Map httpsmaps quakemapcovid1920200520v3grm234900Global earthquake risk map Source GEMGlobal Earthquake Modelin COVID19 cases due to peoplesdisplacement from damaged buildingsOwusu and difference between both is thatprospective neglects historical clusters which may havepreviously occurred before the most current time period ofanalysis with no health threat Kulldorff Thereforethe prospective version of spacetime scan statistic iscommonly used to detect statistically signiï¬cant active orevolving clusters of diseases for the present time periodand when more data become available the tool can be rerun to detect new evolving clusters with update on relativerisks for each affected sites Previously the prospectivespacetime scan statistic was used in thyroid cancerKulldorff shigellosis Jones measlesYin syndromic surveillance Yih and many other diseases However cluster analysis of 0cJournal of the Indian Society of Remote Sensingdiseases can be performed through several packages andlibraries in R Go´mezRubio and Pythonsoftware Yeng The contribution of cluster detections and analysis inCOVID19 pandemic is becoming useful nowadays as itdetects active and emerging clusters of COVID19 andnotify epidemiologist decision makers and public healthcare ofï¬cials which can help in eradicating infections fromaffected sites and improving interventions quarantine andisolation measures The signiï¬cant applications of clustering with respect to infectious diseases modelling aredemonstrated across the world Zarikas forexample India Bhosale and Shinde USA Desjardins Hohl Brazil Martines Italy Cereda China Ji Liu 2020a b Qiu Zhang Singapore Bhosale and Shinde Pung SouthKorea Shim French Alps Danis Germany Pfefferle Sergipe Andrade etcOutlier AnalysisThe outlier is deï¬ned by Hawkins as an observationwhich deviates so much from the other observations as toarouse suspicions thatit was generated by a differentmechanism In other words when data generation processstarts behaving abnormal and reï¬ects the abnormalities orerrors in data such abnormalities are known as outliersBansal However the outliers generally holdadvantageous information about the systems unusual characteristics and entities which impact the data generationprocess Some of the useful applications of outliers in diseases are Cleynen Dai and Bikdash Krishnan Lo Prensner Washington Wu and Krishnan Clusteringalgorithms are optimized to ï¬nd clusters rather than outliersand the accuracy of outlier detection depends on how goodthe clustering algorithm captures the structure of clustersMaximum Entropy Modelling Maxent ApproachIn context of disease systems disease transmission risksdepend on distribution of pathogens species in space andtime in some complex environmental conditions Townsend and as such treatments are focused mainly on spatialdimensions therefore diseases transmission risks are purelyhandled through geographical phenomena Such geographical link with diseases leads to the challenge of spatialmapping of disease transmission which overcame throughthe branches of biodiversity scienceecology and biogeography Such approach of ecological and biogeographical modelling can be seen from various studies on diseasetransmission risks mapping for example Arboleda Deka and Morshed Ferreira Holt Mweya Nakazawa Reeves Samy Qian Zhao Zhu Following recent studies on geographical mapping ofpathogens causing disease transmission machine learningbased maximum entropy method Maxent Elith Phillips is applied on spatial records ofCOVID19 with a set of bioclimatic environmentalvariables from WorldClim Poggio Ramı´rezVillegas and Bueno Cabrera to analyse theirfavourable environmental conditions as shown in Fig and Table required in maintaining its population TheMaxent principle is to estimate the target probability distribution by applying the maximum entropy to distributionwhich is most spread or closest This study is carried out inR software Ihaka and Gentleman and a geographical dataset consists of latitude and longitude of thoseregions which were affected till March Figure depicts the habitat suitability map of virus withprobability range in colour scale to visualize the highsuitability light and dark green colour medium suitabilityyellow and dark brownlow suitability light browncolour and unsuitable grey colour Table lists thefavourable bioclimatic variables and their contribution inpercent in maintaining the suitability of virusSusceptibleInfectiousRecovered SIR ModelEpidemiology deals with the study of pattern and occurrence of diseases in space and time associated with otherfactors such as environment demography and the translation of epidemiology into mathematical equations todescribe the spread of infectious diseases is known asmathematical epidemiology Allen Rayner andBender The mathematical epidemiology model isimplemented to understand the transmission dynamics ofcommunicable diseases by categorizing population intosusceptible infectious and recovered compartments Theï¬rst basic model known as SusceptibleInfectiousRecovered SIR model was proposed by Kermack andMcKendrick to describe the transmission of epidemic diseases from individual to individual The SIRmodel is a set of nonlinear ordinary differential equationswhich is mathematically deï¬ned as follows¼ l N þ Sðà 00 bSI¼ bSI 00 cI 00 lI¼ cI 00 lRdSdtdIdtdRdtð1Ãð2Ãð3à 0cJournal of the Indian Society of Remote SensingFig Predicted suitability of Betacoronavirus using data till March Table Responsible bioclimatic variables in suitability modellingHereS is the class of susceptible individuals who are not yetcontracted to diseaseI is the class of infectious people who are now infectedwith disease and become infectious to infect others¢ R is class of recovered individuals who have recoverednow and are removed from class S¢ N is a total population size N S I R and t istime in days or weeks¢ b is the contact rate of infected person with suspected¢¢¢Bioclimatic variablesPercent contributionMean temperature of coldest quarterPrecipitation of wettest monthMean diurnal rangeIsothermalityAnnual mean temperatureMax temperature of warmest monthPrecipitation of coldest quarterPrecipitation of wettest quarterAnnual precipitationPrecipitation of driest quarterMean temperature of driest quarterMean temperature of wettest quarterPrecipitation seasonalityTemperature seasonalityPrecipitation of warmest quarterMean temperature of warmest quarterTemperature annual rangePrecipitation of driest monthMin temperature of coldest monthperson per dayc is the infectious period and average infectious periodis 1c¢ l is the per capita death rate which is adjusted by birthrate lNThere are many other compartment models derived fromthe basic epidemic model SIR with more compartmentsand transitions SusceptibleExposedInfectiousRecovered SEIR Li and Muldowney SusceptibleInfectiousExposedRecoveredDeadSEIRDPiccolomiini and Zama SusceptibleInfectiousExposedRecoveredSusceptible SEIRS Liu and Zhang SusceptibleInfectiousQuarantineRecoveredSIQR Erdem
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"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genesi reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used housekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employedi cv �xsx where �x and Ïx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isnt a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm Ëcamplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks curls were deparaffinized inxylene at Ëc followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at Ëc for minutes followed by amplification for cyclesdenaturation at Ëc for sec amplification at Ëc for sec and extension at Ëc for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq samplemean cq ntc and relative expression aseδcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithmsgenorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkins bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of adjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgsptcd2 ppp1r3b andfbxw9 have sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting original draft sreejeta mondal sujan k dharwriting review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for doubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092 derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may 101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep 101038leu2016382 pmid bomben r ferrero s dagaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr 103324haematol2017184325pmid dheda k huggett jf chang js kim lu
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" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the consensusclusterplus package was used to cluster in an unbiased and unsupervisedmanner based on the overlapping degs cumulative distribution function cdf and relative change inarea under the cdf curve were used to determine theoptimal number of clusters k then mannwhitney 0chu bmc cancer page of signedrank test was performed to study the differenceof immune cells proportion between the clusters and theviolin plot was established to show the differences ofimmune cells among clusters survival analysis of degsbased on the degs the univariate cox analysis was performed to determine the prognostic value of immunerelated genes then the osrelated genes were validatedin the gse21257 dataset while the dfsrelated geneswere validated in the gse39055 dataset only genes successfully validated were selected for further analysis afterward based on the validated genes the multivariate coxanalysis was performed to establish the prognostic signature for predicting the prognosis of osteosarcoma patientsthe risk score for each patient was calculated based onthe coefficient from the multivariate cox analysis and thecorresponding gene expression meanwhile all patientswere divided into the high and lowrisk groups accordingto the median of the risk score the survival receiver operating characteristic roc curve was used to show the discrimination of signatures and the kaplanmeier survivalcurve with the logrank test was generated to show thedifferences of os and dfs between high and lowriskgroups in addition the risk score of patients in the validation cohort was also calculated according to the aforementioned risk signature the kaplanmeier survivalcurve and survival roc curve were generated to show thepredictive ability of the signature in the validation cohortdevelopment of a nomogram for osteosarcoma patientsnomogram is a tool to visualize the predictive model andconvenient for clinical practice therefore we attemptedto develop a nomogram based on the tmerelated genessignature and clinicopathologic data to predict the prognosis of osteosarcoma patients firstlythe univariatecox analysis was performed to filter prognostic variableswhich will be further included in the multivariate coxanalysis secondly based on independent prognostic variables two nomograms were established for predicting theos and dfs respectively the cindex was used to assessthe discriminatory performance of the nomogram whichrange from to a cindex of means agreement by chance and a cindex of represents perfectdiscriminatory performance the higher value of the cindex the better performance of the nomogram is furthermore the calibration curves of and 3year weredeveloped to evaluate the effectiveness of nomogramsresultsimmune significantly associated with the prognosis ofosteosarcoma patients osteosarcoma patients were included in the presentstudy including males and females the immunescore of the cohort range from to tostudy the relationship between the immune score and theprognosis of osteosarcoma patients patients wereincorporated into the lowimmune score group while theremaining patients were incorporated into the highimmune score group the survival analysis indicated thatpatients with higher immune score had a favorable osand dfs fig 1a and b after adjusted age tumor siteand metastatic status the immune score still was a prognostic variable for both os and dfsfig 1a and b inaddition the relationship between immune score and clinical features was also investigated however there was nosignificant relationship between immune score and clinicalvariables supplementary figure 1a1cdifferential expression analysisaccording to the median of the immune score patients were divided into highscore n and lowfig association between immune score and prognosis in osteosarcoma patients a kaplanmeier survival analysis of overall survival for patientswith high vs low immune score b kaplanmeier survival analysis of diseasefree survival for patients with high vs low immune score 0chu bmc cancer page of score group n there were differentiallyexpressed genes between two groups which include upregulated genes and downregulated genesfig 2a b and supplementary table to furtherunderstand the function of degs go analysisand kegg analysis were performed the top significant results of go analysis among three types wereillustrated in fig 2c interestingly we can find that theresults of go analysis are mostly associated with immunity which further verify that the immunerelated degsare associated with immune features in addition the results of kegg also confirmed it such as phagosomeautoimmune thyroid disease antigen processing andpresentation b cell receptor signaling pathway intestinal immune network for iga production inflammatorybowel disease primary immunodeficiency th1 andth2 cell differentiation th17 cell differentiation natural killer cell mediated cytotoxicity and nfkappa bsignaling pathway fig 2dconsensusunsupervisedevaluation of degs and immune cellsto further understand the molecular heterogeneity ofosteosarcomaanalysis wasperformed to divide patients into subgroups to explorewhether immunerelated genes presented discernable patterns based on the consensus matrix heat map patientswere clearly divided into two clustersfig 3a in additionby comprehensively analyzing the relative change in areaunder the cumulative distribution function two clusterswere determined fig 3bc the immune score betweentwo clusters was significantly different fig 3d in additionthe proportion of types of immune cells in osteosarcomapatients was illustrated in a barplot fig 3e interestinglywe can see that the t cells cd4 memory activated ofcluster is significantly higher than cluster fig 5fprognostic value of tmerelated genesprevious studies indicated that tmerelated genes canserve as the prognostic biomarker for tumor patientsfig differentially expressed genes with the immune score in osteosarcoma patients a heatmap of significantly differentially expressed genesbased on immune score b the volcano figure to show the upregulated and downregulated genes c go analysis of differentially expressedgenes d kegg of differentially expressed genes go gene ontology kegg kyoto encyclopedia of genes and genomes 0chu bmc cancer page of fig the immune landscape of the tumor microenvironment ac unsupervised clustering of all samples based on the overlapping degs dcomparison of immune score between two clusters e the distribution of types of immune cells in osteosarcoma patients f the comparisonof types of immune cells between clusters deg differentially expressed genehence we performed the univariate cox analysis toidentify prognostic degs the results showed that and genes were identified as os and dfsrelateddegs respectively supplementary table and afterward five osrelated genes were successfully validated inthe gse21257 data set and five dfsrelated genes were successfully validated in the gse39055 cohort furthermoremultivariate cox analysis was performed and two prognostic signatures were generated for predicting the os anddfs respectively the risk score for predicting the os wasasrisk score fcgr2b0766 gfap0702 mpp70387 in addition the risk score for predicting thedfs was as follows risk score cyp2s10574 icam3 the auc values of osrelated signature were follows 0chu bmc cancer page of and in and 3year respectively fig 4aand the auc values of dfsrelated signature were and in and 3year respectively fig 5amoreover survival curves showed that patients in the highrisk group had worse os and dfs compared with the lowrisk patients figs 4b and 5b heat maps risk score plotsand survival status were generated to show the distinctionbetween highrisk patients and lowrisk patients figs 4ceand 5ce then both signatures were validated in independent cohorts for os signature the auc values ofvalidation cohort were and at and3year fig 4f for dfs signature the auc values ofvalidation cohort were and at and3year fig 5f additionallyin both validation cohortssurvival curves showed that lowrisk patients were favorableprognosis than highrisk patients figs 4g and 5gheat maps risk score plots and survival status of validation cohorts were also generated to show the distinction between highrisk patients and lowrisk patientsfigs 4hj and f 5hjdevelopment of a nomogram for osteosarcoma patientsto generate a nomogram for clinical use the cox analysiswas performed to select the clinical prognostic variables infig establishment and validation of the prognostic model for overall survival based on significant degs a receiver operating characteristiccurves of prognostic signature in the training cohort b the survival curve showed the different overall survival status between high and lowriskpatients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each sample reordered by riskscore e the scatter plot showed the overall survival status of osteosarcoma patients in the training cohort f receiver operating characteristiccurves of prognostic signature in validation cohort g the survival curve showed the different overall survival status between high and lowriskpatients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reordered by riskscore j the scatter plot showed the overall survival status of osteosarcoma patients in the validation cohort 0chu bmc cancer page of fig establishment and validation of the prognostic model for diseasefree survival based on significant degs a receiver operatingcharacteristic curves of prognostic signature in the training cohort b the survival curve showed the different diseasefree status between highand lowrisk patients c the heat map showed the expression of prognostic genes in the training cohort d the risk curve of each samplereordered by risk score e the scatter plot showed the diseasefree status of osteosarcoma patients in the training cohort f receiver operatingcharacteristic curves of prognostic signature in validation cohort g the survival curve showed the different diseasefree status between high andlowrisk patients h the heat map showed the expression of prognostic genes in the validation cohort i the risk curve of each sample reorderedby risk score j the scatter plot showed the diseasefree status of osteosarcoma patients in the validation cohortthe univariate cox analysis risk score and metastatic status were identified as both os and dfsrelated variablesfig 6a and e afterward risk score and metastatic statuswere determined as both independent os and dfsrelated variables in the multivariate cox analysis fig 6band f based on independent variables two nomogramswere established for predicting the os and dfs in osteosarcoma patients respectively fig 6c and g the cindexvalues were and in os nomogram and dfsnomogram respectively the results of cindex mean thatboth two nomograms have good discrimination meanwhile to evaluate the calibration of nomograms six calibration curves were generated and the results showed thatthe predictive curves were close to the ideal curve fig 6dand h which indicated a good calibrationdiscussionthe relationship between tme and tumor have beenwidely studied in recent years in the present study estimate algorithm was utilized to quantify the immunescore based on gene expression profiles in osteosarcomapatients from target database we confirmed that thetme is significantly associated with the prognosis ofosteosarcoma patientsinadditionfunctional enrichment analyses of tmerelated genes indicated that immunerelated processesincluding os and dfs 0chu bmc cancer page of fig nomograms based on the tumor microenvironment related genes for osteosarcoma patients a univariate cox analysis of overall survivalrelated variables b multivariate cox analysis of overall survivalrelated variables c nomogram for predicting the overall survival in osteosarcomapatients d1 and 3year calibration curves of overall survival nomogram e univariate cox analysis of diseasefree survivalrelated variables fmultivariate cox analysis of diseasefree survivalrelated variables g nomogram for predicting the diseasefree survival in osteosarcoma patientsh1 and 3year calibration curves of diseasefree survival nomogramknown to contribute to tumor progression more importantly degs based on the tme were identified asimportant prognostic biomarkers for osteosarcoma patients and two nomograms were developed for predicting the os and dfs of osteosarcoma patientsrespectivelyin recent years an increasing number of studiesfocused on the carcinogenesis and progression of tumorsbased on the tme and the estimate algorithm is oneof the most important quantitative tools for this researchfield based on the estimate algorithm the association between the prognosis and tme has been initially 0chu bmc cancer page of elucidated in some tumors such as cervical squamouscell carcinoma gastric cancer cutaneous melanomaacute myeloid leukemia bladder cancer and clear cellrenal carcinoma [ ] however previousstudies indicated that tme scores serve as a differentrole in different tumors for example for hepatocellularcarcinoma gastric cancer acute myeloid leukemiabladder cancer and clear cell renal carcinoma patientswith high immune score have a worse prognosis [ ] however for cervical squamous cell carcinoma adrenocortical carcinoma and cutaneous melanoma patients with high immune score have a favorableprognosis [ ] therefore we can find great heterogeneity among different tumors from the perspectiveof tme for osteosarcoma patients the present studyindicated that patients with higher immune score had abetter os and dfs hence the present study indicatedthat immune cells infiltrating tumor tissue may play animportant role in suppressing tumor progressionin our research tmerelated genes were identified by comparing the highscore and lowscore osteosarcoma patients the functional enrichment includinggo and kegg analyses showed that tmerelated geneswere mainly involved in the immune features such asregulation of leukocyte activation mhc protein complex mhc protein and complex binding more importantly the unsupervised cluster analysis based on degswas performed and all patients were divided into twoclusters immune score and t cell cd4 memory activated fraction were significant difference between twoclusters which further elucidated the relationship between degs and immune featuresdue to the poor prognosis of osteosarcoma patientsidentifying robust prognostic biomarker is very importantthe tumor immune microenvironment is closely relatedto the prognosis of bone tumor patients emilie etal performed the first genomewide study to describe therole of immune cells in osteosarcoma and found thattumorassociated macrophages are associated with reduced metastasis and improved survivalin highgradeosteosarcoma recently the prognostic signature based ontmerelated genes have been established for many tumors [ ] but only one study focused on osteosarcoma patients compared with the study performedby zhang we think that our research have someadvantages firstly our signatures were established basedon several validated genes and both two signatures weresuccessfully validated in independent cohorts secondlythe outcome of dfs was not reported in the previousstudy as reported in published studies tumor recurrenceis a terrible medical problem for osteosarcoma patientsand the 5year survival rate for osteosarcoma patients withmetastasis or relapse remains disappointing [ ]hence the dfs nomogram can improve the managementof osteosarcoma patients finally two nomograms incorporated tmerelated signature and clinical variables wereestablished in our research which further facilitated theclinical application of our findingsin our research five genes were incorporated into thefinal prognostic signatures fcgr2b gfap and mpp7were identified and validated as osrelated biomarkerswhile cyp2s1 and icam3 were dfsrelated biomarkersthe role of these genes in tumor prognosis had beenwidely reported in previous studies [] fcgr2bhas been confirmed as an immunerelated gene previously although the relationship between fcgr2band prognosis in sarcoma patients had not been reported the prognostic value of fcgr2b had been widelyconfirmed in other cancerssuch as hepatocellularcarcinoma and glioblastoma [ ] in addition newm etal demonstrated that mpp7 is novel regulatorsof autophagy which was thought to be responsible forthe prognosis of pancreatic ductal adenocarcinomacyp2s1 described as cytochrome p450 family subfamily s member was reported significantly associatedwith colorectal cancer in primary colorectal cancercyp2s1 was present at a significantly higher level ofintensity compared with normal colon more importantly the presence of strong cyp2s1 immunoreactivity was associated with poor prognosis the roleof icam3 in cancer was also widely reported in published studies and the akt pathway plays an importantrole in the impact of icam3 on tumors yg kim etal reported that icam3 can induce the proliferationof cancer cells through the pi3kakt pathway additionally jk park etal showed that the icam3 can enhancethe migratory and invasive potential of human nonsmall celllung cancer cells by inducing mmp2 andmmp9 via akt pathway showed that the icam3can enhance the migratory and invasive potential ofhuman nonsmall celllung cancer cells by inducingmmp2 and mmp9 via akt pathwayalthough the role of tme and tmerelated genes inosteosarcoma patients have been initially studied by bioinformatic and statistical analyses in our research somelimitations should be elucidated firstly the treatmentinformation cannot be obtained from the target database which may influence the prognosis of osteosarcomapatients secondly two nomograms were generated andshowed good performance in our study however externalvalidation by a large cohort is needed thirdly many independent prognostic genes for osteosarcoma patients wereidentified in the present study but the potential mechanism to influence osteosarcoma remains unclear finally inthe training cohort and degs were identified asos and dfsrelated degs respectively however onlyfive os and five dfsrelated genes were identified in thevalidation cohort the different age structures smaller 0chu bmc cancer page of sample sizes and the platform covering only part of thegenes may contribute to this resultreceived february accepted july in tme plays an important role in osteosarcoma patients and related with the progression of thetumor moreover tmerelated genes can serve as prognostic biomarkers in osteosarcoma patients howeverfurther researches are needed to study the potentialmechanism and validate the nomogram that developedin our present studysupplementary informationsupplementary information accompanies this paper at doi101186s12885020072162additional file additional file additional file additional file abbreviationstme tumor microenvironment deg differentially expressed genesos overall survival dfs diseasesfree survival roc receiver characteristiccurve estimate estimation of stromal and immune cells in malignanttumor tissues using expression data target therapeutically applicableresearch to generate effective treatments go gene ontology bp biologicalprocesses mf molecular functions cc cellular components kegg kyotoencyclopedia of genes and genomes cdf cumulative distribution functionacknowledgementsnoneauthors contributionsc h l y sq t c l and yh w conceived of and designed the study c h r sand c l performed literature search r s l y and b c generated the figuresand tables l y hl r x y and jy l analyzed the data c h wrote themanuscript and sq t and l y critically reviewed the manuscript l ysupervised the research all authors have read and approved the manuscriptfundingwe received no external funding for this studyavailability of data and materialsthe data of this study are from target and geo databaseethics approval and consent to participatethe research didnt involve animal experiments and human specimens noethics related issuesconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of joint surgery the affiliated hospital of qingdao universityqingdao china 2department of medical oncology the first hospital ofchina medical university shenyang china 3department of nursing sir runrun shaw hospital affiliated to zhejiang university hangzhou china4wenzhou medical university wenzhou chinareferencesjaffe n bruland os bielack s pediatric and adolescent osteosarcoma vol new york springer science business media vander rg osteosarcoma and its variants orthopedic clin north am biermann js adkins d benjamin r brigman b chow w conrad eu 3rdfrassica d frassica fj gee s healey jh bone cancer j natl comprcancer netw simpson s dunning md de brot s grauroma l mongan np rutland cscomparative review of human and canine osteosarcoma morphologyepidemiology prognosis treatment and genetics acta vet scand chen x cates jm du yc jain a jung sy li xn hicks jm man tkmislocalized cytoplasmic p27 activates pak1mediated metastasis and is aprognostic factor in osteosarcoma mol oncol huang x yang w zhang z shao z dysregulated circrnas serve as prognosticand diagnostic markers in osteosarcoma by sponging microrna to regulatethe downstream signaling pathway j cell biochem liu m yang p mao g deng j peng g ning x yang h sun h long noncoding rna malat1 as a valuable biomarker for prognosis in osteosarcoma asystematic review and metaanalysis int j surg xu k xiong w zhao s wang b microrna106b serves as a prognosticbiomarker and is associated with cell proliferation migration and invasionin osteosarcoma oncol lett zheng w huang y chen h wang n xiao w liang y jiang x su w wens nomogram application to predict overall and cancerspecific survival inosteosarcoma cancer manag res kahlert c kalluri r exosomes in tumor microenvironment influence cancerprogression and metastasis j mol med binnewies m roberts ew kersten k chan v fearon df merad m coussenslm gabrilovich di ostrandrosenberg s hedrick cc understanding thetumor immune microenvironment time for effective therapy nat med yoshihara k shahmoradgoli m martÃnez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a
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Association of variant on the promoter of cluster ofdifferentiation in graves disease and gravesophthalmopathyYuHuei Liu123 ChiouYuan Shen1 and FuuJen Tsai3451Graduate Institute of Integrated Medicine China Medical University Taichung Taiwan 2Drug development center China Medical University Taichung Taiwan 3Department ofMedical Genetics and Medical Research China Medical University Hospital Taichung Taiwan 4Department of Pediatrics China Medical University Hospital Taichung Taiwan5School of Chinese Medicine China Medical University Taichung TaiwanCorrespondence YuHuei Liu yuhueiliumailcmuedutwThe macrophage migration inhibitory factor MIFcluster of differentiation CD74plays a role in immunological functions The present study aims to investigate whethersinglenucleotide polymorphisms SNPs in the MIF and CD74 are risk factors for developing Graves ophthalmopathy GO in patients with Graves disease GD A casecontrolstudy enrolled patients with GD with and without GO and healthy individuals SNPs were discriminated using realtime polymerase chain reaction HardyWeinbergequilibrium as well as frequencies of allele and genotype between GD patients with andwithout GO were estimated using the Chisquare test The effects of CD74 on adipocyteproliferation and differentiation were evaluated using 3T3L1 preadipocytes QuantitativeDNAimmunoprecipitation was used to detect the binding capacity of NR3C1 and FOXP3to AG oligonucleotides The results showed that individuals carrying the GG genotype atrs2569103 in the CD74 had a decreased risk of developing GD P3390 oddsratio OR confidence interval CI however patients with GDcarrying the AG genotype at rs2569103 in the CD74 had an increased risk of developing GOP0009 OR CI The knockdown of CD74 reduced adipocyteproliferation and differentiation NR3C1 had a higher affinity for A whereas FOXP3 had ahigher affinity for G of rs2569103 The results suggested the existence of a link between thegenetic variation of CD74 promoter and the risk for developing GD and GO which shouldbe considered in clinical practiceBackgroundGraves disease GD a complex autoimmune disorder that occurs more often in women is characterized by the presence of autoantibodies and thyroidstimulating immunoglobulins targeting thethyroidstimulating hormone receptor to stimulate both thyroid hormone synthesis and thyroid glandgrowth and results in hyperthyroidism and its accompanying features [] Graves ophthalmopathyGO is one common anspecific complication affecting of patients with GD [] Activation oforbital fibroblasts through proliferation and differentiation into adipocytes and myofibroblasts is thoughtto play a major role in the generation of the extracellular matrix During inflammatory cell infiltrationand edema the activation augments the volume of tissues surrounding the eyes which in turn leads to anincrease in intraocular pressure []Genetic predispositions epigenetic regulations and environmental factors are risk factors for GD andGO [] Representative studies shed new light on the pathogenesis of GD such as thyroid antigensthyroidstimulating hormone receptor and human leukocyte antigen HLA class I and II regions []However the genomewide approaches to determining the relative risks of developing GO are relativelyReceived June Revised July Accepted July Accepted Manuscript online August Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072limited [] Candidate gene approaches revealed that polymorphisms of genes involved in immune response andinflammation might be linked to the development of GO []Cluster of differentiation CD74 encoded by CD74 is an HLA class II histocompatibility antigen gamma chainalso known as HLADR antigenassociated invariant chain and a signaltransducing receptor of macrophage migration inhibitory factor MIF that maintains cell proliferation and survival [] The singlenucleotide polymorphisms SNPs in HLA class II and MIF play a role in the development of GD [] Conversely the chromosome5q3133 region where CD74 is located 5q32 may play a pivotal role in the development of GD and could be thesusceptibility region for developing GD [] Results from mRNASeq also reveal CD74 as a novel signature fD However to our knowledge there is no study on the putative impact of CD74 locus variations on the risk ofGD or GO In an attempt to contribute to the understanding of the pathogenic processes underlying GD and GO acasecontrol study was designed to evaluate the association between SNPs in the upstreamdownstream regulatoryregion of the MIFCD74 axis and the risk of developing GD and GOMethodsPatients healthy individuals and DNA isolationThe study followed the Declaration of Helsinki and was approved by the Medical Ethics Committee of China MedicalUniversity Hospital DMR100IRB144 CMUH103REC2071 A total of patients with GD females100males mean age y range y at enrollment from the China Medical University Hospital and patients had GO and did not All participants provided written informed consent Detailed descriptions of theinclusionexclusion criteria blood drawing and handling genomic DNA storage and quality assurance have beendescribed [] SNP data for ethnicitymatched healthy individuals were obtained from the Taiwan biobankSNP selection and genotypingSNPs were selected based on the following criteria i a threshold minor allele frequency MAF in the Asian population of ii primerprobe set passed by the manufacturer criteria to ensure a high genotyping success rate andiii SNP data for healthy individuals could be obtained without imputation from the Taiwan biobank Four SNPsnamely rs476240 and rs507715 in the downstream region of MIF which is also the upstream region of MIF antisense RNA [MIFAS1] as well as rs13175409 and rs2569103 in the upstream region of CD74 were analyzedGenotyping using specific primerprobe sets have been described previously []Cell cultureThe human HEK293 cells and mouse 3T3L1 preadipocytes were obtained from Bioresource Collection and Research Center BCRC Hsinchu Taiwan and maintained in Dulbeccos modified Eagles medium DMEM Thermo Fisher Scientific Waltham MA USA with fetal bovine serum Uml penicillin and μgml streptomycin and mM Lglutamine at ¦C in a humidified atmosphere of CO2CD74 knockdownShort hairpin RNAs shRNAs obtained from the RNAi core Academia Sinica Taipei Taiwan were used in CD74knockdown experiments For CD74 knockdown confluent 3T3L1 preadipocytes in sixwell dishes were incubated inOptiMEM Thermo Fisher Scientific and transfected with either CD74 shRNA or nonspecific shRNA using Lipofectamine Thermo Fisher Scientific according to the manufacturers protocol After h the medium was replacedwith complete DMEM with a differentiation cocktail μM 3isobutyl1methylxanthine μM dexamethasoneand μM insulin to induce differentiation into mature adipocytes day Western blottingEqual amounts of protein lysates were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis andthen transferred to polyvinylidene fluoride membranes After blocking with skim milk the membranes wereincubated with primary antibodies and subsequently with appropriate peroxidaseconjugated secondary antibodiesPrimary antibodies including targets catalog numbers dilutions and suppliers were as follows antibodies specific toCD74 GTX110477 were from GeneTex Hsinchu Taiwan and antibodies specific to actin MAB1501 were from MilliporeSigma St Louis MI USA The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Adipocyte differentiationThe 2day postconfluency preadipocytes were cultured in complete DMEM with a differentiation cocktail μM3isobutyl1methylxanthine μM dexamethasone and μM insulin On day of differentiation cells wereswitched to complete DMEM with μM insulin for the remaining duration of differentiationCell counting3T3L1 cells were detached from sixwell plates using trypsin Thermo Fisher Scientific resuspended in complete DMEM and counted using a cell counter Millipore every day from day Oil Red O stainingDifferentiated adipocytes were fixed in formalin and stained for min with Oil Red O MilliporeSigma working solution Oil Red O dye in isopropanol Oil Red O was extracted using isopropanol and theabsorbance was measured at nm using a spectrophotometerCell culture and extraction of nuclear proteins from established NR3C1FOXP3 and CD74 transformantsCells were transfected with the pCMV3CMycNR3C1 pCMV3CMycFOXP3 or pCDNA4CD74 usingthe Lipofectamine kit Thermo Fisher Scientific according to the manufacturers protocol The nuclear proteinswere extracted using NEPER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific supplementedwith protease inhibitor cocktail and phosphatase inhibitors Roche Basel Switzerland according to the manufacturers protocolQuantitative DNA immunoprecipitation qDNAIP assayqDNAIP assays were performed on nuclear extracts from established FOXP3 and NR3C1 transformantsDNA binding of FOXP3 or NR3C1 was assessed using the annealed double strand oligonucleotides 5cid3biotinlabeled rs2569103A probes 5cid3CCAAATGGCTGGTTTCAGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCTGAAACCAGCCATTTGG3cid3 as well as 5cid3biotinlabeled rs2569103G probes 5cid3CCAAATGGCTGGTTTCGGGGCTGGAGATGGGGG3cid3 and 5cid3CCCCCATCTCCAGCCCCGAAACCAGCCATTTGG3cid3 PURIGOBiotechnology Taipei Taiwan For the binding reactions μg of nuclear proteins were incubated with or without labeled oligonucleotides in binding buffer [ mM TrisHCl pH mM NaCl mM MgCl2 mMEDTA mM DTT mgml polydIdC and glycerol] for min at ¦C in a final volume of μl FOXP3 or NR3C1nucleotide complexes were crosslinked with formaldehyde final concentration for min at room temperature followed by immunoprecipitation with antibodies specific to Myc tag GTX115046 GeneTex and Protein AG magnetic beads GE Healthcare Immunoprecipitated DNA was detected usinghorseradish peroxidaseconjugated streptavidin The reaction was developed with the 33cid355cid3tetramethylbenzidinereagent Sigma and read at nm with a Microplate reader BioRad Hercules CA USAStatistical analysesThe statistical analyses were performed using the PASW Statistics software from IBM Armonk NY USAA ttest was used to evaluate the associations between GO and age A Chisquare test was used to evaluate the associations between polymorphisms and GD or GO Screening for linkage disequilibrium LD was performed usingHaploview ver [] A twotailed Pvalue less than with Bonferroni correction was considered statistically significant [] Logistic regression with a confidence interval CI was used to estimate odds ratiosORsResultsDemographic data clinical characteristics and their correlations withGO in patients with GDThe frequency distributions of clinical characteristics such as goiter nodular hyperplasia myxedema vitiligo andage in male and female groups were compared between the patients with GD with or without GO As demonstratedin Table gender and age were significantly associated with GO in patients with GD Even myxedema was associatedwith GO in patients with GD however due to a limited number of cases the association needs further investigation The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Demographic data and clinical characteristics of graves disease patients with or without graves ophthalmopathyCharacteristicGDnonGO N GDGO N PNumber of patientsFemale genderAge of diagnosis Year Mean SD[Range]Presence of goiterNo1a1bPresence of nodular hyperplasiaPresence of myxedemaPresence of vitiligoWith radioiodine therapy historyWith thyroid surgery historyWith smoke historyFree T3 pgmlFree T4 ngdlT3 ngdlT4 μgdlTSH μIUmlTRAb positive [] [] Abbreviations GD graves disease GO graves ophthalmopathy N numberaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbSigniï¬cance of age were evaluated by t testP005P00010039a 105b 0165a0539a0039a 0743a0273a0227a0527a0900a0692a0146a0310a0479a0482aThese results adhered to other epidemiological results that GO occurred more commonly in the middleaged femalepopulationLD among SNPs of MIF and CD74Four SNPs of the MIF and CD74 were genotyped to determine whether polymorphisms in these genes influencethe development of GO in patients with GD The distribution of the four SNPs fit the HardyWeinberg equilibriumHWE in patients with GD and healthy individuals However the strong r208 LD r2 values calculated for thetwo SNPs at the CD74 in healthy individuals were not observed in patients with GD with or without GO suggestingthat there is more variation in the extent of LD within CD74 in patients with GD Figure Allele and genotype distributions of CD74 contribute to GDGOdevelopmentNo significant association was found in the examined SNPs of MIF nor was a significant association found betweenthe polymorphisms and the clinical features or the indicators of thyroid function including free triiodothyronineT3 free thyroxine T4 thyroid stimulating hormone TSH and thyrotropin receptor antibodies TRAbs in patients with GD However allele frequencies showed that individuals carrying a G allele at rs2569103 in the CD74 hada reduced risk of developing GD P0005 OR CI Table Genotype frequenciesfurther showed that individuals carrying the GG genotype at rs2569103 in the CD74 had a reduced risk of developing GD P3390 OR CI which was consistent with results from allelefrequencies however the patients with GD carrying the AG genotype at rs2569103 in the CD74 had an increasedrisk of developing GO P0009 OR CI Table The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Linkage disequilibrium LD values between the two polymorphisms rs13175409 and rs2569103 in the CD74region in a TaiwaneseChinese populationThe color scale reflects the strength of LD between the two single nucleotide polymorphisms SNPs A Healthy individuals BPatients with Graves disease GD with and without Graves ophthalmopathy GO C Patients with GD without GO D Patientswith GD with GOTable Allele distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDPaControl vs GDOR 95CINonGO vsGO PaNonGO vsGO OR95CIMIF rs476240AGMIF rs507715ACCD74 rs13175409CTCD74 rs2569103AG 0929bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOdds ratios and CI per genotype were estimated by applying unconditional logistic regressionP005 with Bonferroni correction OR with signiï¬canceKnockdown of the expression of CD74 inhibits 3T3L1 adipocytedifferentiationThe swelling of extraocular orbital fat is one reason that the development of GO is triggered [] To understand thepossible regulation between CD74 and adipocyte differentiation 3T3L1 cells were chosen as an experimental modelThe expression of CD74 in CD74 knockdown CD74KD cells by shRNA was confirmed as compared with those withcontrol of shRNA Figure 2A Cell numbers of CD74KD and control cells were counted every day The knockdownof CD74 decreased cell proliferation from days after induction Figure 2B In addition the degree of Oil Red The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Table Genotype distributions of MIF and CD74GenotypesControl N GDnonGO NGDGO N Control vs GDP aControl vs GDOR 95CINonGO vsGO P aNonGO vsGO OR95CIMIF rs476240AAAGGGMIF rs507715AAACCCCD74 rs13175409CCCTTTCD74 rs2569103AAAG2495cGG b0154b2467bAbbreviations CI conï¬dence interval GD graves disease GO graves ophthalmopathy N number OR odds ratiosaFrequencies of genotypes were determined by the chisquare test using or contingency tablesbOR and CI per genotype were estimated by applying unconditional logistic regressioncOR and CI per genotype were estimated by adjusting with gender age and myxedemaP005 with Bonferroni correctionOR with signiï¬canceFigure Changes in adipocyte differentiation and proliferation after knockdown of CD74A Endogenous expression of CD74 protein in 3T3L1 cells was examined and knockdown of CD74 was examined by Westernblotting Actin was used as an internal control B The downregulation of CD74 inhibits cell growth 3T3L1 cells were detachedfrom sixwell plates and counted P001 P0001 CD74 knockdown vs control cells C Cells were stained with Oil Red Oafter inducing differentiation Quantitative analyses were performed by measurement of optical density OD at nm in extractsfrom Oil Red Ostained cells transfected with CD74 short hairpin RNA shRNA and control shRNA P0001 CD74 knockdownvs control cellsO staining was weaker in CD74KD cells than in control cells on day and on day respectively forCD74 shRNA vs control cells Figure 2C The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoterdepends on SNP rs2569103The CD74 SNP rs2569103 was located within the upstream region of CD74 and showed the strongest associationwith the disease making it a possible target for transcription factors Indeed the putative transcription factorbindingsites were predicted using PROMO [] At SNP rs2569103 the A allele generates motifs for nuclear receptorsubfamily group C member NR3C1 TCAGG whereas the G allele generates a motif for forkhead box P3FOXP3 GTTTCG Bulk RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasetsPRJEB4337 were demonstrated Figure 3A To interpret the possible regulatory mechanisms of these moleculespublished mRNA expression results were explored The mRNA expression of NR3C1 only showed a negative correlation with that of CD74 in thymoma samples Pearsons correlation Spearmans correlation Figure3B whereas the mRNA expression of FOXP3 showed a positive correlation with that of CD74 Pearsons correlation Spearmans correlation in thymoma samples welldifferentiated papillary thyroidcarcinoma and welldifferentiated thyroid cancer respectively Figure 3CE The qDNAIP results supported thatNR3C1 tends to bind to probes with promoter sequence containing AA at rs2569103 whereas FOXP3 tends to bindto probes with promoter sequence containing GG at rs2569103 Figure 3F These results suggested that the CD74expression may be orchestrated by complex transcription factor networks The AA genotype may play a role in response to NR3C1induced CD74 downregulation whereas the GG genotype on rs2569103 on the CD74 promotermay play an additional role in response to FOXP3induced CD74 upregulationDiscussionEnvironmental factors and genetic loci have been thought to be associated with immune regulation [] Here weidentified new candidates CD74 alleles and genotypes for the susceptibility of GD and GO in a TaiwaneseChinesepopulation CD74 is involved in adipocyte differentiation through its differential promoter binding affinity for transcription factors To the best of our knowledge this is the first study to demonstrate novel CD74 polymorphisms inassociation with the development of GD and GO Our results support wholegenome screening studies in that thechromosome 5q32 may play a role in generating GD and GO in humansThe thyroid gland of patients with GD revealed marked enlargement of the gland due to autoantibodies Patientswith accompanying GO exhibited enlargement of the retroorbital connective tissue and extraocular muscles inpart due to the inflammatory deposition of glycosaminoglycans collagen and fat [] Indeed genes involved inthe regulation of cell survival DNA transcription and protein synthesis have been considered risk factors for GDand GO [] Overexpression of CD74 plays a crucial role in preventing hyperreactivity between immature antigens and major histocompatibility complex class II as well as cell growth and survival whereas downregulation ofCD74 is often correlated with autoimmunity and cell apoptosis [] Upon expression of surface CD74 the cellsmay transduce survival signaling through extracellular signalregulated kinase or cJun Nterminal kinase JNKmitogenactivated protein kinase MAPK pathways or AKT pathways in a MIFdependent manner thereby improving cell survival and proliferation [] Due to the limitation to find identical cells expressed GG or AA genotypeon rs2569103 current results we did not show the direct impact of these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients withor without GO although loss the protective GG genotype most of them hold AG heterogenous genotype insteadsuggested the lossofprotect effect on the disease In the present study cellbased experiments showed that CD74 isinvolved in adipocyte differentiation but the link toward GO development remained to be investigated On the otherhand the GG genotype on rs2569103 with a higher frequency in healthy individuals Table increased the bindingof FOXP3 to the CD74 promoter Figure 3F thereby increasing CD74 upregulation and protecting autoimmuneresponses Conversely the AA genotype on rs2569103 increases the binding of NR3C1 to the CD74 promoter whichdownregulates CD74 and increases autoimmune response and manifestations of GDGO Due to the limitation tofind identical cells expressed GG or AA genotype on rs2569103 current results we did not show the direct impactof these transcription factors to the CD74 expression Further evidence such as RNAseq as secondary data was warranted The results showed that GD patients with or without GO although they lost the protective genotype mostof them hold the AG heterogenous genotype instead suggesting the lossofprotection effect of the disease Furtherstudies on the detailed mechanisms through CD74derived adipocyte differentiation are warrantedConversely the ligand of CD74 MIF has previously been reported to be counterregulatory to glucocorticoid secretion [] The glucocorticoidinduced MIF secretion was noted at min after dexamethasone administration[] In addition nonsteroidal antiinflammatory drugs such as aspirin ibuprofen and naproxen have been used torelieve the pain and inflammation of GO This evidence further supports the crucial role of CD74 in the transduction The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072Figure Different binding afï¬nities of NR3C1 and FOXP3 for CD74 promoter depends on singlenucleotide polymorphismSNP rs2569103A RNAseq analysis of NR3C1 and FOXP3 in thyroid and fat tissues from public datasets PRJEB4337 BE Bioinformaticanalysis of mRNA expression correlation between NR3C1 and CD74 or FOXP3 and CD74 The mRNA expression of NR3C1 andCD74 in thymoma samples B and the mRNA expression of FOXP3 and CD74 in thymoma samples C welldifferentiated papillarythyroid carcinoma D and welldifferentiated thyroid cancer E F Probe with promoter sequence containing rs2569103 probe Ahas a higher affinity for NR3C1 whereas G at rs2569103 probe G has a higher affinity for FOXP3 as shown by quantitative DNAimmunoprecipitation qDNAIP assay P001 P0001 probe A vs probe G The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20202072101042BSR20202072of MIF signaling However due to the limited population of the minor polymorphism the present study is unable toreach the interactions among cells and molecules in the orbital microenvironment and their association toward thetarget polymorphism due to the inaccessibility of the orbital tissues The current finding may have further implications for understanding the link between the polymorphismexpression of CD74 and current treatments for GOatherapeutic effect issue that might be of value for future treatment strategies targeting MIF or CD74In conclusion the current study identified new SNPs in the CD74 that were found to be associated with GD and GOin a TaiwaneseChinese population Biological studies provide insights into the genetic information that influencesthe development of GD and GO via adipocyte proliferation and differentiationPerspectives¢The impact of genetic factors on the orbital microenvironment cannot be closely monitored due tothe inaccessibility of the orbital tissue Studies on feasible cellbased models may help elucidate howgenetic factors such as CD74 SNPs modulate the target gene expression¢¢The present study combined clinical observations and cell models to investigate how CD74 polymorphisms affect adipocyte proliferation and differentiationThe present clinical observations suggest that the genetic factors of CD74 should be considered inclinical practiceCompeting InterestsThe authors declare that there are no competing interests associated with the manuscriptFundingThis work is supported by Ministry of Science and Technology Taiwan [grant numbers MOST 1042815C039002B and MOST1072320B039032MY3] the peak project and thematic project of Academia Sinica Taiwan the higher education sproutproject by the Ministry of Education MOE Taiwan via Drug Development Center of China Medical University from The FeaturedAreas Research Center Program and China Medical University [grant numbers CMU105S33 and CMU106S46] TaichungTaiwanAuthor ContributionYHL proposed the concept designed the experiment anized the study wrote and reviewed the manuscript CYS performed the experiments FJT coordinated patient enrollment collected the clinical samples and applied official applicationAcknowledgementsWe thank Taiwan Biobank for providing related data all anonymous for our research The sponsorfunding anization had norole in the design or conduct of this researchAbbreviationsCD74 cluster of differentiation CI confidence interval FOXP3 forkhead box P3 GD graves disease GO graves ophthalmopathy HLA human leukocyte antigen HWE HardyWeinberg equilibrium JNK cJun Nterminal kinase LD linkagedisequilibrium MAPK mitogenactivated protein kinase MIF macrophage migration inhibitory factor NR3C1 nuclear receptorsubfamily group C member OR odds ratio PCR polymerase chain reaction qDNAIP quantitative DNA immunoprecipitation SNP singlenucleotide polymorphism T3 triiodothyronine T4 free thyroxine TRAb thyrotropin receptor antibody TSHthyroid stimulating hormoneReferences Smith TJ and Hegedus L Graves Disease N Engl J Med 101056NEJMra1510030 Brent GA Clinical practice Graves disease N Engl J Med 101056NEJMcp0801880 Ginsberg J Diagnosis and management of Graves disease CMAJ Canadian Med Assoc J J de lAssoc Med Canadienne The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative CommonsAttribution License CC BY 0cBioscience Reports BSR20202072101042BSR20202072 McIver B and Morris JC The pathogenesis of Graves disease Endocrinol Metab Clin North Am 101016S0889852905702991 Bednarczuk T Gopinath B Ploski R and Wall JR Susceptibility genes in Graves ophthalmopathy searching for a needle in a haystackClin Endocrinol Oxf 101111j13652265200702854x Anvari M Khalilzadeh O Esteghamati A Esfahani SA Rashidi A Etemadi A et al Genetic susceptibility to Graves ophthalmopathy therole of polymorphisms in proinï¬ammatory cytokine genes Eye Lond 101038eye2009244 Bahn RS Understanding the immunology of Graves ophthalmopathy Is it an autoimmune disease Endocrinol Metab Clin North Am vi Manji N CarrSmith JD Boelaert K Allahabadia A Armitage M Chatterjee VK et al Inï¬uences of age gender smoking and familyhistory on autoimmune thyroid disease phenotype J Clin Endocrinol Metab 101210jc20061402 Stan MN and Bahn RS Risk factors for development or deterioration of Graves ophthalmopathy Thyroid Ofï¬cial J Am Thyroid Assoc 101089thy20101634 Bahn RS and Heufelder AE Pathogenesis of Graves ophthalmopathy N Engl J Med Tomer Y Barbesino G Greenberg DA Concepcion E and Davies TF Mapping the major susceptibility loci for familial Graves andHashimotos diseases evidence for genetic heterogeneity and gene interactions J Clin Endocrinol Metab Tomer Y Ban Y Concepcion E Barbesino G Villanueva R Greenberg DA et al Common and unique susceptibility loci in Graves andHashimoto diseases results of wholegenome screening in a data set of multiplex families Am J Hum Genet 101086378588 Gianoukakis AG and Smith TJ Recent insights into the pathogenesis and management of thyroidassociated ophthalmopathy Curr OpinEndocrinol Diabetes Obesity 101097MED0b013e32830eb8ab Shiina T Ota M Shimizu S Katsuyama Y Hashimoto N Takasu M et al Rapid evolution of major histocompatibility complex class I genesin primates generates new disease alleles in humans via hitchhiking diversity Genetics 101534genetics106057034 Liu YH Chen YJ Wu HH Wang TY and Tsai FJ Single nucleotide polymorphisms at the PRR3 ABCF1 and GNL1 genes in the HLA class Iregion are associated with Graves ophthalmopathy in a genderdependent manner Ophthalmology 101016jophtha201404027 Wang S Sun H Chen HY Zhao ZF Yang Y Zhao YJ et al Intercellular adhesion molecule gene polymorphisms do not contribute toGraves disease in Chinese patients Endocrine 101007s1202000700329 Liu YH Chen RH Chen WC Tsai Y Wan L and Tsai FJ Disease association of the CD103 polymorphisms in Taiwan Chinese Gravesophthalmopathy patients Ophthalmology 101016jophtha200912037 Bednarczuk T Hiromatsu Y Seki N Ploski R Fukutani T Kurylowicz A et al Association of tumor necrosis factor and human leukocyteantigen DRB1 alleles with Graves ophthalmopathy Hum Immunol 101016jhumimm200402033 Khalilzadeh O Anvari M Esteghamati A Mahmoudi M Tahvildari M Rashidi A et al Graves ophthalmopathy and gene polymorphisms ininterleukin1alpha interleukin1beta interleukin1 receptor and interleukin1 receptor antagonist Clin Exp Ophthalmol Siegmund T Usadel KH Donner H Braun J Walï¬sh PG and Badenhoop K Interferongamma gene microsatellite polymorphisms inpatients with Graves disease Thyroid Ofï¬cial J Am Thyroid Assoc 101089thy199881013 Wong KH Rong SS Chong KK Young AL Pang CP and Chen LJ Genetic Associations of Interleukinrelated Genes with GravesOphthalmopathy a Systematic Review and Metaanalysis Sci Rep 101038srep16672 Bucala R and Shachar I The integral role of CD74 in antigen presentation MIF signal transduction and B cell survival and homeostasis MiniRev Med Chem 1021741389557515666150203144111 Leng L Metz CN Fang Y Xu J Donnelly S Baugh J et al MIF signal transduction initiated by binding to CD74 J Exp Med 101084jem20030286 Liu YH Chen CC Yang CM Chen YJ and Tsai FJ Dual effect of a polymorphism in the macrophage migration inhibitory factor gene isassociated with newonset Graves disease in a Taiwanese Chinese population PLoS ONE e92849 101371journalpone0092849 Nakabayashi
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high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with Åukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in omics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a Åukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a symptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that symptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie Åukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see membership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in fuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of theÅukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see fuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see dendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 [ ] and lettingd be a dissimilarity relation which is obtained from r bydx y rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie Åukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute s if sielse ci sti si ª si ¦ sicid8 si replace si with si si and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ¤ i ¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let a be the identity matrixb for each ci calculate e a a ª a ¦ cic if a is not changed a a and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the Ï2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the Ï2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have Ï2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logranks test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logranks test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ] to determine the number of clusters for a method we used the elbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ] 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrankstest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean log10 of logranks testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg high medium low in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of log10 of logranks testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes ac results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliparthenope tecniche di machine learning e soft computing per lelaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli parthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason ciaramella a cocozza sand clustering of genomic data neural netw iorio f miele g napolitano f pinelli m raiconi g tagliafer
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mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5
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"rapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Elizabeth Ann L Enninga2 Fabrice Lucien Matteoni3 Jacob J Orme Heather Dale4 Edwin Burgstaler4 Susan M Harrington3 Matthew K Ball4 Aaron S Mansfield1 Sean S Park5 Mathew S Block1 Svetomir N Markovic1 Yiyi Yan1 Haidong Dong3 Roxana S Dronca6 Jeffrey L Winters4To cite Orme a0JJ Enninga a0EAL Lucien Matteoni a0F et a0al Therapeutic plasma exchange clears circulating soluble PD L1 and PD L1 positive extracellular vesicles Journal for ImmunoTherapy of Cancer 20208e001113 101136jitc2020001113 º Additional material is published online only To view please visit the journal online http dx jitc Accepted June Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Division of Medical Oncology Mayo Clinic Rochester Minnesota USA2Department of Obstetrics and Gynecology Mayo Clinic Rochester Minnesota USA3Department of Urology Mayo Clinic Rochester Minnesota USA4Department of Laboratory Medicine and Pathology Mayo Clinic Rochester Minnesota USA5Department of Radiation Oncology Mayo Clinic Rochester Minnesota USA6Department of Hematology and Oncology Mayo Clinic Florida Jacksonville Florida USACorrespondence toDr Jacob J Orme orme jacob mayo eduBackground Trans acting programmed death ligand PD L1 derives from malignant cells in three known forms High levels of secreted splice variant PD L1 sPD L1 ADAM10ADAM17 shed sPD L1 and PD L1 positive extracellular vesicles evPD L1 each predict poor prognosis and limited response to PD L1 checkpoint inhibitors in cancer To our knowledge no clinical intervention has reduced any of these circulating forms of extracellular PD L1 Here we explore therapeutic plasma exchange TPE as a treatment to reduce circulating extracellular PD L1Results In patients with melanoma sPD L1 levels above ngmL predicted inferior overall survival In patients undergoing TPE for non malignant indications each TPE session removed a mean sPD L1 and evPD L1 detectable in plasma TPE also reduced total and ADAM10 positive extracellular vesiclesConclusion Here we report the first known clinical intervention to remove either sPD L1 or evPD L1 from plasma in vivo TPE reduces plasma sPD L1 and evPD L1 in vivo and may have a role in treatment with immunotherapy TPE may also prove useful in patients with other extracellular vesicle related conditionsINTRODUCTIONOvercoming initial or acquired resistance to programmed death ligand PD L1 immune checkpoint inhibitors is a major area of unmet need for many cancers1 Although the full scope of mechanisms of resistance to these therapies has yet to be determined different forms of tumor derived extracellular PD L1 have been linked to resistance in multiple clinical studies2in Malignant cells produce trans acting extracellular PD L1 three distinct forms First tumor cells transcribe and secrete soluble PD L1 sPD L1 splice variants4 Second enzymes ADAM10 and ADAM17 shed sPD L1 ectodomain directly from the tumor cell surface6 Both forms of sPD L1 carry known homodimerization domains and can be detected by ELISA sPD L1 can outcompete PD L1 inhibitors kill CD8 T cells and limit the ability of healthy peripheral blood mononuclear cells to kill tumor cells in vitro6 In a third form tumors generate extracellular vesicles EVs bearing surface PD L18 PD L1 positive EVs evPD L1 exhibit similar properties to sPD L1 in systemic circulation9 Each type of trans acting extracellular PD L1 correlates with poor survival in multiple clinical trials online supplementary table While broad spectrum pharmacological inhibitors and genetic manipulation have been shown to reduce release of these forms of PD L1 in culture or animal models none are suitable for clinical use To date we know of no reported clinical intervention that safely and reliably eliminates any of these forms of immunosuppressive systemic extracellular PD L1Therapeutic plasma exchange TPE is a procedure in which blood is passed through an apheresis machine separating plasma from cellular components Removed plasma is discarded and replaced with either colloid eg albumin or crystalloid and colloid solutions Unlike dialysis which removes small ions by diffusion TPE removes plasma restricted substances like antibodies that are too large for rapid diffusion On average each TPE session removes approximately of large non cellular plasma restricted intravascular components10It is unknown whether sPD L1 approximately kDa or PD L1 positive EVs nm are plasma restricted non diffusing and unbound If so we hypothesized that TPE could efficiently remove these substances from patient blood Such an intervention could if effective improve response to immunotherapyOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Figure Soluble programmed death ligand PD L1 suppresses antitumor immunity and predicts overall survival in patients with melanoma A A model of three known tumor derived extracellular PD L1 forms evPD L1 ADAM10ADAM17 cleaved soluble PD L1 sPD L1 ectodomain and secreted splice variant sPD L1that downregulate antitumor immunity and prevent response to PDL1 inhibition B A Kaplan Meier plot shows significantly worse overall survival for patients with melanoma exhibiting high ¥ ngmL versus low ngmL plasma sPD L1 levels p0005 C Patients with melanoma exhibited a higher mean plasma sPD L1 level ngmL in comparison to healthy controls ngmL p0001RESULTSsPDL1 levels predict overall survival in patients with melanomaEach form of extracellular PD L1 acts in trans as a systemic immunosuppressant through PD1 signaling figure 1A online supplementary table To confirm the clinical impact of plasma sPD L1 we measured sPD L1 levels in a retrospective cohort of patients with melanoma Exploratory analysis of overall survival OS determined a working cut off value of sPD L1 ¥ ngmL and baseline characteristics at the time of entry into study were similar online supplementary table Patients with high plasma sPD L1 levels experienced inferior median OS compared with patients with low plasma sPD L1 levels figure 1B vs months p0005 In comparison to healthy age matched controls patients with melanoma exhibited higher mean plasma sPD L1 figure 1C ngmL vs ngmL p0001 In a multivariate Cox proportional hazards analysis high sPD L1 prior to treatment predicted worse survival HR CI to p0025 when accounting for advanced age not significant sex not significant late stage p0002 and high serum LDH p001 online supplementary table TPE significantly reduces plasma sPDL1 levelsWe hypothesized that TPE may remove extracellular PD L1 in its various forms figure 2A To address this question we prospectively enrolled patients undergoing planned TPE figure 2B Twenty eight patients met inclusion criteria of which provided informed consent Baseline patient characteristics are in table One patient was excluded for biotin containing supplement use as biotin interferes with the established sPD L1 detection assay The remaining patients underwent plasma exchange and sample collection before and after the procedure as described Discarded plasma samples from the TPE device waste bag for each session were also collected sPD L1 was measured in each sample and most patients undergoing TPE exhibited sPD L1 levels above the clinically relevant ngmL cut off from the retrospective melanoma studyMost patients undergoing TPE did not have an active cancer diagnosis Baseline sPD L1 levels in all patients were compared with matched normal controls and patients with melanoma online supplementary fig and some patients exhibited sPD L1 above the clinically significant cut off level determined in the retrospective melanoma cohort Patients with high baseline sPD L1 levels were significantly more anemic than patients with lower baseline sPD L1 even when controlling for the higher number of female subjects in the high sPD L1 group female only mean Hgb vs p004 male only mean Hgb vs p003 Groups were otherwise similar TPE significantly reduced plasma sPD L1 levels in patients receiving albumin only ie no Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Therapeutic plasma exchange TPE significantly reduces plasma soluble programmed death ligand sPD L1 levels A A model of the TPE procedure in which patient plasma is separated and replaced to extract non cellular substances confined to the plasma B A diagram of the present study in which patients undergo plasma exchange C All plasma levels of sPD L1 immediately prior to pre and after post TPE using albumin replacement fluid are plotted TPE significantly reduced sPD L1 levels in patient plasma by Wilcoxon signed rank test p00001 D In a typical timeline patient sPD L1 levels are reduced by each successive session of TPE gray bars See also table a0 online supplementary figures FFP replacement fluid figure 2C p00001 Removed sPD L1 was detected in matching plasma samples from the TPE procedure waste bag Each TPE session removed a mean of detectable plasma sPD L1 mean regeneration of sPD L1 between sessions was table TPE sessions were usually separated by daysincluding sessions A representative individual patient treatment course showing sPD L1 reduction over four successive TPE sessions is also shown figure 2D All individual patient TPE courses involving donated human blood products eg fresh frozen plasma or FFP are shown in online supplementary fig Pre TPE and post TPE sPD L1 levels for all sessions are also shown online supplementary fig TPE significantly reduced plasma sPD L1 even when sessions requiring donated FFP were included p00001FFP is sometimes given during TPE for patients with increased risk of bleeding We observed that some patients receiving FFP with low baseline sPD L1 experienced rapid increases in sPD L1 levels after TPE presumably passively acquired from donor plasma as this was not observed in patients receiving albumin replacement alone sPD L1 was not detected in the discarded plasma from the procedure for these patients We observed a mild association between post FFP infusion rises in sPD L1 levels and the blood type of the recipient mainly in patients with O type blood Individuals with group O blood are universal recipients of FFP products and universal donors of cellular products due to a lack of ABO group antigens and the presence of preformed anti A and anti B antibodies respectively Recipients of FFP usually receive a mixture of compatible plasma from multiple donors To determine whether blood type in FFP donors is associated with FFP sPD L1 content we measured sPD L1 by ELISA in plasma from multiple FFP donors online supplementary fig O negative plasma donors showed higher sPD L1 levels than donors with most other blood typesTPE efficiently reduces plasma EV levels in vivoWe postulated that TPE may remove PD L1 positive EVs evPD L1 from patient blood To address this question we measured total EV levels and evPD L1 in each sample by flow cytometry We also determined the impact of TPE on platelet derived CD61 positive EVs one of the most abundant EV Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Table Patient baseline characteristicsCharacteristicHigh sPDL1 n17Low sPDL1 n7StatisticStarting sPD L1Age yearsGender FActive cancer YesImmunotherapyNoneAtezolizumabPembrolizumabPlasma exchange indicationCNS demyelination myelitis MS NMO myelopathyImmune encephalitisMyasthenia gravisParaneoplastic syndrome encephalitis neuropathy pemphigusParaproteinemia Waldenström cryoglobulinemia kappa gammopathySusac syndromeTransplant rejection heart kidneyPre TPE white cell countPre TPE hemoglobinPre TPE creatinine to to to to to to to to to to F1223619 p0001F122035 p0558X2070 p0404 X20 p0967 X2288 p0237 X2288 p0315 F122078 p0385F122860 p0008F122382 p0063Patients undergoing therapeutic plasma exchange TPE are compared by starting soluble programmed death ligand sPD L1 level above or below survival cut off established in patients with melanoma ngmL For categorical variables n is given For continuous variables mean quartiles is givenKruskal Wallis PearsonCNS central nervous system MS multiple sclerosis NMO neuromyelitis opticasubpopulations in blood CD61 is a platelet marker and ADAM10 positive low density EVs ADAM10 has been implicated in exosome loading and pathogenesis11TPE significantly reduced total plasma particle concentration figure 3A average per exchange p00001 TPE sessions requiring FFP or other human blood product were excluded from analysis leaving session pairs PD L1 positive evPD L1 and ADAM10 positive EVs were Table Soluble programmed death ligand sPD L1 reduction and regeneration per exchange Reduction per exchangen44Mean SDMedian min max Regeneration between exchangesMean SDMedian min maxRegeneration per cycle pgmLMean SDMedian min max n44 38k 154kFor each exchange not requiring FFP percent sPD L1 reduction and regeneration between each exchange is calculated n44FFP fresh frozen plasmasignificantly reduced by TPE figure 3BC p0028 and p00001 respectively and were detected in waste plasma data not shown Each TPE session using albumin based replacement fluid with pre TPE levels above one million removed a mean of detectable PD L1 positive EVs from patients online supplementary table Platelet derived CD61 positive EVs while abundant were not significantly reduced by plasma exchange figure 3DIndividual patient courses showing total plasma PD L1 positive ADAM10 positive and CD61 positive EV levels before and after each TPE session are shown in online supplementary fig with exemplary nanoflow plots in online supplementary fig Three successive TPE sessions consistently depleted total PD L1 positive and ADAM10 positive but not CD61 positive EVs These trends were less pronounced when sessions in which patients received donor FFP were included online supplementary fig In normal control FFP donors blood type did not correlate with plasma EV concentrations online supplementary fig DISCUSSIONExtracellular PD L1in the form of splice variant sPD L1 ADAM10ADAM17 cleaved sPD L1 ectodomain or evPD L1 positive EVs evPD L1mediates resistance to PD L1 inhibitors4 These forms are resistant to clinically tested Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c accessFigure Plasma exchange efficiently reduces total programmed death ligand PD L1 positive and ADAM10 positive extracellular vesicle EV levels in vivo Plasma levels of total EVs immediately prior to pre and after post therapeutic plasma exchange TPE are plotted TPE significantly reduced A total p00001 B PD L1 positive p0028 and C ADAM10 positive p00001 but not D CD61 positive EVs p094 by Wilcoxon signed rank test See also online supplementary figures and online supplementary table combinations of chemotherapy and immunotherapies in vitro and in animal models and are associated with poor prognosis in many cancer types In the present study we found that TPE reliably reduces sPD L1 and evPD L1 This reduction was most pronounced over approximately three consecutive single plasma volume treatment sessions Given the dramatic reduction in sPD L1 and evPD L1 TPE may provide a novel approach to combating these mechanisms of resistanceWhile promising the present study was limited to patients receiving TPE mainly for non oncological indications over a short time horizon One patient in the study had melanoma receiving pembrolizumab and exhibited high pre TPE evPD L1 that was reduced on treatment Another patient had a uterine neuroendocrine tumor receiving atezolizumab and exhibited high pre TPE sPD L1 that was reduced on treatment The purpose of TPE in all cases however was to blunt paraneoplastic autoimmunity or treat some other coexisting autoimmune disordernot the underlying malignancy Neither of these patients experienced improvement in their autoimmunity after TPE The source of sPD L1 and evPD L1 in these cases is uncertain as no assay currently differentiates tumor derived and non tumor derived PD L1 We observed that these forms of immunosuppressive extracellular PD L1 exist naturally although at lower levels in healthy subjects than in patients with cancer suggesting a potentially beneficial immunoregulatory role While we observed some regeneration for both sPD L1 and evPD L1 between TPE sessions it is unknown to what degree malignant cells may regenerate and maintain extracellular PD L1 homeostasis Relatedly it is uncertain how other plasma substances removed by TPE may affect response to immunotherapy or more broadly cancer immunity overall Nor is it known at what level sPD L1 andor PD L1 positive EV removal would become clinically relevant These facets will be tested in future studiesImmunotherapy resistance is widespread and costly In most instances PD L1 inhibitors such as pembrolizumab nivolumab atezolizumab durvalumab and avelumab are used in situations in which less than half of tumors are expected to respond Of patients that benefit many do not experience a sustained durable response These treatments represent a major investment the cost of PD L1 checkpoint blockade commonly reaches several hundred thousand dollars over the course of therapyTo our knowledge this is the first report of an intervention to achieve consistent rapid reduction in either sPD L1 or PD L1 positive EVs in a clinical setting TPE is safe and commonly prescribed Thus preimmunotherapy TPE may combat immunotherapy resistance In light of the heavy investment that anti PD L1 therapy entails the added cost of TPE in selected patients may be practical14 While the durability of extracellular PD L1 reduction in malignancy will be explored in future studies the present study suggests that this approach warrants further investigationOrme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c access Beyond evPD L1 this is also to our knowledge the first known intervention to reliably deplete EVs in a clinical setting EVs have been implicated in oncogenesis and metastasis through miRNA carriage and direct protein signaling independent of PD L115 Beyond cancer EVs have also been implicated in autoimmunity17 agingneurodegeneration18 infection19 obesity20 and heart disease21 The selective removal of ADAM10 positive ie likely immune derived versus CD61 positive ie likely platelet derived EVs in this study suggests flexible selective EV depletion may be both possible and expedient in other indications The present study is only a proof of concept and additional exploratory studies in these areas are necessaryIn summary TPE reduces extracellular forms of PD L1 associated with PD L1 checkpoint inhibitor resistance Future studies will explore the potential role of TPE in improving cancer immunotherapyMETHODSRetrospective melanoma outcomes study designIn a retrospective analysis baseline blood samples from patients with melanoma prior to treatment in one of three clinical trials by the North Central Cancer Treatment Group N057e22 N077523 and N087924 between and were tested for sPD L1 of patients were diagnosed with cutaneous melanoma and none received immunotherapy treatments Blood from healthy volunteers undergoing blood donation at Mayo Clinic was also testedProspective TPE study designIn an investigator initiated label single center observational study adults undergoing TPE were approached from December through March In consenting subjects samples of whole blood immediately prior to TPE and on completion of the procedure were collected in ACD vacutainers BD In each case the first mL of blood was discarded to avoid contamination after which an mL sample was obtained in sequence Plasma was isolated by centrifugation A postprocedure blood sample was obtained after completion of the procedure In addition matching samples from discarded plasma from the procedure waste bag were collected Samples were obtained from up to four consecutive procedures for each patient If a patient underwent fewer than four TPE procedures samples were obtained from as many procedures as possiblePatients included were adults able to give consent and undergoing TPE for a variety of hematological neurological and renal diseases as indicated by published guidelines from the American Society for Apheresis ASFA or according to the medical judgment of the referring physicians25 Patients taking biotin supplements were excluded from the study due to biotin interference with the sPD L1 ELISA assay Procedures were performed using centrifugation based cell separators either the Fenwal Amicus Fresenius KABI USA LLC Lake Zurich Illinois USA or the Spectra Optia Terumo BCT Lakewood Colorado USA For each patient a single plasma volume was exchanged using either peripheral intravenous preferred or central lines for vascular access For this study due to the possibility of sPD L1 or PD L1 positive EVs present in donor plasma only TPE sessions using no donor plasma ie fresh frozen plasma FFP in the replacement fluid were included in calculations Anticoagulation consisted of either mL of acid citrate dextrose solution A ACD A or mL of ACD A with units of unfractionated heparin Anticoagulant to blood ratios were when ACD A was used and when ACD Aheparin was used Patients did not receive routine electrolyte replacement but mL of calcium gluconate was administered by slow intravenous push for signs and symptoms of hypocalcemia related to the ACD A anticoagulant in one patientELISAELISA was performed as previously published26 Both secreted splice variant and shed sPD L1 are reliably detected by this ELISA In brief paired mouse IgG2 monoclonal antibody clones H1A and B11 against extracellular human PD L1 were utilized in a capture detection plate assay using biotinylation and HRP streptavidin detection This assay is specific for sPD L1 and does not exhibit cross reactivity to other B7 H homologues nor to evPD L1 Concentrations were determined by optical density measurements along a known standard curve of recombinant human PD L1 ELISAs were performed by team members who were blinded to the identity of the samplesFlow cytometryFlow cytometry for EVs was performed as previously published27 In brief plasma samples were centrifuged twice at 2000g to deplete platelets Resultant platelet free plasma were analyzed using an A60 Micro Plus Nanoscale Flow Cytometer Apogee FlowSystems gating for mid intensity light angle light scatter and markers of interest Anti PD L1 Genentech atezolizumab ADAM10 RD Systems clone and CD61 BioLegend clone VI PL2 antibodies were conjugated to fluorophores Life Technologies Alexa647 PE phycoerythrin and Alexa488 and titrated prior to use Nanoscale flow cytometer calibration was performed using a standard reference bead mix as previously published Flow cytometry was performed by team members blinded to the identity of the samplesStatistical analysisAll statistical analyses were performed using R Statistical Software R Foundation Retrospective progression free survival was analyzed using Kaplan Meier and Cox proportional hazards modeling Optimal cut off values for sPD L1 levels were determined using the greyzoneSurv package for R Wilcoxon signed rank test was used to compare paired pre TPE and post TPE patient sample sPD L1 and EV levels as indicated Baseline clinical characteristics for the study were compared by Kruskal Wallis test for continuous variables and Pearsons Ï2 test for discrete variables as indicated Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0cOtherwise groups were compared by unpaired two sided Students t test Figures comprising box plots show quartile values and individual data points Mean values and CI are indicated in corresponding online supplementary figures and tables P was considered statistically significant In figures p values are denoted with with and with Twitter Jacob J Orme JakeOrmeMDPhDAcknowledgements Statistical guidance was provided generously by Nathan Foster of the Mayo Clinic Center for Clinical and Translational Science Some illustrations were created using Servier Medical Art templates which are licensed under a Creative Commons Attribution Unported License https smart servier com Additional illustrations were provided by Mayo Clinic Media Services The authors thank Daniel Summerfield MD MS for use of his likeness in Fig 2AContributors JO originated hypotheses designed the study oversaw experiments performed analyses and wrote the article EALE performed retrospective melanoma cohort analysis FL M performed nanoflow cytometry HD and EB oversaw and performed TPE study enrollment sample collectionprocessing and blinding SMH performed ELISAs MB AM SP MB SNM YY HD RD and JLW helped develop hypotheses provided clinical samples and reagents and contributed support and oversightFunding R21 5R21CA19787802 Role of Bim and soluble B7 H1 in monitoring T cell responses to anti PD1 therapy in melanoma HD and RD L30 CA23154101 Soluble B7H1 as a PD1 Checkpoint Remote Control in Cancer JJO U10 CA180790 EE K12 CA090628 YY Richard M Schulze Family Foundation HD and RDCompeting interests Intellectual property has been filed addressing discoveries disclosed in this manuscript The authors report no other relevant conflicts of interestPatient consent for publication ObtainedEthics approval All research protocols involving human subjects were approved by Mayo Clinics Institutional Review Board and all human subjects gave written informed consentProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available in a public access repository All data will be available for download at the Science Framework at https osf io qtskd access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See http creativecommons licenses by nc ORCID iDJacob J a0Orme http orcid REFERENCES O'Donnell JS Long GV Scolyer RA et a0al Resistance to PD1PDL1 checkpoint inhibition Cancer Treat Rev Ando K Hamada K Watanabe M et a0al Plasma levels of soluble PD L1 correlate with tumor regression in patients with lung and gastric cancer treated with immune checkpoint inhibitors Anticancer Res Fan Y Che X Qu J et a0al Exosomal PD L1 retains immunosuppressive activity and is associated with gastric cancer prognosis Ann Surg Oncol Zhou J Mahoney KM Giobbie Hurder A et a0al Soluble PD L1 as a biomarker in malignant melanoma treated with checkpoint blockade Cancer Immunol Res access Mahoney KMet a0al A secreted PD L1 splice variant that covalently dimerizes and mediates immunosuppression Cancer Immunol Immunother Orme JJ Jazieh KA Xie T et a0al ADAM10 and ADAM17 cleave PD L1 to mediate PD L1 inhibitor resistance OncoImmunology Romero Y Wise R Zolkiewska A Proteolytic processing of PD L1 by ADAM proteases in breast cancer cells Cancer Immunol Immunother Chen G Huang AC Zhang W et a0al Exosomal PD L1 contributes to immunosuppression and is associated with anti PD1 response Nature Poggio M Hu T Pai C C et a0al Suppression of exosomal PD L1 induces systemic anti tumor immunity and memory Cell Derksen RH Schuurman HJ Meyling FH et a0al The efficacy of plasma exchange in the removal of plasma components J Lab Clin Med Berckmans RJ Nieuwland R Böing AN et a0al Cell Derived microparticles circulate in healthy humans and support low grade thrombin generation Thromb Haemost Crescitelli R Lässer C Jang SC et a0al Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation J Extracell Vesicles Kowal J Arras G Colombo M et a0al Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes Proc Natl Acad Sci U S A 2016113E968 Winters JL Brown D Hazard E et a0al Cost minimization analysis of the direct costs of tpe and IVIg in the treatment of Guillain Barré syndrome BMC Health Serv Res Lee JC Zhao J T Gundara J et a0al Papillary thyroid cancer derived exosomes contain miRNA 146b and miRNA222 J Surg Res Yang J Wei F Schafer C et a0al Detection of tumor cell specific mRNA and protein in exosome like microvesicles from blood and saliva PLoS One 20149e110641 Nakao R Hasegawa H Ochiai K et a0al Outer membrane vesicles of Porphyromonas gingivalis elicit a mucosal immune response PLoS One 20116e26163 Thompson AGet a0al Extracellular vesicles in neurodegenerative disease pathogenesis to biomarkers Nature Reviews Neurology Nature Publishing Group Marcilla A Martin Jaular L Trelis M et a0al Extracellular vesicles in parasitic diseases J Extracell Vesicles Huang Doran I Zhang CY Vidal Puig A Extracellular Vesicles Novel Mediators of Cell Communication In Metabolic Disease Trends in Endocrinology and Metabolism Elsevier Inc Boulanger CM Loyer X Rautou PE et a0al Extracellular vesicles in coronary artery disease Nature Reviews Cardiology Nature Publishing Group Kottschade LA Suman VJ Amatruda T et a0al A phase II trial of nab paclitaxel ABI007 and carboplatin in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group Study N057E1 Cancer Kottschade LA Suman VJ Perez DG et a0al A randomized phase study of temozolomide and bevacizumab or nab paclitaxel carboplatin and bevacizumab in patients with unresectable stage IV melanoma a North Central Cancer Treatment Group study N0775 Cancer McWilliams RR Allred JB Slostad JA et a0al NCCTG N0879 Alliance A randomized phase cooperative group trial of carboplatin paclitaxel and bevacizumab ± everolimus for metastatic melanoma Cancer Padmanabhan A Connelly Smith L Aqui N et a0al Guidelines on the Use of Therapeutic Apheresis in Clinical Practice Evidence Based Approach from the Writing Committee of the American Society for Apheresis The Eighth Special Issue J Clin Apher Frigola X Inman BA Lohse CM et a0al Identification of a soluble form of B7 H1 that retains immunosuppressive activity and is associated with aggressive renal cell carcinoma Clin Cancer Res Gomes J Lucien F Cooper TT et a0al Analytical considerations in nanoscale flow cytometry of extracellular vesicles to achieve data linearity Thromb Haemost Orme a0JJ et a0al J Immunother Cancer 20208e001113 101136jitc2020001113 0c"
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cancer is a complex and heterogeneous disease with many possible genetic and environmentalcauses the same treatment for patients of the same cancer type often results in different outcomes in terms ofefficacy and side effects of the treatment thus the molecular characterization of individual cancer patients isincreasingly important to find an effective treatment recently a few methods have been developed to constructcancer samplespecific gene networks based on the difference in the mrna expression levels between the cancersample and reference samplesmethods we constructed a patientspecific network with multiomics data based on the difference between areference network and a perturbed reference network by the patient a network specific to a group of patients wasobtained using the average change in correlation coefficients and node degree of patientspecific networks of thegroupresultsnetworks with multiomics data the main differences of our method from previous ones are as follows networksare constructed with multiomics mrna expression copy number variation dna methylation and micrornaexpression data rather than with mrna expression data alone networks are constructed with bothnormal samples and cancer samples of the specified type to extract cancerspecific gene correlations and bothpatient individualspecific networks and patient groupspecific networks can be constructed the results of evaluatingour method with several types of cancer show that it constructs more informative and accurate gene networks thanprevious methodss the results of evaluating our method with extensive data of seven cancer types show that thedifference of gene correlations between the reference samples and a patient sample is a more predictive feature thanmrna expression levels and that gene networks constructed with multiomics data show a better performance thanthose with single omics data in predicting cancer for most cancer types our approach will be useful for finding genesand gene pairs to tailor treatments to individual characteristicskeywordsindividualspecific gene network groupspecific gene network cancer multiomics datacorrespondence khaninhaackr1department of computer engineering inha university incheon southkoreafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0clee bmc medical genomics 13suppl page of for the past years we have witnessed the rapid development of targeted cancer therapy targeted therapies forcancer work by targeting specific genes proteins or tissues that contribute to cancer growth and survival manytargeted therapies are effective only for patients with specific genetic alterations known as driver mutations thathelp cancer cells form and grow [ ] thus identifying genetic mutations specific to individual patients is ofutmost importance to determine targeted therapies thatcan effectively cure cancer patients while minimizing sideeffects motivated by a massive amount of data generated byhighthroughput technologies several cancer studies usedgene networks to explore gene expression characteristics [] however constructing a patientspecific genenetwork with a single sample obtained from a patient isdifficult because a gene network requires many samples tocompute genegene relationsrecently a few methods have been proposed to construct cancer samplespecific gene networks based onthe difference in the mrna expression levels betweenthe cancer sample and reference samples for exampleliu proposed a method to construct a samplespecific network by computing the difference between areference network from multiple reference samples anda network perturbed by a new sample however a slightchange to the reference samples can result in a significantly different samplespecific network for the samesample due to the small number of reference samples furthermore their samplespecific networks cannot reflectposttranslational modification and epigenetics becausethe networks are built using mrna expression data onlythis paper presents a new method for constructingcancer patientspecific and groupspecific gene networkswith multiomics data using a samplespecific networkand network propagation method network propagation strategies are widely used in recent cancerrelatedresearch li presented a synergy prediction algorithm using network propagation and predicted the drugsynergy in various cancers zhang introduceda propagation algorithm which learns the mutated subnetworks underlying tumor subtypes using a supervisedapproach and classified tumors to known subtypes onbreast and glioblastoma tumors peng identifiedbladder cancerrelated genes by propagating informationfrom seed genes to candidate genes the primary focusof our method is to construct a gene correlation network specific to cancer with multiomics data thus it isdifferent from a typical gene coexpression network thatrepresents coexpression relations between genes frommrna expression data our gene network is not a generegulatory network because our network does not showregulatory relations between genesthe main differences of our method from previous onesare as follows networks are constructed with multiomics mrna expression copy number variation dnamethylation and microrna expression data rather thanwith mrna expression data alone networks are constructed with both normal samples andcancer samples of the specified type to extract cancerspecific gene correlations and both patient individualspecific networks and patient groupspecific networks canbe constructed as shown later in this paper the resultsof evaluating our method with several types of cancershow that it constructs more informative and accuratetargetspecific networks than previous methodsmethodsat the top level our method consists of the followingsteps data processing constructing individualspecific gene networks and constructing a groupspecific gene networks a highlevel description of themethod is given in fig data collection and preprocessingfrom the broad institute tcga gdac firehose we obtained multiomics data of cancer samples of seventypes breast invasive carcinoma brca colon adenocarcinoma coad head and neck squamous cell carcinomahnsc pankidney cohort kipan liver hepatocellular carcinoma lihc lung adenocarcinoma luad andlung squamous cell carcinoma luscthe multiomics data used in this study includemrna expression mrnaseq copy number variationcnv dna methylation and mature mirna expression mirseq data the mrnaseq data were processedusing quartile normalized rsem and then log2transformed the segmented cnv data were convertedto genelevel data using the ensembl api and thecntools package of bioconductor the methylationdata were filtered to select the probe with the mean signal values for each gene the mirseq data were processedby rpm and log2transformed mrnas and mirnas thatwere not expressed in more than of the total sampleswere excluded in further analysis missing expression values of mrnas and mirnas were replaced by the smallestpositive normalized floatingpoint number realmin ofmatlab the number of samples and genes used in thisstudy are available in additional file individualspecific gene networkin each group of tumor samples and normal samples wefirst computed genegene relations by the pearson correlation coefficient pcc selected highly correlated genepairs ie those with pcc and constructed twosample networks one for each group from the tumorsample network we removed edges common to both 0clee bmc medical genomics 13suppl page of fig overview of constructing an individualspecific network and a groupspecific network with multiomics datatumor and normal sample networks and obtained a template reference network for cancer fig 2a the templatereference network consists of highlycorrelated gene pairsthat are specific to cancerwith n reference samples which may be different fromtumor samples used in the template network we computed pcc for every pair of genes in the template reference network and constructed a reference network forthe reference samples for a patient of interest we constructed a network which is a perturbed network byadding a single sample of the patient to the n reference samples a patientspecific network was obtainedby subtracting the reference network from the perturbednetwork 01pcc pccn1 pccnwe computed the difference in the absolute value ofpcc between the perturbed reference network and reference network by eq we also carried out a ztest ofpccn1pccn by eq for a large n we can approximatethe mean μ and standard deviation Ï of pccn1 pccn as and pcc2nn respectively pccchange pccn1 pccnz score pccchange μpccchangeÏ pccchange pccchangepcc2nnthe edges of the patientspecific network were classified into four types correlationgained edges fene pairs whose pccs are increased from the referencenetwork to the patientspecific network correlationlost edges for gene pairs whose pccs are decreased fromthe reference network to the patientspecific network correlationreversed edges for gene pairs whose signs ofpccs are changed from positive to negative or negativeto positive and correlationinvariant edges for genepairs with little change in pccs between the reference andpatientspecific networks ie those with zscore fig 2bthe edges were classified in the following way we firstselected gene pairs with zscore as correlationinvariant type and then selected gene pairs which havedifferent signs of pccs between the reference networkand the patientspecific network as correlationreversedtype the remaining gene pairs were classified into eithercorrelationgained or correlationlost type depending onwhether their pccs are increased correlationgained ordecreased correlationlost from the reference network tothe patientspecific network thus zscore ¥ in bothcorrelationgained and correlationlist gene pairsgroupspecific gene networka groupspecific gene network is useful when analyzinga large number of patientspecific gene networks afterconstructing patientspecific gene networks we obtained 0clee bmc medical genomics 13suppl page of fig process of constructing a patientspecific gene network a template of the cancer reference network obtained by removing edges common toboth networks b patientspecific gene network and four types of edges in the network edges with a zscore represent correlationinvariantgene pairs and edges with zscore ¥ and different signs of pccn and pccn1 represent correlationreversed gene pairs edges with zscore ¥ and 01pcc are correlatedgained gene pairs and those with zscore ¥ and 01pcc are correlationlost gene pairsa gene network specific to a group of patients based onthe average 01pcc and node degree of the patientspecificnetworks fig if the dominant type for a particular edge is correlationgained positive 01pcc in thepatientspecific networks the edge is represented in redin the groupspecific network in contrast if the dominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edgeis represented in blue in the groupspecific network inthe groupspecific network only the dominant type isshown for each edge if nondominant types are shownin addition to the dominant type for each edge the network becomes cluttered and unreadable the node sizeof a groupspecific gene network is proportional to theaverage degree of the nodeintegration of multiomics datato integrate multiomics data we first computed intergene correlations by pcc with four different types ofsingle omics data mrna expression cnv dna methylation and mirna expression separately and selectedsignificant intergene correlations only in mrna expression cnv and dna methylation data we select thetop pcc with pvalue in mirna expression data we selected the top pcc with pvalue due to a smaller number of mirnas inthe data the intergene correlations selected in eachsingle omics data are represented in four correlationmatrices mexpr mcnv mmethyl and mmirna andnormalizedusing the proteinprotein interactions ppis of thestring database we constructed separate weightednetworks from each omics data by eq in eq wexprwcnv and wmethyl denote the weighted networks andppiexpr ppicnv and ppimethyl are subnetworks of a ppinetwork consisting of genes present in each omics datasince the ppi network does not contain information onmirna a weighted network for mirna was not constructedcid3wexpr cid2 ppiexprwcnv mcnv à ppicnv wmethyl cid2 ppimethylwe then integrated the multiomics data by linear xmethylregression using eq in eq yi xcnvand xmirnadenote gene is expression level cnv levelmethylation level and mirna regulator expression levelrespectively βcnvdenote the regression coefficients of gene is expression level on cnv and methylation respectively βmirnais the regression coefficientof gene is expression level on its mirna regulator jsexpression leveland βmethyl mexpr mmethylcid3 à cid2cid3 à cid2cid3iiijiiijyi βcnvii βmethylxcnvixmethyli ncid4j1βmirnaijxmirnaij 01 0clee bmc medical genomics 13suppl page of fig process of constructing a groupspecific gene network from multiple patientspecific gene networks in the groupspecific gene network thenode size and node color are proportional to the average degree and average 01pcc of the node respectively if the dominant type for a particularedge is correlationgained positive 01pcc in the patientspecific networks the edge is represented in red in the groupspecific network if thedominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edge is represented in blue in thegroupspecific networkfrom the regression coefficients and the weighted networks a weight matrix w was derived and normalizedinto w eqs and the weight matrix w is symmetricso wij wji w11 w22 w33 and w44 represent wexprwcnv wmethyl and mmirna respectively the submatrices w21 and w31 contain regression coefficients βcnvand βmethylfor every gene i respectively w41 representsiβmirnaij the submatrices w32 w42 and w43 are emptyi¡¢¢£w w11 w12 w13 w14w21 w22 w23 w24w31 w32 w33 w34w41 w42 w43 w44¤¥¥¦eq and updated iteratively by eq in this iterative process the influence of the seed is propagated tothe neighbors until a mean squared error of st and st¤ à §ªªªª¨ªªªª©if v is a nonseed nv ¥ αif v is a nonseed nv αif v is a seednvnvenvα à nvnvdv st λ à st à w λ à d wheres1 d where nv is the number of neighbors of node v and nv isthe number of seeds in the neighbors the parameter αwhich is a threshold for nv was set to and λ was set to genes with the top st were used in findingcancerrelated genes and in classifying tumor samples andnormal samplesw i j w i jcid11cid12cid12cid13mcid4k1w i k à mcid4k1w k jin network propagation seed genes have greater impactthan nonseed genes on their neighbors thus only thegenes with a high average 01pcc were selected as seedgenes for a groupspecific network and their mirnasregulators extracted from mirtarbase were used asseed mirnas we calculated the cancerrelevance st ofeach gene to reflect the effect of the seed genes and mirnas on neighbors the initial score d was calculated byresultspatientspecific and groupspecific gene networksin this study we constructed patientspecific genenetworks for seven cancer types additional file foreach cancer type we also constructed groupspecific genenetworks as an example fig shows a groupspecificgene network derived from lung squamous cell carcinoma lusc patients 0clee bmc medical genomics 13suppl page of there are three distinct subnetworks in the networkfor the lusc group the subnetwork enclosed in box aof fig contains many hub genes large green nodesthe subnetwork in box b consists of correlationgainededges dark red edges whereas the subnetwork in box ccontains many correlationlost edges dark blue edgescomparison of multiomics data and singleomics datawe performed leaveoneout cross validation loocvto evaluate cancerrelevance score st of a gene and thecontribution of multiomics data to finding cancerrelatedgenes for comparison the cancerrelevance scores werecomputed with multiomics data and single omics dataseparately each seed gene was regarded as a nonseed anda new cancerrelevance score was calculated for the geneseed genes and nonseed genes were considered as positive and negative respectively seed genes included in thetop n genes were considered as true positives and seedgenes not included in the top n genes were consideredas false negatives similarly nonseed genes included infig groupspecific gene network for lung squamous cell carcinoma lusc patients a subnetwork of multiple hub genes large greennodes b subnetwork of correlationgained edges dark red edges c subnetwork with many correlationlost edges dark blue edges 0clee bmc medical genomics 13suppl page of the top n genes and nonseed genes not included in thetop n genes were considered as false positives and truenegatives respectivelywe carried out loocv with different ratios of seedgenes to nonseed genes figure shows the receiver operating characteristic roc curve and the area under thecurve auc of loocv of the cancer relevance of geneson data of breast cancer samples with various seedratios ranging from to enlarged plots of fig are available in additional file for comparative purposes we also computed the cancer relevance of geneswith single omics data as shown in fig multiomicsdata consistently exhibited better performance than singleomics data with any seed ratio between to forlater analysis the seed ratio was set to by default theaverage 01pcc and class label of each gene are available inadditional file indeed the superiority of multiomics data over singleomics data in determining the cancer relevance scoreof genes was observed in all seven types of canceradditional file in seven types of cancer the cancerrelevance score of genes computed with multiomics dataexhibited a good performance auc ¼ thecancer relevance score of genes computed with mrnaexpression data showed the second best performanceauc ¼ in particular the cancer relevancescore computed with mrna expression data showed avery similar performance to that with multiomics inbreast cancer brca the performance of the cancer relevance score computed with cnv auc ¼and dna methylation data auc ¼ alonewas lower than that with mrna expression data auc ¼fig roc curves of the leaveoneout cross validation of the cancer relevance score st of genes with different ratios of seed genes breastcancer samples were used in the leaveoneout cross validation the performance of multiomics data is always better than that of single omics data 0clee bmc medical genomics 13suppl page of evaluation of gene correlations and networksmany networkbased approaches to cancer research havefocused on finding genes that show differential expressions between tumor samples and normal samples genegene correlations ie intergene correlations may bemore helpful than individual genes because intergenecorrelations depend on the expression of neighbor genesin a gene regulatory network to compare the effect ofusing individual genes to that of intergene correlationsie 01pcc we constructed a support vector machinesvm model for classifying cancer samples and normalsamples the svm model was implemented using csvcand rbf kernel and the parameter values of the modelwere determined by the grid search algorithm mrnaexpression levels and 01pccs were used as features ofthe svm models for rigorous validation the test dataused in testing the models were not used in training themadditional file as shown in fig 6a 01pcc showed a better performance than mrna expression levels for six cancertypes except lusc the classification model with 01pccshowed mcc above in six cancer types except hnscwe also examined the effect of different networks on individualspecific networks in the workby liu ppi data with high confidence scoresin the string database were used to construct a network however the ppi data of stringdoes not reflect cancer typespecific characteristicsfigure 6b shows the performance of the classificationmodel with two different networks network from ppi data of string the approachby liu and cancer network ourapproach 01pcc was used as a feature of the classification model except for coad the performance ofthe classification model with the cancer network was better than the model with the string reference network in particular the classification model showed a significant difference for breastcancer brca mcc of vs mcc of detailed results of the classification model are available inadditional file discussionin the analysis of finding cancerrelated genes and genepairs we focused on a subnetwork of genes with a 01pcctable shows the top genes with a high average 01pcc in each groupspecific network of seven cancertypes in breast invasive carcinoma brca fam171a1showed the highest average 01pcc in the groupspecificnetwork fam171a1 is known as a potential biomarkerin triplenegative breast cancer foxc1 is involvedin tumor development and metastasis and associated withpoor prognosis in basallike breast cancer il33 isoverexpressed in various cancers and the serum concentration of il33 is a valuable indicator of poor prognosis inbreast cancer mamdc2 is significantly correlatedwith diseasefree survival of breast cancer patients mterfd1 is closely related to breast cancer recurrencefig results of evaluating features and networks by a validation set a comparison of mrna expressions of genes and 01pcc of genepairs b comparison of the cancer network with the network from ppi data 0clee bmc medical genomics 13suppl page of table top genes with a high average 01pcc in a groupspecific network for seven cancer typesbrcakipanlihccoadhnscfam171a1foxc1il33mamdc2mterfd1hoxa7cttnbp2znf204pjam3frem1gfra2scnn1bddx27rnps1ube2imdficctnnbl1cdk5rap1esf1trmt6cyp2j2barx2znf135ppfia1parlfaddcst6cobloraov1xpo7tfcp2l1kcnq1arl15kctd1oaz2tmem45bhpcal1tmem91sema5begln3slc19a3ecm1cyp2b6fbp1gpaa1f9pahagxt2hsd17b6rnase4luadcldn18adamts8pecam1sftpa1gimap6akr1c1mmeatad2cyp3a5chaf1bluscnsun2cct5fbxo45gpr116slc39a8fxr1inmtveph1crtamwdr53 and hoxa7 plays a critical role in regulating theproliferation of erpositive cancer cells in colon adenocarcinoma coad gfra2 showed thehighest average 01pcc in the groupspecific network itis known to be crucial for enteric neuron survival scnn1b and ddx27 are significantly related to colorectal cancer [ ] no direct relation of rnps1 withcolorectal cancer is known but rnps1 is essential tononsensemediated mrna decay that plays complexfunctions in cancer knockdown of sumo conjugating enzyme ube2i also known ubc9 or e2 inhibitsmaintenance and selfrenewal of colorectal cancer stemcell while overexpression of ube2i increases colorectalcancer cell stemness among the top genes with a high average 01pccin lung adenocarcinoma luad several genes such ascldn18 adamts8 pecam1 and sftpa1 have beenknown to be associated with luad in previous studies[] no direct relation of nsun2 and slc39a8 withlung squamous cell carcinoma lusc has been known sofar however recent studies [ ] reported that nsun2is correlated with survival in other types of squamous cellcarcinomas gao also showed that the epigeneticsilencing of slc39a8 expression by dna methylation isinvolved in the acquisition of resistance against cadmiumin lung cells and the relation between cadmium andlung cancer has received much attention many othergenes in table found in the groupspecific networksfor head and neck squamous cell carcinoma hnscpankidney cohort kipan and liver hepatocellular carcinoma lihc are also directly or indirectly related tocancerin addition to individual genes we identified genepairs of the same type ie either correlationgained orcorrelationlost in most patientspecific networks of thesame type table shows the most frequent gene pairs in breast cancer samples the most frequent gene pairsin other types of cancer are listed in additional file it isinteresting to note that all the gene pairs shown in table include at least one gene in the gene pair mamdc2hoxa7 and that they are correlationgained edges in thegroupspecific network for breast cancer figure showsa subnetwork containing mamdc2 and hoxa7 in thegroupspecific network of breast cancer the subnetworkwas obtained by selecting the edges for which the proportion of the same edge type ie correlationgained or lostis above in the total individualspecific networks ofbreast cancer patients it is interesting to note that all thegene pairs in table are included in the subnetworkto date the actual role of the mamdc2 gene in cancer is not clear but meng reported mamdc2as one of three genes mamdc2 tshz2 and cldn11that are significantly correlated with diseasefree survival of breast cancer patients mamdc2 is known as atarget of mir196a in head and neck squamous cell carcinoma as a member of the family of homeoboxgenes hoxa7 is associated with cell proliferation nerveinvasion distant metastasis and degree of tumor differentiation in several cancers [ ] hoxa7 is regulatedtable the most frequent gene pairs in breast cancersamples all the gene pairs are of a correlationgained type thegenes of table are shown in bold the proportion represents theratio of the gene pairs of the same type ie correlationgained orlost to the total number of patientspecific networksgene pairgene pairsproportion of the gene pairsin total cancer samplesmamdc2hoxa7mamdc2ccl14mamdc2znf204pmamdc2klmamdc2svep1mamdc2coro2bhoxa7meox2hoxa7hoxa9mamdc2sobpmamdc2hoxa9 0clee bmc medical genomics 13suppl page of fig a subnetwork of the groupspecific network of brca which contains mamdc2 and hoxa7 genes in the most frequent gene pairs shown intable are enclosed by a circleby several mirnas including mir196 [] thusboth mamdc2 and hoxa7 are related with mir196but a clear relation among them is to be uncoveredso far most approaches to constructing individualspecific gene networks have been constructed based onthe differential expressions between a small number ofreference samples and a sample of interest howeversuch networks cannot reflect posttranslational modification and epigenetics and are not reliable because a slightchange to the reference samples can result in a significantly different samplespecific network for the samesamplein this paper we presented a new approach to constructing cancer patientspecific and groupspecific networks with multiomics data the main differences ofour method from previous ones are as follows gene networks are constructed with multiomics mrnaexpression copy number variation dna methylationand microrna expression data rather than with mrnaexpression data alone networks can beconstructed with cancer samples of the specified type and both patient individualspecific networks and patientgroupspecific networks can be constructed the resultsof testing our method with several cancer types showedthat it constructs more informative and accurate genenetworks than existing methodsevaluation of our method with extensive data of sevencancer types showed that changes in gene correlations 01pcc between the reference samples and a patient sample is a more predictive feature than mrna expressionlevels and that gene networks constructed with multiomics data are more powerful than those with singleomics data in predicting cancer for most cancer typesmore work is required to validate the genes and genepairs identified in the cancer patientspecific and groupspecific networks however the method for constructingnetworks specific to individual patients or patient groupswith multiomics data should be useful aids in determining effective treatments to individual characteristicssupplementary informationsupplementary information accompanies this paper athttpsdoi101186s12920020007367additional file number of samples and genes in types of canceradditional file roc curve and auc of the cancerrelevance score ofbrca by various seed ratiosadditional file average 01pcc and class label of each gene in typesof canceradditional file roc curve of the cancerrelevance score of each cancertype with the seed ratio of additional file performance of classification of tumor samples andnormal samplesadditional file top gene pairs for each cancer type 0clee bmc medical genomics 13suppl page of abbreviationsauc area under the curve brca breast invasive carcinoma cnv copynumber variation coad colon adenocarcinoma gdac genome dataanalysis center hnsc head and neck squamous cell carcinoma kipan pankidney cohort lihc liver hepatocellular carcinoma loocv leaveoneoutcross validation luad lung adenocarcinoma lusc lung squamous cellcarcinoma mcc matthews correlation coefficient pcc pearson correlationcoefficient ppi proteinprotein interactions roc receiver operatingcharacteristic svm support vector machine tcga the cancer genome atlasacknowledgementsthe authors thank the anonymous reviewers for the constructive commentsthat contributed to improving the final version of the paperabout this supplementthis has been published as part of bmc medical genomics volume supplement selected s from the 15th international symposiumon bioinformatics research and applications isbra19 medical genomicsthe full contents of the supplement are available online at httpsbmcmedgenomicsbiomedcentralcomssupplementsvolume13supplement6authors contributionswl designed and performed all about this study and prepared the initialmanuscript dh helped integrating multiomics data of breast cancer khsupervised the work and wrote the manuscript all authors read and approvedthe final manuscriptauthors informationwook lee is currently working toward his phd degree at the department ofcomputer engineering inha university korea deshuang huang is a directorof the institute of machines learning and systems biology tongji universitychina kyungsook han is a professor at the department of computerengineering inha university koreafundingthis work was supported by the national research foundation of korea nrfgrant funded by the ministry of science and ict nrf2017r1e1a1a03069921and inha university research grant publication of this was funded bythe nrf grant nrf2017r1e1a1a03069921 the funders had no role in thedesign of the study data collection and analysis or writing the manuscriptavailability of data and materialsadditional files are available at httpbclabinhaackrcancernetworkethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of computer engineering inha university incheon southkorea 2institute of machine learning and systems biology school ofelectronics and information engineering tongji university shanghaichinareceived may accepted june published august references widakowich c de castro g de azambuja e dinh p awada a reviewside effects of approved molecular targeted therapies in solid cancersoncologist liu s kurzrock r toxicity of targeted therapy implications for responseand impact of genetic polymorphisms cancer treat rev verma m personalized medicine and cancer j personalized medbarabasi al gulbahce n loscalzo j network medicine a networkbasedapproach to hum
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among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patients health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patients quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal disquadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology 20e1 lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202adenocarcinomastech coloproctol 0c'
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Scars Burns HealingVolume 1011772059513120940499 reuse guidelinessagepubcomjournalspermissions The Authors journalssagepubcomhomesbhKeloids are pathological scars that grow over time and extend beyond the initial site of injury after impaired wound healing These scars frequently recur and rarely regress They are aesthetically disfiguring can cause pain itching discomfort as well as psychological stress often affecting quality of life Many treatment modalities including surgical and nonsurgical have been explored and have been reported to be beneficial however none have been absolutely satisfactory or optimal for the treatment of all keloid subtypes to date This poses a major challenge to clinicians Often a combinational therapeutic approach appears to offer the best results with higher patient satisfaction compared to monotherapy The aetiopathogenesis of keloids is not fully elucidated however with recent advances in molecular biology and genetics insight is being gained on the complex process of scar formation and hence new therapeutic and management options for keloids In this paper we explore the literature and summarise the general concepts surrounding keloid development and review both current corticosteroids surgical excision siliconebased products pressure therapy radiotherapy cryotherapy laser therapy imiquimod and 5fluorouracil and emerging stem cell therapy mitomycin C verapamil interferons bleomycin botulinum toxin type A and angiotensinconverting enzyme inhibitors treatments Increased knowledge and understanding in this area may potentially lead to the discovery and development of novel therapeutic options that are more efficacious for all keloid typesKeywordsKeloids scar recurrence wound healing treatment managementLay SummaryKeloids are problematic scars that are difficult to treat and manage The aetiopathogenesis of keloids is not clear however recent advances in molecular biology and genetics are beginning to shed light on the underlying mechanisms implicated in keloid scar formation which will hopefully lead to the development of treatment options for all keloid types This review summarises current and emerging therapiesIntroductionWound healing is an intricate and complex series of processes comprising overlapping phases of inflammation granulation tissue formation and tissue remodelling and results in tissue structure integrity and damage being restored1 Abnormal wound healing can give rise to keloids which are benign dermal fibroproliferative nodular lesions that tend to recur after excision Keloid scars Faculty of Medical Sciences The University of the West Indies Cave Hill Campus Bridgetown Barbados West Indies Pine Medical Centre 3rd Avenue Belleville St Michael Barbados West IndiesCorresponding authorNkemcho Ojeh Faculty of Medical Sciences The University of the West Indies Cave Hill Campus PO Box St Michael Bridgetown BB Barbados West Indies Email nkemchoojehcavehilluwieduCreative Commons Non Commercial CC BYNC This is distributed under the terms of the Creative Commons AttributionNonCommercial License creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0c Scars Burns Healinginfluence in keloid aetiology9 Although no one specific gene has been associated with the development of keloids a number of genes and gene loci have been identified610 Genomewide association studies and admixture mapping studies have identified singlenucleotide polymorphisms across certain loci genetically linked to keloid development including the NEDD4 gene which encodes E3 ubiquitin ligase enzyme and the myosin genes MY01E and MYO7A10 Studies have also reported the involvement of several human leucocyte antigen HLA alleles p53 bcl2 and fas genes1014 Furthermore rare genetic disorders have been reported to present with spontaneous keloids including Dubowitz syndrome Bethlem myopathy RubinsteinTaybi syndrome Noonan syndrome Geominne syndrome and others1017 These lines of evidence suggest that genetic factors play a role on keloid predispositionPathophysiology of keloidsKeloid pathology is complex involving both genetic and environmental factors Keloids form as a result of abnormal wound healing and excessive dermal fibrosis Development of keloids has been linked to overproliferation and reduced apoptosis of dermal fibroblasts overproduction of collagen fibres and other extracellular matrix ECM components as well as abnormal ECM production and remodelling1 Various cytokines growth factors and proteolytic enzymes have been implicated in the formation of keloids including transforming growth factor TGF epidermal growth factor EGF vascular endothelial growth factor VEGF plateletderived growth factor PDGF connective tissue growth factor CTGF tumour necrosis factorα TNFα insulinlike growth factor1 IGF1 fibroblast growth factor FGF interleukin6 IL6 and matrix metalloproteinases MMPs31418 Furthermore signalling pathways such as Tolllike receptor signalling SMAD signalling and fibronectin have been reported to be associated with keloid development1819Histopathology of keloidsHistologically keloids comprise an abundance of unordered dermal collagen and vasculature with high inflammatorycell infiltrate and overactive mesenchymal cells1415 In addition to collagen elastin fibronectin and proteoglycans are deposited in excess amounts in keloid scars9 Collagen creates frequent crosslinks in ordinary wounds whereas collagen is irregularly anised in keloids forming nodules in the dermis20 During normal Figure Earlobe keloids as a consequence of ear piercingarise from skin trauma or inflammation and may develop years after the initial insult and rarely regress2 The scar tissue extends beyond the original wound site and can be disfiguring and cause psychosocial issues impairing quality of life3 In addition patients may present with symptoms such as burning pain pruritus movement limitation and hyperaesthesia2AetiologyThe aetiology of keloids is still poorly understood The most common regions of the skin for keloids include upper arms skin overlying joints chest shoulders and headneck regions particularly the ear lobes Figure The anatomical location of a keloid appears to alter its morphological characteristics Some keloids can develop spontaneously however most occur years after local trauma and other events including inflammation surgery burns elective cosmesis foreign body reactions acne insect bites vaccinations or mechanical force45Epidemiology and keloid geneticsThe incidence of keloids is highest among darkerpigmented persons of African Asian and Hispanic descent and is estimated to be in the range of Males and females have an equal risk of developing keloids6 although incidence is slightly increased in females likely attributable to them having more cosmetic procedures like ear piercing7 Persons aged years are also at a higher risk of developing keloids Additional risk factors include having blood type A hyperIgE and hormonal peaks during pregnancy or puberty8Familial keloid case studies and twin studies support the notion that genetic factors have an 0cOjeh for the treatment of keloids2627 Intralesional TAC injections have been shown to reduce scar volume and height improve scar pliability and diminish associated scar pain and itching8 as well as prevent recurrence3 Corticosteroids have antiinflammatory and antimitotic properties1 Several other mechanisms have been reported by which corticosteroids reduce keloid scar including inhibition of fibroblast growth attenuation of procollagen and glycosaminoglycan synthesis reduction of endothelial budding and enhancement of collagen and fibroblast degeneration2829 Corticos teroids inhibit TGF1 expression and induce apoptosis in fibroblasts inhibit VEGF and alphaglobulins which are involved in the wound healing process28 VEGF which promotes angiogenesis was reported to be highly expressed in keloid fibroblasts compared to controls but exogenous addition of the glucocorticoid dexamethasone suppressed its expression in vitro32 Furthermore VEGF expression was overexpressed in keloid tissue which later reduced following intralesional TAC injections in vivo33TAC is typically administered at intervals of weeks until pruritic and painassociated symptoms diminish and the scar flattens34 The dose of TAC is in the range of mgmL depending on the size and anatomical location of the lesion and the age of the patient34 TAC is used either alone as a monotherapy or in combination with other treatment modalities13 The response rates to corticosteroid injections vary clinically with regression rates in the range of reported after one year and recurrence rates in the range of reported after five years3 Combined therapy comprising surgical excision followed by TAC treatment also varied with reported recurrence rates in the range of Previous clinical studies where TAC was used alone reported efficacy and good clinical outcome with this treatment including reduced keloid height length width related pruritus and erythema and improved pliability3637 A recent randomised parallelgroup study that compared the role of intralesional TAC fractional CO2 laser or intralesional verapamil in the treatment of keloid in patients showed reduction in scar height vascularity and pliability in all three groups using the Vancouver Scar Scale score however pigmentation was not completely resolved with any of the treatments The response was fastest with TAC followed by verapamil and laser and this was statistically significant38 However intralesional TAC in combination with other treatment modalities such as 5fluorouracil 5FU pulsed dye laser PDL surgery interferon IFNα2b verapamil and Figure Current and emerging treatment strategieswound healing the early wound immature collagen type III can be modified into mature collagen type I In keloid tissue it mostly comprises disanised collagen types I and III made up of palestaining hypocellular collagen clusters lacking nodules or surplus myofibroblasts21 Furthermore recent research has provided four distinct findings only present in keloid specimens presence of keloidal hyalinised collagen presence of a tonguelike advancing edge underneath normalappearing epidermis and papillary dermis a horizontal cellular fibrous band in the upper reticular dermis and a prominent fascialike band22Treatment of keloidsCurrently various forms of treatment for keloids exist however no single treatment has proven to be the most effective This review will explore and discuss current and emerging treatment modalities Figure Some of the ongoing or completed clinical trials of keloid therapy registered on clinicaltrialsgov and accessed on May are summarised in Table Studies with the status terminated suspended or withdrawn were excludedCurrent treatmentsCorticosteroidsSeveral corticosteroids can be used for the treatment of scars including triamcinolone acetonide TAC hydrocortisone acetate dexamethasone and methyl prednisolone24 However since TAC has been the most widely used corticosteroid 0c Scars Burns HealingTCNVI esahPTCNlebacilppa toN vogslairTlacinilCesahpydutS noitnevretnIsnoitidnoC yaMnodessecca vogslairTacinlil Cnoypareht doek fo slairt lliacinilC elbaTeltit ydutSreifitnediTCNlebacilppa toN ybdewoll iednotecaenoonicmairt llanoiseartnliilsdoeK fo tnemtaerTeht n iof resal OC lanoitcarFidoeKli sdoretS lanoiseartnl iI svyparehTdoretSdetsissA resaL lanoitcarFTCNlebacilppa toNTCNlebacilppa toNTCNVI esahP TRS ypareht noitadar liaicifrepuS iEnmativ enocilis enositrocordyh yparehtyhcarbetaresod ihgh tnavudahtij wnoisicxe lacigruS cihportrepyh idoeKlidoeKlidoeKl TRS yparehTnoitadaRi l laicifrepuS fonoitauavEevitcepsorteRA ilsdoeK fo tnemtaerT rof yparehtyhcarBetaResoDhgHi isracSdoeKdnal dnaytili llbareoTeht gnitauavEydutSevitarapmoCdezimodnaRA TCNlebacilppa toN llamsaphcir teetap suogootuAllidoeKl llllamsaPhciR teetaP suogootuAyb racSdoeK fo tnemtaerTli TCB®tiKnegeRhti wdenatboiTCBtiKnegeRhti wdenatbOi l ieg tcartxenono ro draugracS noitolamredeM xirtaciCracScihportrepyh ildna sdoeK fo tnemtaerTeht rof separehTi l acipoTowT foycaciffE sracScihportrepyHTCNI esahP muiclac fonoitcenj i laruomutartnIidoeKlrefsnartortceeybdewol llof edirohclTCNIII esahP dnaenoonicmairt llanoiseartnlIidoeKl ienodnefriP l ilsdoeK fo tnemtaerTeht rof noitaroportceEmuiclaCl acipoTdnaenoonicmairTl lanoiseartnl I foycaciffEienodnefrip lacipotnoitcenji UFidoeKllygooisyhpohtaPdna tnemtaerT gnirracSdoeKli iIOALSDAL sracSdoeK fo tnemtaerT roflTCNII esahP I esahP inks gnviltneavuqeiil dereyalib fargilpAsresalidoeKl iil tenragmunmuamuirttymumydoenideslupgno l dna OC lanoitcarFidoeKl fo tnemtaerTn ii sresaL tenraGmunmuAmuirttymumydoeNli fonoitneverPdna tnemtaerTeht rof farg ilpA foydutS toli PAisdoeKl ilsdoeKdesicxE foecnerruceR desluPgnoLdnaedixoDnobraCi l anoitcarFgnisUydutSA deunitnoC cihportrepyhTCNlebacilppa toNyparehtoyrc lanoiseartnlI xirtaciC idoeKl eht rof eciveDdesaB saGnogrAnahti WyparehtoyrC lanoiseartnlI sracScihportrepyHdnadoeK fo tnemtaerTli TCNIII esahPxirtaciC fonoitacil ppa lacipoT sracs cihportrepyHcihportrepyhidoekl ilsracS sdoeKdna sracScihportrepyH fo tnemtaerTeht n i IXRTACCI sracScihportrepyHdnadoeK fo tnemtaerTli TCNlebacilppa toNyparehtoyrc lanoiseartnlI xirtaciC idoeKl rof yparehtoyrC lanoiseartnl I foesUeht fonoitauavEevitcepsorPl 0cOjeh TCNVI esahPresal PTKmneht foycaciffE idoekl cihportrepyheahcun siliadoekl xirtacic xirtacic racs lacigrus racS reifitnedi vogslairTlacinilCesahpydutS noitnevretnIsnoitidnoCdeunitnoC elbaTeltit ydutSTCNlebacilppa toNresal eyddesluP cihportrepyh idoeKl desluPmnagnisU tnemtaerT racSnohtdWesluP fo tceffEi TCNlebacilppa toN thgil AVUderutcafunamnamreG idoeklsracs amredorelcSsnoitidnoC rali imSdnaamredorelcS fo tnemtaerT rof thgLAVUi resaLeyD ecivedgnitti me gnisorbif rehtosnoitidnocTCNlebacilppa toN ecivederusserPTCNVI esahPl egenocilis lacipoT cihportrepyh idoeKlilERUSSERP sdoeK raE fo tnemtaerTeht n i eciveDerusserPidoeKl fo tnemtaerTeht no l eGenocili iSgnyrdfleS tnerapsnarT fo tceffEsracssracS l ianmodbAcihportrepyHTCNlebacilppa toNypareht noitadaRiidoeKl yrtsigeRnoitadaRdoeKlii TCNIII esahPnosahtemateB fonoitacil ppa lacipoT sracs cihportrepyHsretehtaC suoneV lartneC retfA sracSTCNI esahPII esahPTCN I esahPII esahP dna sll dicacidisuf dnaetareavl isdoeklidocitrococuglnoitadarri iBVU amredorelcs idoeKl inkS fo tnemtaerTeht n i yparehT thgL BVUil B teovartlUi leraunnaamounargl sracs siliadoekl enca desilacolxirtaM l amreDderetlAhti w snoitidnoC lecFVS suogootua fo snoitcenjI axal situc racs inkSCSDAxirtacic idoekl idevireDesopdA suogootuAhtil waxaL situCdna sracS foyparehT sll eCmetS lamyhcneseMTCNlebacilppa toN gnitti meAVUderutcafunamnamreG amredorelcs idoeKlsnoitidnoC ralimetsys thgil lamounarg sracs enca leraunna imSdnaamredorelcS rof thgLAVUi ilteovartlu VU FVS sll ecnoitcarf raucsavl lamorts FVS etahpsohp lynatit muissatop PTK sll ecmets l amyhcnesemdeviredesopdai hserF fo tnemtaerTeht rof resaLPTKmn l evoNa foydutS to liP CSDA licaruoroulf UFsracS lacigruS 0c Scars Burns HealingA variety of methods can be used for the surgical removal of keloids depending on the size of the keloid anatomical location skin type and age of patient52 These include linear closure and flap coverage excision with grafting Wplasty and Zplasty53 To reduce risk of keloid recurrence the surgeon performing the excision should establish tensionfree wound closure As a general rule closure of the wound should be accomplished with minimal tension and sutures leaving everted wound borders Zplasties threelayered sutures subcutaneousfascial tensile reduction sutures or local ï¬ap surgery can be employed on a casebycase basis5455 The final outcome of the scar is often positively correlated with the experience of the operating surgeon and technique utilised as well as the patients active participation in their wound care52Siliconebased productsSince the 1980s siliconebased products have been used in the treatment of keloids and hypertrophic scars with silicone gel or silicone gel sheeting considered as firstline therapy for minor keloids and hypertrophic scars2635 There are various other forms of silicone including creams sprays gel cushion and liquid24 The precise mechanism of action of silicone products is not fully understood but it is proposed that they enhance hydration and create an occlusive environment53 which influences fibroblast regulation and decreases collagen synthesis56 Silicone gel sheeting has been shown to have minimal side effects including local irritation which can be resolved quickly24 Studies have shown that the beneficial effects of silicone gel sheets include pain reduction tenderness and pruritus and flattening the keloid57 The silicone gel sheeting is recommended to be worn from two weeks after primary wound treatment for h for months58Studies have demonstrated an improvement of up to in keloid scars after using silicone gel sheeting35 and a decrease in the incidence rates of keloids and hypertrophic scars after surgery59 In addition controlled studies have reported the clinical effectiveness of silicone gel and silicone gel sheeting in the prevention and treatment of keloids3558 However a recent metaanalysis review60 and Cochrane review61 found that even though studies published data in support of the efficacy of silicone gel sheeting in the treatment and prevention of keloid and hypertrophic scarring they provided weak evidence and were of poor quality60 Therefore given Figure Auricular keloid surgical excision used as monotherapy Auricular keloid before a and after b surgical excisonsurgery all yielded significant improvements compared to treatment with TAC monotherapy339Intralesional steroid injections can cause several adverse side effects such as telangiectasis atrophy steroid acne pigmentary changes necrosis ulcerations and systemic side effects3 There is also significant pain associated with intradermal corticosteroid injections that can be reduced when local anaesthetic lidocaine is administered to control the pain44 Furthermore the side effects have been reported to diminish when intralesional TAC is used in combination with 5FU45Surgical excisionSurgical excision is a traditional method of removing keloids Figure However excision creates a new wound and can result in a similar or larger keloid47 Therefore surgical excision is not recommended as a monotherapy as it results in high recurrence rates in the range of For better postoperative surgical outcomes surgical excision is often combined with other forms of treatment including radiotherapy intralesional corticosteroid injections IFN injection bleomycin cryotherapy pressure therapy and silicone gel or sheeting82649 Successful use of dermal substitutes and epidermal skin grafting with keloid excision has also been reported50 A recent case series study in which patients with anterior chest wall keloids were given a treatment protocol consisting of complete excision Zplasty postoperative adjuvant radiotherapy and postsurgical wound selfmanagement reported excellent outcomes with a recurrence rate of only The use of steroid tape and injections helped to resolve the recurrence of keloids51 0cOjeh the lack of substantial evidence welldesigned clinical trials and studies are required to gain a better understanding of the effectiveness of siliconebased products in preventing and treating keloidsPressure therapyPressure therapy has been used to treat and manage keloids and hypertrophic scars for decades3563 It has been routinely employed as firstline treatment in the treatment of hypertrophic scarring resulting from burns64 The underlying mechanisms of action of compression techniques remain unclear however several hypotheses exist some of which include increased pressure to the scar surface reduces perfusion and decreased oxygen to the location of injury reduces collagen synthesis It is also thought that pressure increases apoptosis reduces scar hydration stabilising mast cells and decreases angiogenesis165 The application of pressure can be achieved using a variety of materials such as adhesive plaster moulds pressure earrings and customfitted splints16 which have improved scar cosmesis and rates of keloid recurrence66 A continuous pressure of mmHg preferably at the lower range soon after wound reepithelialisation for h per day for months is recommended166869 The efficacy of pressure therapy depends mainly on the anatomical location of the scar with trunk and limb areas being more appropriate sites for pressure therapy In addition a pressure garment is predominantly used for auricular keloids where pressure clips are commonly utilised after surgery6670 As an adjuvant therapy this form of pressure garment has also been successfully used to prevent the recurrence of keloids6667 In contrast a metaanalysis review that analysed the effectiveness of pressure garment therapy for the prevention of abnormal scarring after burn injury was unable to demonstrate any beneficial effects of pressure garment therapy on prevention or treatment of abnormal scarring59 Notwithstanding success rates of pressure therapy are contingent upon patient compliance69 which can sometimes be low due to discomfort Overall pressure therapy is tolerated better and is devoid of the pain often associated with intralesional therapies and hence can be considered as a good adjuvant therapy for keloid scarsRadiotherapyIn the treatment of keloids using superficial Xray irradiation was first described71 Since then it has been used less frequently as a monotherapy and more widely as an effective adjunct treatment after surgical excision72 with success rates in the range of and recurrence rates of about Radioactive skin patches have also been used in combination with other treatment modalities for keloids7879 Radiotherapy is most commonly used h after surgical excision7480 and acts by suppressing angiogenesis and inhibiting ï¬broblast activity1 Decreased fibroblast proliferation induced cell senescence and apoptosis leading to reduction in collagen production and suppression of keloid development have also been reported81Different radiotherapy modalities have been used after surgical excision including electron beam radiotherapy brachytherapy superï¬cial and orthovoltage radiotherapy with varying degrees of success84 Mankowski et a0 al conducted a literature review of studies to compare the clinical outcome of different forms of radiation treatment used for the management of keloids77 The metaanalysis demonstrated that radiation used as monotherapy yielded higher rates of recurrence compared to combinational therapy with postsurgery excision Comparison between the different radiationbased treatments revealed that the lowest rate of recurrence was observed with brachytherapy followed jointly by Xray and electron beam The authors also reported that the rate of recurrence was dependent on anatomical site of the keloid with chest keloids having the highest recurrence rate73The adverse effects of radiotherapy often linked with dose of radiation used can be grouped into acute skin reactions and late complications Acute reactions arise as early as seven days after keloid treatment and include oedema necrosis ulceration desquamation erythema and pigmentary changes with the latter two being the most common Late complications which include changes in pigment atrophy telangiectasis and alopecia may present several weeks after radiotherapy Emollient and steroid ointment used after radiotherapy can help alleviate the side effects19 A recommended radiation dose Gy over several sessions can also minimise adverse effects1980 Radiotherapy carries a risk of malignancy8586 Therefore caution should be used in radiationvulnerable sites such as the head neck thyroid and breast and in patients aged years2 Protecting fragile ans and selecting the most appropriate sitedependent dose protocol can help minimise further complications of radiotherapy87 0c Scars Burns HealingCryotherapyCryotherapy is a lowtemperature treatment that causes vascular damage resulting in tissue necrosis88 It has been used to treat keloids as a monotherapy or in combination with other treatment methods such as intralesional steroid injections89 Various delivery methods used for cryotherapy include spray and contact probes or the intralesionalneedle cryoprobe method Compared to contact and spray methods intralesionalneedle cryoprobe was found to be the most effective method in treating keloid scars90 Positive outcomes were observed in a number of studies that used liquid nitrogen and cryotherapy to treat keloids with success rates in the range of External cryotherapy has been associated with several side effects including hypopigmentation blistering pain delayed healing and infection9093 Moreover larger keloids have been shown to need multiple cryotherapy sessions9093 To minimise side effects intralesional cryotherapy was introduced and there are now a number of nitrogenbased cryodevices that have been described for the treatment of keloid scars with two commercially available a liquid nitrogenbased device88 and an argon gasbased device94 The intralesional cryotherapy was designed to overcome the hypopigmentation seen mostly in darkskinned individuals with external cryotherapy It works by destroying the core of the keloid sparing the surface epithelial cells including melanocytes9596 As a result it enhances volume decrease while minimising the risk of hypopigmentation and other surface reactions90 A recent comprehensive review based on the preferred reporting items for systematic reviews and metaanalysis was performed to investigate the efficacy of intralesional cryotherapy on keloid scars90 The review of eight studies that met the inclusion criteria revealed that average scar volume decreased in the range of but complete eradication of the scar on average was lacking Recurrence of keloid scars was in the range of The authors also reported that patients complaints of pain and pruritus was considerably reduced however hypopigmentation was seen mostly in Fitzpatrick skin type patients after treatment90Laser therapyLaser therapy for keloid treatment was introduced in the 1980s97 Since then different systems have been used for the treatment of keloid and hypertrophic scars4898 These lasers target skin chromophores like haemoglobin and melanin based on the principle of selective photothermolysis99 Lasers can be classified as ablative and nonablative The most common ablative lasers include the 2940nm erbiumdoped yttrium aluminium garnet ErYAG laser and the 10600nm carbon dioxide CO2 laser These emit a laser beam that is absorbed by water in the skin leading to local tissue destruction and reduction of lesion volume3 Common examples of nonablative lasers include 585nm or 595nm PDLs 1064nm neodymiumdopedyttriumaluminiumgarnet NdYAG neodymiumdopedvanadate NdVan laser and nm Qswitched NdYAG laser with low fluence100 These lasers induce thermal injury to the scars microvasculature leading to thrombosis and ischaemia which result in collagen denaturation and collagen fibre realignment101532nm laser Laser therapy requires several treatments at intervals of weeks depending on scar type and type of laser used98104 with possible side effects including itching pigmentary changes blister formation and postoperative purpura98 The use of the nonfractional vascular nm PDL in the treatment of keloid and hypertrophic scars has been welldocumented105 and has response rates in the range of PDL monotherapy has been shown to be effective108110 as well as CO2 laser monotherapy38111112 In a clinical study where patients with moderate to severe keloids were treated with highenergy pulsed CO2 laser the treatment was efficacious and welltolerated with minimal side effects112 In other studies where CO2 laser ablation was compared with other forms of treatment CO2 laser was as efficient as the other forms38111 It must be noted however that these studies are small and randomised controlled studies are lacking98Laser therapy such as PDL CO2 and NdYAG have been associated with a high rate of recurrence at months111113 However optimal results can be achieved with combination treatment especially with intralesional TAC injections116 Kumar and coworkers conducted a cohort study on patients with keloids previously treated with an NdYAG laser and reported complete scar resolution and flattening in seven patients only when intralesional TAC was used after laser therapy41 Moreover combined therapy with PDL and TAC119 and PDL TAC and 5FU36 were shown to produce better clinical results In a recent study that evaluated and compared the efficacy of combination therapy of 0cOjeh scars statistically fractional CO2 laser and intralesional TAC injection or TAC injection alone in keloid and hypertrophic significant improvements were reported in overall scar quality with the combined treatment options comto TAC monotherapy120 Moreover pared combined CO2 laser and IFNα2b injections given to patients with auricular keloids resulted in no recurrence in of patients three years after treatment121 Laser therapy can also be combined with other laser treatment topical corticosteroids and cyanoacrylate glue98 and have shown promising results however larger controlled clinical studies are needed to further evaluate their efficacy and safetyRecently lasers are also being explored as tools for assisted drug delivery Kraeva et a0al proposed an alternative technique of corticosteroid administration of laserassisted drug delivery of topical TAC This was shown to be effective when used on a keloid on the posterior scalp of a patient after each CO2 laser session122 More efficient intraepidermal drug delivery options are also being investigated Singhal et a0al developed TACcontaining polymeric microps that were prepared using a cryomilling technique for freezing fracture After ablation with a fractional ErYAG laser these microps can be deposited in cutaneous micropores and provide highdose intraepidermal reservoir systems with minimal transdermal permeation leading to sustained and targeted local drug delivery123It must be noted that one of the biggest limitations in studies available at present is the lack of histological definition between keloid and hypertrophic scars so conclusions are not valid on efficacyImiquimod creamImiquimod cream is approved for the treatment of basal cell carcinoma actinic keratoses and genital warts21 As an immuneresponse modifier it stimulates the production of proinflammatory cytokines such as TNFα interleukins and IFNs by activated Tcells124 thereby changing the expression of genes associated with apoptosis125 and reducing collagen production16 Studies have reported conflicting findings regarding efficacy of imiquimod cream postoperatively following keloid excision likely due to keloid location Many studie
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thyroid cancer THCAprognosis and construct a polygene risk prediction model for prognosis predictionand improvementMethods The HTSeqCounts data of THCA were accessed from TCGA databaseincluding cancer samples and normal tissue samples edgeR package wasutilized to perform differential analysis and weighted gene coexpression networkanalysis WGCNA was applied to screen the differential coexpression genes associatedwith THCA tissue types Univariant Cox regression analysis was further used for theselection of survivalrelated genes Then LASSO regression model was constructed toanalyze the genes and an optimal prognostic model was developed as well as evaluatedby KaplanMeier and ROC curvesResults Three thousand two hundred seven differentially expressed genes DEGs wereobtained by differential analysis and coexpression genes COR P were gained after WGCNA analysis In addition eight genes significantly related to THCAsurvival were screened by univariant Cox regression analysis and an optimal prognostic3gene risk prediction model was constructed after genes were analyzed by the LASSOregression model Based on this model patients were grouped into the highrisk groupand lowrisk group KaplanMeier curve showed that patients in the lowrisk group hadmuch better survival than those in the highrisk group Moreover great accuracy of the3gene model was revealed by ROC curve and the remarkable correlation between themodel and patients prognosis was veriï¬ed using the multivariant Cox regression analysisConclusion The prognostic 3gene model composed by GHR GPR125 and ATP2C2three genes can be used as an independent prognostic factor and has better predictionfor the survival of THCA patientsKeywords THCA WGCNA prognostic 3gene risk prediction model prediction prognosisINTRODUCTIONThyroid cancer THCA derived from parafollicular cells or thyroid follicular cells is the mostcommon endocrine malignancy accounting for about of all kinds of human cancers Papillary PTC follicular anaplastic and medullary thyroid carcinomas are the four subtypes ofTHCA among which papillary and follicular carcinomas are common and have better prognosisEdited byChristoph ReinersUniversity HospitalW¼rzburg GermanyReviewed byTrevor Edmund AngellUniversity of Southern CaliforniaUnited StatesRoberto VitaUniversity of Messina ItalyCorrespondenceHaixing Fanghaixing01231163comSpecialty sectionThis was submitted toThyroid Endocrinologya section of the journalFrontiers in EndocrinologyReceived November Accepted June Published August CitationZhao H Zhang S Shao S and Fang H Identiï¬cation of a Prognostic3Gene Risk Prediction Model forThyroid CancerFront Endocrinol 103389fendo202000510Frontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancer while anaplastic carcinoma is rare to be seen with extremelypoor prognosis Therefore its very important to ï¬nd eï¬ectiveapproaches for the improvement of the overall THCA prognosisAt present the conventional prognostic model of THCA inclinical practice is constructed according to predictive factorslike age tumor size and lymph nodule metastasis Withthe development of highthroughput sequencing technologymRNA expression proï¬les of speciï¬c cancers are easy to obtainwhich helps us better ï¬nd more robust prognostic signals For instance microarraybased gene expression analysis enablesus to identify the important genes during tumor progressionand helps to deï¬ne and diagnose prognostic characteristics In this way many THCA prognostic biomarkers have beenveriï¬ed However these markers are almost single genes and havenot been widely accepted Polygenic combination has beenreported to possess better predictive ability for cancer prognosisthan single genes Therefore recent studies have involvedin the identiï¬cation of the biomarkers for THCA prognosis However restricted by research methods novel biologicalalgorithm needs to be explored to construct more accuratediagnostic or prognosis modelsIn the present study a large number of mRNA expressionproï¬les of THCA patients were accessed from TCGA databaseand modules associated with THCA were identiï¬ed by WGCNAA 3gene risk prediction model was constructed using Cox andLASSO regression models which could help us better predictTHCA prognosisMATERIALS AND METHODSData ResourceExpression proï¬les of THCA mRNA and corresponding clinicaldata were accessed from TCGA database cancergenomenihgovsamples and normaltissue samples The study was in line with the guidelinesreleased by TCGA httpcancergenomenihgovpublicationspublicationguidelinesincluding cancerIdentiï¬cation and Conï¬rmation ofTHCAAssociated GenesedgeR packagebioconductorpackagesreleasebiochtmledgeRhtml was used to perform diï¬erential analysisbetween cancer tissues and normal tissues Genes met thecriteria logFC and P were considered to havesignificant diï¬erencesModule Selection With WGCNAThe mechanism of WGCNA is the research for coexpressionmodules and the exploration of the correlation between the genenetwork and the phenotypes which is motivated by the analysesof scalefree clustering and dynamic tree cut on expressionproï¬les In the present study modules that were most relatedto THCA tissue types in the coexpression network constructedby WGCNA package cranrprojectwebpackagesWGCNAindexhtml were selected and genes meeting P and COR were extracted for further studyConstruction of the Prognostic RiskPrediction ModelTHCA prognosisassociated genes werescreened usingunivariant Cox regression analysis Then a prognostic modelwas constructed using the least absolute shrinkage and selectionoperator LASSO According to this model risk score of eachsample was calculated and patients were divided into thehighrisk group and lowrisk group with the median risk scoreas the threshold KaplanMeier was used to evaluate the survivalof the two groups The ROC curve was drawn for the evaluationof the prognosis performance of the model and the area underthe curve AUC was calculated Furthermore multivariantCox regression analysis was performed to assess the correlationbetween the risk score and patients prognosis KaplanMeierand ROC curves of each gene in this model were plotted to makea comparison with those curves of the modelStatistical AnalysisUnivariant and multivariant Cox regression analyses wereboth performed in TCGA dataset glmnet package of theR software wwwrproject was used for LASSOstatistic algorithm IBM SPSS statistical software IBMCorp Armonk NY USA was applied for statistical analysis P was considered statistically significantRESULTSIdentiï¬cation of THCAAssociatedModulesAs shown in A a total of DEGs were identiï¬edlogFC P WGCNA was used to screen THCArelated modules and appropriate adjacency matrix weightparameter power was selected to ensure the scalefreedistribution of the coexpression network as possible In therange of log k and log Pk were calculated for linearmodels construction respectively is the squared value of thecoeï¬cient R As shown in B the soft threshold poweris higher with the elevated R2 suggesting that the network closelyapproaches to scalefree distribution In the present study R2 for the ï¬rst time was selected to ensure the realizationof scalefree distribution as possible and make the values on thecurve approach to the minimum threshold When themean connectivity of RNA in the network was Cwhich was consistent with the smallworld network in the scalefree one Then cluster dendrogram was constructed Dand dynamic tree cut was performed deep split Modulesobtained were merged with the minimum size of and modules were eventually developedThe correlation and signiï¬cance between the modulecharacteristics and sample phenotypes were calculated Amongthe modules genes in blue brown pink and turquoisemodules were veriï¬ed to be most associated with THCAprognosis E THCA tissue typeassociated geneswere obtained from the four modules taking the P andCOR as the threshold FFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE Identiï¬cation of the THCA tissue typeassociated RNA functional modules A Volcano plot of DEGs B Analysis of scaleindependence index for varioussoft threshold powers Horizontal axis is the soft threshold power and vertical axis is the scalefree topology ï¬tting indices R2 The red line refers to the standardcorresponding to the R2 of C Analysis of the mean connectivity under different soft threshold powers D Cluster dendrogram of all DEGs clustered based on adissimilarity measure E Distribution of average gene signiï¬cance and errors in the modules associated with the progression of THCA F Venn diagram of the genesin the four modules for coexpression genes selectionFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerConstruction of a Prognostic 3Gene RiskPrediction Model for THCAUnivariant Cox regression was performed for analysis ofthe coexpression genes suggesting that eight genes weresignificantly correlated with survival as shown in Table LASSO regression model was constructed to analyze thegenes and an optimal prognostic risk prediction modelwasScore GHR GPR125 Atp2c2 Risk prediction wasperformed according to this model and patients were rangedFigure 2A RiskeventuallydevelopedTABLE Basic information of the eight prognostic genesidHRHR95LHR95HPvaluebased on the risk scores Figure 2B The median risk score wasused as the critical value to group the patients into the highriskgroup n and lowrisk group n As shown inthe KaplanMeier curve in Figure 2C patients in the highriskgroup had worse overall survival OS than those in the lowriskgroup ROC curve was plotted to predict the 3year survival andthe results showed in Figure 2D revealed that AUC of the 3genemodel was which indicated the good performance of therisk score in survival prediction Multivariant Cox proportionalhazards regression analysis was then performed combined withclinical factors and the correlation between the risk score andprognosis of patients was veriï¬ed Figure 2E From the heatmaps of the expression proï¬les of these three genes Figure 2Fthe expression levels of GHR GPR125 and Atp2c2 were found tobe positively correlated with the risk score and all of them wereregarded as highrisk genesAtp2c2GPR125GHRCLMNCYTH3PLA2R1RYR2C8orf88Evaluation of the 3Gene Risk PredictionModelKaplanMeier curves of the three genes were drawn using thelog rank test As shown in Figures 3AC THCA patients withlow expression of GHR GPR125 and Atp2c2 had longer survivaltime indicting that these three genes were highrisk genes whichwas in agreement with the results predicted by univariant Coxregression analysis Furthermore ROC curves Figures 3DFFIGURE Construction of a 3gene risk prediction model A LASSO regression model B 3gene based distribution of risk scores C Survival analysis of realhub genes in the TCGATHCA dataset D ROC curve of real hub genes in the TCGATHCA dataset E The correlation between the risk score and patientsprognosis F Heatmap of the genes expression proï¬lesFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid CancerFIGURE The evaluation of the 3gene risk prediction model AC Survival analyses of GHR GPR125 and Atp2c2 in the TCGATHCA dataset DF ROCanalyses of GHR GPR125 and Atp2c2 in the TCGATHCA datasetrevealed that the AUC of GHR GPR125 and Atp2c2 was and respectively all of which were smaller than thatof the 3gene risk prediction model Findings above demonstratethat risk score is a good indicator for prognosis and the 3genemodel has a higher accuracyDISCUSSIONWith the development of the microarray and RNA sequencingtechnologies new era of large data on biology is coming It hasbeen reported that microarraybased gene expression analysiscould achieve characterization in human cancers identiï¬cationof the important genes during tumorigenesis and the deï¬nitionas well as the diagnosis of prognostic features However therole of genes as prognosis factors has been few investigated In the present study a large amount of RNAseq proï¬les andclinical prognosis data of THCA patients were accessed fromTCGA database and coexpression gene modules were screenedusing WGCNA Studies have shown that gene modules are muchreliable in cancer prognosis than biomarkers While there arefew studies on the crosstalk among the modules and someimportant modules might be ignored Therefore in ourstudy gene coexpression network was constructed via WGCNAand was used to identify THCA tissue typeassociated genemodules including blue brown pink and turquoise Twentythree common genes were obtained from the four modulesand an optimal prognostic 3gene risk prediction model wasthen constructed by univariant Cox and LASSO regressionanalyses Along with the LASSO model all independent variablescan be processed simultaneously verifying the more accurateperformance than the stepwise regression model GHRGPR125 and Atp2C2 were the three genes in this model GHR isa kind of proteincoding gene coding transmembrane receptorsof the growth hormone In prior studies GHR has been veriï¬edto be a oncogene in some cancers such as breast cancer pancreatic ductal carcinoma and melanoma but therole in THCA prognosis is ï¬rstly reported GPR125 a 57KDafactor for transmembrane signal transduction is considered toplay a key role in cell adhesion and signal transduction Itsreported that GPR125 is upregulated in human cerebral cancertissues and promotes cell adhesion as well as the formationof myelosarcoma In our study GHR and GPR125 wereveriï¬ed as highrisk genes in THCA which was consistent withthe previous studies Moreover we found that these two genescould be used as independent risk predictive factors but theaccuracy was lower than that of the 3gene risk prediction modelwhich was further veriï¬ed by ROC and KaplanMeier curvesAs the expression proï¬les of THCA and clinical informationare just from one dataset of TCGA the samples for analyzingthe prognostic 3gene model are limited In addition the modelconstructed in this study might be not available when it comesto other databases and its necessary to improve the model withFrontiers in Endocrinology wwwfrontiersinAugust Volume 0cZhao et alA Prognostic 3Gene Model for Thyroid Cancermore datasets In a word a 3gene model is constructed to be anindependent predictor in this study which provides novel viewand approach for the prognosis of THCA patientsDATA AVAILABILITY STATEMENTAll datasets generated for this study are included in thesupplementary materialAUTHOR CONTRIBUTIONSHZ contributed to the study design and gave the ï¬nalapproval of the version to be submitted SZ conducted theliterature search and performed data analysis and draftedSS acquired the data and revised the HF wrote the All authors contributed to the and approved thesubmitted versionREFERENCES Hedayati M Zarif Yeganeh M Sheikholeslami S Afsari F Diversityof mutations in the RET protooncogene and its oncogenic mechanismin medullary thyroid cancer Crit Rev Clin Lab Sci Carling T Udelsman R Thyroid cancer Annu Rev Med 101146annurevmed061512105739 Dralle H Machens A Basa J Fatourechi V Franceschi S Hay IDet al Follicular cellderived thyroid cancer Nat Rev Dis Primers 101038nrdp201577 SmallridgeRCCoplandand emergingAnaplasticJAtherapies Clin Oncolthyroidcarcinoma pathogenesis 101016jclon201003013 Shaha AR Implications of prognostic factors and risk groups in themanagement of diï¬erentiated thyroid cancer Laryngoscope Zhao QJ Zhang J Xu L Liu FF Identiï¬cation of a ï¬velong noncoding RNA signature to improve the prognosis prediction for patientswith hepatocellular carcinoma World J Gastroenterol 103748wjgv24i303426et Hebrant A Dom G Dewaele M Andry G Tr©sallet C LeteurtreEthyroidcarcinoma molecular anatomy of a killing switch PLoS ONE 7e37807 101371journalpone0037807al mRNA expression in papillaryand anaplastic Brennan K Holsinger C Dosiou C Sunwoo JB Akatsu H Haile R et alDevelopment of prognostic signatures for intermediaterisk papillary thyroidcancer BMC Cancer 101186s1288501627716 ZuoS Dai G Ren XIdentiï¬cation ofa6genepredicting prognosis 101186s1293501807247for colorectal cancer Cancer CellsignatureInt Cui ZJ Zhou XH Zhang HY DNA methylation module networkcancer Genestyping ofbased prognosisand molecular 103390genes10080571 Gu JX Zhang X Miao RC Xiang XH Fu YN Zhang JY et alrecurrencefreein hepatocellular carcinoma World J GastroenterolSixlongsurvival 103748wjgv25i2220noncodingsignaturepredictsRNA Arumugam A Subramani R Nandy SB Terreros D Dwivedi AK Saltzstein Eet al Silencing growth hormone receptor inhibits estrogen receptor negativebreast cancer through ATPbinding cassette subfamily G member Exp MolMed 101038s1227601801978 Subramani R LopezValdez R Salcido A Boopalan T Arumugam A NandyS et al Growth hormone receptor inhibition decreases the growth andmetastasis of pancreatic ductal adenocarcinoma Exp Mol Med 46e117 101038emm201461 Proudfoot NJ Gil A Whitelaw E Studies on messenger RNA endformation in globin genes a transcriptional interference model for globin geneswitching Prog Clin Biol Res Wu Y Chen W Gong L Ke C Wang H Cai Y Elevated Gprotein receptor GPR125 expression predicts good outcomes in colorectal cancer andinhibits Wntbetacatenin signaling pathway Med Sci Monit 1012659MSM910105 Pickering C Hgglund M SzmydyngerChodobska J Marques F Palha JAWaller L et al The adhesion GPCR GPR125 is speciï¬cally expressed in thechoroid plexus and is upregulated following brain injury BMC Neurosci Fu JF Yen TH Chen Y Huang YJ Hsu CL Liang DC et alInvolvement of Gpr125 in the myeloid sarcoma formation inducedby cooperating MLLAF10OMLZ and oncogenic KRAS in a mousebone marrow transplantation model 101002ijc28195J CancerInt Li X Dai D Wang H Wu B Wang R Identiï¬cation of prognostic signaturesassociated with longterm overall survival of thyroid cancer patients basedon a competing endogenous RNA network Genomics 101016jygeno201907005 Li J Yu X Liu Q Ou S Li K Kong Y et al Screening of importantadenocarcinomalncRNAsbased on integrated bioinformatics analysis Mol Med Rep 103892mmr201910061associated with the prognosis oflung Tavares C Melo M CameselleTeijeiro JM Soares P Sobrinhothyroid cancer 174R117 101530EJESimoes M Endocrine tumours genetic predictors ofoutcome EurJ EndocrinolConï¬ict of Interest The authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestCopyright Zhao Zhang Shao and Fang This is an openaccess distributed under the terms of the Creative Commons Attribution License CC BYThe use distribution or reproduction in other forums is permitted provided theoriginal authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction is permitted which does not comply with thesetermsFrontiers in Endocrinology wwwfrontiersinAugust Volume 0c'
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the incidence and death rate of nonsmall cell lung cancer nsclc in china ranks the first among the malignant tumors circular rna circrna was reported to be involved in the progression of nsclc our study aimed to investigate the underlying mechanism of circ_0020123 in nsclc progressionmethods quantitative realtime polymerase chain reaction qrtpcr was used to detect the expression of circ_0020123 mir5905p and thrombospondin thbs2 in nsclc tissues and cells cell proliferation and migration were examined by cell counting kit8 cck8 assay and transwell assay respectively flow cytometry assay was used to detect the apoptosis of nsclc cells the protein levels of ki67 matrix metalloprotein9 mmp9 cleavedcaspase9 cleavedcasp9 and thbs2 were detected by western blot the targets of circ_0020123 and mir5905p were predicted by starbase and targetscan and then confirmed by dualluciferase reporter assay and rna immunoprecipitation rip assay the animal experiment showed the effect of circ_0020123 on tumor growth in vivoresults the expression of circ_0020123 was upregulated in nsclc tissues and cells functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of nsclc cells interestingly circ_0020123 directly targeted mir5905p and inhibition of mir5905p reversed the knockdown effects of circ_0020123 on nsclc cells more importantly thbs2 was a target of mir5905p and thbs2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in nsclc cells finally suppression of circ_0020123 inhibited tumor growth in vivo through mir5905pthbs2 axis circular rna circ_0020123 regulated thbs2 by sponging mir5905p to promote cell proliferation and migration and inhibit cell apoptosis in nsclc cellskeywords nsclc circ_0020123 mir5905p thbs2highlights circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in nsclc cellscorrespondence bskrju163comdepartment of thoracic surgery lianyungang second peoples hospital no hailian east road haizhou district lianyungang jiangsu china circ_0020123 directly targeted mir5905p and mir5905p downregulation reversed the knockdown effects of circ_0020123 on nsclc progression thbs2 acted as a target of mir5905p and overthe effects of expression of thbs2 reversed circ_0020123 knockdown on nsclc progression downregulation of circ_0020123 suppressed tumor growth in vivo through mir5905pthbs2 axis the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwang a0et a0al cancer cell int page of lung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis small cell lung cancer sclc accounts for about of all lung cancer cases however nonsmall cell lung cancer nsclc accounts for of lung cancer and the a0years overall survival rate os is only about therefore it is important to find the effective treatment and potential molecular targets of nsclc progressioncircular rna circrna is a single stranded rna molecule with a closed circular structure recently amounts of circular dna have been discovered and most of which were thought to be the byproducts of typical splicing [ ] previous reports indicated that the expression of circrna was tissuespecific and the change of its expression intensity was associated with some diseases [] furthermore circrna was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] for example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging mir34a5p to regulate cc chemokine ligand ccl22 meanwhile hsa_circ_0043256 participated in the progression of nsclc cells by mediating the cinnamaldehyde treatment a previous report suggested that circ_0020123 acted as an oncogene in nsclc and circ_0020123 regulated zincfingerenhancer binding protein zeb1 and enhancer of zeste homolog ezh2 by competitively binding with mir144 to induce cell progression and migration these reports suggested that circ_0020123 was a vital factor in the pathogenesis of nsclc and its function and molecular mechanism need to be further studiedas a small endogenous rna microrna mirna is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers recently some aggregated mirnas have been found in prostate cancer such as mir221222 mir143145 mir23b27b241 and mir1133a which were downregulated and had tumor inhibiting functions a previous study found that circulating mir5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy besides overexpression of mir5905p reduced the development of nsclc cells and regulated the expression of epithelialmesenchymal transformation emtrelated proteins by targeting the signal transducers and activators of transcription stat3 however the precise mechanism by which mir5905p affects nsclc needs further investigationthrombospondin thbs2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer colorectal cancer and nsclc a previous report suggested that thbs2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by mir20a tian et a0al found the expression and clinicopathological features of thbs2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis however the molecular function of thbs2 in nsclc remains poorly definedin this study the targeting relationship between circ_0020123 and mir5905p was firstly detected the effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesmaterials and a0methodspatients and a0specimensnsclc tissues and the adjacent healthy lung tissues were taken from nsclc patients in the lianyungang second peoples hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at a0 °c for further experiments this research was approved by the ethics committee of lianyungang second peoples hospitalcell culture and a0cell transfectiontwo nsclc cell lines a549 and h1299 and one normal lung cell line imr90 were obtained from the beijing concorde cell library beijing china a549 h1299 and imr90 cells were cultivated in dulbeccos modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0 with co2small interfering rna sirna targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin rna shrna targeting circ_0020123 shcirc_0020123 mir5905pinhibitors sirna negative control sinc shnc and ncinhibitors were all obtained from biomics biotech jiangsu china full length of thbs2 cdna sangon biotech shanghai china was subcloned into pcdna31 plasmid ekbioscience shanghai china then cell transfection was performed by lipofectamine thermo fisher scientific waltham ma usa 0cwang a0et a0al cancer cell int page of rna isolation and a0quantitative realtime polymerase chain reaction qrtpcrthe trizol reagent invitrogen carlsbad ca usa was used for extracting the total rnas next the reversed transcription was carried out by rtpcr kit invitrogen the qrtpcr was performed using the abi sybr green master mix invitrogen the primers in our study were as follows f5²ttc gga cga ccg tca aac at3² and r5²agg atc cct gca cca caa tg3² for circ_0020123 f5²tga aag acg tga tgg cac ac3² and r5²ctt cca ttt tgg for mir5905p f5²aga agg ggt ttt tgg3 ² ctg ggg ctc att tg3² r5²agg ggc cat cca cag tct tc3² for glyceraldehyde3phosphate dehydrogenase gapdh f5²gcg gct ggg tct att tgt c3² and r5²gca gga ggt gaa gaa cca tc3² for thbs2 f5²att gga acg ata cag aga agatt3² and r5²gga acg ctt cac gaa ttt g3² for u6 gapdh and u6 were the internal parameterscell counting kit cck assaythe proliferation viability of a549 and h1299 cells were detected by the cellcounting kit8 msk wuhan china a549 and h1299 cells were cultivated into a 96well plate with a density of à a0cellswell and incubated in a0°c for or a0h then a0μl fresh medium and cck8 solution was added after incubation at a0°c for a0h the od values were detected by the multiskan ascent microplate reader abcam cambridge ma usatranswell assaytranswell chamber corning life sciences corning ny usa was used to detect cell migration firstly the serumfree dmem thermo fisher scientific was fixed with cell suspension cells and seeded into the upper chamber and the dmem containing serum was put into the lower chamber after incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed finally the migrated cells were stained with crystal violet corning life sciences and observed by using a microscopeflow cytometryfirstly a549 and h1299 cells were cultured and pbs was used for washing cells then the binding buffer was used to resuspend cells and the annexin vfluorescein isothiocyanate vfitcpropidium iodide pi apoptosis detection kit thermo fisher scientific was used to stain cells finally cell apoptosis was detected by flow cytometry thermo fisher scientificwestern blot analysisthe total proteins of nsclc tumors or cells were collected by ripa lysis buffer sangon biotech then the proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis sdspage and transferred to polyvinylidene fluoride pvdf membranes thermo fisher scientific the skimmed milk was added and incubated with primary antigapdh antibody invitrogen carlsbad ca usa antiβactin antibody invitrogen antiki67 antibody invitrogen antimatrix metalloprotein9 mmp9 antibody invitrogen anticleavedcaspase9 cleavedcasp9 antibody invitrogen or antithbs2 antibody invitrogen at a0°c overnight finally the membranes were incubated with the secondary antibody for a0 h at room temperature the results were viewed using kodak film developer fujifilm japandualluciferase reporter assaysthe wild type circ_0020123 sequences circ_0020123wt mutant circ_0020123 sequences circ_0020123mut wild type thbs2 ²utr sequences thbs2wt mutant thbs2 ²utr sequences thbs2mut were cloned into pgl3 luciferase reporter plasmid promega madison wi usa then the plasmid and mir5905p or mirnc were cotransfected into a549 and h1299 cells by lipofectamine thermo fisher scientific after transfection for a0h the dualluciferase reporter assay system promega was performed to detect the luciferase activityrna immunoprecipitation ripfirstly the magna rip rnabinding protein immunoprecipitation kit gzscbio guangzhou china was performed to verify the relationship between circ_0020123 and mir5905p in brief the magnetic beads and antiago2 antibody abcam were added into cells and incubated for a0h then the proteinase k and the phenolchloroformisoamyl alcohol reagent were added for purifying rnas finally qrtpcr was used to measure circ_0020123 enrichmentanimal experimentsthe 4weekold balbc male nude mice vitalriver beijing china were raised in a sterile environment for 0cwang a0et a0al cancer cell int page of experiments then pbs was used to suspend a549 cells à transfected with shcirc_0020123 or shnc next the nude mice were divided into two groups n a549 cells transfected with shcirc_0020123 or shnc were shcirc_0020123 or shnc inoculated into the nude mice the tumor volume was detected every a0 days after a0days the nude mice were euthanatized and the tumor weight was detected besides the tumor tissues from each group were collected to detect the expression of circ_0020123 mir5905p and thbs2 the animal experiment was approved by the animal experimentation ethics committee of lianyungang second peoples hospitalstatistical analysisthe software graphpad prism was performed for statistical analysis the data was displayed as mean ± standard deviation sd the significant difference was calculated by students t test and oneway analysis of variance anova p was considered as statistically significantresultscirc_0020123 was a0upregulated in a0nsclc tissues and a0cellsto begin with qrtpcr was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in nsclc tissues compared with the adjacent healthy tissues fig a0 1a similarly the expression of circ_0020123 in nsclc cells a549 and h1299 was markedly higher than that in normal cells imr90 fig a01b from these data it is speculated that circ_0020123 might be acted as an oncogene in nsclcfig circ_0020123 was upregulated in nsclc tissues and cells a qrtpcr was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b the expression of circ_0020123 in normal cell line imr90 and nsclc cell lines a549 and h1299 was detected by qrtpcr p downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0nsclc cellsto investigate the functional effects of circ_0020123 on nsclc cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into a549 and h1299 cells firstly the transfection efficiency was detected by qrtpcr fig a02a next cck8 was used to detect the proliferation and the results showed that the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced fig a0 2b moreover the migration of a549 and h1299 cells was significantly downregulated by circ_0020123 knockdown fig a02c in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with sinc fig a02d finally the protein levels of cell proliferationrelated protein ki67 and cell migrationrelated protein mmp9 were inhibited while cell apoptosisrelated protein cleavedcasp9 was upregulated in nsclc cells transfected with sicirc_00201231 or sicirc_00201232 fig a02e these data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in nsclc cellscirc_0020123 directly targeted mir5pby searching in the online software starbase the potential binding sites between circ_0020123 and mir5905p were detected fig a0 3a to confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123wt reporter plasmid was reduced by mir5905p mimic while the circ_0020123mut reporter plasmid activity was not changed in a549 and h1299 cells fig a03b furthermore the expression of mir5905p was lower in a549 and h1299 cells compared with that in imr90 cells fig a0 3c in contrast mir5905p expression was elevated in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a0 3d finally the rip assay was also used to confirm the targeting relationship between circ_0020123 and mir5905p and the results showed that circ_0020123 and mir5905p were enriched in antiago2 group fig a03emir5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0nsclc cellsto further explore the functional effects between circ_0020123 and mir5905p mir5905pinhibitor was established qrtpcr was used to detect the transfection efficiency fig a0 4a interestingly mir5905p was upregulated in a549 and h1299 cells transfected with sicirc_00201231 while the expression of mir5905p was recovered in cells transfected with 0cwang a0et a0al cancer cell int page of fig downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of nsclc cells a the transfection efficiency of sicirc_00201231 and sicirc_00201232 in a549 and h1299 cells was detected by qrtpcr b cck8 assay was used to detect the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 c the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by transwell assay d flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 e the protein levels of cell proliferation related protein ki67 cell migration related protein mmp9 and cell apoptosis related protein cleavedcasp9 were detected by western blot p fig a0sicirc_00201231 mir5905pinhibitors 4b moreover circ_00201231 knockdown inhibited cell proliferation and migration while the mir5905p inhibitor reversed these effects fig a0 4c d in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 was increased which was abolished by mir5905pinhibitor fig a0 4e similarly mir5905p inhibitors reversed the effects on the protein levels of ki67 mmp9 and cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 fig a0 4f these results that mir5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of nsclc cellsindicated mir5p targeted thbs2 in a0nsclc cellsthe thbs2 ²utr was predicted to contain the binding sites of mir5905p through the online software targetscan fig a05a then the dualluciferase reporter assay was used to confirm the targeting relationship the results showed that cotransfection of mir5905p and thbs2wt significantly limited the luciferase activity in both a549 and h1299 cells the luciferase activity was not altered in cells cotransfected with mir5905p and thbs2mut fig a05b importantly the mrna and protein level of thbs2 was enahnced in nsclc cells fig a05c d we further explored whether circ_0020123 affected the functions of thbs2 in nsclc cells the mrna and protein expression of thbs2 were repressed in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a05e f 0cwang a0et a0al cancer cell int page of fig circ_0020123 directly targeted mir5905p a the binding site between circ_0020123 and mir5905p was detected by the online software starbase b the luciferase activity of circ_0020123wt or circ_0020123mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p was detected by dualluciferase reporter assay c qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells d the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr e rip assay was used to confirm the relationship between circ_0020123 and mir5905p p thbs2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0nsclc cellsbased on the work ahead of us the pcdna31thbs2 was constructed then the qrtpcr and western blot were used to detect the transfection efficiency and the thbs2 expression was increased in a549 and h1299 cells transfected with pcdna31thbs2 fig a0 6a b in addition the proliferation and migration rates of a549 and h1299 cells transfected with sicirc_00201231 pcdna31thbs2 were higher than that transfected with sicirc_00201231 fig a0 6c d meanwhile a similarly phenomenon was also observed in cell apoptosis the pcdna31thbs2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis fig a0 6e furthermore the effects of circ_0020123 deletion on ki67 mmp9 and cleavedcasp9 protein levels were also reversed by thbs2 overexpression fig a0 6f these data suggested that overexpression of thbs2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisreduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123mir5pthbs2 axisto further explore the function of circ_0020123 in nsclc cells the shcirc_0020123 was constructed and the xenograft tumor was established then a549 cells transfected with shcirc_0020123 or shnc were injected into the nude mice the xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shnc group fig a07a moreover tumor weight was inhibited by circ_0020123 knockdown fig a0 7b furthermore the expression circ_0020123 and thbs2 was decreased while the mir5905p was increased in xenograft tumor transfected with shcirc_0020123 fig a0 7c western blot assay also revealed that the protein level of thbs2 was repressed by circ_0020123 knockdown fig a07d finally the digital tomosynthesis dts was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group fig a07e the results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivodiscussionclinically only a few nsclc patients were diagnosed at an early stage and treated by surgical resection more than of nsclc patients were diagnosed with the advanced stage or metastatic tumors thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of nsclcrecently circrna was no longer considered as a random product in the rna shearing process and its biological significance and function in malignant tumors 0cwang a0et a0al cancer cell int page of fig mir5905p downregulation reversed circ_0020123 knockdown effects in nsclc cells a qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells transfected with mir5905pinhibitors b the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was detected by qrtpcr c the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was tested by cck8 assay d transwell assay was used to measure the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors e flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors were detected by western blot p had received more and more attention previous reports revealed that circ_0020123 was involved in the development of nsclc moreover the level of circ_0020123 was elevated in nsclc cells consistently we found that the expression of circ_0020123 was markedly higher in nsclc tissues and cells moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of nsclc cells in a0 vitro besides circ_0020123 promoted tumor growth in a0vivoendogenous circrnas could act as microrna sponges to inhibit their function and some studies linked mirna sponges to human diseases including cancer a previous study indicated that circrna ctransferrin receptor ctfrc regulated tfrc by sponging mir107 to facilitate bladder carcinoma development mir5905p was studied in different cells such as airway smooth muscle cells colon epithelial cells and nsclc cells however the potential relationship between mir5905p and circrna has not been researched in this study circ_0020123 directly targeted mir5905p and mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc progression these data provided a clue to the therapeutic strategy for nsclc 0cwang a0et a0al cancer cell int page of fig mir5905p targeted thbs2 in nsclc cells a the potential binding site between thbs2 ²utr and mir5905p was predicted by the online software targetscan b dualluciferase reporter assay was used to measure the luciferase activity of thbs2wt or thbs2mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p c qrtpcr was used to detect the mrna expression of thbs2 in nsclc cells d the protein level of thbs2 in nsclc cells was tested by western blot e the mrna expression of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr f western blot was used to measure the protein level of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 p our study also confirmed that mir5905p could target thbs2 directly in nsclc cells thbs2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase mmp genes involved in tissue formation and repair [ ] a previous document suggested that thbs2 acted as a target of mir2213p and participated in lymph node metastasis in cervical cancer the data in this research showed that the expression of thbs2 in nsclc cells was markedly higher than normal healthy cells furthermore overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells suggesting that circ_0020123 promoted the progression of nsclc cells through mir5905pthbs2 axisin our research showed that the expression of circ_0020123 was higher in nsclc tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of nsclc cells and also suppressed tumor growth in a0 vivo moreover circ_0020123 directly targeted mir5905p while mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc cells more importantly circ_0020123 regulated the expression of thbs2 by sponging mir5905p and upregulation of thbs2 reversed the effects of circ_0020123 knockdown on nsclc cells therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cwang a0et a0al cancer cell int page of fig overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells a b the mrna and protein expression of thbs2 in a549 and h1299 cells transfected with pcdna31thbs2 was detected by qrtpcr and western blot c cck8 assay indicated the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 d the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was measured by transwell assay e the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was detected by flow cytolysis assay f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 were detected by western blot p apoptosis of nsclc cells by sponging mir5905p to regulate thbs2results and develop the manuscript all authors read and approved the final manuscriptabbreviationsnsclc nonsmall cell lung cancer circrna circular rna qrtpcr quantitative realtime polymerase chain reaction cck8 cell counting kit8 mmp9 matrix metalloprotein9 cleavedcasp9 cleavedcaspase9 cleavedcasp9 cleavedcaspase9 rip rna immunoprecipitation zeb1 zincfingerenhancer binding protein ezh2 zeste homolog stat3 signal transducers and activators of transcription thbs2 thrombospondin acknowledgementsnot applicableauthors contributionslw collaborated to design the study lz were responsible for experiments analyzed the data yw wrote the paper all authors collaborated to interpret fundingnoneavailability of data and materialsplease contact corresponding author for data requestsethics approval and consent to participatethis research was approved by the ethics committee of lianyungang second peoples hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second peoples hospitalconsent for publicationall listed authors have actively participated in the study and have read and approved the submitted manuscript 0cwang a0et a0al cancer cell int page of fig reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123mir5905pthbs2 axis a a total of à a549 cells transfected with shcirc_0020123 or shnc were injected into nude mice to establish the xenograft tumor tumor volume was measured every d after injection b tumor weight was measured on d c the expression of circ_0020123 mir5905p and thbs2 in xenograft tumor was measured by qrtpcr d the protein level of thbs2 in xenograft tumor was evaluated by western blot e the number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis dts p competing intereststhe authors declare that they have no competing interestsreceived april accepted july references bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin abe h takase y sadashima e fukumitsu c murata k ito t kawahara a naito y akiba j insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value cancer cytopathol li c zhang l meng g wang q lv x zhang j li j circular rnas pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer j cancer res clin oncol belousova ea filipenko ml kushlinskii ne circular rna new regulatory molecules bull exp biol med zhang z xie q he d ling y li y li j zhang h circular rna new star new hope in cancer bmc cancer li l chen y nie l ding x zhang x zhao w xu x kyei b dai d zhan s guo j zhong t wang l zhang h myodinduced circular rna cdr1as promotes myogenic differentiation of skeletal muscle satellite cells biochim biophys acta gene regul mech greco s cardinali b falcone g martelli f circular rnas in muscle function and disease int j mol sci weng xd yan t liu cl circular rna_larp4 inhibits cell migration and invasion of prostate cancer by targeting foxo3a eur rev med pharmacol sci deng n lei d huang j yang z fan c wang s circular rna expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsi
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in china esophageal cancer ec is the fourth most frequently diagnosed form of malignant tumor inmales and the fifth most commonly diagnosed form in females approximately and casesoccurred in respectively the incidence of ec in eastern asia is in the top five worldwide including china esophageal squamous cell carcinoma escc is a major histological subtype accountingfor of all ec cases the complex interaction of economical and environmental conditions with individuals hereditary factors may lead to ec development the etiology and development of ec is notfully understood despite many investigations have payed close attention to the importance of immunity recently it was hypothesized that some important variants in immunerelated genes may influencethe susceptibility of esccreceived november revised july accepted july accepted manuscript online august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895interleukin4 il4 coded by the il4 gene is an important regulator of the inflammation pathways il4 apleiotropic cytokine may be correlated with survival and growth of lymphocytes il4 is produced by mast cellprecursors and by the tcell thymocyte populations it is important for bcell activation proliferation and differentiation it is reported that il4 is necessary for producing immunoglobulin e and implicated in immune diseasesin the process of innate immune responses il4 may activate m2 macrophage and then play a specific role it hasantiinflammatory effect which is relevant to the development of escc recently a number of studies have focusedon the relationship of il4 with cancer development il4 single nucleotide polymorphisms snps have alsobeen explored for an association with susceptibility to cancer [] the rs2070874 tc located in the 5cid3utrregion of the il4 gene is an important snp in cancer development some metaanalyses have indicated that il4rs2070874 may be associated with cancer development in asian populations [] kim reported that il4rs2070874 might affect the role of aspirin in regulating il4 expression rs2243263 gc polymorphism is anintron snp of il4 gene this intron snp might play a role in splicing although the exact role of this intron snpis unknown the associations of il4 rs2243263 gc snp with the human disease have been explored a previousstudy suggested that il4 rs2243263 was associated with the reverse seroconversion of hepatitis b virus hbv this snp was also studied for the relationship of the susceptibility to cancer a previous report investigated the correlation of the il4 rs2243263 locus with colorectal cancer although in this study a null association was identifiedhowever lan in a large simple size study found that the il4 rs2243263 gc snp might increase the susceptibility to nonhodgkin lymphoma currently the associations of il4 the rs2070874 tc and rs2243263 gcpolymorphisms with escc development are unknownthe il10 gene is located in chromosome 1q322 il10 another immune regulator serves as an inhibitor of dendritic cells and macrophages and inhibits the production of many inflammatory cytokines eg tumor necrosis factorα il1 il6 il12 and others il10 is a vital antiinflammatory regulator after il10 combineswith its receptor il10r signal transducer and activator of transcription is triggered which plays a vital role inantiapoptosis and proliferation an investigation found that the upregulated mrna expression of the il10gene and higher serum levels of il10 were found among subjects who carried the rs1800896 gallele thers1800872 snp a promotor variant could influence the level of il10 protein some investigations have suggested that the il10 rs1800896 ag and rs1800872 ac variants may influence thesusceptibility to escc of late a metaanalysis indicated that these il10 snps increased the risk of ec however in this earlier metaanalysis the sample size was very limited ec patients and controls includedthe association of the il10 rs1800896 ag and rs1800872 ac polymorphisms with ec development should befurther studiedherpesvirus entry mediator hvem also known as tnfrsf14 plays a major role in the immune response[] hvem has been found to be expressed in lymphoid cells as well as in other cells a previous study suggestedthat the hvemb and tlymphocyte attenuatorlymphotoxincd160 network in immune reaction to infection andinflammation could play a bidirectional regulatory role several investigations have focused on the role of hvemin cancer survival [] zhu reported that higher expression of hvem may promote apoptosis and herald agood prognosis for bladder cancer patients additionally a previous study has indicated that hvem is implicatedin the development of breast cancer bc a snp in the hvem gene the g to a of rs2234167 in the exon regionwas found to influence the development of bc however the association of hvem rs2234167 ga snp withthe expression of hvem is unknown recently migita found that hvem is critical for both tumor survival andthe escape of the host immune system in escc cases thus it could be a useful target for escc therapy to dateinvestigation has not been performed to identify a relationship of the hvem rs2234167 ga polymorphism withescc susceptibilitytherefore in this investigation the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 andrs1800872 polymorphisms were selected and investigated for their effect on escc development in a chinese hanpopulationmaterials and methodssubjectsour casecontrol study was performed in fujian union hospital fuzhou china and the no1 peoples hospital of zhenjiang city zhenjiang china this investigation was approved by jiangsu university registration idk20160036y and fujian medical university registration id 2016zqn25 participants were recruited betweenfebruary and april our study included escc cases and controls these escc patients werehistopathologically confirmed and were from to years old controls were cancerfree individuals from to the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895 years old the controls were not related to any escc case using a prestructured questionnaire we collectedepidemiological data from participants the escc patients and normal controls signed consent formsdna extraction and genotyping of hvem rs2234167 il4 rs2070874 andrs2243263 and il10 rs1800896 and rs1800872 lociwe collected a blood sample ml from each participant dna was extracted carefully as described in a previousstudy using an snpscan¢ assay genesky biotechologies inc shanghai china we determined the genotypesof hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 polymorphisms to confirm the accuracy of genotyping samples were selected and retested the genotypes of hvem rs2234167 il4rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 loci were reanalyzed by another technician thegenotypes of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 snps wereunchangedstatistical analysisthe difference in alcohol consumption body mass index bmi gender cigarette use and age were tested by using Ï2 test mean age was calculated by using a students t test we used a chisquare test Ï2 or fishers exact testto determine whether the frequencies of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896and rs1800872 variants in escc cases and controls were different a multivariate logistic regression analysis methodwas used to calculate the crude and adjusted odds ratios ors and confidence intervals cis sas softwarepackage sas institute inc cary nc usa the relationship of hvem rs2234167 il4 rs2070874 and rs2243263and il10 rs1800896 and rs1800872 polymorphisms with escc development was determined by ors and cisthe statistical significance of all analyses was p005 twosided an internetbased hardyweinberg equilibriumhwe test httpihggsfdecgibinhwhwa1pl was also harnessed to assess whether the distribution of hvemrs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes could represent the included populationresultsbaseline characteristicsin total escc cases and controls were recruited table of these escc cases were females and were males average age was years in the control group there were females and maleswith an average age of years there was no difference in terms of mean age p0413 the categoricalvariables age and gender were wellmatched p005 however the distribution of other categorical variables egtobacco use bmi and drinking status were significantly different all p0001 among escc cases there were with lymphatic metastasis the ajcc version criteria was used to determine the escc stage and escc cases were stage iii and were stage iiiiv after genotyping the participants the association ofhvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes with escc riskwas assessedthe minor allele frequencies mafs of hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896and rs1800872 loci are shown in table they are similar to the data of chinese population as presented in table the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypes in controlsaccorded with hwerelationship of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with escctable shows the hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872 genotypesthe frequencies of il4 rs2070874 tt tc and cc genotypes were and inescc cases and and in controls when the reference was il4 rs2070874 ttgenotype we found the il4 rs2070874 cc genotype significantly decreased the risk of escc p0023 when thereference was il4 rs2070874 tttc genotype the il4 rs2070874 cc genotype also significantly decreased the riskof escc p0028 adjustment for bmi smoking drinking age and gender the decreased susceptibility was alsoidentified cc vs tt p0008 cc vs tttc p0010hvem rs2234167 il4 rs2243263 and il10 rs1800896 and rs1800872 genotypes are shown in table both crudeand adjusted comparisons indicated that hvem rs2234167 il4 rs2243263 and il10 rs1800896 and rs1800872 lociwere not associated with the risk of escc table the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0ctable distribution of selected demographic variables and risk factors in escc cases and controlsbioscience reports bsr20193895101042bsr20193895p1variableage yearsage yearssex¥malefemaletobacco usenevereveralcohol usenevereverbmi kgm2¥lymph node statuspositivenegativetmn stageiiiiiiivgradeg1g2g3cases n721n controls n1208n bold values are statistically signiï¬cant p005 abbreviation tmn tumorlymph nodemetastasis1twosided Ï2 test and students t testtable primary information for the included snpsgenotyped polymorphismschromosomeposition regionmaf1 in database 1000g chinese hanpopulatonsmaf in our controls n1208pvalue for hwe2 test in our controls genotyping value1maf2hwehvemrs2234167 gail4 rs2070874tcil4 rs2243263gcil10 rs1800872tgil10 rs1800896tc3cid3utr5cid3utrintron variant5cid3ï¬anking5cid3ï¬ankingadditionally a subgroup analysis was conducted by escc stage we identified an association between il4rs2070874 tc snp and the decreased susceptibility of escc in stage iii subgroup cc vs tt p0022 cc vstttc p0025 table however this association could not been identified for other snps the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table the frequencies of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872polymorphisms in different escc subgroupsstage iii patientsstage iiiiv patientsn328n393controls n1208ngenotypehvem rs2234167 gagggaaaa alleleil4 rs2070874 tctttcccc alleleil4 rs2243263 gcgggcccc alleleil10 rs1800872 tgtttgggg alleleil10 rs1800896 tctttcccc alleleoverall casesn721nnnrelationship of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with escc in stratiï¬ed analysesin a stratified analysis the il4 rs2070874 genotypes are listed in table after an adjustment we suggested that il4rs2070874 c allele was a protective factor for escc in five subgroups male subgroup cc vs tt p0028 cc vstttc p0031 ¥ years old subgroup cc vs tt p0026 cc vs tttc p0029 never smoking subgroupcc vs tt p0041 cctc vs tt p0013 and tc vs tt p0042 drinking subgroup cc vs tt p0025cc vs tttc p0024 and bmi kgm2 subgroup cc vs tt p0010 cc vs tttc p0012 in othersubgroups no association of l4 rs2070874 with escc risk was found table the il4 rs2243263 gc genotypes in the stratified analysis are listed in table after adjustment we identifiedthat il4 rs2243263 gc polymorphism was a risk factor for escc development in the bmi ¥ kgm2 subgroupgc vs gg p0030 and gccc vs gg p0018 table in other stratified analyses adjustment comparisons suggested that hvem rs2234167 and il10 rs1800872 andrs1800896 loci did not confer a risk of escc data not shownassociation of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with lymphatic metastasis in escccasesamong the escc patients patients had lymphatic metastasis as presented in table we found a null association of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872 snps with differentlymph node status the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cilcenseccbytheauthorsthisisanopenaccessarticelpublishedbyportlandpressilmitedonbehalfofithebochemcailsocetyianddistributedunderthecreativecommonsattributiontable logistic regression analyses of association of hvem rs2234167 il4 rs2070874 rs2243263 and il10 rs1800896 and rs1800872polymorphisms with risk of esccgenotypehvem rs2234167 gaga vs ggaa vs gggaaa vs ggaa vs gggail4 rs2070874 tctc vs ttcc vs tttccc vs ttcc vs tttcil4 rs2243263 gcgc vs cccc vs gggccc vs ggcc vs gggcil10 rs1800872 tgtg vs ttgg vs ttggtg vs ttgg vs tttgil10 rs1800896 tctc vs ttcc vs tttccc vs ttcc vs tttcpatientsn721vscontrolsoveralln1208crude or cipadjustedor1 ci pstage iii patients n328 vs controlsn1208crude or ciadjustedor1 ci ppstage iiiiv patients n393 vs controlsn1208crude or ciadjustedor1 ci pp 1adjusted for age sex smoking status alcohol use and bmi status bold values are statistically signiï¬cant p005iiboscencereportsbsrbsr 0cbioscience reports bsr20193895101042bsr20193895table stratiï¬ed analyses between il4 rs2070874 tc polymorphism and crc risk by sex age bmi smokingstatus and alcohol consumptionil4 rs2070874 tccasecontrol1tttccctttcadjusted or2 ci pcctc cccc vs tcttvariablesexmalefemaleage years¥smoking statusnevereveralcohol consumptionnevereverbmi kgm2¥ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1for il4 rs2070874 tc the genotyping was successful in crc cases and controls2adjusted for multiple comparisons [age sex bmi smoking status and alcohol consumption besides stratiï¬ed factors accordingly] in a logisticregression modelbold values are statistically signiï¬cant p005association of hvem rs2234167 il4 rs2070874 and rs2243263 andil10 rs1800896 and rs1800872 loci with tumor grade of escc casesas presented in table patients had welldifferentiated tumors had moderately differentiated tumors and has poorly differentiated tumors we found an association of the il10 rs1800872 tg snp with a worse differentiation tg vs tt p0048 and ggtg vs tt p0032 table discussionimmunotherapy is altering how we comprehend malignancies and offers new methods to treat them ec is a representative model of immune and inflammationrelated cancer recently some studies indicated that the snps ininflammation and immunerelated genes might influence the risk of ec in this study we explored the role ofimmunerelated gene snps hvem rs2234167 il4 rs2070874 and rs2243263 and il10 rs1800896 and rs1800872to escc development we observed that il4 rs2070874 tc could decrease a risk to escc even in the stage iiisubgroup however in bmi ¥ kgm2 subgroup il4 rs2243263 gc might increase the risk of escc we alsofound an association of the il10 rs1800872 tg snp with a worse differentiationil4 is an important regulator of immune and inflammation pathways some reports have suggested that il4 levelsare higher in untreated escc patients than in controls [] it is considered that il4 levels may be implicated inthe development of escc the il4 rs2070874 tc polymorphism is a 5cid3utr snp in a highrisk gastric cancergc region a previous study suggested that rs2070874 c allele in the il4 gene might decrease the susceptibilityto gc in a chinese population lu reported that the rs2070874 c allele increased the risk of hcc in amale subgroup however chang and wang found that the il4 rs2070874 polymorphism might notinfluence the susceptibility of cancer in chinese population in this study we included subjects andinvestigated the correlation of this snp to escc susceptibility we found that il4 rs2070874 tc polymorphism the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0ctable stratiï¬ed analyses between il4 rs2243263 gc polymorphism and crc risk by sex age bmi smokingstatus and alcohol consumptionbioscience reports bsr20193895101042bsr20193895il4 rs2243263 gccasecontrol1gggcccgggcadjusted or2 ci pccgccccc vs gcggvariablesexmalefemaleage¥smoking statusnevereveralcohol consumptionnevereverbmi kgm2¥ p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1for il4 rs2243263 gc the genotyping was successful in crc cases and controls2adjusted for multiple comparisons [age sex bmi smoking status and alcohol consumption besides stratiï¬ed factors accordingly] in a logisticregression modelbold values are statistically signiï¬cant p005seemed to be a protective factor for escc development our findings were similar to a previous metaanalysis thatsuggested that the il4 rs2070874 c allele could be associated with a decreased susceptibility of gastrointestinal cancer a functional study indicated that the il4 rs2070874 allele c could promote a higher level of il4 in plasma il4 has an antiinflammatory effect and may decrease the risk of escc by inhibiting the inflammation fitzgerald reported that the il4 rs2070874 allele c could decrease the risk of prostate cancer specific mortality consistent with that report we identified an association between the il4 rs2070874 tc snp and a decreasedsusceptibility to escc in the stage iii subgroup however we did not find an association of il4 rs2070874 tcpolymorphism with lymphatic metastasis this might be due to the limited sample sizes in the future the relationshipof the rs2070874 snp in il4 gene with progress and prognosis should be further exploredrs2243263 gc an intron snp in the il4 gene was studied for the relationship of this snp to some diseasesthis snp might decrease the risk of asthma in the african american children while this relationship was not identified in caucasians hsiao reported that the il4 rs2243263 c allele was a protective factor for hbv surfaceantigen reverse seroconversion in nonhodgkin lymphoma cases undergoing rituximab treatment a previous studyinvestigated the relationship of il4 rs2243263 gc with colon and rectal cancer risk but no association wasfound however in a large simple size study lan found that the il4 rs2243263 gc snp increased the susceptibility to nonhodgkin lymphoma in this study we found that the il4 rs2243263 gc might increase therisk of escc in obese and overweight subjects table it was reported that the il4 level in mothers was inverselylinked to overweight in early childhood and might influence the metabolic profile of childhood in addition thelevel of il4 decreased with antipsychoticinduced weight gain it is suggested that the level of il4 could influence obesity and overweight introns are regulatory sequences that can affect the expression of genes here we foundthat the rs2243263 gc polymorphism a snp in il4 intron region might alter the risk of escc it is presumed the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table logistic regression analyses of association between hvem rs2234167 il4 rs2070874 rs2243263 andil10 rs1800896 and rs1800872 polymorphisms and lymph node status in escc patientsgenotypepositive n405negative n316crude or cipadjusted or1 ciphvem rs2234167 gagggaaaga aagggaaaa alleleil4 rs2070874 tctttccccctctttcccc alleleil4 rs2243263 gcgggccccgccgggcccc alleleil10 rs1800872 tgtttgggggtgtttgggg alleleil10 rs1800896 tctttccccctctttcccc allelenn1adjusted for age sex smoking alcohol use and bmi status that the rs2243263 gc polymorphism influences the level of il4 by regulating gene transcription in the future afunctional study should be considered to explore the potential mechanismthe il10 rs1800872 tg is a promotor snp torrespoveda reported that the expression of il10 mrnaand the level of serum il10 were significantly higher in subjects with the il10 rs1800872 t allele a recent studyfound that il10 rs1800872 tg snp promoted the risk of ec a metaanalysis also confirmed this association in our casecontrol study we did not find the association of il10 rs1800872 tg snp with the developmentof ec even in stratified analyses and reviewing different lymph node status additionally liu reported thatil10 rs1800872 gg genotypes predicted the worse survival of diffuse large bcell lymphoma patients treated withrituximabchop cyclophosphamide doxorubicin vincristine and prednisone in this study we found that the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895table logistic regression analyses of association between hvem rs2234167 il4 rs2070874 rs2243263 andil10 rs1800896 and rs1800872 polymorphisms and grades of esccgenotypeg2g3 n579hvem rs2234167 gagggaaaga aagggaaaa alleleil4 rs2070874 tctttccccctctttcccc alleleil4 rs2243263 gcgggccccgccgggcccc alleleil10 rs1800872 tgtttgggggtgtttgggg alleleil10 rs1800896 tctttccccctctttcccc allelenng1 n142crude or cipadjusted or1 cip 1adjusted for age sex smoking alcohol use and bmi statusbold values are statistically signiï¬cant p005the il10 rs1800872 g allele was associated with poorly differentiated tumor thus in the future the association ofthe il10 rs1800872 tg snp and the survival of escc cases should be further studiedlimitations in the present study should be acknowledged first in the present study we only included five functional snps and explored the association of the risk to escc second there were other environmental risk factorseg vegetable and fruit intake aspirin and nsaids use and physical exercise which we did not consider for theirinfluence to the development of escc third the number of escc patients was limited and our study may be the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commons attributionlicense cc by 0cbioscience reports bsr20193895101042bsr20193895underpowered in some subgroups fourth in this investigation the protein expression levels of the suspect factors were not measured finally considering the low penetrance of snp the other functional polymorphisms in thehvem il4 and il10 genes should not be ignoredin summary the present study suggests that the il4 rs2070874 tc polymorphism is a protective factor for esccdevelopment while the il4 rs2243263 gc increases a risk to escc in obese and overweight subjects additionallyit is highlighted that the il10 rs1800872 g allele is associated with poorly differentiated tumorcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis work was supported in part by the young and middleaged talent training project of health development planning commission in fujian province [grant number 2016zqn25] the program for new century excellent talents in fujian province university[grant number ncetfj2017b015] and the joint funds for the innovation of science and technology fujian province [grantnumber 2017y9099]author contributionall authors contributed significantly to the present study conceived and designed the experiments wt and mk performed theexperiments sc rc and cl analyzed the data wt and mk contributed reagentsmaterialsanalysis tools mk wrote themanuscript sc and rc other please specify noneacknowledgementswe appreciate all subjects who participated in the present study we wish to thank dr yan liu genesky biotechnologies incshanghai china for technical supportabbreviationsajcc american joint committee on cancer bc breast cancer bmi body mass index ci confidence interval ecesophageal cancer escc esophageal squamous cell carcinoma gc gastric cancer hbv hepatitis b virus hvem herpesvirus entry mediator hwe hardyweinberg equilibrium il interleukin nsaid nonsteroidal antiinflammatory drug or odds ratio snp single nucleotide polymorphismreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin 103322caac21492 matejcic m and iqbal parker m geneenvironment interactions in esophageal cancer crit rev clin lab sci 1031091040836320151020358 qin jm yang l chen b interaction of methylenetetrahydrofolate reductase c677t cytochrome p4502e1 polymorphism andenvironment factors in esophageal cancer in kazakh population world j gastroenterol pmcid pmc2773864103748wjg146986 lin ew karakasheva ta hicks pd the tumor microenvironment in esophageal cancer oncogene pmcidpmc5003768 101038onc201634 park r williamson s kasi a immune therapeutics in the treatment of advanced gastric and esophageal cancer anticancer res 1021873anticanres12891 nelms k keegan ad zamorano j the il4 receptor signaling mechanisms and biologic functions annu rev immunol 101146annurevimmunol171701 rush js and hodgkin pd b cells activated via cd40 and il4 undergo a division burst but require continued stimulation to maintain divisionsurvival and differentiation eur j immunol 101002152141412001043143c1150aidimmu11503e30co2v suzuki a leland p joshi bh targeting of il4 and il13 receptors for cancer therapy cytokine 101016jcyto201505026 francipane mg alea mp lombardo y crucial role of interleukin4 in the survival of colon cancer stem cells cancer res 10115800085472can076874 tan n song j yan m association between il4 tagging single nucleotide polymorphisms and the risk of lung cancer in china molgene genom med e00585 pmcid pmc6465665 101002mgg3585 the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc by 0cbioscience reports bsr20193895101042bsr20193895 cardenas dm sanchez ac rosas da preliminary analysis of singlenucleotide polymorphisms in il10 il4 and il4ralpha genesand proï¬le of circulating cytokines in patients with gastric cancer bmc gastroenterol pmcid pmc6288868101186s1287601809139 shamoun l skarstedt m andersson re association study on il4 il4ralpha and il13 genetic polymorphisms in swedish patientswith colorectal cancer clin chim acta 101016jcca201809024 jia y xie x shi x associations of common il4 gene polymorphisms with cancer risk a metaanalysis mol med rep pmcid pmc5561993 103892mmr20176822 cho ya and kim j association of il4 il13 and il4r polymorphisms with gastrointestinal cancer risk a metaanalysis j epidemiol pmcid pmc5394226 101016jje201606002 zhenzhen l xianghua l qingwei w three common polymorphisms in the il4 gene and cancer risk a metaanalysis involving cases and controls tumour biol 101007s1327701307618 kim bs park sm uhm tg effect of single nucleotide polymorphisms within the interleukin4 promoter on aspirin intolerance inasthmatics and interleukin4 promoter activity pharmacogenet genomics hsiao lt wang hy yang cf human cytokine genetic variants associated with hbsag reverse seroconversion in rituximabtreatednonhodgkin lymphoma patients medicine baltimore e3064 pmcid pmc4839912 101097md0000000000003064 bondurant kl lundgreen a herrick js interleukin genes and associations with colon and rectal cancer risk and overall survival intj cancer pmcid pmc3470814 101002ijc27660 lan q wang ss menashe i genetic variation in th1th2 pathway genes and risk of nonhodgkin lymphoma a pooled analysis ofthree populationbased casecontrol studies br j haematol pmcid pmc3075370101111j13652141201008424x kwasniak k czarnikkwasniak j maziarz a scientiï¬c reports concerning the impact of interleukin interleukin and transforminggrowth factor beta on cancer cells central eur j immunol pmcid pmc6745546 105114ceji201876273 dandrea a asteamezaga m valiante nm interleukin il10 inhibits human lymphocyte interferon gammaproduction bysuppressing natural killer cell stimulatory factoril12 synthesis in accessory cells j exp med pmcid pmc2191152101084jem17831041 miteva ld stanilov ns deliysky ts signiï¬cance of 1082ag polymorphism of il10 gene for progression of colorectal cancer andil10 expression tumour biol 101007s1327701425892 pereira apl trugilo kp okuyama ncm il10 c592ca rs1800872 polymorphism is associated with cervical cancer j cancerres clin oncol 101007s00432020032560 yang y and fa x role of il10 gene polymorphisms on the susceptibility for esophageal cancer and its association with environmental factorsint j clin exp pathol pmcid pmc4583954 sun jm li q gu hy interleukin rs1800872 tg polymorphism was associated with an increased risk of esophageal cancer in achinese population asian pac j cancer prev zhao x lu c chu w microrna214 governs lung cancer growth and metastasis by targeting carboxypeptidased dna cell biol 101089dna20163398 breloer m hartmann w blankenhaus b cutting edge the btlahvem regulatory pathway interferes with protective immunity tointestinal helmin
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Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young at diagnosis patients who received curative resection for breastcancer The signiï¬cance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identiï¬ed variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI P and OS HR CI P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identiï¬ed as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI and CI respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identiï¬ed for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to beneï¬t prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and Chinascontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkins lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classiï¬cation system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassiï¬ed as luminal Alike ER PR¥ HER2 andKi6715luminal Blike ER andor PR HER2HER2enriched ER PR HER2 or triplenegative ERPR HER2 subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have deï¬niteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insuï¬cient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was deï¬ned as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was deï¬ned asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the KaplanMeier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asigniï¬cance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the rms package of R The discriminationofthe nomogram models was estimated by the Harrellsconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank à statistics with the Xtileprogram Pearsons chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the ï¬ndings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsuï¬cientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patientsFIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical signiï¬cancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The models explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI and CI respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS patients intermediateriskgroup TPS patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS patientsintermediaterisk group TPS patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the ï¬rsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentiï¬ed as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE KaplanMeier curves for A diseasefree survival and B overallsurvival stratiï¬ed by HBsAg status in very young patients with breast canceridentiï¬ed as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE KaplanMeier curves for A diseasefree survival and B overall survival stratiï¬ed by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may beneï¬t patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insuï¬cientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population conï¬nes the universality of our resultsFurthermore the information was insuï¬cient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE KaplanMeier curves for A diseasefree survival and B overall survival stratiï¬ed by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the ï¬rst evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to conï¬rm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the ï¬nal versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al Very young women years with operable breastGcancer 101093annoncmdf039at presentation Ann Oncolfeaturesof disease Anders CK Hsu DS Broadwater G Acharya CR Foekens JA Zhang Y et alYoung age at diagnosis correlates with worse prognosis and deï¬nes a subsetof breast cancers with shared patterns of gene expression J Clin Oncol 101200JCO2007142471 Narod SA Breast cancer in young women Nat Rev Clin Oncol 101038nrclinonc2012102 Trepo C Chan HL Lok A Hepatitis B virus infection Lancet 101016S0140673614602208 de Martel C Gees D Bray F Ferlay J Cliï¬ord GM Global burden of cancerattributable to infections in a worldwide incidence analysis LancetGlobal Health 8e180 101016S2214109X19304887 Lu FM Li T Liu S Zhuang H Epidemiology and prevention of hepatitisJ Viral Hepatitis 17Suppl B virus infection in China 101111j13652893201001266x Liu J Zhang S Wang Q Shen H Zhang M Zhang Y et al Seroepidemiologyof hepatitis B virus infection in million men aged years in ruralChina a populationbased crosssectional study Lancet Infect Dis 101016S1473309915002182 Zhang Y Fang W Fan L Gao X Guo Y Huang W et al HepatitisB surface antigen prevalence among rural women of childbearingage in Hainan Province China a crosssectional study Virol J 1011861743422X1025 Cui Y Jia J Update on epidemiology of hepatitis B and C in China JGastroenterol Hepatol 28Suppl 101111jgh12220 Chan SL Wong VW Qin S Chan HL Infection and cancer the case ofHepatitis B J Clin Oncol 101200JCO2015615724 Fwu CW Chien YC You SL Nelson KE Kirk GD Kuo HS et al Hepatitis Bvirus infection and risk of intrahepatic cholangiocarcinoma and nonHodgkinlymphoma a cohort study of parous women in Taiwan Hepatology 101002hep24150 Kamiza AB Su FH Wang WC Sung FC Chang SN Yeh CC Chronichepatitis infection is associated with extrahepatic cancer developmenta nationwide populationbased study in Taiwan BMC Cancer 101186s1288501629185 Nath A Agarwal R Malhotra P Varma S Prevalence of hepatitis B virusinfection in nonHodgkin lymphoma a systematic review and metaanalysisInternal Med J 101111j14455994200902060x Luo G Hao NB Hu CJ Yong X Lu MH Cheng BJ et al HBV infectionincreases the risk of pancreatic cancer a metaanalysis Cancer Causes Control 101007s1055201201442FUNDINGThis work was supported by the National Natural ScienceFoundation of China No SUPPLEMENTARY MATERIALThe Supplementary Materialonline202001403fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncat Wei XL Qiu MZ Jin Y Huang YX Wang RY Chen WW et al Hepatitis Bvirus infection is associated with gastric cancer in China an endemic area ofboth diseases Br J Cancer 101038bjc2014406 Ye YF Xiang YQ Fang F Gao R Zhang LF Xie SHHepatitis B virusin southern China Cancer Epidemiol Biomarkers Prevent 10115810559965EPI150344et alinfection and risk of nasopharyngeal carcinoma Peng JW Liu DY Lin GN Xiao JJ Xia ZJ Hepatitis B virusinfection is associated with poor prognosis in patients with advancednon small cell 107314APJCP201516135285lung cancer Asian Paciï¬c J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in Vickers AJ Elkin EB Decision curve analysis a novel method for evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res 10115810780432CCR040713Conï¬ict of Interest The authors declare that the research was conducted in theabsence of any commercial or ï¬nancial relationships that could be construed as apotential conï¬ict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice No use distribution or reproduction is permitted which does not complywith these termsFrontiers in Oncology wwwfrontiersinAugust Volume 0c'
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"Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective tailored therapies. Until recently personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing such as the semiconductor-based Ion Torrent sequencing platform has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS EGFR and TP53 genes in the breast cancer samples of various histologic types. Thus this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual breast cancer-specific mutations. This research was supported by the grants from National Natural Science Foundation of China the Wu Jieping Foundation and the National Institute of Health (R01 CA90427 & R01 AI084811 to SY Chen). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Lung cancer is the most common malignancy worldwide and also the leading cause of cancer related deaths. In 2008 an estimated 1.61 million new cases were reported globally accounting for 12.7% of all new cancers [1]. Additionally roughly 1.38 million deaths (18.2% of total cancer deaths) were reported around the world [2]. In China lung cancer has the highest incidence of all new cancer cases in both men and women (21.7% in 2008) with more than a 24.9% mortality rate [2]. Women in China reported only a slightly higher incidence of lung cancer over breast cancer this same year; however the mortality rate of lung cancer is more than 3 times higher than that of breast cancer (20.2% vs. 6.1% respectively) [2]. Lung cancer often exhibits non-specific symptoms and diagnosis often occurs at an advanced stage or after metastasis has already occurred [3]. While efforts continue to improve early diagnosis and treatment of lung cancer the staggering incidence poor prognosis and considerable mortality rate prevails. The leading cause of lung cancer is cigarette smoking and increased exposure is directly correlated with an increased risk of developing lung cancer [4]. 8590% of lung cancer deaths are associated with smoking and current smokers are 15 times more likely to die from lung cancer than never-smokers [5]. There are two major forms of lung cancer: non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC). NSCLC which accounts for roughly 85% of all lung cancers can be further divided into three major histologic subtypes: squamous-cell carcinoma (SCC) adenocarcinoma and large-cell lung cancer. While smoking can be attributed to all forms of lung cancer it is most commonly linked to SCLC and SCC. Never-smokers on the other hand are most commonly diagnosed with adenocarcinoma [3] [5]. Interestingly only 1024% of smokers develop lung cancer indicating the importance of other environmental and individual genetic factors [6] [7]. Aside from tobacco smoke other etiologic agents and risk factors have been identified including occupation exposure to second-hand smoke asbestos radon gas and air pollution in addition to genetic factors [8][10]. Roughly 1015% of lung cancers arise in patients that report never having smoked and these cancers do so spontaneously with an accumulation of genetic and epigenetic changes [5]. Despite ongoing efforts to improve screening and treatment of lung cancers the prognosis of patients with most forms of lung cancer remains poor [3]. Because the genetic and environmental factors causing lung cancer vary widely each tumor has the potential to exhibit a unique gene mutation profile. As such accumulating evidence suggests that individualized tailored therapies are essential for effective treatment against lung cancers. This can be accomplished by profiling an individual's cancer genome in order to dissect the oncogenic mechanisms that regulate the progression of the cancer. Recently a new technology based on semiconductor sequencing called Ion Torrent sequencing [11] is tackling many of the issues associated with other sequencing methods namely the cost time and overall practicality of individualized genome sequencing. In this study we have used Ion Torrent sequencing to analyze 76 clinical lung cancer samples to identify the genetic mutations in 737 loci of 45 known cancer-related genes. Results Mutation analysis of human lung cancer tumors with Ion Ampliseq Cancer Panel A total of 76 Lung cancer samples () was analyzed using Ion Torrent Ampliseq Cancer Panel to identify mutations in 737 loci of 45 oncogenes and tumor suppressor genes in human lung cancers. These Lung cancer samples were all from Chinese patients ranging from 2880 years old represented by 40 men with a mean age of 62 years and 36 women with a mean age of 59 years. .0095228.t001 Detected mutations (including Missense point mutations/deletion/insertion) in 45 genes (737 loci) of 76 human lung cancer samples."
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Protocol Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trialMarcelo Marreira Lidiane Rocha Mota Daniela F¡tima Teixeira Silva Christiane Pavani To cite Marreira a0M Rocha Mota a0L Silva a0DFT et a0al Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trial BMJ 202010e036684 101136bmj 2019036684 º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received December Revised July Accepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJBiophotonics Applied to Health Sciences Universidade Nove de Julho Sao Paulo BrazilCorrespondence toDr Christiane Pavani chrispavani gmail comIntroduction The search for non invasive procedures to reduce localised adiposity in aesthetics clinics has recently been increasing In this context procedures such as cryolipolysis ultracavitation photobiomodulation PBM and other techniques have been proposed Some studies have shown that PBM can be used in body contouring However there is no standardisation of the protocol More than that as in other techniques for reducing adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences to the general health of an individual This work will aim to compare the light wavelengths when using PBM as a technique for reducing the abdominal waist circumference while also evaluating the efficacy of the method Changes in the lipid profile in the blood with a long term follow up will also be appraisedMethods and analysis This will be a controlled randomised double blind single centred clinical trial patients will be recruited at the Nove de Julho University Brazil and then divided into three groups Group ARED PBM Group BINFRARED PBM Group CPLACEBO Sham treatment The treatments will consist of eight sessions two times a week for weeks At each session the participants will receive minutes PBM using a radiant exposure of Jcm2 with an abdominal strap containing LED clusters with devices each following the indication of randomisation All of the groups will receive min of Aussie Current at kHz modulated at Hz mA The main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness changes in the local microcirculation and the quality of life and self esteem The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Ethics and dissemination The Ethics Committee of the Nove de Julho University Brazil approved the modified version of this project under No on June This study is not yet recruiting The results obtained Strengths and limitations of this study º The use of the same dosimetry at different wavelengths will allow for a real comparison between red and infrared as being the most suitable wavelength for body contouring º Analyses of body contouring will be performed by non invasive methods º The waist circumference measurement will not discriminate the factors underlying the volume modifications º The habits of the participants such as diet and exercise routines may affect the results º Gender may affect the results and be dependent on the number of participants of each gender These differences may not be considered by the statistical analysiswill be published in a peer reviewed journal in the related fieldTrial registration number Brazilian Registry of Clinical TrialsReBec RBR 9bwxcxINTRODUCTIONFat storage is intended to protect the human body in cases of prolonged fasting intense physical activity and temperature regulation Once freed from these situations fat is stored unnecessarily putting the individual at the risk of health problems together with a greater pr sity for pathologies such as systemic arterial hypertension diabetes mellitus metabolic syndrome and even some types of neoplasms1Another type of negative impact that is related to excessive fat storage is body dissatisfaction This naturally leads to a decrease in the individuals self esteem4 Studies have shown that aesthetic treatments significantly increase a patients quality of life They Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access are associated with improved self esteem6 There are some traditional surgical methods for the reduction of abdominal adiposity However the methods are invasive techniques which may present a high number of complications such as bruising seroma pain perforated ans and viscera as well as with an increased risk of deep vein thrombosis10 The demand for minimally invasive procedures that are aimed at reducing abdominal fat has increased by about while the demand for surgical procedures has decreased by around Among the minimally invasive techniques one can mention low level laser therapy which has recently also been called photobiomodulation PBM PBM has many novel advantages when compared with traditional techniques such as surgical procedures since it can guarantee the preservation of the noble adjacent structures such as the nerves the blood vessels and the skin15 PBM has been widely studied for several applications due to its important biochemical cellular consequences and its few side effects16 Some manuscripts have described erythema and oedema as the main side effects of PBM but importantly these side effects may have been higher as a result of the patient using any drug that increased photosensitivitySome other studies have shown that PBM can be used in body contouring18 Sadly there is no standardisation of the protocol The treatments vary in terms of the number of sessions their frequency times per week and wavelength nm nm nm nm while other dosimetry information such as irradiance Wcm2 and radiant exposure Jcm2 are frequently not mentioned Recently Croghan and coworkers showed that two times a week was the best frequency when compared with one or three times a week This was in terms of improving the patients quality of life and body satisfaction as well as their weight waist circumference body mass index BMI and body fat mass reduction18 However more studies are needed in order to standardise the wavelength the dosimetry and the application time as well as the durability of the results achievedOne of the proposed mechanisms for a PBM effect in adipose tissue is the formation of transient pores in the adipocyte membranes thus allowing for the lipids to escape15 Adipocyte apoptosis activation has also been proposed The production of reactive oxygen species is also possible due to the action of PBM and this is related to the mitochondrial activation on account of the radiation absorption by the cytochrome c oxidase molecules This is followed by an increased ATP synthesis and with an increased cyclic adenosine monophosphate messenger which can trigger the activation of the lipases that perform the hydrolysis of the triglycerides into fatty acids and glycerol23Some reports have affirmed that the results obtained by the use of PBM for reducing waist circumference are modest and that the reduction is temporary which deserves much greater attention from researchers for a better understanding of this factor These effects may be associated with the mechanisms of action and the dosimetric parameters being used24 When taken to the tissues the free fatty acids are used as an energy source during beta oxidation for the production of ATP In some literature reports PBM is associated with aerobic or resistance exercise while other reports have mentioned waist and arm circumference reductions with the use of PBM displaying an absence of diet restrictions or exercise requirements25 It is also reported that the amount of fat mobilised during a PBM session is similar to the amount that is consumed during a meal in such a way that it can be absorbed by normal body energy requirements andor exercise routine while at the same time the risk of atherosclerosis is not increased by the treatment30 On the other hand if not consumed these fatty acids may be re esterified and redistributed throughout the body30 causing no final changes in waist circumference Since neuromuscular electrical stimulation increases energy expenditure in a similar way to that associated with exercise the protocol will be complemented with the Aussie current application31As for other techniques for reducing the adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences for the general health of the individual Some studies have shown that these important treatments may affect the serum lipid levels while others affirm that there are no changes in the serum lipid levels32 Given these scenarios this work will aim to compare the different light wavelengths when using PBM as a technique for the reduction of abdominal waist circumference while at the same time evaluating the efficacy of the method and by following the changes in the lipid profile in the blood as well as with reviewing the long term follow upMETHODSStudy designThis will be a controlled randomised double blind single centred clinical trial designed in accordance with the criteria as established by the Standard Protocol Items Recommendations for Interventional Trials It will be conducted at the Nove de Julho University located in the city of S£o Paulo Brazil The recruitment will be performed from September to November through the university website Thus the selection of sites includes urban locations the city of S£o Paulo and its neighbourhoods After verbal and written explanations regarding the procedures the risks and the benefits by MM a coauthor of this protocol those individuals who agree to participate in the study will sign an informed consent form Based on an anamnesis questionnaire the researchers will check if the participants meet the inclusionexclusion criteria The anamnesis questionnaire will include identification data anthropometric data clinical history and daily living habits especially dietary intake physical activity assessments and menstrual period appraisals Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cSince dietary intake and physical activity may have direct effects on the results at each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measured The enrolment period will be extended until the sample size is reachedPatient and public involvement statementThe patients andor the public will not be involved in the design the recruitment or in the conduct of the studyInclusion criteriaThis study will include men and women aged years with a BMI of between and kgm2 normotrophic and overweight together with hyperplasia of the abdominal fat tissue abdominal skin folds higher than mm Those who agree to participate in this research will sign an informed consent form see online supplementary file Exclusion criteriaThe following people will be excluded from this survey those participants who are undergoing aesthetic treatments to reduce waist circumference those who have been previously submitted to abdominoplasty or liposuction surgeries those who are on a diet in order to reduce their measurements those people who engage in a physical activity more than two times a week those who are using or have taken drugs or food supplements in last days in order to reduce their measurements and their weight which may affect their lipid metabolism appetite or nutrients absorption those who have been submitted previously to oophorectomy those with signs andor symptoms of climacteric at the menopause pregnant or lactating women those participants who are not regular in attending the sessions those participants who present metabolic dysfunctions diabetes and thyroid disorders cardiovascular problems hypertension cardiac insufficiency arrhythmia thrombosis pacemaker use respiratory issues asthma chronic obstructive pulmonary disease haematological disturbances anaemia renal non alcoholic fatty liver disease dermatological or digestive disorders gastritis ulcers those with a history of oncological pathology those with cognitive deficitsSample calculationIn order to calculate the sample size a study showing the therapeutical effects of PBM when associated with aerobic plus resistance training was used26 The researchers used the highest and the lowest abdominal circumference values as well as the SD of the measurements The highest and lowest abdominal circumference values for the PBM group were and respectively and the highest SD of the measurements was with being the number of intervention groups in the study The effect size hence was calculated when using these values as described below biggersmaller Ïn2 accessWhen using the effect size value as calculated above the sample size was calculated using GPower software V3192 Dusseldorf Germany Two way Analysis of Variance ANOVA was used for the interactions within and between the groups in order to evaluate the differences between the three groups studied as well as for the five evaluations during the treatments and the follow up The test power was α005 The sample size was calculated on participantsRandomisationThe randomisation will be performed by DFTS a coauthor of this work who is not directly linked to the treatments or the evaluation of the participants by using the Excel program Microsoft USA The participants will be randomised into blocks of and into groups designated as A B and C Opaque envelopes will be identified by sequence numbers and they will receive a paper containing the information about which treatment will be performed on the participants abdomen according to the draw The sealed envelopes will be safely kept with the researcher who generated the randomisation DFTS Before the beginning of the procedures the researcher responsible for the procedure LRM a coauthor of this protocol will receive each envelope and proceed with the treatment as indicated according to its allocation group A team of undergraduate students previously trained and prepared is going to be part of the research group and for the treatment or evaluation of the participants This study will be a double blind study since the participants will not be aware of the group in which heshe is participating only the researcher who will perform the procedure will know The data collection and analyses will be performed by another researcher MM a coauthor of this study who will also be unaware of the allocationsInterventionThe abdomen of the participants will be cleaned by using a neutral cleansing soap They will receive eye protection using goggles for safety This will also help with the blindness of the study PBM will be applied when using abdominal straps as developed by Cosmedical Mau¡ S£o Paulo Brazil following the parameters as described in table The abdomen strap will be covered with a sheet and that will also help with the blindness of the study All of the participants will receive min of PBM with an abdominal strap containing LED clusters with devices following the indication of the randomisation being per group Group ARED ± nm Group BINFRARED ± nm Group CPLACEBO The treatment will consist of eight sessions that will occur two times a week totalling month of treatments The placebo group will use a strap with no light emission but it will emit the same sounds like that of the active device In order to increase the oxidation of the free fatty acids the participants will receive min of Aussie Current at kHz modulated at Hz mA for min Tensor Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access Table Dosimetry for the studyParameterRed LEDContinuousCentre wavelength nmSpectral width nmOperating modeAverage radiant powerone LED mWAperture diameterone LED cmPower density at aperture mWcm2Beam spot size at targetone LED cm2Total number of LEDsArea irradiated cm2Irradiance at target mWcm2 Exposure duration sRadiant exposure Jcm2Energy density at aperture Jcm2Radiant energy kJApplication techniqueNumber and frequency of treatment sessionsContactweek for a month a total of sessionsInfrared LEDContinuousContactweek for a montha total of sessionsDGM Electronica Santo Andr© S£o Paulo Brazil33 None of the PBM devices will significantly increase the temperature at the target area causing no burns or skin damage No modifications in the intervention will be performed for any reason However the participants who withdraw their consent or the ones who are not assiduous to the sessions will be removed from the studyThe dosimetric parameters that will be used in this protocol as presented in table were measured andor calculated The centre wavelength and the spectral width of the devices were measured by a Spectrophotometer USB2000XR1 Ocean Optics Florida USA The radiant power of one LED was measured by a Power Meter FieldMaxII TO Coherent Santa Clara California USA The abdominal straps will be composed of LED clusters having LEDs each totalling LEDs units and distributed in a cm cm area cm2 each cm2 total see figure The effective irradiated area will be cm2 times the beam spot size at the target The irradiance at the target was determined by the ratio between the average radiant power mW and the beam spot site at the target cm2 The radiant exposure was determined by multiplying the irradiance at the target mWcm2 by the exposure duration s The radiant energy was calculated by multiplying the average radiant power of one LED mW by the total number of LEDs and by the exposure duration sFigure Photobiomodulation PBM application PBM device off A and on B patient receiving the experimental protocol in dorsal decubitus min per session C and DOutcomesThe main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness as measured by ultrasound changes in the local microcirculation quality of life and self esteem The participants will be evaluated at the same time of the day at all times throughout the studyThe anthropometric data that will be collected will be body weight height and BMI skin fold thickness and bioimpedance Blood will be collected for analyses of the lipid profile total cholesterol high density lipoprotein HDL Low density lipoprotein LDL triglycerides and liver function serum glutamic oxaloacetic transaminase SGOT serum glutamic pyruvic transaminase SGPT All of this will be processed and analysed at SCS Medicina Diagn³stica S£o Caetano do Sul Brazil a partner laboratory The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Abdominal ultrasound will be performed to assess the fat layer thickness before and after the treatments For the recording of the local temperature a technique that is widely used is infrared thermography using a Compact Thermal Camera C2 FLIR Systems Oregon USA The thermal camera by means of infrared emission from the body or from the material analysed has the ability to calculate the temperature of a given surface It is possible through this method that the study will infer the changes in local microcirculation34The quality of life questionnaire The WHO Quality of Life WHOQOL BREF as well as the Body Shape Questionnaire BSQ34 Self Image Scale will be used for the participants These questionnaires have been translated and submitted to cross cultural adaption into Brazilian Portuguese37 The Brazilian Portuguese version of these questionnaires will be applied by MM The questionnaires will take around min to be completed The quality of life and the self image questionnaires will be applied at D0and again at the end of the last session D30 The flow chart of the study is presented in figure Adverse events will be collected during the treatment sessions and they will be reported to the regulatory agency and again Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cRecruitment recruitmen t accessEvaluated for eligibility Elected patients n174 Anamnesis application of the quality of life and selfesteem questionnaire anthropometry blood collection ultrasonography Randomised n Allocation Group A LED with devices ± nm with Wcm2 Group B LED with devices ± nm with Wcm2 Group C Sham Each session minutes being measured at the end of each session waist circumference and IRT Total Sessions Analysis at the end of treatment Application of the quality of life and selfesteem questionnaire anthropometry blood collection IRT ultrasonography FollowUp days Anthropometry and blood collection days Anthropometry and blood collection days Anthropometry blood collection ultrasonography Figure Flow chart describing the study design the sample composition and the experimental protocol IRT infrared thermographyat the final publication of the results Since the participants are enrolled and randomised the investigators will make efforts to keep the participants together during the follow up by making phone calls emailWhatsApp contact with the patients and with relevant instructions regarding healthcare and beautyAs a strategy to improve adherence at each session the participant will schedule the next visit and receive a card with some instructions regarding preparation for the evaluation day and the appointment date of the next visit When a participant misses a session heshe will receive a phone call in order to reschedule the missed sessionData analysis planThe data that will be collected from this study will only be administered by the principal investigators the authors of this document Since the study will be of short duration and with known minimal risks this trial will not need a formal data monitoring committee After the data collection the data will be anised using Microsoft Office Excel by DFTS coauthor of this protocol and then stored on a protected by password computer at the university The data will be analysed by descriptive and inferential statistics and then compiled into tables andor graphs using SPSS V240 For testing the normality of the data the Shapiro Wilk test will be performed If the data show a non parametric behaviour a mathematical function will be used in order to normalise the data Two way ANOVA tests followed by the Bonferroni post test will be performed in order to compare the treatments along with the time points being evaluated Some parameters Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access may affect the results of the therapy and they are going to be analysed as co variables for example skin phototype by the Fitzpatrick scale the stage of the menstrual cycle total cholesterol and triglycerides altered or not α005 will be considered the level of significance for all of the tests used Since this trial is part of a PhD thesis study an interim analysis will be performed for the qualification examination and this can be used at trial adaptations such as for a sample size re estimation or for stopping the trial The trial protocol and the full study report will be fully available at the end of the study after the manuscript of the results has been published At the end of the study the participants from the placebo group who received the treatment will experience no adverse effects and they will have received the most effective treatmentDISCUSSIONStudies have shown that lasers used in PBM typically operate at powers of mW or less They can produce energy in the visible spectrum wavelengths nm and near the infrared regions nm Light penetration in the soft tissues is known to be directly related to wavelength that is the longer the wavelength the greater the penetration The reports on PBM for reducing local adiposity include the use of green nm red and nm and infrared and nm However there is no comparison available regarding the best wavelength for this purpose18 Based on the localisation of fat tissue more profound when compared with epidermis and dermis this study will choose red and infrared light for the comparisons The use of the same dosimetry at these different wavelengths will allow for the evaluation of the most suitable wavelength for body contouringSince the waist circumference measurements will not discriminate against the factors underlying the volume modifications a placebo group will be included This will allow for the measurement of the differences in waist circumference due to daily habits or hormonal variations as in the menstrual cycle of women The measurements of the skin folds and bioimpedance will complement the evaluation in terms of body fat At each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measuredGender may also affect the results Sexual dimorphism in adipogenesis is already known as well as sex hormones in the white adipose tissue function and in adipose metabolism39 If the sample consists of a huge difference in men and women this factor cannot be considered in the statistical analysisThe development of a non invasive protocol for PBM together with an Aussie current for the reduction of adiposity may present an important novel tool for the reduction of health risk problems as well as for increasing an individuals self esteemETHICS AND DISSEMINATIONThe Ethics Committee of the Nove de Julho University S£o Paulo Brazil approved the modified version of this project and the Patient Informed Consent Form under No on June according to the guidelines of the Brazilian National Ethics Committee The protocol of this study has already been registered in the Brazilian Registry of Clinical Trials being first registered on November and modified on August providing full public access to the protocol information including all items from the WHO Trial Registration Data Set MM and DFTS will be the data curators with the data stored on a protected by password computer at the university The results acquired within this project will be presented in conferences and published in a journal in the related field The authorship of the results paper and the conference abstracts will include the authors of this protocol together with other researchers who may contribute to the procedures or to the analysis of the data Any modifications of this protocol will require a formal amendment and they will be approved by the Ethics Committee of the Nove de Julho University The modifications will be properly reported and justified in the manuscript for the publication of the results The main results obtained will be sent to the participants by mailAcknowledgements The authors would like to thank the Nove de Julho University UNINOVE S£o Paulo Brazil for the availability of its laboratories the company Cosmedical for the development of the equipment for PBM and the SCS Medicina Diagn³stica Laboratory S£o Caetano do Sul Brazil for their partnership in the analyses of the laboratory testsContributors MM LR M DFTS and CP designed the study MM and LR M will conduct the experiments and will be making the data acquisitions DFTS and CP will perform data analysis and interpretation MM and LR M drafted the work while CP and DFTS revised it critically for important intellectual content All of the authors approved the final version of the manuscriptFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewed access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See a0http creativecommons licenses by nc ORCID iDsDaniela F¡tima Teixeira a0Silva http orcid Christiane a0Pavani http orcid REFERENCES Silva Figueiredo P Carla Inada A Marcelino G et a0al Fatty acids consumption the role metabolic aspects involved in obesity and its associated disorders Nutrients Landecho MF Tuero C Valent V et a0al Relevance of leptin and other adipokines in obesity associated cardiovascular risk Nutrients Kong Y Zhang S Wu R et a0al New insights into different adipokines in linking the pathophysiology of obesity and psoriasis Lipids Health Dis Jim©nez Flores P Jim©nez Cruz A Bacardi Gasc³n M Body image dissatisfaction in children and adolescents a systematic review Nutr Hosp Weinberger N A Kersting A Riedel Heller SG et a0al Body Dissatisfaction in individuals with obesity compared to normal weight Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cindividuals a systematic review and meta analysis Obes Facts Kouris A Platsidaki E Christodoulou C et a0al Patients self esteem before and after chemical peeling procedure J Cosmet Laser Ther Stundzaite Barsauskiene G Tutkuviene J Barkus A et a0al Facial perception self esteem and psychosocial well being in patients after nasal surgery due to trauma cancer and aesthetic needs cluster analysis of multiple interrelations Ann Hum Biol Ribeiro F Steiner D Quality of life before and after cosmetic procedures on the face a cross sectional study in a public service J Cosmet Dermatol Bensoussan J C Bolton MA Pi S et a0al Quality of life before and after cosmetic surgery CNS Spectr Appleton SE Ngan A Kent B et a0al Risk factors influencing transfusion rates in DIEP flap breast reconstruction Plast Reconstr Surg Lievain L Aktouf A Auquit Auckbur I et a0al [Abdominoplasty complications particularities of the post bariatric patients within a patients series] Ann Chir Plast Esthet Sterodimas A Boriani F Nicaretta B et a0al Revision Abdominoplasty with truncal Liposculpting a year experience Aesthetic Plast Surg Al Dujaili Z Karcher C Henry M et a0al Fat reduction complications and management J Am Acad Dermatol Krueger N Mai SV Luebberding S et a0al Cryolipolysis for noninvasive body contouring clinical efficacy and patient satisfaction Clin Cosmet Investig Dermatol Neira R Arroyave J Ramirez H et a0al Fat liquefaction effect of low level laser energy on adipose tissue Plast Reconstr Surg Karu T Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP Photomed Laser Surg Feng J Zhang Y Xing D Low power laser irradiation LPLI promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERKSp1 pathway Cell Signal Croghan IT Hurt RT Schroeder DR et a0al Low level laser therapy for weight reduction a randomized pilot study Lasers Med Sci Thornfeldt CR Thaxton PM Horn
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"Background Complement receptor 1 (CR1) the receptor for C3b/C4b complement peptides plays a crucial role in carcinogenesis. However the association of genetic variants of CR1 with susceptibility to lung cancer remains unexplored. Methods This case-control study included 470 non-small cell lung cancer (NSCLC) patients and 470 cancer-free controls. Based on the Chinese population data from HapMap database we used Haploview 4.2 program to select candidate tag SNPs. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed by logistic regression to evaluate the association of each tag SNP with NSCLC. Results Multivariate regression analysis indicated that the rs7525160 CC genotype was associated with an increased risk of developing NSCLC (OR?=?1.52 95% CI?=?1.02-2.28; P?=?0.028) compared with the GG genotype. When stratified by smoking status the risk of NSCLC was associated with the rs7525160 C allele carriers in smokers with OR (95% CI) of 1.72 (1.15-2.79) but not in non-smokers with OR (95% CI) of 1.15 (0.81-1.65). When the interaction between smoking status and rs7525160 G?>?C variant was analyzed with cumulative smoking dose (pack-year). Similarly GC or CC genotype carriers have increased risk of NSCLC among heavy smokers (pack-year???25) with OR (95% CI) of 2.01 (1.26-3.20) but not among light smokers (pack-year <25) with OR (95% CI) of 1.32 (0.81-2.16). Conclusion CR1 rs7525160 G?>?C polymorphism was associated with an increased risk of developing NSCLC in Chinese population. The association displays a manner of gene-environmental interaction between CR1 rs7525160 tagSNP and smoking status. CR1 Polymorphism Tag SNPs Lung cancer Background The complement system plays a critical role in the process of carcinogenesis. Despite of significant research controversial viewpoints remain on the exact relationship of complement system with cancer. Classically the complement system fights against cancer by exerting the effects of immunosurveillance in the immunologic microenvironment of tumors [1]. Recently it was found that complement may contribute to tumor growth by a wide variety of mechanisms including dysregulation of mitogenic signaling pathways sustained cellular proliferation angiogenesis insensitivity to apoptosis invasion and migration and escape from complement cytotoxicity [2]. This suggested complement just like a double-edged sword plays a dual role in carcinogenesis. In particular component C3 and its receptors have been demonstrated to be a key link between innate and adaptive immunity [3]. Complement receptor type 1 (CR1 CD35) is a multifunctional polymorphic glycoprotein which binds to C3b fragment of C3 and to C4b with lower affinity [45]. CR1 belongs to the regulators of complement activation (RCA) family of proteins and is expressed in a wide spectrum of cells and involved in T-cell and B-cell mediated immune regulation [67]. CR1 also modulates the complement cascade activation by preventing formation of classical and alternative pathway convertases and by acting as a cofactor for factor I mediated inactivation of C3b and C4b [89]. It has been demonstrated that chronic inflammation can predispose to cancer development and spread [10] as a fundamental component of innate immunity the complement cascade consists of potential proinflammatory molecules especially C3 and C5. Moreover complement activation and abnormal expression in tumor tissues has been demonstrated [11]. Considering the important role of CR1 in complement activation innate immunity and chronic inflammation CR1 has emerged as a molecule of immense interest in gaining insight into the susceptibility to cancer. CR1 gene is located on the Chromosome 1 at the locus 1q32 [12]. Various polymorphisms have been studied including the intronic and exonic density polymorphism for their ability to alter the density of erythrocyte CR1 on the cell membranes [13-15]. There are also the molecular weight variants due to insertion-deletion polymorphisms [16]. Up to now there have been very few studies on the association of genetic variants of CR1 with susceptibility to autoimmune and inflammatory diseases. It has been proposed that genetic variant at CR1 gene (rs6656401) might influence the susceptibility to late-onset Alzheimers disease [17]. CR1 expression in Peripheral Blood Mononuclear Cells (PBMCs) may be a new biomarker for prognosis of nasopharyngeal carcinoma and a potential therapeutic target [18]. Recently it has been indicated that CR1 A3650G (His1208Arg) polymorphism plays a critical role in conferring genetic susceptibility to gallbladder cancer in north Indian population [19]. However the association of genetic variants of CR1 with risk of lung cancer remains unexplored. Worldwide lung cancer is the most common cancer in terms of both incidence and mortality [20]. NSCLC is the most common subtype of lung cancer and less aggressive and metastic than SCLC. Although cigarette smoking is the predominant risk factor for lung cancer inherited genetic characteristics are presumed to account in part for this interindividual variation in lung cancer susceptibility. Recently several genome-wide association studies have demonstrated the common genetic variations associated with susceptibility to lung cancer [21-24]. Given the involvement of the complement system in coordinating innate immunity and inflammatory response [25] further examination of the potential association between genetic variation of CR1 genes and lung cancer is warranted. In the current study we conducted a case-control study to investigate the association of tag SNPs in CR1 gene with the risk of NSCLC and effect of the interaction of gene-environment on the risk of NSCLC. Results Subject characteristics The frequency distributions of select characteristics in cases and control subjects were shown in . The mean age (±SD) was 59.6?±?10.5 years for the cancer patients and 57.2?±?13.3 years for the controls. No significant difference was found in the mean age between cases and controls (P?=?0.470). There was no significant difference in proportion of sex and smoking status between cases and controls (P?=?0.832 and P?=?0.321 respectively). However there was significant difference between cases and controls when compared by pack-year smoked (P = 0.001). The heavy smokers (?25 pack-year) accounted for 61.5% in cases but only 45.5% in controls which suggested that cigarette smoking was a prominent contributor to the risk of lung cancer. Of the 470 case patients 178 (37.9%) were diagnosed as adenocarcinoma 238 (50.6%) as squamous cell carcinoma and 100 (%) as other types including large cell carcinoma (n?=?49) and mixed cell carcinoma (n?=?5). Distributions of select characteristics in cases and control subjects Variables ???Cases (n?=?470) ???Controls (n?=?470) No (%) No (%) P a ???Sex 0.832 ???Male 324 68.9 328 69.8 ???Female 146 31.1 142 30.2 ???Age 0.470 ???<50 84 17.9 96 20.4 ???50-59 177 37.7 187 39.8 ???60-69 129 27.4 111 23.6 ????70 80 17.0 76 16.2 ???Smoking status 0.321 ???Non-smoker 265 56.4 281 59.8 ???Smoker 205 43.6 189 40.2 ???Pack-year smoked 0.001 ???<25 75 36.6 96 50.8 ????25 130 63.4 93 49.2 aTwo-sided ?2 test. Association of CR1 tag SNP with NSCLC risk Total 13 selected tag SNPs of CR1 in HapMap database among Chinese population were analyzed. Except for rs9429782 polymorphism the genotype distributions of other SNPs in controls were consistent to Hardy-Weinberg equilibrium. Therefore we excluded the rs9429782 from further analysis. In order to screen the genetic variants that confer the susceptibility to lung cancer 12 candidate tagSNPs were genotyped in a case-control study consisting of 470 lung cancer patients and 470 cancer-free controls as shown in . Importantly genotype frequency of one intronic SNP (rs7525160 G?>?C) in cases was found to be significantly different from those of controls (?2?=?6.339 P=0.042). Further multivariate regression model with adjustment for age gender and smoking status was used to assess the association between rs7525160 G?>?C polymorphism and the risk of NSCLC. The results indicated that the rs7525160 CC genotype was associated with an increased risk of developing NSCLC with OR (95% CI) of 1.52 (1.02-2.28) compared with the GG genotype. Other tagSNPs of CR1 were not significantly associated with the risk of NSCLC in our study population (P >0.05). Genotype frequencies of CRI among cases and controls and their association with non-small cell lung "
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cancer is a complex and heterogeneous disease with many possible genetic and environmentalcauses the same treatment for patients of the same cancer type often results in different outcomes in terms ofefficacy and side effects of the treatment thus the molecular characterization of individual cancer patients isincreasingly important to find an effective treatment recently a few methods have been developed to constructcancer samplespecific gene networks based on the difference in the mrna expression levels between the cancersample and reference samplesmethods we constructed a patientspecific network with multiomics data based on the difference between areference network and a perturbed reference network by the patient a network specific to a group of patients wasobtained using the average change in correlation coefficients and node degree of patientspecific networks of thegroupresultsnetworks with multiomics data the main differences of our method from previous ones are as follows networksare constructed with multiomics mrna expression copy number variation dna methylation and micrornaexpression data rather than with mrna expression data alone networks are constructed with bothnormal samples and cancer samples of the specified type to extract cancerspecific gene correlations and bothpatient individualspecific networks and patient groupspecific networks can be constructed the results of evaluatingour method with several types of cancer show that it constructs more informative and accurate gene networks thanprevious methodss the results of evaluating our method with extensive data of seven cancer types show that thedifference of gene correlations between the reference samples and a patient sample is a more predictive feature thanmrna expression levels and that gene networks constructed with multiomics data show a better performance thanthose with single omics data in predicting cancer for most cancer types our approach will be useful for finding genesand gene pairs to tailor treatments to individual characteristicskeywordsindividualspecific gene network groupspecific gene network cancer multiomics datacorrespondence khaninhaackr1department of computer engineering inha university incheon southkoreafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0clee bmc medical genomics 13suppl page of for the past years we have witnessed the rapid development of targeted cancer therapy targeted therapies forcancer work by targeting specific genes proteins or tissues that contribute to cancer growth and survival manytargeted therapies are effective only for patients with specific genetic alterations known as driver mutations thathelp cancer cells form and grow [ ] thus identifying genetic mutations specific to individual patients is ofutmost importance to determine targeted therapies thatcan effectively cure cancer patients while minimizing sideeffects motivated by a massive amount of data generated byhighthroughput technologies several cancer studies usedgene networks to explore gene expression characteristics [] however constructing a patientspecific genenetwork with a single sample obtained from a patient isdifficult because a gene network requires many samples tocompute genegene relationsrecently a few methods have been proposed to construct cancer samplespecific gene networks based onthe difference in the mrna expression levels betweenthe cancer sample and reference samples for exampleliu proposed a method to construct a samplespecific network by computing the difference between areference network from multiple reference samples anda network perturbed by a new sample however a slightchange to the reference samples can result in a significantly different samplespecific network for the samesample due to the small number of reference samples furthermore their samplespecific networks cannot reflectposttranslational modification and epigenetics becausethe networks are built using mrna expression data onlythis paper presents a new method for constructingcancer patientspecific and groupspecific gene networkswith multiomics data using a samplespecific networkand network propagation method network propagation strategies are widely used in recent cancerrelatedresearch li presented a synergy prediction algorithm using network propagation and predicted the drugsynergy in various cancers zhang introduceda propagation algorithm which learns the mutated subnetworks underlying tumor subtypes using a supervisedapproach and classified tumors to known subtypes onbreast and glioblastoma tumors peng identifiedbladder cancerrelated genes by propagating informationfrom seed genes to candidate genes the primary focusof our method is to construct a gene correlation network specific to cancer with multiomics data thus it isdifferent from a typical gene coexpression network thatrepresents coexpression relations between genes frommrna expression data our gene network is not a generegulatory network because our network does not showregulatory relations between genesthe main differences of our method from previous onesare as follows networks are constructed with multiomics mrna expression copy number variation dnamethylation and microrna expression data rather thanwith mrna expression data alone networks are constructed with both normal samples andcancer samples of the specified type to extract cancerspecific gene correlations and both patient individualspecific networks and patient groupspecific networks canbe constructed as shown later in this paper the resultsof evaluating our method with several types of cancershow that it constructs more informative and accuratetargetspecific networks than previous methodsmethodsat the top level our method consists of the followingsteps data processing constructing individualspecific gene networks and constructing a groupspecific gene networks a highlevel description of themethod is given in fig data collection and preprocessingfrom the broad institute tcga gdac firehose we obtained multiomics data of cancer samples of seventypes breast invasive carcinoma brca colon adenocarcinoma coad head and neck squamous cell carcinomahnsc pankidney cohort kipan liver hepatocellular carcinoma lihc lung adenocarcinoma luad andlung squamous cell carcinoma luscthe multiomics data used in this study includemrna expression mrnaseq copy number variationcnv dna methylation and mature mirna expression mirseq data the mrnaseq data were processedusing quartile normalized rsem and then log2transformed the segmented cnv data were convertedto genelevel data using the ensembl api and thecntools package of bioconductor the methylationdata were filtered to select the probe with the mean signal values for each gene the mirseq data were processedby rpm and log2transformed mrnas and mirnas thatwere not expressed in more than of the total sampleswere excluded in further analysis missing expression values of mrnas and mirnas were replaced by the smallestpositive normalized floatingpoint number realmin ofmatlab the number of samples and genes used in thisstudy are available in additional file individualspecific gene networkin each group of tumor samples and normal samples wefirst computed genegene relations by the pearson correlation coefficient pcc selected highly correlated genepairs ie those with pcc and constructed twosample networks one for each group from the tumorsample network we removed edges common to both 0clee bmc medical genomics 13suppl page of fig overview of constructing an individualspecific network and a groupspecific network with multiomics datatumor and normal sample networks and obtained a template reference network for cancer fig 2a the templatereference network consists of highlycorrelated gene pairsthat are specific to cancerwith n reference samples which may be different fromtumor samples used in the template network we computed pcc for every pair of genes in the template reference network and constructed a reference network forthe reference samples for a patient of interest we constructed a network which is a perturbed network byadding a single sample of the patient to the n reference samples a patientspecific network was obtainedby subtracting the reference network from the perturbednetwork 01pcc pccn1 pccnwe computed the difference in the absolute value ofpcc between the perturbed reference network and reference network by eq we also carried out a ztest ofpccn1pccn by eq for a large n we can approximatethe mean μ and standard deviation Ï of pccn1 pccn as and pcc2nn respectively pccchange pccn1 pccnz score pccchange μpccchangeÏ pccchange pccchangepcc2nnthe edges of the patientspecific network were classified into four types correlationgained edges fene pairs whose pccs are increased from the referencenetwork to the patientspecific network correlationlost edges for gene pairs whose pccs are decreased fromthe reference network to the patientspecific network correlationreversed edges for gene pairs whose signs ofpccs are changed from positive to negative or negativeto positive and correlationinvariant edges for genepairs with little change in pccs between the reference andpatientspecific networks ie those with zscore fig 2bthe edges were classified in the following way we firstselected gene pairs with zscore as correlationinvariant type and then selected gene pairs which havedifferent signs of pccs between the reference networkand the patientspecific network as correlationreversedtype the remaining gene pairs were classified into eithercorrelationgained or correlationlost type depending onwhether their pccs are increased correlationgained ordecreased correlationlost from the reference network tothe patientspecific network thus zscore ¥ in bothcorrelationgained and correlationlist gene pairsgroupspecific gene networka groupspecific gene network is useful when analyzinga large number of patientspecific gene networks afterconstructing patientspecific gene networks we obtained 0clee bmc medical genomics 13suppl page of fig process of constructing a patientspecific gene network a template of the cancer reference network obtained by removing edges common toboth networks b patientspecific gene network and four types of edges in the network edges with a zscore represent correlationinvariantgene pairs and edges with zscore ¥ and different signs of pccn and pccn1 represent correlationreversed gene pairs edges with zscore ¥ and 01pcc are correlatedgained gene pairs and those with zscore ¥ and 01pcc are correlationlost gene pairsa gene network specific to a group of patients based onthe average 01pcc and node degree of the patientspecificnetworks fig if the dominant type for a particular edge is correlationgained positive 01pcc in thepatientspecific networks the edge is represented in redin the groupspecific network in contrast if the dominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edgeis represented in blue in the groupspecific network inthe groupspecific network only the dominant type isshown for each edge if nondominant types are shownin addition to the dominant type for each edge the network becomes cluttered and unreadable the node sizeof a groupspecific gene network is proportional to theaverage degree of the nodeintegration of multiomics datato integrate multiomics data we first computed intergene correlations by pcc with four different types ofsingle omics data mrna expression cnv dna methylation and mirna expression separately and selectedsignificant intergene correlations only in mrna expression cnv and dna methylation data we select thetop pcc with pvalue in mirna expression data we selected the top pcc with pvalue due to a smaller number of mirnas inthe data the intergene correlations selected in eachsingle omics data are represented in four correlationmatrices mexpr mcnv mmethyl and mmirna andnormalizedusing the proteinprotein interactions ppis of thestring database we constructed separate weightednetworks from each omics data by eq in eq wexprwcnv and wmethyl denote the weighted networks andppiexpr ppicnv and ppimethyl are subnetworks of a ppinetwork consisting of genes present in each omics datasince the ppi network does not contain information onmirna a weighted network for mirna was not constructedcid3wexpr cid2 ppiexprwcnv mcnv à ppicnv wmethyl cid2 ppimethylwe then integrated the multiomics data by linear xmethylregression using eq in eq yi xcnvand xmirnadenote gene is expression level cnv levelmethylation level and mirna regulator expression levelrespectively βcnvdenote the regression coefficients of gene is expression level on cnv and methylation respectively βmirnais the regression coefficientof gene is expression level on its mirna regulator jsexpression leveland βmethyl mexpr mmethylcid3 à cid2cid3 à cid2cid3iiijiiijyi βcnvii βmethylxcnvixmethyli ncid4j1βmirnaijxmirnaij 01 0clee bmc medical genomics 13suppl page of fig process of constructing a groupspecific gene network from multiple patientspecific gene networks in the groupspecific gene network thenode size and node color are proportional to the average degree and average 01pcc of the node respectively if the dominant type for a particularedge is correlationgained positive 01pcc in the patientspecific networks the edge is represented in red in the groupspecific network if thedominant type for a particular edge is correlationlost negative 01pcc in the patientspecific networks the edge is represented in blue in thegroupspecific networkfrom the regression coefficients and the weighted networks a weight matrix w was derived and normalizedinto w eqs and the weight matrix w is symmetricso wij wji w11 w22 w33 and w44 represent wexprwcnv wmethyl and mmirna respectively the submatrices w21 and w31 contain regression coefficients βcnvand βmethylfor every gene i respectively w41 representsiβmirnaij the submatrices w32 w42 and w43 are emptyi¡¢¢£w w11 w12 w13 w14w21 w22 w23 w24w31 w32 w33 w34w41 w42 w43 w44¤¥¥¦eq and updated iteratively by eq in this iterative process the influence of the seed is propagated tothe neighbors until a mean squared error of st and st¤ à §ªªªª¨ªªªª©if v is a nonseed nv ¥ αif v is a nonseed nv αif v is a seednvnvenvα à nvnvdv st λ à st à w λ à d wheres1 d where nv is the number of neighbors of node v and nv isthe number of seeds in the neighbors the parameter αwhich is a threshold for nv was set to and λ was set to genes with the top st were used in findingcancerrelated genes and in classifying tumor samples andnormal samplesw i j w i jcid11cid12cid12cid13mcid4k1w i k à mcid4k1w k jin network propagation seed genes have greater impactthan nonseed genes on their neighbors thus only thegenes with a high average 01pcc were selected as seedgenes for a groupspecific network and their mirnasregulators extracted from mirtarbase were used asseed mirnas we calculated the cancerrelevance st ofeach gene to reflect the effect of the seed genes and mirnas on neighbors the initial score d was calculated byresultspatientspecific and groupspecific gene networksin this study we constructed patientspecific genenetworks for seven cancer types additional file foreach cancer type we also constructed groupspecific genenetworks as an example fig shows a groupspecificgene network derived from lung squamous cell carcinoma lusc patients 0clee bmc medical genomics 13suppl page of there are three distinct subnetworks in the networkfor the lusc group the subnetwork enclosed in box aof fig contains many hub genes large green nodesthe subnetwork in box b consists of correlationgainededges dark red edges whereas the subnetwork in box ccontains many correlationlost edges dark blue edgescomparison of multiomics data and singleomics datawe performed leaveoneout cross validation loocvto evaluate cancerrelevance score st of a gene and thecontribution of multiomics data to finding cancerrelatedgenes for comparison the cancerrelevance scores werecomputed with multiomics data and single omics dataseparately each seed gene was regarded as a nonseed anda new cancerrelevance score was calculated for the geneseed genes and nonseed genes were considered as positive and negative respectively seed genes included in thetop n genes were considered as true positives and seedgenes not included in the top n genes were consideredas false negatives similarly nonseed genes included infig groupspecific gene network for lung squamous cell carcinoma lusc patients a subnetwork of multiple hub genes large greennodes b subnetwork of correlationgained edges dark red edges c subnetwork with many correlationlost edges dark blue edges 0clee bmc medical genomics 13suppl page of the top n genes and nonseed genes not included in thetop n genes were considered as false positives and truenegatives respectivelywe carried out loocv with different ratios of seedgenes to nonseed genes figure shows the receiver operating characteristic roc curve and the area under thecurve auc of loocv of the cancer relevance of geneson data of breast cancer samples with various seedratios ranging from to enlarged plots of fig are available in additional file for comparative purposes we also computed the cancer relevance of geneswith single omics data as shown in fig multiomicsdata consistently exhibited better performance than singleomics data with any seed ratio between to forlater analysis the seed ratio was set to by default theaverage 01pcc and class label of each gene are available inadditional file indeed the superiority of multiomics data over singleomics data in determining the cancer relevance scoreof genes was observed in all seven types of canceradditional file in seven types of cancer the cancerrelevance score of genes computed with multiomics dataexhibited a good performance auc ¼ thecancer relevance score of genes computed with mrnaexpression data showed the second best performanceauc ¼ in particular the cancer relevancescore computed with mrna expression data showed avery similar performance to that with multiomics inbreast cancer brca the performance of the cancer relevance score computed with cnv auc ¼and dna methylation data auc ¼ alonewas lower than that with mrna expression data auc ¼fig roc curves of the leaveoneout cross validation of the cancer relevance score st of genes with different ratios of seed genes breastcancer samples were used in the leaveoneout cross validation the performance of multiomics data is always better than that of single omics data 0clee bmc medical genomics 13suppl page of evaluation of gene correlations and networksmany networkbased approaches to cancer research havefocused on finding genes that show differential expressions between tumor samples and normal samples genegene correlations ie intergene correlations may bemore helpful than individual genes because intergenecorrelations depend on the expression of neighbor genesin a gene regulatory network to compare the effect ofusing individual genes to that of intergene correlationsie 01pcc we constructed a support vector machinesvm model for classifying cancer samples and normalsamples the svm model was implemented using csvcand rbf kernel and the parameter values of the modelwere determined by the grid search algorithm mrnaexpression levels and 01pccs were used as features ofthe svm models for rigorous validation the test dataused in testing the models were not used in training themadditional file as shown in fig 6a 01pcc showed a better performance than mrna expression levels for six cancertypes except lusc the classification model with 01pccshowed mcc above in six cancer types except hnscwe also examined the effect of different networks on individualspecific networks in the workby liu ppi data with high confidence scoresin the string database were used to construct a network however the ppi data of stringdoes not reflect cancer typespecific characteristicsfigure 6b shows the performance of the classificationmodel with two different networks network from ppi data of string the approachby liu and cancer network ourapproach 01pcc was used as a feature of the classification model except for coad the performance ofthe classification model with the cancer network was better than the model with the string reference network in particular the classification model showed a significant difference for breastcancer brca mcc of vs mcc of detailed results of the classification model are available inadditional file discussionin the analysis of finding cancerrelated genes and genepairs we focused on a subnetwork of genes with a 01pcctable shows the top genes with a high average 01pcc in each groupspecific network of seven cancertypes in breast invasive carcinoma brca fam171a1showed the highest average 01pcc in the groupspecificnetwork fam171a1 is known as a potential biomarkerin triplenegative breast cancer foxc1 is involvedin tumor development and metastasis and associated withpoor prognosis in basallike breast cancer il33 isoverexpressed in various cancers and the serum concentration of il33 is a valuable indicator of poor prognosis inbreast cancer mamdc2 is significantly correlatedwith diseasefree survival of breast cancer patients mterfd1 is closely related to breast cancer recurrencefig results of evaluating features and networks by a validation set a comparison of mrna expressions of genes and 01pcc of genepairs b comparison of the cancer network with the network from ppi data 0clee bmc medical genomics 13suppl page of table top genes with a high average 01pcc in a groupspecific network for seven cancer typesbrcakipanlihccoadhnscfam171a1foxc1il33mamdc2mterfd1hoxa7cttnbp2znf204pjam3frem1gfra2scnn1bddx27rnps1ube2imdficctnnbl1cdk5rap1esf1trmt6cyp2j2barx2znf135ppfia1parlfaddcst6cobloraov1xpo7tfcp2l1kcnq1arl15kctd1oaz2tmem45bhpcal1tmem91sema5begln3slc19a3ecm1cyp2b6fbp1gpaa1f9pahagxt2hsd17b6rnase4luadcldn18adamts8pecam1sftpa1gimap6akr1c1mmeatad2cyp3a5chaf1bluscnsun2cct5fbxo45gpr116slc39a8fxr1inmtveph1crtamwdr53 and hoxa7 plays a critical role in regulating theproliferation of erpositive cancer cells in colon adenocarcinoma coad gfra2 showed thehighest average 01pcc in the groupspecific network itis known to be crucial for enteric neuron survival scnn1b and ddx27 are significantly related to colorectal cancer [ ] no direct relation of rnps1 withcolorectal cancer is known but rnps1 is essential tononsensemediated mrna decay that plays complexfunctions in cancer knockdown of sumo conjugating enzyme ube2i also known ubc9 or e2 inhibitsmaintenance and selfrenewal of colorectal cancer stemcell while overexpression of ube2i increases colorectalcancer cell stemness among the top genes with a high average 01pccin lung adenocarcinoma luad several genes such ascldn18 adamts8 pecam1 and sftpa1 have beenknown to be associated with luad in previous studies[] no direct relation of nsun2 and slc39a8 withlung squamous cell carcinoma lusc has been known sofar however recent studies [ ] reported that nsun2is correlated with survival in other types of squamous cellcarcinomas gao also showed that the epigeneticsilencing of slc39a8 expression by dna methylation isinvolved in the acquisition of resistance against cadmiumin lung cells and the relation between cadmium andlung cancer has received much attention many othergenes in table found in the groupspecific networksfor head and neck squamous cell carcinoma hnscpankidney cohort kipan and liver hepatocellular carcinoma lihc are also directly or indirectly related tocancerin addition to individual genes we identified genepairs of the same type ie either correlationgained orcorrelationlost in most patientspecific networks of thesame type table shows the most frequent gene pairs in breast cancer samples the most frequent gene pairsin other types of cancer are listed in additional file it isinteresting to note that all the gene pairs shown in table include at least one gene in the gene pair mamdc2hoxa7 and that they are correlationgained edges in thegroupspecific network for breast cancer figure showsa subnetwork containing mamdc2 and hoxa7 in thegroupspecific network of breast cancer the subnetworkwas obtained by selecting the edges for which the proportion of the same edge type ie correlationgained or lostis above in the total individualspecific networks ofbreast cancer patients it is interesting to note that all thegene pairs in table are included in the subnetworkto date the actual role of the mamdc2 gene in cancer is not clear but meng reported mamdc2as one of three genes mamdc2 tshz2 and cldn11that are significantly correlated with diseasefree survival of breast cancer patients mamdc2 is known as atarget of mir196a in head and neck squamous cell carcinoma as a member of the family of homeoboxgenes hoxa7 is associated with cell proliferation nerveinvasion distant metastasis and degree of tumor differentiation in several cancers [ ] hoxa7 is regulatedtable the most frequent gene pairs in breast cancersamples all the gene pairs are of a correlationgained type thegenes of table are shown in bold the proportion represents theratio of the gene pairs of the same type ie correlationgained orlost to the total number of patientspecific networksgene pairgene pairsproportion of the gene pairsin total cancer samplesmamdc2hoxa7mamdc2ccl14mamdc2znf204pmamdc2klmamdc2svep1mamdc2coro2bhoxa7meox2hoxa7hoxa9mamdc2sobpmamdc2hoxa9 0clee bmc medical genomics 13suppl page of fig a subnetwork of the groupspecific network of brca which contains mamdc2 and hoxa7 genes in the most frequent gene pairs shown intable are enclosed by a circleby several mirnas including mir196 [] thusboth mamdc2 and hoxa7 are related with mir196but a clear relation among them is to be uncoveredso far most approaches to constructing individualspecific gene networks have been constructed based onthe differential expressions between a small number ofreference samples and a sample of interest howeversuch networks cannot reflect posttranslational modification and epigenetics and are not reliable because a slightchange to the reference samples can result in a significantly different samplespecific network for the samesamplein this paper we presented a new approach to constructing cancer patientspecific and groupspecific networks with multiomics data the main differences ofour method from previous ones are as follows gene networks are constructed with multiomics mrnaexpression copy number variation dna methylationand microrna expression data rather than with mrnaexpression data alone networks can beconstructed with cancer samples of the specified type and both patient individualspecific networks and patientgroupspecific networks can be constructed the resultsof testing our method with several cancer types showedthat it constructs more informative and accurate genenetworks than existing methodsevaluation of our method with extensive data of sevencancer types showed that changes in gene correlations 01pcc between the reference samples and a patient sample is a more predictive feature than mrna expressionlevels and that gene networks constructed with multiomics data are more powerful than those with singleomics data in predicting cancer for most cancer typesmore work is required to validate the genes and genepairs identified in the cancer patientspecific and groupspecific networks however the method for constructingnetworks specific to individual patients or patient groupswith multiomics data should be useful aids in determining effective treatments to individual characteristicssupplementary informationsupplementary information accompanies this paper athttpsdoi101186s12920020007367additional file number of samples and genes in types of canceradditional file roc curve and auc of the cancerrelevance score ofbrca by various seed ratiosadditional file average 01pcc and class label of each gene in typesof canceradditional file roc curve of the cancerrelevance score of each cancertype with the seed ratio of additional file performance of classification of tumor samples andnormal samplesadditional file top gene pairs for each cancer type 0clee bmc medical genomics 13suppl page of abbreviationsauc area under the curve brca breast invasive carcinoma cnv copynumber variation coad colon adenocarcinoma gdac genome dataanalysis center hnsc head and neck squamous cell carcinoma kipan pankidney cohort lihc liver hepatocellular carcinoma loocv leaveoneoutcross validation luad lung adenocarcinoma lusc lung squamous cellcarcinoma mcc matthews correlation coefficient pcc pearson correlationcoefficient ppi proteinprotein interactions roc receiver operatingcharacteristic svm support vector machine tcga the cancer genome atlasacknowledgementsthe authors thank the anonymous reviewers for the constructive commentsthat contributed to improving the final version of the paperabout this supplementthis has been published as part of bmc medical genomics volume supplement selected s from the 15th international symposiumon bioinformatics research and applications isbra19 medical genomicsthe full contents of the supplement are available online at httpsbmcmedgenomicsbiomedcentralcomssupplementsvolume13supplement6authors contributionswl designed and performed all about this study and prepared the initialmanuscript dh helped integrating multiomics data of breast cancer khsupervised the work and wrote the manuscript all authors read and approvedthe final manuscriptauthors informationwook lee is currently working toward his phd degree at the department ofcomputer engineering inha university korea deshuang huang is a directorof the institute of machines learning and systems biology tongji universitychina kyungsook han is a professor at the department of computerengineering inha university koreafundingthis work was supported by the national research foundation of korea nrfgrant funded by the ministry of science and ict nrf2017r1e1a1a03069921and inha university research grant publication of this was funded bythe nrf grant nrf2017r1e1a1a03069921 the funders had no role in thedesign of the study data collection and analysis or writing the manuscriptavailability of data and materialsadditional files are available at httpbclabinhaackrcancernetworkethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of computer engineering inha university incheon southkorea 2institute of machine learning and systems biology school ofelectronics and information engineering tongji university shanghaichinareceived may accepted june published august references widakowich c de castro g de azambuja e dinh p awada a reviewside effects of approved molecular targeted therapies in solid cancersoncologist liu s kurzrock r toxicity of targeted therapy implications for responseand impact of genetic polymorphisms cancer treat rev verma m personalized medicine and cancer j personalized medbarabasi al gulbahce n loscalzo j network medicine a networkbasedapproach to hum
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"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genesi reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used housekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employedi cv �xsx where �x and Ïx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isnt a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm Ëcamplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks curls were deparaffinized inxylene at Ëc followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at Ëc for minutes followed by amplification for cyclesdenaturation at Ëc for sec amplification at Ëc for sec and extension at Ëc for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq samplemean cq ntc and relative expression aseδcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithmsgenorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkins bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of adjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgsptcd2 ppp1r3b andfbxw9 have sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting original draft sreejeta mondal sujan k dharwriting review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for doubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092 derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may 101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep 101038leu2016382 pmid bomben r ferrero s dagaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr 103324haematol2017184325pmid dheda k huggett jf chang js kim lu
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mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate ï¬ux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate ï¬ux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signalingcancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdacisheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspeciï¬cnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspeciï¬c mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we ï¬nd that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our ï¬ndingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientiï¬c research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetriï¬uoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulï¬de bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmica and mica vectors containingthe coding sequences of mica or mica alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micaï¬reï¬y luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c ocallaghan university of oxfordoxford united kingdom from tocris biosciencepuriï¬cation of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoys 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoys 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions¦c and co2 and were passaged every daysfor proliferation assay wt and mgat5 ko cells wereseeded in or cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc ï¬nger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were conï¬rmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturers protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were conï¬rmed by sequencing at euroï¬ns mwgoperons luxembourg colonies were ampliï¬ed and plasmidswere puriï¬ed with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were ampliï¬ed and dna waspuriï¬ed using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at ¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a ï¬nal volume of µl the dna mixture was complexed with µl 2x hbsunder constant air ï¬ow and the transfection mix was addeddropwise to hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged mica and micaconstructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbrieï¬y dnarna were prepared in µl cacl2 25mand adjusted to a ï¬nal volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at ¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by ï¬ow cytometry using accuri c6 ï¬ow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for h gfp expression of target cellswas assessed with accuri c6 ï¬ow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturers protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio of each mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 ï¬ow cytometerwas assessed by accurate mass and retention time amrt plusfragment identiï¬cation at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending conï¬rmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturers protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in cells harvested withtrypsinization and pelleted by centrifugation for g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at ¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite proï¬lingto determine intracellular metabolite levels cell pellets from cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at g for min at ¦c µl supernatants were collected transferred to ultrafreemccentrifugal ï¬lter devices merck millipore ltd cork irelandand centrifuged at g for min at ¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of ï¬ight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm mm µm p size coupled to a guardcolumn mm mm µm p size and an inlineï¬lter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the ï¬owrate was mlmin injection volume was µl and the columnoven was maintained at ¦c the acquisition was obtainedwith a mass range of mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsï¬les were independently processed with an inhouse developedpcdl library for polar metabolites using proï¬nder version b06agilent technologies identiï¬cation of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular ï¬ux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density cellswell ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at ¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at g for min ¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen ï¬ow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modiï¬ed procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientiï¬cwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientiï¬c for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at ¦c proteinextracts were denatured at ¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturers protocol membranes wereblocked in tbst blocking buï¬er licor biosciences probed with primary antibodies in tbs w tween and bsa overnight on a shaker at ¦c and washed intbs tween secondary antibody was from licorlicor biosciences and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 ï¬ow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of ï¬uorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake cells seeded the day prior toexperiment were washed once in pbs and incubated for minat ¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2dfc receptor 1299nk rd systemsand igg1fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean ï¬uorescent intensity mfi values aredisplayed in ï¬gures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and puriï¬ed on directzol spincolumns zymoresearch irvine ca united states according to manufacturersprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with ï¬reï¬y luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and ï¬reï¬y luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectioneï¬ciency ï¬reï¬y luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcrampliï¬ed sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 ï¬ow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were ï¬ltered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identiï¬ed using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantiï¬cation and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical signiï¬cance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in ï¬gure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical signiï¬cance was determined by p p and p p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to conï¬rm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspeciï¬cally to mgat5modiï¬ed nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modiï¬ednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modiï¬cation of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d speciï¬city supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a signiï¬cant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspeciï¬c since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconï¬rming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deï¬ciency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles mica and micaare distinctly regulated posttranslationally and althoughmica was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding ofï¬uorescently labeled lpha mgat5 modiï¬cations or epha mgat3 modiï¬cations on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by ï¬ow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean ï¬uorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signiï¬es nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5
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" leukotriene receptor antagonists ltras are broadly used for the management of allergic asthmaand have recently been indicated to inhibit carcinogenesis and cancer cell growth in colorectal cancer crcchemoprevention studies the occurrence of adenoma or crc itself is generally set as the trial endpoint althoughthe occurrence rate of crc is the most confident endpoint it is inappropriate for chemoprevention studies becausecrc incidence rate is low in the general population and needed for longterm monitoring aberrant crypt fociacf defined as lesions containing crypts that are larger in diameter and darker in methylene blue staining thannormal crypts are regarded to be a fine surrogate biomarker of crc therefore this prospective study was designedto explore the chemopreventive effect of ltra on colonic acf formation and the safety of the medicine in patientsscheduled for a poly resection as a pilot trial leading the crc chemoprevention trialmethods this study is a nonrandomized openlabel controlled trial in patients with colorectal acf and polypsscheduled for a polypectomy participants meet the inclusion criteria will be recruited and the number of acf inthe rectum will be counted at the baseline colonoscopic examination next the participants will be assigned to theltra or no treatment group participants in the ltra group will continue mg of oral montelukast for weeksand those in the no treatment group will be observed without the administration of any additional drugs at theend of the 8week ltra intervention period a polypectomy will be conducted to evaluate the changes in thenumber of acf and cell proliferation in the normal colorectal epithelium will be analyzeddiscussion this will be the first study to investigate the effect of ltras on colorectal acf formation in humanstrial registration this trial has been registered in the university hospital medical information network uminclinical trials registry as umin000029926 registered november keywords colorectal cancer chemoprevention leukotriene receptor antagonist aberrant crypt foci correspondence takuma_hyokohamacuacjpdepartment of gastroenterology and hepatology yokohama city universityschool of medicine fukuura kanazawa yokohama kanagawa japan the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chigurashi bmc cancer page of cancer is a one of the major health issues and a leadingcause of death globally the incidence prevalence andmortality rates of colorectal cancer crc continue torise all over the world [ ] the majority of crc casesare derived from adenomatous polyps and their resection has been shown to reduce the risk of the futuredevelopment of advanced adenomas and crc [ ]thereby preventing crcrelated deaths however patients with polyps adenomatous polyps andor hyperplastic polyps represent a highrisk group for theoccurrence of metachronous colorectal polyps andorcrc then a paradigm is shifting from surveillancefor the early detection of advance adenomas or crcsand resection to novel tactics for prevention is requiredto reduce the mortality rate of this disease a number oflargescale epidemiologic andor clinicaltrials haveassessed the prophylactic agentsincluding vitamin dcalcium fiber and nonsteroidal antiinflammatory drugsnsaids such as aspirin and selective cyclooxygenase cox2 inhibitors in preventing against crc development we previously reported that the nsaidsulindac suppressed the development of sporadic colorectal adenomas to this point nsaids particularlycox2 inhibitors have been proven to offer the greatestpossibility for reducing crc risk either alone or in combination with other drugs while it has been reportedan elevated risk of serious cardiovascular events relatedwith the administration of cox2 inhibitors [ ] inview of these adverse cardiovascular effects and the lackof efficacy of other drugs that initially looked promisingthe development of novel agents that meet both safetyand efficacy in preventing crc is essentialleukotriene receptor antagonists ltras such asmontelukast and zafirlukast are commonly used for thetreatment of allergic asthma [ ] and tsai mj reported that ltras reduced cancer risk in a dosedependent manner in asthma patients it was alsoreported that the cancer incidence rate was significantlylower in ltra users than in nonltra users vs per patientsyear this means that apart fromits role in asthma ltra has also been associated withcarcinogenesis and tumormediated immunosuppression for example the overexpression of cysteinyl leukotriene receptor cyslt1r has been observed in crcand montelukast leads the apoptosis of crc cancer cells[ ] previous in vivo studies have shown the chemopreventive effect of ltras [ ] but the chemopreventive effects of ltra have not been studied in clinicalpractice therefore we designed this study to ivestigatethe chemopreventive effect of ltras in clinical practicein crc chemoprevention trials the occurrence of adenomas or crc itself is generally set as the trial endpointthough the occurrence of crc is the most confidentendpoint it is not recommended for chemopreventionstudies because crc incidence rate is low in the generalpopulation and needed for longterm monitoring furthermore there are ethical concerns about conductinglongterm trials to determine whether a test agent is effective or notaberrant crypt foci acf defined as lesions containing crypts that are larger in diameter and darker inmethylene blue staining than normal crypts [] areregarded to be a fine surrogate biomarker of crc our group has previously reported that acf is usefulbiomarker for crc [ ] and study endpoint for achemoprevention study [ ] the advantages of chemoprevention studies with the number of colorectalacf as the trial endpoint are that longterm observationis not needed to investigate the agent efficacy and thenumber of acf can be quantitatively estimated therefore we set the acf count as a good endpoint for thisstudy to the knowledge of us this is the first clinicalstudy investigating the use of ltras as chemopreventive agents against colorectal acf in humansmethodsstudy design and settingthis study was designed as a nonrandomized openlabel controlled trial to be conducted in patients withcolorectal acf it will be performed at the departmentof gastroenterology and hepatology yokohama cityuniversity ycu hospital japan the coordinating office will be at the ycu hospital and patient registrationwill be conducted at the ycu center for novel and exploratory clinical trials ynext and data collectionwill be done using electronic data caputureethical considerations and trial registrationthe trial protocol complies with the declaration ofhelsinki and the ethical guidelines for clinical research of the ministry of health labour and welfarejapan ethical approval of this trial was obtainedfrom the ethics committee of ycu hospital on may the study protocol and informed consent documents were approved by the ycu hospital ethics committee this trial has been approved in the clinical trialact in japan and registered in the japan registry of clinical trials jrct as jrcts031180094 and the universityhospital medical information network umin clinicaltrials registry as umin000029926 all study participants will submit a written study participation informedconsent formparticipation criteriawe will recruit the patients with colorectal acf and resectable polyps for this trial the inclusion criteria are asfollows patients with resectable polyps [adenoma 0chigurashi bmc cancer page of hyperplastic polyp and sessile serrated adenomapolypssap] patients with more than five rectal acfand submit written study participation informed consent formthe exclusion criteria are as follows patients withlesions suitable for early removal a history of ltrause within months before study participation a history of malignant disease within years before studyparticipation a history of heart renal liver failure orliver cirrhosis a history offamilial adenomatouspolyposis hereditary nonpolyposis crc or inflammatory bowel disease pregnancy or the possibility ofpregnancy prohibitions of montelukast allergiesto montelukast regular use of nsaids metforminand pioglitazone and participants considered as unsuitable for the study by the researchersinterventionall eligible participants will be assigned to the ltra orno treatment group because this is an openlabel trialpatients will be assigned to the no treatment group afterthe inclusion of patients in the ltra group participantsin the ltra group will receive mg of oral montelukast for weeks and those in the no treatment groupwill be observed without the administration of any additional drugs at the end of the 8week ltra treatmentperiod a polypectomy will be performed to evaluate thechanges in the number of acf and cell proliferation inthe normal colorectal epithelium will be analyzedoutcome measurementsthe primary endpoint will be the change in the numberof colorectal acf after weeks of treatment a magnifying colonoscope pcfq290azi hz290 olympus cotokyo japan will be used in all cases procedure preparation for the colonoscopy will begin day before theprocedure each participant will be informed to take alowresidue diet and mg oral sodium picosulfate onthe evening before the procedure on the day of the procedure each participant will be given ml polyethylene glycol peg if the stools are not clear enough anadditional ml peg will be given to ensure adequatebowel cleansing in most cases conscious sedation withmidazolam mg and pentazocine mg willbe use at the start of the colonoscopy subcutaneousscopolamine or glucagon will be administered for colonic movements reduction at the time of the first colonoscopy the endoscopists will insert into the cecumand the observe entire colorectum as the endoscope ispulled back one colonic mucosal sample will be collected the number of rectal acf will be counted usinga magnifying endoscope at the end of the 8week ltratreatment period the same endoscopists will performthe polypectomies and countthe number of acfendoscopists will record all procedures on a hard diskdrive and take photograph all acf the number of acfin each participant will first be counted by the endoscopists during the procedure to provide additional validation the number of acf will be recounted by threeblinded expert endoscopists aj ht and ak by observing the recorded hard disk drive cases that these evaluators deem colonoscopy to be inappropriate will beexcluded from the final analysisthe secondary outcomes will be as follows drugsafety adverse events aes will be graded according tothe national cancer institute common toxicity criteriafor adverse events ncictcae version all trialparticipants will be provided with a trial record for thedaily dose of the trial agent and aes participants whodevelop serious ae of grade or higher will be discontinued at that time in the study the effects ofltras on cell proliferation in the rectal mucosa onecolonic mucosal sample will be collected from the samestudy patient by performing a biopsy at the time of thebaseline colonoscopy and polypectomy a biopsy will beobtained from all participants cell proliferation will beevaluated by ki67 staining briefly we will randomly select six crypts and count the number of ki67positivecells per crypt in total cells will be counted at amagnification of à using a brightfield microscopethe results will be presented as the percentage of ki67positive cells all participants will receive laboratory testsand a physical examination at the point of the baselinecolonoscopy and polypectomydrug supplymontelukast capsules will be purchased from kyorincorporation ltd tokyo japan participants will be informed to take one tablet of the study drug every nightbefore bed medication adherence will be monitored bycounting the empty medication sheets returned by theparticipants at the time of their polypectomy the participants will also be interviewed and monitored to confirm thatthey have not used any prohibited drugsaspirin metformin andor other nsaids aes will bemonitored by the investigator and graded according tothe ncictcae version if serious aes or less than drug adherence are confirmed in a participant thisparticipant will be withdrawn from the trialsample size estimation and allocationwe previously reported the administration of mgmetformin per a day for weeks reduced the number ofacf in that trial the mean number of acf per patientdecreased significantly from ± at baseline to ± at weeks p although this study is exploratory research and the accurate chemopreventive efficacy of montelukastis unknown we assume that 0chigurashi bmc cancer page of montelukast may have an effect that is equivalent to of that observed for metformin on the reduction of acfnubmer therefore we estimate that the acf numberwill change by about to on average we determined that a sample size of individuals in theltra group was needed to detect a significant reduction in the number of acfs in the ltra group using apaired ttest with a twosided significance level of and power assuming some dropouts we propose to recruit participants in the ltra group to confirmthat the number of acf does not change during thestudy period we propose to recruit patients in the notreatment group after consecutively accumulating patients in the ltra group therefore we propose to recruit patientsstatistical analysisthe change in the number of acf which is the primaryendpoint will be compared before and after the 8weekstudy period between the ltra and no treatment groupsby the paired ttest drug safety will be assessed by thechisquare test and the remaining results will be compared by the ttest or mannwhitney u test between thetwo groups p will be defined as statistically significant all statistical analyses will be conducted using jmp® software sas institute inc cary nc usatrial steering committee and data monitoring committeethe trial steering committee and data monitoringthe ynext thecommittee will be located atmanagement team will perform central monitoring ofthe trial status and data collection every monthstudy flowa study flow is shown in fig discussionto the knowledge of us this is the first clinical trial proposed to investigate the efficacy of ltras on colorectalacf formation ltras are broadly used for the treatment of allergic asthma and rhinitis [ ] and ltrasare reported to decrease the risk of cancer in asthma patients in a dosedependent manner previous basicresearch has reported that cyslt1r is overexpressed incrc and that montelukast induces the apoptosis ofcrc cells [ ] cyslts have recently been focusedon as significant regulators of gut homeostasis with endogenous cyslt production mediating the proliferationand survival of gut mucosal cells recent evidence focuses on the effect of leukotrienec4 ltc4 in accelerating oxidative dna damage if notadequately repaired can contribute to increase mutationrates and genomic instability dna damage andgenomic instability are major drivers of carcinogenesis cyslts also acts as leukocyte chemoattractants inaddition cyslt1 mediates th17 cell migration the storage of which associates with the progression ofinflammationassociated cancers chronic inflammation is a risk factor for cancer initiation and progression as observed in patients with inflammatory bowelfig study flow chart 0chigurashi bmc cancer page of disease furthermore leukotriene d4 ltd4 antagonists suppress chronic inflammation in a rodent modelof acute enteritis and this may be effective in preventinginflammationassociated crc ltras are leukotriene pathway inhibitors and thus they may have potentialas chemotherapy andor chemoprevention agents to reduce the effect of leukotrienes previous in vivo studieshave elucidated the chemopreventive effect of leukotriene pathway inhibitors [ ] and showed the potentialuse of ltras for chemoprevention therefore we willconduct this trial to explore the chemopreventive effectof ltras in clinical settingthis trial may have some limitations as follow firstacf are believed to be a fine surrogate biomarker ofcrc though its biological significance in humans is stillcontroversial in crc chemoprevention studies typically set the occurrence of adenomas or the crc itselfas endpoint of the study though the occurrence of crcis the most appropriate endpoint it is inappropriate forchemoprevention studies because crc incidence rate islow in the general population and needed for longtermmonitoring our group has previously reported thatacf is useful biomarker for crc and conducted a chemoprevention study for colorectal acf [ ] therefore we designed this study using the number of acf asthe primary endpoint to investigate the chemopreventiveeffect of ltras second an intervention duration of weeks may be too short to reliably detect differences between two groups since our group reported in a previous trial that oral administration of metformin for weeks reduced the number of colorectal acf in humans an intervention period of weeks should beenough to assess the changes in the number of acf ifltras have a chemopreventive effectour group previously conducted a shortterm chemoprevention study of metformin for colorectal acfand reported the preventive effect of the agent on theformation of acf then we conducted a oneyearmetformin chemoprevention studycolorectalpolyps we propose to repeat the same steps as inour metformin study for the chemoprevention studyusing montelukastforif ltras were proved to have efficacy for crc prevention the impact would be significant therefore webelieve it will be very interesting to assess whetherltras inhibit the formation of human colorectal acfabbreviationscrc colorectal cancer nsaids nonsteroidal antiinflammatory drugs cox cyclooxygenase2 ltras leukotriene receptor antagonistscyslt1r cysteinyl leukotriene receptor acf aberrant crypt fociycu yokohama city university umin university hospital medicalinformation network ssap sessile serrated adenomapolyp ncictcae national cancer institute common toxicity criteria for adverseevents peg polyethylene glycol aes adverse events ltc4 leukotriene c4ltd4 leukotriene d4acknowledgmentsthe authors would like to thank the staff for their support in recruitingeligible patients and the patients who participated in this study and theirfamily we thank cathel kerr bsc phd and melissa crawford phd fromedanz group httpsenauthorservicesedanzgroupcom for editing a draftof this manuscriptauthors contributionsth ja and an conceived the study th and ja equally contributed to thismanuscript ja th ka nm ty tm af and ho will perform the baselinecolonoscopies and polypectomies ja th and ka will conduct the secondcount of acf using a hard disk drive recording to ensure validity tt nm tyaf and ho will recruit participants and followup with them at an outpatientclinic the analysis and interpretation of data will be conducted by ja thand ka all authors have read the final manuscript and approved its submission for publicationfundinga grant for this research from the kanagawa institute of industrial scienceand technology kistec was awarded to th we declare that the fundingbody has no role in the design data collection analysis interpretation andwriting of the studyavailability of data and materialsthe datasets used andor analyzed during the current study will be availablefrom the corresponding author on reasonable requestethics approval and consent to participateethical approval of this trial was obtained from the ethics committee of ycuhospital on may the study protocol and informed consentdocuments were approved by the ycu hospital ethics committee this trialhas been registered in the university hospital medical information networkumin clinical trials registry as umin000029926 all study participants willsubmit a written study participation informed consent formconsent for publicationnot applicablecompeting intereststhe authors declare that they have no conflicts of interestsreceived may accepted august referencestorre la bray f siegel rl global cancer statistics ca cancer jclin anderson wf umar a brawley ow colorectal carcinoma in black andwhite race cancer metastasis rev vogelstein b fearon er hamilton sr genetic alterations duringcolorectaltumor development n engl j med winawer sj zauber ag ho mn obrien mj gottlieb ls sternberg ss wayejd schapiro m bond jh panish jf prevention of colorectal cancer bycolonoscopic polypectomy the national polyp study workgroup n engl jmed citarda f tomaselli g capocaccia r barcherini s crespi m italianmulticentre study group efficacy in standard clinical practice ofcolonoscopic polypectomy in reducing colorectal cancer incidence gutzauber ag winawer sj obrien mj lansdorpvogelaar i van ballegooijenm hankey bf shi w bond jh schapiro m panish jf stewart et waye jdcolonoscopic polypectomy and longterm prevention of colorectalcancerdeaths n engl j med maisonneuve p botteri e lowenfels ab fiveyear risk of colorectalneoplasia after negative colonoscopy n engl j med author reply das d arber n jankowski ja chemoprevention of colorectal cancerdigestion epub oct review matsuhashi n nakajima a fukushima y yazaki y oka t effects of sulindacon sporadic colorectal adenomatous polyps gut 0chigurashi bmc cancer page of drazen jm cox2 inhibitorsa lesson in unexpected problems n engl jmed epub feb meyskens fl jr mclaren ce pelot d fujikawabrooks s carpenter pmhawk e kelloff g lawson mj kidao j mccracken j albers cg ahnen djturgeon dk goldschmid s lance p hagedorn ch gillen dl gerner ewdifluoromethylornithine plus sulindac for the prevention of sporadiccolorectal adenoma a randomized placebocontrolled doubleblind trialcancer prev res phila scott jp petersgolden m antileukotriene agents for the treatment of lungdisease am j respir crit care med shen z genomic instability and cancer an introduction j mol cell biolkim hs lee g the cysteinyl leukotriene receptor cysltr1 mediates th17cell migration j immunol bernstein cn blanchard jf kliewer e wajda a cancer risk in patients withinflammatory bowel disease a populationbased study cancer nishikawa m hikasa y hori k tanida n shimoyama t effect of leukotrienec4d4 antagonist on colonic damage induced by intracolonic administrationof trinitrobenzene sulfonic acid in rats j gastroenterol publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations dahlen se dahlen b drazen jm asthma treatment guidelines meet the realworld n engl j med tsai mj wu ph sheu cc hsu yl chang wa hung jy corrigendumcysteinyl leukotriene receptor antagonists decrease cancer risk in asthmapatients sci rep wang d dubois rn eicosanoids and cancer nat rev cancer nielsen ck the leukotriene receptor cyslt1 and 5lipoxygenase areupregulated in colon cancer adv exp med biol burke l butler ct murphy a moran b gallagher wm osullivan j kennedybn evaluation of cysteinyl leukotriene signaling as a therapeutic target forcolorectal cancer front cell dev biol savari s liu m zhang y sime w sjolander a cyslt1r antagonists inhibittumor growth in a xenograft model of colon cancer plos one e73466 bellamkonda k satapathy sr douglas d chandrashekar n selvanesan bcliu m savari s jonsson g sjolander a montelukast a cyslt1 receptorantagonist reduces colon cancer stemness and tumor burden in a mousexenograft model of human colon cancer cancer lett roncucci l stamp d medline a cullen jb bruce wr identification andquantification of aberrant crypt foci and microadenomas in the humancolon hum pathol roncucci l medline a bruce wr classification of aberrant crypt foci andmicroadenomas in human colon cancer epidemiol biomark prev pretlow tp barrow bj ashton ws aberrant crypts putativepreneoplastic foci in human colonic mucosa cancer res pretlow tp oriordan ma pretlow tg stellato ta aberrant crypts in humancolonic mucosa putative preneoplastic lesions j cell biochem suppl takayama t katsuki s takahashi y ohi m nojiri s sakamaki s kato jkogawa k miyake h niitsu y aberrant crtpt foci of the colon as precursorsof adenoma and cancer n engl j med sakai e takahashi h kato s uchiyama t hosono k endo h maeda syoneda m taguri m nakajima a investigation of the prevalence andnumber of aberrant crypt foci associated with human colorectal neoplasmcancer epidemiol biomarkers prev epub jul ohkubo h takahashi h yamada e sakai e higurashi t uchiyama t hosonok endo h taguri m nakajima a natural history of human aberrant cryptfoci and correlation with risk factors for colorectal cancer oncol rep takahashi h yoneda m tomimoto a endo h fujisawa t iida h mawatarih nozaki y ikeda t akiyama t yoneda m inamori m abe y saito snakajima a nakagama h life stylerelated diseases of the digestive systemcolorectal cancer as a life stylerelated disease from carcinogenesis tomedical treatment j pharmacol sci hosono k endo h takahashi h sugiyama m sakai e uchiyama t suzuki kiida h sakamoto y yoneda k koide t tokoro c abe y inamori mnakagama h nakajima a metformin suppresses colorectal aberrant cryptfoci in a shourtterm clinical trial cancer prev res phila the world medical association wma declaration of helsinki ethicalprinciples for medical research involving human subjects the ministry of health labour and welfare ethics guidelines for clinicalresearch paruchuri s mezhybovska m juhas m sjolander a endogenous productionof leukotriene d4 mediates autocrine survival and proliferation via cyslt1receptor signaling in intestinal epithelial cells oncogene dvash e hartal m barak s meir o rubinstein m leukotriene c4 is themajor trigger of stressinduced oxidative dna damage nat commun 0c"
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Epidemiologic and clinical features of patients with COVID19 in BrazilCaractersticas epidemiolgicas e clnicas dos pacientes com COVID19 no BrasilVanessa Damazio Teich1 Sidney Klajner1 Felipe Augusto Santiago de Almeida1 Anna Carolina Batista Dantas1 Claudia Regina Laselva1 Mariana Galvani Torritesi1 Tatiane Ramos Canero1 Ot¡vio Berwanger1 Luiz Vicente Rizzo1 Eduardo Pontes Reis1 Miguel Cendoroglo Neto1 Hospital Israelita Albert Einstein S£o Paulo SP Brazil 1031744einstein_journal2020AO6022 Objective This study describes epidemiological and clinical features of patients with confirmed infection by SARSCoV2 diagnosed and treated at Hospital Israelita Albert Einstein which admitted the first patients with this condition in Brazil Methods In this retrospective singlecenter study we included all laboratory confirmed COVID19 cases at Hospital Israelita Albert Einstein S£o Paulo Brazil from February until March Demographic clinical laboratory and radiological data were analyzed Results A total of patients with a confirmed diagnosis of COVID19 were included in this study Most patients were male with a mean age of years A history of a close contact with a positivesuspected case was reported by of patients and had a history of recent international travel The most common symptoms upon presentation were fever nasal congestion cough and myalgiaarthralgia Chest computed tomography was performed in patients and of those showed abnormal results Hospitalization was required for patients and were admitted to the Intensive Care Unit Regarding clinical treatment the most often used medicines were intravenous antibiotics chloroquine and oseltamivir Invasive mechanical ventilation was required by of Intensive Care Unit patients The mean length of stay was days for all patients and days for patients requiring or not intensive care respectively Only one patient died during followup Conclusion These results may be relevant for Brazil and other countries with similar characteristics which are starting to deal with this pandemicKeywords Communicable diseases Lung diseasesepidemiology SARSCoV2 COVID19 Coronavirus infections Epidemiology RESUMOObjetivo Descrever as caractersticas epidemiolgicas e clnicas de pacientes com infec§£o confirmada pelo SARSCoV2 diagnosticados e tratados no Hospital Israelita Albert Einstein que admitiu os primeiros pacientes com essa condi§£o no Brasil Mtodos Neste estudo retrospectivo de centro ºnico inclumos todos os casos com confirma§£o laboratorial de COVID19 no Hospital Israelita Albert Einstein em S£o Paulo SP de fevereiro a mar§o de Foram analisados dados demogr¡ficos clnicos laboratoriais e radiolgicos Resultados Foram includos pacientes com diagnstico confirmado de COVID19 A maioria dos pacientes era do sexo masculino com mdia de idade de anos Foi relatada histria de contato prximo com um caso positivosuspeito por dos pacientes e tinham histria de viagens internacionais recentes Os sintomas mais comuns foram febre congest£o nasal tosse e mialgiaartralgia A tomografia computadorizada de trax foi realizada em pacientes e deles apresentaram How to cite this Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Epidemiologic and clinical features of patients with COVID19 in Brazil einstein S£o Paulo 202018eAO6022 httpdx1031744einstein_journal2020AO6022Corresponding authorVanessa Damazio Teich Avenida Albert Einstein MorumbiZip code S£o Paulo SP Brazil Phone Email vanessateicheinsteinbrReceived onJuly Accepted onJuly Conflict of interest noneCopyright This content is licensed under a Creative Commons Attribution International LicenseORIGINAL ISSN eISSN 23176385Official Publication of the Instituto Israelita de Ensino e Pesquisa Albert Einsteineinstein S£o Paulo 0cresultados anormais A hospitaliza§£o foi necess¡ria para pacientes e foram admitidos na Unidade de Terapia Intensiva Quanto ao tratamento clnico os medicamentos mais utilizados foram antibiticos intravenosos cloroquina e oseltamivir A ventila§£o mec¢nica invasiva foi necess¡ria em dos pacientes na Unidade de Terapia Intensiva O tempo mdio de interna§£o foi dias para todos os pacientes e dias para pacientes que necessitaram ou n£o de cuidados intensivos respectivamente Apenas um paciente morreu durante o acompanhamento Conclus£o Estes resultados podem ser relevantes para o Brasil e outros pases com caractersticas semelhantes que come§aram a lidar com essa pandemiaDescritores Doen§as transmissveis Pneumopatiasepidemiologia SARSCoV2 COVID19 Infec§µes por coronavrus Epidemiologia INTRODUCTIONSince December several cases of pneumonia of unknown origin have been reported in Wuhan China1 The pathogen was further identified as a novel RNA coronavirus currently named as severe acute respiratory syndrome coronavirus SARSCoV22 Huang et al reported the first cases in China with a common clinical presentation of fever cough myalgia fatigue and dyspnea with an dysfunction eg acute respiratory distress syndrome ARDS shock acute cardiac and kidney injuries and death in severe cases3Afterwards in January the World Health anization WHO declared the outbreak a Public Health Emergency of International Concern PHEIC and next in March it was characterized as a pandemic4 As of April a total of cases had been reported in countries and regions across all five continents with deaths worldwide5 More recently the Chinese Center for Disease Control and Prevention published data on patients with classified as confirmed cases of coronavirus disease COVID19 Most patients were aged to years with mild clinical presentation ie nonpneumonia and mild pneumonia and overall casefatality rate of increased in elderly population with casefatality rate of in those aged years and older6On February the first Brazilian patient had a confirmed diagnosis of COVID19 at Hospital Israelita Albert Einstein HIAE Hospital Israelita Albert Einstein is a philanthropic hospital in the city of S£o Paulo SP Brazil with twelve health care units including a quaternary hospital with beds and four outpatient emergency care units By the end of this study on March of patients with confirmed COVID19 in Brazil had been diagnosed at HIAE Given the rapid spread of the COVID19 clinical and epidemiological data of several countries are being published on a daily basis79 However no studies have been reported to date presenting the characteristics of COVID19 patients diagnosed in Brazil OBJECTIVETo describe epidemiological and clinical features of patients with confirmed infection by SARSCoV2 diagnosed and treated at Hospital Israelita Albert Einstein which admitted the first patients with this condition in Brazil METHODSStudy design and oversightThis was a retrospective observational singlecenter study which included all consecutive patients with a confirmed diagnosis of COVID19 at HIAE between February and March The study was supported by an internal grant from HIAE and designed by the investigators The study was approved by the Research Ethics Committee of the anization protocol number CAAE and the National Commission for Research Ethics PatientsThe diagnosis of the COVID19 disease was performed according to the WHO interim guidance10 A confirmed case of COVID19 was defined as a positive result of realtime reverse transcriptase polymerase chain reaction RTPCR assay of nasal and pharyngeal swab specimens11 All cases included in the current analysis had laboratory confirmationData sourcesThe data were obtained from patients electronic medical records EMR including inpatients and outpatients with laboratoryconfirmed COVID19 Data collected included demographic clinical laboratorial and radiological information and was anonymized so that patients could not be identified Demographic characteristics included age sex tobacco smoking weight and body mass index BMI Clinical information included medical travel Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cand exposure history signs symptoms underlying comorbidities continuous medication use and treatment measures ie antiviral therapy steroid therapy respiratory support and kidney replacement therapy Laboratory assessment consisted of complete blood count assessment of renal and liver function and measurements of electrolytes Ddimer procalcitonin lactate dehydrogenase Creactive protein and creatine kinase Radiologic abnormality was defined based on the medical report documented in the EMR Disease duration from onset of symptoms hospital and Intensive Care Unit ICU length of stay LOS were also documented Statistical analysis Continuous variables were expressed as means with standard deviations medians minimum and maximum values Categorical variables were summarized as counts and percentages No imputation was made for missing data All statistics are deemed to be descriptive only considering that the cohort of patients in our study was not derived from random selection All analyses were performed using Microsoft Excel RESULTSDemographic and clinical characteristicsBetween February and March a total of patients were diagnosed with COVID19 at HIAE This study included patients for whom data regarding demographics clinical symptoms laboratory and imaging findings were available in the EMR The remaining patients had only used the hospital laboratory facilities and were followedup by physicians not working in our service network Patients demographic and clinical characteristics are shown in table A total of had a recent international travel history and had been at the same marriage celebration in Bahia a state in the Northeast region of Brazil patients had a history of close contact either with a positive or suspected case of COVID19 Most patients were male and the mean age was years Only of patients were younger than years old and were older than yearsFever was present in only of patients upon admission but had a reported history of fever followed by nasal congestion cough Table Clinical and epidemiological characteristicsCharacteristicAge years Mean±SDMedianMinimumMaximumNumber of patientsAge distribution years ¥Sex MaleFemaleTravel historyTotal patients n510Total patientsNonhospitalized patients n438Hospitalized patients n72±±± European Union and United Kingdom United States of America and Canada Middle East and IranChina and Japan Latin America Other countries Bahia Brazilian stateNo travel history Exposure source of transmission contact with confirmed or suspected casesExposureNo exposureHealthcare professionalYesNoSmoking historyCurrent smokerFormer smokerNever smokedFever on admission YesNoMedianTemperature distribution on admission°C °C°C°CSymptomsNasal congestionHeadacheCoughSore throatSputum production continueEpidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0cContinuationTable Clinical and epidemiological characteristicsContinuationTable Clinical and epidemiological characteristicsCharacteristicFatigueDyspneaNausea or vomitingDiarrheaMyalgia or arthralgiaChillsFeverConjunctival congestionOther symptomsNo symptomsSymptoms duration daysMean±SDMedianMinimumMaximumSigns of infectionThroat congestionTonsil swellingSkin rashOther alterationsNo alterationsCoexisting disordersAny coexisting disorderAsthma or chronic pulmonary obstructive disorderDiabetesHypertensionCoronary heart disease or other heart conditionsCerebrovascular diseaseHepatitis B C HIV or other immunodeficiencyCancerChronic renal diseasean transplantPregnancy Other coexisting disordersNo coexisting disordersMean BMI±SDChronicuse medicationsAny medicationStatinMultivitaminAntidepressantAntihypertensiveAntiplatelet or anticoagulantThyroid hormonesTotal patients n510 Total patientsNonhospitalized patients n438 Hospitalized patients n72 ± ± ± ±± ± continueCharacteristicAntidiabeticPain killersAntibioticsCorticosteroidInhaled medicationsOther medicationsNo use of medicationsTotal patients n510 Total patientsNonhospitalized patients n438 Hospitalized patients n72 Chronicuse medications number of medications distributionOnly type of medication types of medications types of medications or more types of medications polypharmacy ESI on arrival Destiny after first evaluation Discharge to homeAdmission on general wardAdmission on ICU Return to the emergency room after first evaluation SimN£oResults expressed by total nn if not otherwise indicatedSD standard deviation BMI body mass index ESI Emergency Severity Index ICU intensive care unitand myalgia or arthralgia The mean duration of symptoms was days which was the same for patients hospitalized or not Upon admission the majority of patients had no significant changes on physical examination Considering all included patients had at least one comorbidity This rate however was far higher in the hospitalized group when compared with the nonhospitalized group the most common comorbidities were hypertension and diabetes The distribution of patients in the Emergency Severity Index ESI differs between the two groups analyzed with the hospitalized group showing a higher rate of ESI indicating that the initial severity was greater in this group since the onset of symptoms Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cRadiologic and laboratory findingsTable demonstrates the radiologic and laboratory findings upon admission Only of patients were initially evaluated with chest radiographs whereas were submitted to computed tomography CT Of the radiographs performed had some abnormality while of CT scans showed abnormal results The most common patterns on chest CT were groundglass opacity and bilateral patchy shadowing Table Radiologic and laboratory findingsCharacteristicsRadiologic findings in chest radiographChest radiograph performedAbnormalities on chest radiograph Groundglass opacity Local patchy shadowing Bilateral patchy shadowing Interstitial abnormalities Radiologic findings in chest CT Chest CT performed Abnormalities on chest CT Ground glass opacity Local patchy shadowing Bilateral patchy shadowing Interstitial abnormalitiesLaboratory findingsMedian PaO2FiO2 ratio IQRWhite blood cell countMedian per mm3 Distribution per mm3 Lymphocyte countMedian per mm3 Distribution per mm3 Platelet countTotal number of patients n510 Total number of patientsNonhospitalized patients n438Hospitalized patients n72 Median per mm3Distribution per mm3 Median hemoglobin gdLDistribution of other findings Creactive protein 5mgL Procalcitonin 05ngmLLactate dehydrogenase 214UL Aspartate aminotransferase 40UL Alanine aminotransferase 40UL Total bilirubin 12mgdL Creatine kinase UL Creatinine 1mgdLDdimer 500ngmL Mean sodium mmolLMean potassium mmolL Results expressed by total nn if not otherwise indicatedCT computer tomography PaO2FiO2 oxygen partial pressurefractional inspired oxygen IQR interquartile rangeleukopenia Upon admission lymphocytopenia was identified in of patients thrombocytopenia in and in Most patients had elevated levels of both Creactive protein and lactate dehydrogenase Less common findings were elevated levels of Ddimer aspartate aminotransferase and alanine aminotransferase The hospitalized group had more patients with higher levels of Creactive protein procalcitonin and lactate dehydrogenase The other results do not show any major difference between groups A viral panel was collected in patients and it was positive for rhinovirus in nine cases influenza B in two cases and influenza A in one caseTreatment and complicationsAs shown in table patients had been hospitalized at HIAE by the time of the analysis Among Table Treatments complications and clinical outcomesTotal number of patients n510 CharacteristicDisease severitySevereNot severeIntensive care use during hospital stayYesNoHospital treatments medicationsIntravenous antibioticsOseltamivirLopinavir and ritonavirChloroquineCorticosteroidsHospital treatments support treatmentsOxygen therapyMechanical ventilationInvasiveNoninvasiveExtracorporeal membrane oxygenationContinuous renal replacement therapyComplicationsSeptic shockAcute respiratory distress syndromeAcute kidney injuryPneumoniaMean length of stay daysLOS all patientsPatients requiring ICU daysICUInpatients unitsPatients not requiring ICU inpatients units daysResults expressed by total nn if not otherwise indicated LOS length of stay ICU intensive care unitEpidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0cthose patients required intensive care during their hospital stay in that were referred from the emergency room to the ICU and eight presented worsening of the clinical condition at inpatients units and were transferred to the ICU The majority of patients received intravenous antibiotic therapy received chloroquine and oseltamivir Oxygen therapy was necessary in of hospitalized patients required mechanical ventilation invasive and noninvasive and extracorporeal membrane oxygenation ECMO was used in only one case Considering patients admitted to the ICU invasive mechanical ventilation was required by of them During hospital admission most patients were diagnosed with pneumonia followed by acute kidney injury and ARDS The mean LOS was days considering only patients requiring intensive care the mean ICU LOS was days and the mean total LOS was days whereas for patients not admitted to the ICU the mean LOS was days Only one patient died in this series that is mortality rate DISCUSSION It took months from the first diagnosed case of COVID19 in China until diagnosis of patient zero in Brazil on February at HIAE During days after the first diagnosis all cases had a history of recent international travels On March the first case of local transmission was confirmed also at HIAE A relevant proportion of all patients with confirmed COVID19 infection had been diagnosed at HIAE by the time of the analysisThe patients in our series had a mean age of years and were mostly male The studies describing demographic characteristics in the infected general population showed a median age of years712 and the proportion of males was in the Chinese report7 and in the Singapore report The respiratory symptoms were similar to those of patients described in reports from China United States and Europe7913 However the mean days of symptoms was far lower in our series days versus days in Singapore12 days in the United States13 and days in China3 Although fever was reported by the majority of patients it was only present in of patients at the initial assessment at hospital suggesting not only it might not be considered to determine severity of illness but also that diagnostic algorithms using fever for testing may mask the total number of cases and delay diagnosis The prevalence of chronic diseases was far higher in the hospitalized group as compared to nonhospitalized group This prevalence was even higher in the subgroup admitted to the ICU The mean age of hospitalized patients was higher than nonhospitalized patients versus years and the required hospitalization increased with age for patients aged to years for to years and for patients older than years In this Brazilian case series hospitalization was required for patients and of them demanded critical care accounting for of total admissions a number far greater than the Chinese series in which only required ICU7The majority of patients were admitted to the ICU because of acute hypoxemic respiratory failure that required ventilatory support Invasive mechanical ventilation was needed in of ICU patients of total hospitalizations whereas were managed with noninvasive mechanical ventilation The necessity of invasive mechanical ventilation was similar to an ICU series reported from the United States of Washington13 lower than that reported in an Italian publication of Lombardy9 but higher than the Chinese reports and of Wuhan half of these treated with extracorporeal membrane oxygenation31415 Considering the use of noninvasive ventilation the rate was again similar to that reported in Washington and lower than the rates in China and of Wuhan including patients receiving highflow nasal cannula31415 A total of three patients of patients admitted to the ICU developed acute kidney injury and required continuous renal replacement therapy Among those only one patient had chronic kidney disease The prevalence of chronic kidney disease was among hospitalized patients in the Chinese report14 and among patients admitted to the ICU in the series from the United States This study has important limitations First part of the cases had incomplete information documented in the medical records and patient clinical history documentation was not homogeneous among all patients This is a common limitation in retrospective observational studies taking into account that data Teich VD Klajner S Almeida FA Dantas AC Laselva CR Torritesi MG Canero TR Berwanger O Rizzo LV Reis EP Cendoroglo Neto Meinstein S£o Paulo 0cgeneration was clinically driven and not in systematic fashion Second since many patients remained at the hospital and the outcomes were unknown at the time of data collection we censored the data regarding their clinical outcomes as of the time of the analysis Third only patients hospitalized at HIAE were included in the hospitalization group and there is no documentation of hospital admissions outside of our service network Finally this study only included patients attended as outpatients or inpatients at HIAE therefore asymptomatic and mild cases who did not seek medical care were not considered Hence our study cohort may represent more severe COVID19 cases CONCLUSIONTo date there is no study in Brazil reporting the characteristics of patients diagnosed with COVID19 Brazil is the country in the south hemisphere with the highest number of confirmed cases this disease and Hospital Israelita Albert Einstein is the center where the first patient was diagnosed with a representative sample of all confirmed COVID19 cases in the country The results presented in this study may be relevant for Brazil and other countries with similar characteristics which are starting to deal with this pandemic CONTRIBUTION OF AUTHORSData were analyzed and interpreted by the authors All authors reviewed the manuscript and checked the exactness and completeness of data AUTHORS INFORMATIONTeich VD httporcid0000000285396037Klajner S httporcid0000000341201047 Almeida FA httporcid0000000171310039 Dantas AC httporcid0000000195056784 Laselva CR httporcid0000000182859633 Torritesi MG httporcid0000000236236475 Canero TR httporcid0000000273994718Berwanger O httporcid0000000249722958Rizzo LV httporcid0000000199499849 Reis EP httporcid000000015110457X Cendoroglo Neto M httporcid0000000281634392 REFERENCES Lu H Stratton CW Tang YW Outbreak of pneumonia of unknown etiology in Wuhan China The mystery and the miracle J Med Virol Zhu N Zhang D Wang W Li X Yang B Song J Zhao X Huang B Shi W Lu R Niu P Zhan F Ma X Wang D Xu W Wu G Gao GF Tan W China Novel Coronavirus Investigating and Research Team A novel coronavirus from patients with pneumonia in China N Engl J Med Huang C Wang Y Li X Ren L Zhao J Hu Y et al Clinical features of patients infected with novel coronavirus in Wuhan China Lancet Erratum in Lancet Jan World Health anization WHO Coronavirus disease COVID19 outbreak [Internet] Geneva WHO [cited July ] Available from httpswwwwhointwesternpacificemergenciescovid19 The Johns Hopkins Coronavirus Resource Center CRC COVID19 Dashboard by the Center for Systems Science and Engineering CSSE at Johns Hopkins University [Internet] CRC USA [cited July ] Available from httpscoronavirusjhuedumaphtml Wu Z McGoogan JM Characteristics of and important lessons from the coronavirus disease COVID19 outbreak in China Summary of a report of cases from the Chinese Center for Disease Control and Prevention JAMA Feb 101001jama20202648 Guan WJ Ni ZY Hu Y Liang WH Ou CQ He JX Clinical characteristics of coronavirus disease in China N Engl J Med Holshue ML DeBolt C Lindquist S Lofy KH Wiesman J Bruce H Spitters C Ericson K Wilkerson S Tural A Diaz G Cohn A Fox L Patel A Gerber SI Kim L Tong S Lu X Lindstrom S Pallansch MA Weldon WC Biggs HM Uyeki TM Pillai SK Washington State 2019nCoV Case Investigation Team First case of novel coronavirus in the United States N Engl J Med Grasselli G Zangrillo A Zanella A Antonelli M Cabrini L Castelli A Baseline characteristics and outcomes of patients infected with SARSCoV2 admitted to ICUs of the Lombardy region Italy JAMA World Health anization WHO Clinical management of severe acute respiratory infection when novel coronavirus 2019nCoV infection is suspected interim guidance [Internet] Geneva WHO [cited July ] Available from httpswwwwhointdocsdefaultsourcecoronaviruseclinicalmanagementofnovelcovpdf Brasil Ministrio da Saºde Centro de Opera§µes de Emergªncias em Saºde Pºblica Coronavirus Covid19 Boletim Di¡rio [Internet] Braslia DF Ministrio da Saºde [citado Jul ] Disponvel em httpswwwsaudegovbrimagespdf2020marco2929COVIDpdf Young BE Ong SW Kalimuddin S Low JG Tan SY Loh J Ng OT Marimuthu K Ang LW Mak TM Lau SK Anderson DE Chan KS Tan TY Ng TY Cui L Said Z Kurupatham L Chen MI Chan M Vasoo S Wang LF Tan BH Lin RT Lee VJ Leo YS Lye DC Singapore Novel Coronavirus Outbreak Research Team Epidemiologic features and clinical course of patients infected with SARSCoV2 in Singapore JAMA Mar 101001jama20203204 Bhatraju PK Ghassemieh BJ Nichols M Kim R Jerome KR Nalla AK Covid19 in critically Ill patients in the Seattle region Case series N Engl J Med Wang D Hu B Hu C Zhu F Liu X Zhang J Clinical characteristics of hospitalized patients with novel coronavirusinfected pneumonia in Wuhan China JAMA Feb 101001jama20201585 Yang X Yu Y Xu J Shu H Xia J Liu H Clinical course and outcomes of critically ill patients with SARSCoV2 pneumonia in Wuhan China a singlecentered retrospective observational study Lancet Respir Med Erratum in Lancet Respir Med 202084e26Epidemiologic and clinical features of patients with COVID19 in Brazileinstein S£o Paulo 0c'
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inhibitor with Temozolomide results in significant apoptosis in glioblastoma via the p65 and actin cytoskeleton regulatory pathwaysNaze G Avci1 Sadaf EbrahimzadehPustchi1 Yasemin M Akay1 Yoshua Esquenazi2 Nitin Tandon JayJiguang Zhu Metin Akay1Glioblastoma GBM is the most malignant brain tumor characterized by intrinsic or acquired resistance to chemotherapy GBM tumors show nuclear factorκB activity that has been associated with tumor formation growth and increased resistance to therapy We investigated the effect of inhibitor BAY with Temozolomide TMZ on the signaling pathways in GBM pathogenesis GBM cells and patientderived GBM cells cultured in 3D microwells were cotreated with BAY and TMZ or BAY and TMZ alone and combined experiments of cell proliferation apoptosis wound healing assay as well as reversephase protein arrays western blot and immunofluorescence staining were used to evaluate the effects of drugs on GBM cells The results revealed that the cotreatment significantly altered cell proliferation by decreasing GBM viability suppressed pathway and enhanced apoptosis Moreover it was found that the cotreatment of BAY and TMZ significantly contributed to a decrease in the migration pattern of patientderived GBM cells by modulating actin cytoskeleton pathway These findings suggest that in addition to TMZ treatment can be used as a potential target to increase the treatments outcomes The drug combination strategy which is significantly improved by inhibitor could be used to better understand the underlying mechanism of GBM pathways in vivo and as a potential therapeutic tool for GBM treatmentGlioblastoma multiforme GBM is the most malignant primary brain tumor in the central nervous system Current standard of care therapy includes surgery followed by radiotherapy and concomitant and adjuvant chemotherapy with the alkylating agent Temozolomide TMZ which provides survival benefits for patients with GBM1 However even with the advances in surgical resection combined with TMZ therapy and irradiation the prognosis for newly diagnosed GBM patients remains poor In fact due to its rapid proliferation increased invasion and migration capacity and chemoresistance to the alkylating agents a0the median survival is only a0months with the Stupp regimen radiation with daily TMZ a0weeks followed by cyclic TMZ2 and 5year survival rate is less than which is the lowest longterm survival rate of malignant brain tumors3 TMZ methylates DNA at the O6 positions of guanine and DNA repair enzyme O6methylguanine methyltransferase MGMT removes alkyl groups from O6 position of guanine in DNA making cells resistant to TMZ6 Therefore new therapies are necessary to prevent cell proliferation and induce apoptosis for GBM patientsNuclear factorkappa B NFκB is a regulatory transcription factor of the Rel gene family including p50 cRel RelB or p65 subunits It is involved in the control of tumor cell proliferation migration immune response and apoptosis7 Studies have shown that NFκB gene was involved in the regulation pathways of different cancer types such as thyroid cancer head and neck squamous cell carcinoma and colorectal cancer711 Increased 1Department of Biomedical Engineering University of Houston Cullen Blvd Houston TX USA 2UTHealth Neurosurgery McGovern Medical School Memorial Hermann at Texas Medical Center The University of Texas Health Science Center at Houston Houston TX USA email makayuheduScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cactivation of NFκB has also been identified in GBM tumors where the expression of NFκB was much higher in GBM tissue compared with nonGBM tissue1415 NFκB also promotes chemoresistance to TMZ and regulates MGMT activity in GBM by promoting MGMT gene expression through NFκB binding sites within the MGMT promoter16 NFκB inhibitors such as parthenolide do not completely eradicate tumors therefore they are mostly used in combination with other drugs17 When used in combination with TMZ NFκB inhibitor parthenolide has been shown to activate mitochondrial apoptosis signaling in U87MG and U373 GBM cells which lead to cell death18 and had a combined effect on cell cytotoxicity in LN18 and T98G glioma cells19 NFκB inhibitor CBL0137 has been shown to bind DNA leading the functional inactivation of the Facilitates Chromatin Transcription FACT complex a chromatin remodeling complex regulating transcription replication and DNA repair2021 In a0vitro evaluation of the CBL0137 on FACT p53 and NFκB has been done using U87MG and A1207 GBM cells It was shown that CBL0137 induced loss of chromatinunbound FACT activated p53 and inhibited NFκB dependent transcription21 In a0vivo studies showed that CBL0137 was effective in increasing survival rates in TMZresistant orthotopic mouse models21 Moreover Wang et a0al indicated that NFκB inhibitor BAY suppresses the expression of MGMT and enhances the TMZinduced apoptosis in TMZ resistant U251 cells22 However there is still a lack of characterization of the precise pattern of NFκB activation in combination with TMZ in GBM cell populations that have been a0surgically resected from patientsIn vitro and in a0vivo identifications and validations of molecular targets of GBM are important as they can progress into clinical studies Studies reported that combining multiple gene targets may prevent tumor growth and improve the treatment strategy for GBM23 Both Bay and TMZ exert antitumoral activities individually in different tumor types28 Therefore in this study we aimed to analyze functionally the combined effect of Bay and TMZ in different GBM cells For this purpose first we used our 3D PEGDAbased hydrogel microwell platform31 to provide reliable preclinical models that can recapitulate in a0vivo features of the GBM tumors We cultured GBM cells U87 and LN229 and patientderived GBM cells in 3D microwells for a more precise and personalized treatment approach We then treated GBM cells with Bay and TMZ in combination or alone Our results indicated that the cotreatment of Bay and TMZ significantly reduced cell viability in all three cell lines in correlation with a significant decrease in the spheroid size The levels of NFκB protein and its subunits p65 and p50 were also significantly decreased compared with the control and single drug applications Similar a0decreases in the cell viability and protein levels were observed in all three GBM cells Tumor biopsy samples could give more realistic information about how tumors respond to drugs when they are used for in a0vitro or in a0vivo studies35 Therefore we decided to continue our experiments with only using our patientderived GBM cells We treated patientderived GBM cells with Bay and TMZ or alone and analyzed specific cellular proteins along with their posttranslational modifications via reversephase protein arrays RPPA to elucidate the mechanism of action of the proteins3839 We observed that several cell signaling pathways including cell metabolism proliferation apoptosis were significantly affected by the combination of the drugs which were consistent with the literature4041 Furthermore our RPPA data revealed that there was a significant change in the modulation of actin cytoskeleton and following experiments including western blot analysis for the expression of FAK protein and wound healing assay for cell migration patterns confirmed the RPPA results We observed a significant decrease in both actin fluorescence intensity and migration pattern in the a0cotreated patientderived GBM cells To the best of our knowledge the effect of cotreatment of Bay and TMZ has never been studied previously on the actin modulation of patientderived GBM cells These results suggested that Bay and TMZ induced alteration in the a0actin filament anization by reducing the level of focal adhesion protein which might implicate in cell apoptosis The effect of Bay with TMZ necessitates further exploration to better understand its mechanism of action in GBM and potential therapeutic tools for GBM treatmentResultsCotreatment of Bay and TMZ reduced viability of GBM cells We used our previously a0published data to select the most effective drug concentrations for this study42 We cultured LN229 U87 and patientderived cells in the microwells for a0days where they formed 3D spheroids and we added a0µM of Bay and a0µM of TMZ in combination or alone Then we cultured the spheroids for more days with or without drug Control group did not receive any treatment The cell viability assay was performed on day after drug administration The results showed that the a0cotreatment significantly reduced cell viability of GBM cells LN229 and U87 and patientderived GBM cells cultured in 3D PEGDA microwells respectively as shown in Fig a01ac When they were used alone TMZ reduced cell viability to and p and Bay reduced cell viability to and in LN229 U87 and patientderived GBM cells respectively compared to control groups Fig a01d However when they were used in combination the viability of the cells significantly decreased to and in LN229 U87 and patientderived GBM cells respectively compared to control groups p Fig a01d Tumor cells are generally less sensitive to drug treatments in 3D cultures than in 2D cultures4344 This could reflect reduced compound access or differences in the response to cell death To confirm that cotreatment was more effective compared to single drug use we quantified the size of the spheroids using ImageJ45 Our data showed that after a0days of drug treatment the spheroids sizes were significantly reduced in the cotreatment by and in LN229 Fig a01e U87 Fig a01f and patientderived GBM cells p Fig a01g respectively compared to control group p When we compared the spheroids sizes of the cotreatment with TMZ alone there was a reduction of and in LN229 U87 and patientderived GBM cells respectively p Finally the spheroids sizes of the cotreatment compared with Bay alone showed a decrease of and in LN229 U87 and patientderived GBM cells respectivelyScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Representative images of the GBM tumor cells cultured in the PEGDA microwells ac LN229 U87 and patientderived GBM cells were cultured in the microwells for a0days respectively After day Bay and TMZ were applied either alone or in combination onto the cell spheroids Control group did not receive any treatment The cells were cultured with or without drugs additional more days The images were taken on Day Day and Day after the drug application to observe the disruption in the spheroids Dotted black lines represent the edge of the tumor spheroid Scale bars a0µm d Bar graph showing trypan blue staining for cell viability of LN229 U87 and patientderived GBM cells eg Spheroid size quantification was done using ImageJ for LN229 U87 and Patientderived GBM cells respectively Twotailed ttest followed by Wilcoxon test were done GraphPad Prism v5 Data represent the mean ± SD of three biological replicates p and p Suppression of activity in GBM cells by cotreatment of Bay and TMZ As a readout of NFkB activity after drug treatment we first quantitatively assessed the cytoplasmic activation of phosphorylated NFκB p65 subunit in both treated and untreated groups in all GBM cells NFκB pp65 subunit activity was observed in the control groups of all three GBM cells Fig a02a NFκB pp65 subunit activity decreased to and when TMZ applied alone and and when Bay was applied alone in LN229 U87 and patientderived cells respectively However the decrease in NFκB pp65 subunit was reduced to when LN229 U87 and patientderived cells respectively were cotreated p Fig a02a Bay specifically inhibits NFκB activation by blocking phosphorylation of IκBα46 In independent experiments we analyzed the abundance of phosphorylated NFκB p65 NFκB p50 and IκBα in all three GBM cells Qualitative and quantitative western blot analysis revealed that the exposure to Bay with TMZ significantly downregulated the abundance of NFκB p65 NFκB p50 and IκBα compared with control and Bay or TMZ alone Fig a02b Please note that loading controls were used for each experiment but only the representative loading control for p and tP65 and p and tP50 was presented Fig a02b The cell viability assay cells size and protein expressions in all three GBM cells revealed similar results without any dramatic change Therefore considering the importance of using patientderived tumor cells to elucidate the mechanism of drugs and respective signaling pathways35 we further continued our experiments using patientderived GBM cellsApoptosis was promoted by cotreatment of Bay and TMZ RPPA technology is designed for multiplexed antibodybased relative quantification where each array is tested with a validated antibody specific to a particular protein along with their particular posttranslational modifications47 In the attempt to elucidate the mechanism of action of Bay with TMZ by which NFκB subunits were modulated and to identify downstream signaling molecules we employed RPPA platform using our drug treated or untreated patientderived GBM cells RPPA results showed that many oncogenic pathways were altered by the drug treatments but more specifically by the cotreatment Fig a03a Decreased expression of NFκB was not only associScientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 NFkB activity in LN229 U87 and patientderived GBM cell lines a NFkB p65 subunit activity in LN229 U87 and patientderived GBM cell lines respectively The cells cultured with or without drugs for a0days were collected from the microwells and subjected to ELISA Data represent the mean ± SD of three biological replicates p and p b Representative immunoblots LN229 U87 and patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels bottom panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p ated with changes in the a0NFκB pathway but also with apoptosis cell metabolism and proliferation which were confirmed by the analysis of downregulated RPPA proteins in Enrichr KEGG libraries4849 Fig a03c p One of the specific pathways given by RPPA was apoptosis Apoptosis is one of the important mechanisms that regulates cell death and suppress tumorigenesis Studies have demonstrated that Bcl2 family proteins can positively and negatively regulate apoptosis by regulating antiapoptotic protein Bcl2 and proapoptotic protein Bax4050 Our RPPA data using patientderived GBM cells showed that the fold change of Bcl2 relative to control was times higher in cotreated group TMZ alone Bay alone respectively Fig a03b In order to further confirm whether the expression of a0these proteins were downregulated by the cotreatment we performed western blot analysis Our results showed a similar decrease in Bcl2 protein expression in the cotreatment compared with the control and single drug a0treatment Fig a03d In contrast Bax protein fold change relative to control was times higher in cotreated group TMZ alone Bay respectively where we observed a significant increase after the cotreatment of Bay with TMZ compared with the control p Fig a03b Bcl2Bax ratio is a key indicator in susceptibility of the cells to apoptosis Western blot results confirmed the change in Bcl2Bax ratio in the cotreatment compared with the control group and single a0drug treatment Fig a03d Our RPPA data also showed a significant increase in the cleavedcaspase protein Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 The effect of Bay and TMZ on signaling pathways in patientderived GBM cells a Heat map presentation of RPPA analysis showing the changes in the protein expression RPPA was performed on lysates treated with Bay and TMZ alone or in combination All relative protein level data points were normalized to the a0control group Red and green indicate up and down regulations respectively in the heat map The samples were run in duplicate n b Fold change of the a0selected proteins relative to the a0control group via RPPA Data represent the mean ± SD of two biological replicates p p Wilcoxon rank sum test c Analysis of downregulated RPPA proteins shows a a0significant activation in numerous Enrichr KEGG pathways The pathways were a0sorted by p value ranking d Representative immunoblot validation of significantly altered proteins involved in different KEGG pathways Patientderived GBM cells were cultured with or without drugs for a0days lysed and immunoblotted with the indicated antibodies Quantification of the foldchanges in protein levels right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p p fold change relative to control times higher in the cotreatment compared with times higher in TMZ alone and times higher in Bay alone p Fig a03b To confirm if cotreatment triggered apoptosis correlated with caspase activation we performed western blot analysis with procaspase3 cas3 and cleavedcaspase3 Ccas3 We observed that Bay and TMZ induced apoptosis was associated with cas3 Fig a03d Please note that loading controls were used for each experiment but only the representative loading control for Bax cas3 and Ccas3 was presented Fig a03d Moreover another important mechanism of NFκB activation in GBM regulates through AKT phosphorylation of IκB Our RPPA data showed relative fold changes of in the cotreated group TMZ alone and Bay alone respectively p Fig a03b The western blot results also confirmed a significant decrease in the abundance of AKT pT308 Fig a03dTo further investigate whether cotreatment of Bay with TMZ can lead to glioma cell apoptosis and to confirm our RPPA and western blot results we performed apoptosis assay TUNEL The patientderived GBM cells were cotreated with Bay with TMZ or single drug treated and subjected to TUNEL assay to detect DNA damage Fig a04a The results indicated that TUNEL cells in the cotreatment were increased tenfold compared with control and and 24folds compared with TMZ alone and Bay alone respectively p Fig a04b Additionally in some TUNEL cells we observed a typical ring type chromatin aggregation underneath the nuclear membrane which suggested an early stage apoptosis51 Fig a04a red arrows There were also a few TUNEL cells that lacked the typical apoptotic ringlike nuclear structure indicating that they were either at a different stage of apoptosis or alternatively undergoing necrosis52 that we have not investigated furtherCotreatment of Bay with TMZ changed actin anization by inhibiting FAK phosphorylation and cell migration Actin filaments Factin are one of the main components of the cellular cytoskeleton which regulates actin dynamics and migration process in the cells The disruption of the actin cytoskeleton inhibits cell migration and adhesion53 Depolymerization or cleavage of actin lamins and other cytoskeletal proteins have been also found to be involved in cell apoptosis54 To confirm the RPPA results showing changes in the actin modulation pathway and to understand the mechanism that regulates cytoskeletal Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Apoptosis assay TUNEL a Fluorescent images of TUNEL cells in patientderived GBM cells TUNEL assay was performed on cells treated with Bay and TMZ in combination or alone in the microwells Cells were collected from the microwells trypsinized and replated into 8well chamber slides TUNEL cells green with ringlike nuclear stain are indicated with red arrows Nuclei were counterstained with DAPI blue b Numbers of TUNEL cells are presented as mean ± SD of three biological replicates p and p X20 objective Scale bars a0µmanization we treated patientderived GBM cells co treated with Bay with TMZ or single drug treated 3D spheroids collected from the microwells were stained with phalloidin green and DAPI blue Staining cells with fluorescently conjugated phalloidin is considered the most reliable method of accurately labeling Factin in fixed cells57 In the control group intact cells formed finemeshed networks with a distinct Factin anization on both day Fig a05a upper panel and day Fig a05a bottom panel In single drug treated cells actin was still found to be polymerized to filaments as it can be seen by its interaction with phalloidin at both days and However the cells which were cotreated with Bay and TMZ lost their Factin anization and their shape compared with the control and the single drug treated groups at day Fig a05a bottom panel Changes in the a0actin distribution within the cells were quantified by measuring the staining intensity using Fiji Macro ImageJ as described previously5859 At day we observed a a0significant decrease in the fluorescence intensity of phalloidin when the cells were cotreated with Bay and TMZ compared with the a0control and single drug treated groups p Fig a05b To investigate the drug related Factin mechanism we examined the levels of FAK protein following cotreatment or single drug treatment As shown in Fig a05c cotreatment significantly decreased the level of phosphorylated FAK compared with both control and single drug applications p Furthermore we investigated cell migration patterns of the patientderived cells that were cotreated with Bay and TMZ or single drug treated We collected 3D spheroids from microwells after drug treatment and replated them in 24well plate to perform scratch wound healing assay We noted a significant increase in cell density in the scratch area in both control and Bay alone after and a0h of scratch formation p Fig a06a Although compared with the a0control cells both cotreatment and TMZ alone groups showed a decrease in the cell migration into the scratch area after a0h we observed that after a0h the migration rate of the cotreated cells was significantly slower than the cells that were treated with TMZ alone p Fig a06b These results indicated that the disanization of actin microfilaments was concomitant with the cell apoptosis after the a0cotreatment of Bay with TMZDiscussionDespite the increase in the median survival of GBM patients from to months4 the clinical efficacy of standard of care therapy including TMZ chemotherapy combined with surgery and radiotherapy is still limited Due to challenges in treating GBM significant attempts have been made to develop single or combined drug treatments60 However given the cost long time frame and risks of failure associated with developing a new drug repurposing available drugs may be the most effective alternative therapeutic strategy Therefore it is important to evaluate potential drug combinations for GBM treatmentScientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Changes in the actin cytoskeleton and migration pattern in patientderived GBM cells cotreated with Bay and TMZ or single drug treated in the microwells a Upper panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin green and DAPI blue Bottom panel representative images of the patientderived GBM cells cotreated with Bay and TMZ or single drug treated at day stained with phalloidin and DAPI Scale bars a0µm b Intensity of staining obtained with phalloidin was measured in each cell using ImageJ and displayed as boxplots with to confidence intervals A twoway ANOVA with Dunnetts multiple comparisons test was performed to determine statistical relevance Three biological replicates n p p c Representative immunoblots show the levels of FAK pTyr397 and total FAK in patientderived GBM cell lysates cotreated with Bay and TMZ or single drug treated for a0days in the microwells The levels of the proteins were quantified using ImageJ right panel Data were normalized to Bactin Data represent the mean ± SD of three biological replicates p Due to the cell repellent property of PEGDA hydrogel tumor cells can form aggregates at the bottom of the microwells and selfassemble into spheroids in each well within a0days following cell seeding313363 Compared with 2D monolayer cell culture 3D spheroids have an important advantage their larger size Thus often drug effects can easily be monitored over time by measuring the size and shape of spheroids4344 Additionally using 3D in a0vitro tumor models can better recapitulate in a0vivo features of the tumors We used PEGDA hydrogelbased microwell platform313363 in order to culture different types of a0GBM cells commercially available GBM cell lines LN229 U87 and a0patientderived GBM cells However we investigated the effect of the drugs on the patientderived GBM cells more in detail since growing tumors from tumor biopsy samples could give very detailed information about how tumors respond to drugs35 Considering the precious nature of the patient samples this platform which requires fewer cells compared with 2D monolayer cultures provides us with a robust tool to recapitulate in vivo features of GBM tumors and to test our drug combinationsNFκB is one of the major transcription factors associated with GBM and responsible for activating a series of cellular responses including cell proliferation survival invasion and apoptosis6465 Previous studies have shown that NFκB can activate Akt and promote cell survival and proliferation by downregulating the expression of phosphatase and tensin homolog deleted on chromosome ten1866 NFκB pathway can inhibit cell apoptosis by inhibiting a stressactivated protein kinase and a mitogenactivated protein kinase signaling pathway67 It can also be activated in response to treatment with cytotoxic drugs such as vinca alkaloids and topoisomerase inhibitors Several studies have demonstrated the activation of NFκB in GBM patientderived stemlike cells cultures96869 Moreover alkylating agents TMZ can activate NFκB through DNA damage pathway activation7071 The combination effect of Bay and TMZ have been showed in our previous study where we determined the most effective drug concentrations on GBM cells using our microfluidics platform42 Another study that investigated the combined effect of NFκB inhibitor BAY with TMZ showed that combined drug application induced TMZ resistant in U251 GBM cells22 However the characterization of the precise pattern of NFκB activation in different GBM cell populations from surgically resected tissues still remains elusive Therefore in this study we investigated the interaction of Bay with TMZ and their effects on the LN299 and U87 GBM cell lines as well as patientderived GBM cells in order to recapitulate NFκB activation as in a0vivo features of the GBM and its signaling pathways We applied a0µM of Bay and a0µM of TMZ3442 in combination or alone for all three GBM cell types First we observed a significant decrease in both cell viability and size of the spheroids in the cotreatment compared with control and single drug application Then we showed quantitatively and Scientific RepoRtS 101038s41598020703925Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Cell migration of patientderived GBM cells by wound healing assay a Patientderived cells were cotreated with Bay and TMZ or single drug treated in the microwells trypsinized and replated in 24well plates After they reached to their confluency a scratch wound was formed with a 200μl tip and cells were incubated for the next a0h Images were taken 4x at a0h a0hr and a0hr Scale bars a0µm b The wound width was measured with ImageJ and the average wound width was shown Data represent the mean ± SD of three biological replicates p and p oneway ANOVA with Tukeys post hoc testqualitatively the expression of NFκB in all three GBM cell types a0We noted a significant decrease in the cotreated group compared with control and single drug application Our western blot data also confirmed the decrease in the abundance of pP65 pP50 and pIKBa that Bay has been shown to inhibit its phosphorylation46 However in the cotreated group the decrease was significantly higher compared to both control and single drug application This data showed that cotreatment of Bay and TMZ has more effect on the inhibition of NFκB pathway than Bay or TMZ alone and suggests a a0decreased downstream transcription of oncogenic proteins72 Although there were slight differences in the NFκB expression patterns in three different GBM cell types a0we focused on the patientderived cells in the rest of the study due to their ability to better recapitulate the genomic similarities to primary disease7374Proteins that interact with each other activate multiple pathways which can result in apoptosis according to tissue type and pathological condition Glioblastoma tumors express high levels of antiapoptotic BCL2 family proteins such as Bcl2 and BclxL which may cause glioblastoma cells to resist apoptosis75 The proapoptotic members of Bcl2 family such as Bax and Bak are necessary for their proapoptotic effect Interactions and the ratio between antiapoptotic Bcl2 and proapoptotic Bax are decisive factors in the induction of apoptosis7677 Active NFκB can prevent cells from apoptosis by stimulating the expression of genes and promoting cell proliferation Although patientderived GBM samples have been shown to be highly resistant to apoptosis77 our data revealed changes in the expression of various members of Bcl2 family and NFκB signaling pathway after cotreatment of Bay and TMZ Our RPPA results outlined distinct molecular profiles in which apoptotic P53 signaling and NFκB signaling pathways were significantly affected after the a0cotreatment These results supported that the inhibition of NFκB expression could inhibit the expression of Bcl2 and promote the expression of Bax thus promote apoptosis Our data also suggested the possible interaction between Bcl2 and p53 in Scientific RepoRtS 101038s41598020703925Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Proposed schematic of the a0signaling pathways involved in Bay and TMZmediated inhibition in GBM patientderived cells The effect of combined therapy of Bay and TMZ was achieved through the inhibition of SrcFAKVinculin which regulate the cytoskeleton anization through MAPKs JNK and PI3KAKT signaling pathways Exposure to both Bay and TMZ also leads to receptormediated activation of Bax but not Bcl2 in the subsequent inhibition of the downstream NFκB transcription factor Inhibition of NFκB in turn causes cell deathregulating cell survival and death7778 The activation of extrinsic and intrinsic molecular pathways can lead to the proteolytic activation caspases The extrinsic pathway is triggered by proapoptotic ligands that activate cell surface death receptors and procaspase8 which in turn leads to the cleavage of caspase3 and apoptosis79 Our results determined that the a0cotreatment significantly inhibited the expression of caspase3 while the expression of cleaved caspase3 was increased Additionally TUNEL assay which detects DNA strand breaks which could occur as an event in the apoptosis showed a dramatic increase in the TUNEL cells after the cotreatment compared with the a0control and single drug application Altogether these results suggested that the inhibition of cell proliferation Bcl2 and caspase3 by a0the cotreatment of Bay and TMZ may occur through the NFκB mediated apoptosis and they might be tightly coupled8081The literature provides evidence that supports crosstalk between PI3KAktmTOR signaling pathway and NFκB which is downstream of Akt NFκB activation in GBM regulates through AKT phosphorylation of IκB resulting in an activated NFκB that translocates to nucleus8283 Our data showed that when Bay was used with TMZ there was a decrease in the abundance of PI3Kp110 AktpS473 AktpT308 and mTORpS2448 This preliminary data is important to suppo
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Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAccumulating evidence has supported an increased risk of osteoporotic fracture in postmenopausal women and elderly mendiagnosed with diabetes mellitus However it is not uncommon for young and middleaged male patients diagnosed with type diabetes mellitus T2DM to suï¬er from oste ia or osteoporosis Few studies focused on this population group are availableThe aim of this study is to evaluate bone metabolic status and investigate the uence of T2DM on bone metabolism in yearold men Anthropometric assessment and blood samples were obtained from patients with T2DM and nondiabeticvolunteers Serum parathyroid hormone PTH and bone turnover markers BTMs including serum procollagen type I Nterminal peptide PINP osteocalcin OC and crosslinked Ctelopeptide of type I collagen CTX were analysed Nosignificant diï¬erences were observed based on age body mass index systolic blood pressure serum calcium phosphoruscreatinine total protein and albumin levels when comparing T2DM and control groups Fasting blood glucose HbA1ctriglyceride TG total cholesterol and lowdensity lipoprotein cholesterol were significantly increased while highdensitylipoprotein cholesterol was significantly decreased in the T2DM group Compared with controls diabetic patients showed lowerserum PINP OC and PTH levels whereas serum CTX levels were similar between the two groups Moreover HbA1c levelswere positively correlated with PINP and inversely associated with PTH levels TG levels were negatively correlated with OC orCTX levels Furthermore multiple linear regression revealed a positive correlation between HbA1c and PINP levels Theseresults also revealed a negative association between HbA1c and PTH and between TG and OC levels even after adjusting forexpected confounder factors Collectively these ï¬ndings indicated that young and middleaged male patients with T2DMshowed a lower turnover state resulting from bone formation inhibition Glucose and lipid metabolic disorders may aï¬ect boneformation through diï¬erent pathways IntroductionType diabetes mellitus T2DM is a common chronic metabolic disease caused by insuï¬cient insulin secretion andoractivity leading to chronic hyperglycaemia Its high prevalence has resulted in a heavy burden on social ï¬nancialand health care systems [] There is a large amount of evidence revealing an increased risk of fracture in diabeticpatients particularly hip fracture [ ] Recent metaanalyses indicated that hip fracture risk increases times in patients with T2DM [ ] In addition studies haddemonstrated that severe vertebral fracture in patients withT2DM was associated with increased allcause mortality [] Osteoporotic fracture has been increasingly recognizedas another complication of T2DM High morbidity and mortality make the two diseases be more serious global healthproblem The association between osteoporosis and T2DMshould be paid close attentionOsteoporosis is a skeletal chronic metabolic disease characterized by low bone mass and destroyed bone microarchitecture resulting in the high risk of fragility fracture []Therefore bone metabolism should be further studied inpatients with T2DM Bone metabolism is a dynamic cyclicalprocess where osteoblasts are involved in bone formationand osteoclasts are involved in bone resorption [] Metabolites known as bone turnovers markers BTMs are generated 0cJournal of Diabetes Researchfrom bone tissue and cells during the dynamic process andreï¬ect bone metabolism during a relatively short period oftime [] and are thus better at predicting more recentchanges Speciï¬cally procollagen type I Nterminal peptidePINP is the degradation product during the formation oftype I collagen secreted by osteoblasts serum osteocalcinOC is released by osteoblasts during bone formation crosslinked Ctelopeptide of type I collagen CTX is abreakdown product during the degradation of mature typeI collagen secreted by osteoclasts [] Consequently PINPand OC are key markers of bone formation and CTX is akey marker for bone resorption The International Osteoporosis Foundation IOF recommends PINP and CTX as thereference markers for bone formation and bone resorptionrespectively due to their high sensitivity and speciï¬city[] Recently these BTMs have been used to assess bonemetabolism evaluate the clinical eï¬cacy of osteoporosistherapies and predict fracture risk [] Additionally BTMsare shown to be associated with energy metabolism []which is closely related to glucose metabolism Studying theeï¬ect of glucose metabolism disorders on BTMs is importantto evaluate bone metabolic status in T2DMMost research has focused on studying postmenopausalwomen and elderly men since these two groups of individualsare at a high risk for fractures especially those diagnosedwith T2DM Bone formation and bone mass are highest inthe third decade and then decrease with age [ ] However oste ia or osteoporosis in young and middleagedmale patients with T2DM is not uncommon in clinical practice Yet only a few studies focused on these populationgroups are available It is important to study how bonemetabolism disorders aï¬ect younger patients with T2DMTherefore young and middleaged male patients withT2DM were recruited as the subjects in the study presentedhere We aim to assess bone metabolism by determiningserum PINP OC CTX and parathyroid hormone PTHlevels and investigate the association among these markersand glucose metabolism The goal is to explore the uenceof T2DM on bone metabolism which may allow for an accurate assessment of fracture risk and an earlier management ofbone metabolism disorders Materials and Methods Participants The study presented here is a crosssectional study conducted in men aged years oldPatients with T2DM who were admitted to the TianjinMetabolic Diseases Hospital from December to February were included in the T2DM group Nondiabeticmale volunteers from the physical examination centre wererecruited and included in the control group during thesame periodbloodfastingThe diagnosis of T2DM was based on the guidelinesprovided by the World Health anization [] includ°FBG level ¥ mmolling mgdl or h blood glucose ¥ mmoll mgdlduring an oral glucose tolerance test OGTT Diabeticpatients were treated with oral antidiabetic agents or incombination with insulin Exclusion criteria included theglucosepresence of kidney disease eGFR mLmin173 m2hepatic disease ALT or AST ¥ times than the upperreference cancer rheumatic diseases rheumatic arthritisand rheumatoid arthritis other bone metabolic diseasesosteitis and osteomalacia hypercalcemia or other endocrine diseases Cushings syndrome primary hyperparathyroidism and thyroid dysfunction Participants takingmedications that may uence bone metabolism were alsoexcluded These medications included glucocorticoids calcium vitamin D antiosteoporosis drugs steroids and thyroid hormonesThis study was conducted following the Declaration ofHelsinki and was approved by the Ethics Committee of the Tianjin Medical University Chu HsienI Memorial Hospital Each participant signed a written informedconsent form Clinical Measurements Anthropometric and biochemical assessments were performed in all participants Diabeticduration height weight body mass index BMI and bloodpressure data were collected BMI was calculated by the formula as weight in kg divided by height squared in m2All overnight fasting blood samples were obtained in themorning Serum samples were separated by centrifugationand stored at °C Blood calcium phosphorus total protein albumin alanine aminotransferase aspartate aminotransferase alkaline phosphatase ALP serum creatinineuric acid urea nitrogen haemoglobin A1c HbA1c FBGinsulin CpeptidetriglycerideTG highdensity lipoprotein cholesterol HDLc andlowdensity lipoprotein cholesterol LDLc were measuredusing standard methods Serum parathyroid hormonePTH and BTM levels including PINP OC and CTXwere measured using an IDSiSYS automated analyserRoche Germany The intraassay and interassay coeï¬cients of variation CVs of BTMs were below and respectivelycholesterolTCtotal Statistical Analyses The statistical analyses were performed with SPSS SPSS Inc Chicago IL USA Normality testing was conducted in all continuous variablesVariables with normal distributions were described as mean± standard deviation and the diï¬erences were determinedusing Students ttest between the two groups Those withskewed distributions were expressed as median interquartilerange and diï¬erences between groups were assessed usingthe MannWhitney U test The Pearson or Spearman correlation analysis was used to determine the correlation betweenblood glucose or lipid and bone metabolism markers Multiple linear regression analyses were conducted to evaluate theassociation between HbA1c TG and BTMs P value was considered statistically significant ResultsA total of diabetic patients were included in the T2DMgroup The mean age of these patients was ± yearsand the mean diabetic duration was years ranging from to years The mean FBG was ± mmoll 0cJournal of Diabetes ResearchTable Comparison of characteristics between diabetic patientsand controlsVariablesPatients with T2DMn ± Nondiabeticcontrols n P ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Age yDiabeticduration yHeight cmWeight kgBMI kgm2SBP mmHgDBP mmHgFBG mmollHbA1c INS mIUlCP ngmlTG mmollTC mmollHDLcmmollLDLcmmollCa mmollP mmollTP glALB glALT IUlAST IUlALP IUlCr μmollSUA μmollBUNmmolly years T2DM type diabetes mellitus BMI body mass index SBP systolicblood pressure DBP diastolic blood pressure FBG fasting blood glucoseHbA1c haemoglobin A1c INS fasting insulin CP fasting Cpeptide TGtotaltriglyceride TCcholesterol HDLc highdensity lipoproteinlowdensity lipoprotein cholesterol Ca calcium Pcholesterol LDLcalaninephosphorus TPtotalaminotransferase ASTalkalinephosphatase Cr serum creatinine SUA serum uric acid BUN blood ureanitrogen P value was considered significant ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± protein ALBaspartatealbumin ALTaminotransferase ALPand the mean HbA1c value was ± A total of nondiabetic volunteers were recruited in the control groupthat had a mean age of ± years and a mean FBG of ± mmollBaseline clinical characteristics of the two groups areshown in Table No significant diï¬erences were observedbetween the control or T2DM groups for age P height P weight P BMI P orsystolic blood pressure P There were also no significant diï¬erences between the two groups for serum calciumP phosphorus P creatinine P total protein P albumin P or ALPP As expected patients in the T2DM groupshowed significantly higher FBG levels P comparedwith the control group In addition significantly higher TGP TC P and LDLc P levelsand significantly lower HDLc P levels wereobserved in diabetic patients compared with controlsComparison of BTMs and PTH between diabetic patientsand controls is shown in Table There were significantdecreases in serum PINP P OC P andPTH P levels in patients with T2DM comparedwith controls In contrast serum CTX levels were similarbetween the two groups P Moreover univariate correlation analyses were performed to investigate the association between blood glucoseor lipid and bone metabolism markers The results revealedthat HbA1c was positively correlated with PINP rs P and inversely associated with PTH r P There was a significant negative correlationbetween OC or CTX and TG rs P rs P levels Figure There was no significantassociation observed between PINP and TG or between OCand HbA1c levels Age was negatively correlated with PINPrs P OC rs P andPTH r P but not with CTX levels TheBTMs and PTH levels did not correlate with BMI bloodpressure calcium or phosphorous levelstheFurthermore multiple linear regression analyses wereperformed to examinecrosssectional associationbetween blood glucose or lipid and BTMs after adjustingfor expected confounder factors Serum PINP OC orPTH levels were used as dependent variables while HbA1cor TG levels were used as independent variables Theseï¬ndings indicated that HbA1c was positively correlatedwith PINP P and inversely associatedwith PTH P after adjusting for ageBMI systolic blood pressure TG HDLc LDLc calciumand phosphorus Our results also showed a significantnegative correlation between TG and OC P after adjusting for age BMI systolic blood pressure HbA1c HDLc LDLc calcium and phosphorusTable All independent variables used in multiple linearanalyses are shown in Table S1 DiscussionMost previous studies investigating postmenopausal womenand elderly men have shown that the markers for bone formation andor resorption were reduced in patients withT2DM compared with nondiabetic individuals [] indicating a lower bone turnover state It is unclear whetheryoung and middleaged diabetic patients shared similarresults In this study we focused on young and middleaged male patients with T2DM Results demonstrated thatdiabetic patients had significantly lower serum PINP andOC levels compared with the control individuals In contrast serum CTX levels were not significantly diï¬erentbetween the two groups Results indicated that inhibition 0cJournal of Diabetes ResearchTable Comparison of BTMs and PTH between diabetic patients and controlsPatients with T2DM VariablesPINP ngmlOC ngmlCTX ngmlPTH pgmlT2DM type diabetes mellitus PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone P value was considered significant ± Nondiabetic controls ± Plmgn PNIPlmgn COrs P HbA1c ars P lmgp HTPlmgn XTCð½r P HbA1c brs P TG mmolldTG mmollcFigure Correlation between serum glucose or lipid levels and BTMs or PTH a Correlation between PINP and HbA1c b Correlationbetween PTH and HbA1c c Correlation between OC and TG d Correlation between CTX and TG HbA1c haemoglobin A1c TGtriglyceride PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone r Pearsons correlation coeï¬cient rs Spearmans correlation coeï¬cient P value was considered significantTable Multiple linear regression analyses between serum glucose or lipid and bone metabolism markersIndependent variablesDependent variablePINPOCPTHHbA1c haemoglobin A1c TG triglyceride PINP procollagen type I Nterminal peptide OC osteocalcin PTH parathyroid hormone P value wasconsidered significantUnstandardized coeï¬cients Standardized coeï¬cients HbA1cTGHbA1cPof bone formation phase rather than resorption led to alower bone turnover state in young and middleaged malepatients with T2DM Moreover this study demonstratedthat HbA1c was an independent factor for PINP suggesting the uence of glycaemic control on PINP in youngand middleaged male patients with T2DM Early glycaemic control may reduce the risk of fracture by delayingbone formation reduction 0cJournal of Diabetes ResearchReduced serum OC levels were previously reported inmale patients with T2DM [] Bezerra dos SantosMagalhaes further demonstrated a weak negative correlation between FBG and OC levels [] Whereas serumPINP was not available in these studies A recent studyrevealed a decrease in serum PINP levels in patients withimpaired fasting glucose and diabetes [] which was in linewith our research Further analyses revealed that serum PINPlevels were significantly reduced in younger diabetic patients years old compared with the older patients ¥ yearsold but serum CTX was also significantly decreased []The controversial conclusions may be related to diï¬erencesin age race diabetic duration and glycaemic control Astudy by Kulkarni [] shared a similar relationshipbetween HbA1c and PINP levels Additionally a largescale crosssectional study performed in Germany indirectly supported this conjecture The authors revealed thatchances for metabolic syndrome or T2DM significantlydecreased with the higher serum PINP and CTX levelsin men aged years old [] However two largescale studies performed in China one involving men andwomen aged years old [] and the other includingmen aged years old [] indicated that serum OCwas negatively correlated with chances for T2DM evenafter adjusting age BMI waist circumference blood pressure FBG and TG As described by these studies theclose relationship between glucose and BTMs has beeninvestigated but needs further understandingIn addition compared with controls diabetic patientsshowed higher TG TC and LDLc and lower HDLc levelswhich may represent a high probability of lipid metabolismdisorders in patients with T2DM Further analyses investigating the correlation between lipid and BTMs revealed a significant negative correlation between serum TG and OClevels High TG levels may reduce serum OC levels andinhibit bone formation in young and middleaged malepatients with T2DM These observations were similar towhat was found in a recent male populationbased studywhere serum TG levels were also inversely correlated withOC levels [] Some research investigating male diabeticpatients showed no relationship between serum OC levelsand blood lipid metabolism [ ] These ï¬ndings are contradictory to one another Diï¬erences in age race and metabolic status may account for these controversial resultsThe impact of blood glucose and lipid metabolism disorders on BTMs needs further studies to elucidate mechanismsIt is known that hyperglycaemia can lead to osmotic diuresiswhich causes renal calcium leakage and a negative calciumbalance Improved blood glucose control contributes to thereduction of urinary calcium levels [] The calciumsensing defect and secondary chronic hypomagnesaemiainduced by osmotic diuresis may be responsible for impairedPTH secretion [] The pathological regulation of PTH onBTMs in patients with T2DM is not clear In this studyserum PTH levels were decreased and were negatively associated with HbA1c levels in T2DM implying that diabeticpatients especially those with poor glycaemic control hadlower PTH levels These observations were in line with previous studies [ ] Relative hypoparathyroidism may disrupt bone metabolism in patients with T2DM Previousstudies demonstrated that low PTH levels directly inhibitedosteoblast activity and contribute to bone demineralizationIn the nondiabetic population PTH inhibited transcriptionalsuppression of sclerostin produced by osteocytes As a Wntantagonist sclerostin inhibited Wntcatenin signallingand osteoblast activity However the regulation of PTH onsclerostin may be impaired in diabetes [] As mentionedabove the negative relationship between blood glucose andbone metabolism is probably explainedOtherwise chronic ammatory conditions and turbulence of adipokines increased the risk of osteoporosis inpatients with T2DM [] Advanced glycation endproductsAGEs were accumulated in diabetes and played a key rolein chronic ammatory complications [] Previous studieshave shown that BTMs were suppressed by hyperinsulinemiaand the accumulation of AGEs [] AGEs promoted theproduction of both ammatory cytokines and reactive oxygen species ROS in the body by activating ligands furthertriggering chronic ammation and bone resorption []In vitro studies reported that AGE2 and AGE3 inhibitedthe maturation of human marrow mesenchymal stem cellsMSCs and their diï¬erentiation into cartilage and bone tissues resulting in decreased osteoblasts [] Moreover theformation and accumulation of AGEs inhibited synthesis ofosteocalcin and osteoblastic ossein matrix [] increasednonenzymatic crosslinked folding of the collagen ï¬bres[] and disturbed osteoblast development A recent studyindicated that hyperglycaemia directly inhibited the diï¬erentiation of osteoblasts and then decreased bone formationenhanced osteoclast activity and increased bone absorptioneventually leading to a reduction of bone mass [ ] Glucose and insulin signalling involved receptor activation of thenuclear factor κB ligandosteoprotegerin RANKLOPGpathway [ ] Analyses revealed thatlower serumRANKL levels were associated with higher TG levels []This inverse relationship may explain the results generatedin this study Furthermore adiponectin a recently uncoveredadipocytokineis produced exclusivity in adipose tissueResearch shows that adiponectin stimulated osteoblast proliferation diï¬erentiation and mineralization [] Howeverserum adiponectin concentrations decreased in patients withT2DM [] The turbulence of adipocytokines may lead to animbalance of bone metabolismAntidiabetic agents may have diï¬erent eï¬ects on bonemetabolism Agents that may have an eï¬ect include thiazolidinediones TZDs sodiumglucoselinked transporter2SGLT2 inhibitors insulin and glucagonlike peptide1receptor agonists GLP1 RA A previous work shows thatrosiglitazone a type of TZDs promoted osteoblastosteocyteapoptosis and led to a negative balance in bone metabolism[] Analyses demonstrated a gender diï¬erence when itcame to the eï¬ects of TZDs on fracture in patients withT2DM and conï¬rmed that TZDs only increased fracture riskin female patients and not male patients [] SGLT2 inhibitors improved blood glucose levels by promoting urinaryglucose excretion which may aï¬ect urinary calcium excretionand bone metabolism Canagliï¬ozin treatment was associatedwith a higher fracture rate in patients with T2DM [] A 0cJournal of Diabetes Researchmetaanalysis indicated no relationship between three SGLT2inhibitors canagliï¬ozin dapagliï¬ozin and empagliï¬ozin andfracture risk Clinical studies on adverse skeletal events ofSGLT2 inhibitors are still lacking Few studies have assessedthe association between insulin injection and BTMs Severalstudies reported an increased fracture risk in insulintreatedpatients with T2DM [] A high incidence of hypoglycaemicevents and falling [] may be the main reasons in olderadults Longterm disease and the presence of multiple diabetic complications may also disrupt bone metabolism Nosignificant diï¬erences were observed between diabetic patientsunder treatment with n or without n TZDswith n or without n SGLT2 inhibitors andwith n or without n insulin in this study Liraglutide and exenatide two GLP1 RAs may improve skeletalblood supply increase bone mineral density BMD andreduce the risk of osteoporosis and fracture in animal andhuman studies [ ] However the bone protective eï¬ectsbehind this require clinical studies There were no significantdiï¬erences observed between diabetic patients under treatment with n or without n GLP1 RAs in ourstudy In addition there were also no significant diï¬erencesbetween patients under treatment with n or withoutn dipeptidyl peptidase4 DPP4 inhibitors withn or without n insulin secretagogues withn or without n metformin and with n or without n alphaglucosidase inhibitors AGI in thepresent study Table S2 As a multiple metabolic diseasethe treatment of T2DM is complex and requires additionalclinical studies to evaluate the uence of these therapies onbone metabolismOne advantage of this study is that it focused on male diabetic patients aged years old where BTMs varied withsmall changes and there was a restriction on gender and agebeing an uence on the results With this it was easier toinvestigate the relationship between blood glucose lipidsand bone metabolism However this study still faces somelimitations First the crosssectional design prevents onefrom drawing a causal relationship and failed to explorechanges in BTMs after improving blood glucose and lipidmetabolism disorders Further prospective research mayoï¬er additional information about this Second the samplesize number between the two groups was unequal and thenumber of controls used was inadequate Besides this studywas a singlecentre study that only analysed patients with relatively severe diabetes Therefore the results presented heremay not be generalizable to all young and middleaged malepopulations diagnosed with T2DM Largescale and multicentre studies remained to verify these issues Third theuence of antidiabetic agents on bone metabolism remainscontradictory Consequently potential confounder factorsmay exist Fourth serum levels of bonespeciï¬c alkalinephosphatase BAP vitamin D or steroids all of which uence bone metabolism were not determined in this studySerum ALP is mainly derived from liver isoform LAPand its speciï¬city for bone metabolism is lacking [] BAPis a more bonespeciï¬c marker of bone formation while thecurrent immunoassays available for BAP still show up to crossreactivity toward LAP [] As recommended bythe IOF [] serum PINP was preferred for bone formationbecause of high speciï¬city in our study Vitamin D promotesthe absorption of calcium and may aï¬ect bone metabolismHowever relatively limited data about the eï¬ect of vitaminD on BTMs are available A prospective partial interventionstudy in postmenopausal women with T2DM shows that25OHD was positively correlated with PINP especially inpatients taking alfacalcidol [] The MINOS study a prospective study of men aged years revealed thatserum 25OHD was not associated with BTMs in men under years of age n [] The relationship between vitamin D and BTMs still needs further research Fifth we didnot take BMD into consideration BMD altogether withBTMs may be helpful to evaluate bone metabolism BMDreï¬ects mineral density of bone and is the cumulative resultof longterm bone metabolic activities This study mainlyfocused on the impact of T2DM on BTMs and evaluatedthe recent changes of bone metabolism Further studiesshould be conducted to investigate the longterm eï¬ect ofT2DM on BMD ConclusionsThis study demonstrated that young and middleaged malepatients with T2DM showed a lower turnover state resultingfrom bone formation inhibition HbA1c levels were positively correlated with PINP levels and inversely associatedwith PTH levels These ï¬ndings also revealed a negative correlation between TG and OC levels even after adjusting forexpected confounder factors Glucose and lipid metabolismdisorders may aï¬ect bone formation through diï¬erent pathways The study presented here provides evidence of T2DMuencing bone metabolism in young and middleagedmen The improvement of blood glucose and lipids may bebeneï¬cial to bone metabolism and reduce fracture risk inpatients with T2DMData AvailabilityThe data used to support the ï¬ndings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that there is no conï¬ict of interestregarding the publication of this paperAcknowledgmentsThis work was supported by Scientiï¬c Research Funding ofTianjin Medical University Chu HsienI Memorial Hospitalgrant numbers 2018ZDKF07 We gratefully acknowledgethe participants in this studySupplementary MaterialsTable S1 multiple linear regression between serum glucoseor lipid levels and BTMs or PTH Table S2 the informationaboutthe patients with T2DMSupplementary Materialsthe medications of 0cJournal of Diabetes ResearchReferences[] K Ogurtsova J D da Rocha Fernandes Y Huang IDFDiabetes Atlas Global estimates for the prevalence of diabetesfor and Diabetes Research and Clinical Practicevol pp [] A V Schwartz D E Sellmeyer K E Ensrud Olderwomen with diabetes have an increased risk of fracture a prospective study Journal of Clinical Endocrinology and Metabolism vol no pp [] L Forsen H E Meyer K Midthjell and T H Edna Diabetesmellitus and the incidence of hip fracture results from theNordTr¸ndelag Health Survey Diabetologia vol no pp [] Y Fan F Wei Y Lang and Y Liu Diabetes mellitus and riskof hip fractures a metaanalysis Osteoporosis Internationalvol no pp [] M Janghorbani R M Van Dam W C Willett and F B HuSystematic review of type and type diabetes mellitus andrisk of fracture American Journal of Epidemiology vol no pp [] I KostoglouAthanassiou P Athanassiou A Gkountouvasand P Kaldrymides Vitamin D and glycemic control in diabetes mellitus type Therapeutic Advances in Endocrinologyand Metabolism vol no pp [] H Miyake I Kanazawa and T Sugimoto Association ofbone mineral density bone turnover markers and vertebralfractures with allcause mortality in type diabetes mellitusCalciï¬ed Tissue International vol no pp [] E S Siris R Adler J Bilezikian The clinical diagnosis ofosteoporosis a position statement from the National BoneHealth Alliance Working Group Osteoporosis Internationalvol no pp [] M B Greenblatt J N Tsai and M N Wein Bone turnovermarkers in the diagnosis and monitoring of metabolic bonedisease Clinical Chemistry vol no pp [] S Vasikaran for the IOFIFCC Bone Marker Standards Working Group R Eastell Markers of bone turnover for theprediction of fracture risk and monitoring of osteoporosistreatment a need for international reference standards Osteoporosis International vol no pp [] H W S Cabral B F G Andolphi B V C Ferreira Theuse of biomarkers in clinical osteoporosis Revista da Associa§£o M©dica Brasileira vol no pp [] P Iglesias F Arrieta M Pi±era Serum concentrationsof osteocalcin procollagen type Nterminal propeptide andbetaCrossLaps in obese subjects with varying degrees of glucose tolerance Clinical Endocrinology vol no pp [] J M Wishart A O Need M Horowitz H A Morris andB E C Nordin Eï¬ect of age on bone density and bone turnover in men Clinical Endocrinology vol no pp [] P Szulc P Garnero F Munoz F Marchand and P D Delmas Crosssectional evaluation of bone metabolism inmen Journal of Bone and Mineral Research vol no pp [] K G M M Alberti P Z Zimmet and WHO ConsultationDeï¬nition diagnosis and classiï¬cation of diabetes mellitusand its complications Part diagnosis and classiï¬cation ofdiabetes mellitus provisional report of a WHO consultationDiabetic Medicine vol no pp [] J StarupLinde and P Vestergaard Biochemical bone turnover markers in diabetes mellitus a systematic review Bonevol pp [] L Achemlal S Tellal F Rkiouak Bone metabolism inmale patients with type diabetes Clinical Rheumatologyvol no pp [] S V Kulkarni S Meenatchi R Reeta R Ramesh A R Srinivasan and C Lenin Association of glycemic status with boneturnover markers in type diabetes mellitus InternationalJournal of Applied Basic Medical Research vol no pp [] K B dos Santos Magalh£es M M Magalh£es E T DinizC S Lucena L Griz and F Bandeira Metabolic syndromeand central fat distribution are related to lower serum osteocalcin concentrations Annals of Nutrition and Metabolismvol no pp [] K L HollowayKew L L F De Abreu M A Kotowicz M ASajjad and J A Pasco Bone turnover markers in men andwomen with impaired fasting glucose and diabetes Calciï¬edTissue International vol no pp [] E Lerchbaum VSchwetz M Nauck H V¶lzkeH Wallaschofski and A Hannemann Lower bone turnovermarkersthepopulationbased study of health in Pomerania NutritionMetabolism and Cardiovascular Diseases vol no pp in metabolicsyndromediabetesand[] H Shu Y Pei K Chen and J Lu Signiï¬cant inverse association between serum osteocalcin and incident type diabetesin a middleaged cohort DiabetesMetabolism Research andReviews vol no pp [] A Tan Y Gao X Yang Low serum osteocalcin level is apotential marker for metabolic syndrome results from a Chinese male population survey Metabolism vol no pp [] X Y Ma F Q Chen H Hong X J Lv M Dong and Q YWang The relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type diabetes mellitus the role of osteocalcin in energy metabolism Annals of Nutrition and Metabolism vol no pp [] Y Chen Q Zhao G Du and Y Xu Association betweenserum osteocalcin and glucoselipid metabolism in ChineseHan and Uygur populations with type diabetes mellitus inXinjiang two crosssectional studies Lipids in Health andDisease vol no p [] N C Thalassinos P Hadjiyanni M Tzanela C Alevizaki a
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Protocol Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trialMarcelo Marreira Lidiane Rocha Mota Daniela F¡tima Teixeira Silva Christiane Pavani To cite Marreira a0M Rocha Mota a0L Silva a0DFT et a0al Study protocol for the use of photobiomodulation with red or infrared LED on waist circumference reduction a randomised double blind clinical trial BMJ 202010e036684 101136bmj 2019036684 º Prepublication history and additional material for this paper are available online To view these files please visit the journal online http dx bmj Received December Revised July Accepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJBiophotonics Applied to Health Sciences Universidade Nove de Julho Sao Paulo BrazilCorrespondence toDr Christiane Pavani chrispavani gmail comIntroduction The search for non invasive procedures to reduce localised adiposity in aesthetics clinics has recently been increasing In this context procedures such as cryolipolysis ultracavitation photobiomodulation PBM and other techniques have been proposed Some studies have shown that PBM can be used in body contouring However there is no standardisation of the protocol More than that as in other techniques for reducing adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences to the general health of an individual This work will aim to compare the light wavelengths when using PBM as a technique for reducing the abdominal waist circumference while also evaluating the efficacy of the method Changes in the lipid profile in the blood with a long term follow up will also be appraisedMethods and analysis This will be a controlled randomised double blind single centred clinical trial patients will be recruited at the Nove de Julho University Brazil and then divided into three groups Group ARED PBM Group BINFRARED PBM Group CPLACEBO Sham treatment The treatments will consist of eight sessions two times a week for weeks At each session the participants will receive minutes PBM using a radiant exposure of Jcm2 with an abdominal strap containing LED clusters with devices each following the indication of randomisation All of the groups will receive min of Aussie Current at kHz modulated at Hz mA The main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness changes in the local microcirculation and the quality of life and self esteem The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Ethics and dissemination The Ethics Committee of the Nove de Julho University Brazil approved the modified version of this project under No on June This study is not yet recruiting The results obtained Strengths and limitations of this study º The use of the same dosimetry at different wavelengths will allow for a real comparison between red and infrared as being the most suitable wavelength for body contouring º Analyses of body contouring will be performed by non invasive methods º The waist circumference measurement will not discriminate the factors underlying the volume modifications º The habits of the participants such as diet and exercise routines may affect the results º Gender may affect the results and be dependent on the number of participants of each gender These differences may not be considered by the statistical analysiswill be published in a peer reviewed journal in the related fieldTrial registration number Brazilian Registry of Clinical TrialsReBec RBR 9bwxcxINTRODUCTIONFat storage is intended to protect the human body in cases of prolonged fasting intense physical activity and temperature regulation Once freed from these situations fat is stored unnecessarily putting the individual at the risk of health problems together with a greater pr sity for pathologies such as systemic arterial hypertension diabetes mellitus metabolic syndrome and even some types of neoplasms1Another type of negative impact that is related to excessive fat storage is body dissatisfaction This naturally leads to a decrease in the individuals self esteem4 Studies have shown that aesthetic treatments significantly increase a patients quality of life They Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access are associated with improved self esteem6 There are some traditional surgical methods for the reduction of abdominal adiposity However the methods are invasive techniques which may present a high number of complications such as bruising seroma pain perforated ans and viscera as well as with an increased risk of deep vein thrombosis10 The demand for minimally invasive procedures that are aimed at reducing abdominal fat has increased by about while the demand for surgical procedures has decreased by around Among the minimally invasive techniques one can mention low level laser therapy which has recently also been called photobiomodulation PBM PBM has many novel advantages when compared with traditional techniques such as surgical procedures since it can guarantee the preservation of the noble adjacent structures such as the nerves the blood vessels and the skin15 PBM has been widely studied for several applications due to its important biochemical cellular consequences and its few side effects16 Some manuscripts have described erythema and oedema as the main side effects of PBM but importantly these side effects may have been higher as a result of the patient using any drug that increased photosensitivitySome other studies have shown that PBM can be used in body contouring18 Sadly there is no standardisation of the protocol The treatments vary in terms of the number of sessions their frequency times per week and wavelength nm nm nm nm while other dosimetry information such as irradiance Wcm2 and radiant exposure Jcm2 are frequently not mentioned Recently Croghan and coworkers showed that two times a week was the best frequency when compared with one or three times a week This was in terms of improving the patients quality of life and body satisfaction as well as their weight waist circumference body mass index BMI and body fat mass reduction18 However more studies are needed in order to standardise the wavelength the dosimetry and the application time as well as the durability of the results achievedOne of the proposed mechanisms for a PBM effect in adipose tissue is the formation of transient pores in the adipocyte membranes thus allowing for the lipids to escape15 Adipocyte apoptosis activation has also been proposed The production of reactive oxygen species is also possible due to the action of PBM and this is related to the mitochondrial activation on account of the radiation absorption by the cytochrome c oxidase molecules This is followed by an increased ATP synthesis and with an increased cyclic adenosine monophosphate messenger which can trigger the activation of the lipases that perform the hydrolysis of the triglycerides into fatty acids and glycerol23Some reports have affirmed that the results obtained by the use of PBM for reducing waist circumference are modest and that the reduction is temporary which deserves much greater attention from researchers for a better understanding of this factor These effects may be associated with the mechanisms of action and the dosimetric parameters being used24 When taken to the tissues the free fatty acids are used as an energy source during beta oxidation for the production of ATP In some literature reports PBM is associated with aerobic or resistance exercise while other reports have mentioned waist and arm circumference reductions with the use of PBM displaying an absence of diet restrictions or exercise requirements25 It is also reported that the amount of fat mobilised during a PBM session is similar to the amount that is consumed during a meal in such a way that it can be absorbed by normal body energy requirements andor exercise routine while at the same time the risk of atherosclerosis is not increased by the treatment30 On the other hand if not consumed these fatty acids may be re esterified and redistributed throughout the body30 causing no final changes in waist circumference Since neuromuscular electrical stimulation increases energy expenditure in a similar way to that associated with exercise the protocol will be complemented with the Aussie current application31As for other techniques for reducing the adipose tissue the availability of triacylglycerol may affect the lipid profile in the blood bringing consequences for the general health of the individual Some studies have shown that these important treatments may affect the serum lipid levels while others affirm that there are no changes in the serum lipid levels32 Given these scenarios this work will aim to compare the different light wavelengths when using PBM as a technique for the reduction of abdominal waist circumference while at the same time evaluating the efficacy of the method and by following the changes in the lipid profile in the blood as well as with reviewing the long term follow upMETHODSStudy designThis will be a controlled randomised double blind single centred clinical trial designed in accordance with the criteria as established by the Standard Protocol Items Recommendations for Interventional Trials It will be conducted at the Nove de Julho University located in the city of S£o Paulo Brazil The recruitment will be performed from September to November through the university website Thus the selection of sites includes urban locations the city of S£o Paulo and its neighbourhoods After verbal and written explanations regarding the procedures the risks and the benefits by MM a coauthor of this protocol those individuals who agree to participate in the study will sign an informed consent form Based on an anamnesis questionnaire the researchers will check if the participants meet the inclusionexclusion criteria The anamnesis questionnaire will include identification data anthropometric data clinical history and daily living habits especially dietary intake physical activity assessments and menstrual period appraisals Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cSince dietary intake and physical activity may have direct effects on the results at each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measured The enrolment period will be extended until the sample size is reachedPatient and public involvement statementThe patients andor the public will not be involved in the design the recruitment or in the conduct of the studyInclusion criteriaThis study will include men and women aged years with a BMI of between and kgm2 normotrophic and overweight together with hyperplasia of the abdominal fat tissue abdominal skin folds higher than mm Those who agree to participate in this research will sign an informed consent form see online supplementary file Exclusion criteriaThe following people will be excluded from this survey those participants who are undergoing aesthetic treatments to reduce waist circumference those who have been previously submitted to abdominoplasty or liposuction surgeries those who are on a diet in order to reduce their measurements those people who engage in a physical activity more than two times a week those who are using or have taken drugs or food supplements in last days in order to reduce their measurements and their weight which may affect their lipid metabolism appetite or nutrients absorption those who have been submitted previously to oophorectomy those with signs andor symptoms of climacteric at the menopause pregnant or lactating women those participants who are not regular in attending the sessions those participants who present metabolic dysfunctions diabetes and thyroid disorders cardiovascular problems hypertension cardiac insufficiency arrhythmia thrombosis pacemaker use respiratory issues asthma chronic obstructive pulmonary disease haematological disturbances anaemia renal non alcoholic fatty liver disease dermatological or digestive disorders gastritis ulcers those with a history of oncological pathology those with cognitive deficitsSample calculationIn order to calculate the sample size a study showing the therapeutical effects of PBM when associated with aerobic plus resistance training was used26 The researchers used the highest and the lowest abdominal circumference values as well as the SD of the measurements The highest and lowest abdominal circumference values for the PBM group were and respectively and the highest SD of the measurements was with being the number of intervention groups in the study The effect size hence was calculated when using these values as described below biggersmaller Ïn2 accessWhen using the effect size value as calculated above the sample size was calculated using GPower software V3192 Dusseldorf Germany Two way Analysis of Variance ANOVA was used for the interactions within and between the groups in order to evaluate the differences between the three groups studied as well as for the five evaluations during the treatments and the follow up The test power was α005 The sample size was calculated on participantsRandomisationThe randomisation will be performed by DFTS a coauthor of this work who is not directly linked to the treatments or the evaluation of the participants by using the Excel program Microsoft USA The participants will be randomised into blocks of and into groups designated as A B and C Opaque envelopes will be identified by sequence numbers and they will receive a paper containing the information about which treatment will be performed on the participants abdomen according to the draw The sealed envelopes will be safely kept with the researcher who generated the randomisation DFTS Before the beginning of the procedures the researcher responsible for the procedure LRM a coauthor of this protocol will receive each envelope and proceed with the treatment as indicated according to its allocation group A team of undergraduate students previously trained and prepared is going to be part of the research group and for the treatment or evaluation of the participants This study will be a double blind study since the participants will not be aware of the group in which heshe is participating only the researcher who will perform the procedure will know The data collection and analyses will be performed by another researcher MM a coauthor of this study who will also be unaware of the allocationsInterventionThe abdomen of the participants will be cleaned by using a neutral cleansing soap They will receive eye protection using goggles for safety This will also help with the blindness of the study PBM will be applied when using abdominal straps as developed by Cosmedical Mau¡ S£o Paulo Brazil following the parameters as described in table The abdomen strap will be covered with a sheet and that will also help with the blindness of the study All of the participants will receive min of PBM with an abdominal strap containing LED clusters with devices following the indication of the randomisation being per group Group ARED ± nm Group BINFRARED ± nm Group CPLACEBO The treatment will consist of eight sessions that will occur two times a week totalling month of treatments The placebo group will use a strap with no light emission but it will emit the same sounds like that of the active device In order to increase the oxidation of the free fatty acids the participants will receive min of Aussie Current at kHz modulated at Hz mA for min Tensor Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access Table Dosimetry for the studyParameterRed LEDContinuousCentre wavelength nmSpectral width nmOperating modeAverage radiant powerone LED mWAperture diameterone LED cmPower density at aperture mWcm2Beam spot size at targetone LED cm2Total number of LEDsArea irradiated cm2Irradiance at target mWcm2 Exposure duration sRadiant exposure Jcm2Energy density at aperture Jcm2Radiant energy kJApplication techniqueNumber and frequency of treatment sessionsContactweek for a month a total of sessionsInfrared LEDContinuousContactweek for a montha total of sessionsDGM Electronica Santo Andr© S£o Paulo Brazil33 None of the PBM devices will significantly increase the temperature at the target area causing no burns or skin damage No modifications in the intervention will be performed for any reason However the participants who withdraw their consent or the ones who are not assiduous to the sessions will be removed from the studyThe dosimetric parameters that will be used in this protocol as presented in table were measured andor calculated The centre wavelength and the spectral width of the devices were measured by a Spectrophotometer USB2000XR1 Ocean Optics Florida USA The radiant power of one LED was measured by a Power Meter FieldMaxII TO Coherent Santa Clara California USA The abdominal straps will be composed of LED clusters having LEDs each totalling LEDs units and distributed in a cm cm area cm2 each cm2 total see figure The effective irradiated area will be cm2 times the beam spot size at the target The irradiance at the target was determined by the ratio between the average radiant power mW and the beam spot site at the target cm2 The radiant exposure was determined by multiplying the irradiance at the target mWcm2 by the exposure duration s The radiant energy was calculated by multiplying the average radiant power of one LED mW by the total number of LEDs and by the exposure duration sFigure Photobiomodulation PBM application PBM device off A and on B patient receiving the experimental protocol in dorsal decubitus min per session C and DOutcomesThe main outcome of this study will be waist circumference reduction The secondary variables will be anthropometric data lipid profile liver function and adipose tissue thickness as measured by ultrasound changes in the local microcirculation quality of life and self esteem The participants will be evaluated at the same time of the day at all times throughout the studyThe anthropometric data that will be collected will be body weight height and BMI skin fold thickness and bioimpedance Blood will be collected for analyses of the lipid profile total cholesterol high density lipoprotein HDL Low density lipoprotein LDL triglycerides and liver function serum glutamic oxaloacetic transaminase SGOT serum glutamic pyruvic transaminase SGPT All of this will be processed and analysed at SCS Medicina Diagn³stica S£o Caetano do Sul Brazil a partner laboratory The analyses will be performed at four stages of the research D0 end of the eighth session D30 days after the last session FU15 days after the last session FU90 and days after the last session FU180Abdominal ultrasound will be performed to assess the fat layer thickness before and after the treatments For the recording of the local temperature a technique that is widely used is infrared thermography using a Compact Thermal Camera C2 FLIR Systems Oregon USA The thermal camera by means of infrared emission from the body or from the material analysed has the ability to calculate the temperature of a given surface It is possible through this method that the study will infer the changes in local microcirculation34The quality of life questionnaire The WHO Quality of Life WHOQOL BREF as well as the Body Shape Questionnaire BSQ34 Self Image Scale will be used for the participants These questionnaires have been translated and submitted to cross cultural adaption into Brazilian Portuguese37 The Brazilian Portuguese version of these questionnaires will be applied by MM The questionnaires will take around min to be completed The quality of life and the self image questionnaires will be applied at D0and again at the end of the last session D30 The flow chart of the study is presented in figure Adverse events will be collected during the treatment sessions and they will be reported to the regulatory agency and again Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cRecruitment recruitmen t accessEvaluated for eligibility Elected patients n174 Anamnesis application of the quality of life and selfesteem questionnaire anthropometry blood collection ultrasonography Randomised n Allocation Group A LED with devices ± nm with Wcm2 Group B LED with devices ± nm with Wcm2 Group C Sham Each session minutes being measured at the end of each session waist circumference and IRT Total Sessions Analysis at the end of treatment Application of the quality of life and selfesteem questionnaire anthropometry blood collection IRT ultrasonography FollowUp days Anthropometry and blood collection days Anthropometry and blood collection days Anthropometry blood collection ultrasonography Figure Flow chart describing the study design the sample composition and the experimental protocol IRT infrared thermographyat the final publication of the results Since the participants are enrolled and randomised the investigators will make efforts to keep the participants together during the follow up by making phone calls emailWhatsApp contact with the patients and with relevant instructions regarding healthcare and beautyAs a strategy to improve adherence at each session the participant will schedule the next visit and receive a card with some instructions regarding preparation for the evaluation day and the appointment date of the next visit When a participant misses a session heshe will receive a phone call in order to reschedule the missed sessionData analysis planThe data that will be collected from this study will only be administered by the principal investigators the authors of this document Since the study will be of short duration and with known minimal risks this trial will not need a formal data monitoring committee After the data collection the data will be anised using Microsoft Office Excel by DFTS coauthor of this protocol and then stored on a protected by password computer at the university The data will be analysed by descriptive and inferential statistics and then compiled into tables andor graphs using SPSS V240 For testing the normality of the data the Shapiro Wilk test will be performed If the data show a non parametric behaviour a mathematical function will be used in order to normalise the data Two way ANOVA tests followed by the Bonferroni post test will be performed in order to compare the treatments along with the time points being evaluated Some parameters Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0c access may affect the results of the therapy and they are going to be analysed as co variables for example skin phototype by the Fitzpatrick scale the stage of the menstrual cycle total cholesterol and triglycerides altered or not α005 will be considered the level of significance for all of the tests used Since this trial is part of a PhD thesis study an interim analysis will be performed for the qualification examination and this can be used at trial adaptations such as for a sample size re estimation or for stopping the trial The trial protocol and the full study report will be fully available at the end of the study after the manuscript of the results has been published At the end of the study the participants from the placebo group who received the treatment will experience no adverse effects and they will have received the most effective treatmentDISCUSSIONStudies have shown that lasers used in PBM typically operate at powers of mW or less They can produce energy in the visible spectrum wavelengths nm and near the infrared regions nm Light penetration in the soft tissues is known to be directly related to wavelength that is the longer the wavelength the greater the penetration The reports on PBM for reducing local adiposity include the use of green nm red and nm and infrared and nm However there is no comparison available regarding the best wavelength for this purpose18 Based on the localisation of fat tissue more profound when compared with epidermis and dermis this study will choose red and infrared light for the comparisons The use of the same dosimetry at these different wavelengths will allow for the evaluation of the most suitable wavelength for body contouringSince the waist circumference measurements will not discriminate against the factors underlying the volume modifications a placebo group will be included This will allow for the measurement of the differences in waist circumference due to daily habits or hormonal variations as in the menstrual cycle of women The measurements of the skin folds and bioimpedance will complement the evaluation in terms of body fat At each evaluation before the treatment and the follow up sessions days of food records and physical activity levels will be measuredGender may also affect the results Sexual dimorphism in adipogenesis is already known as well as sex hormones in the white adipose tissue function and in adipose metabolism39 If the sample consists of a huge difference in men and women this factor cannot be considered in the statistical analysisThe development of a non invasive protocol for PBM together with an Aussie current for the reduction of adiposity may present an important novel tool for the reduction of health risk problems as well as for increasing an individuals self esteemETHICS AND DISSEMINATIONThe Ethics Committee of the Nove de Julho University S£o Paulo Brazil approved the modified version of this project and the Patient Informed Consent Form under No on June according to the guidelines of the Brazilian National Ethics Committee The protocol of this study has already been registered in the Brazilian Registry of Clinical Trials being first registered on November and modified on August providing full public access to the protocol information including all items from the WHO Trial Registration Data Set MM and DFTS will be the data curators with the data stored on a protected by password computer at the university The results acquired within this project will be presented in conferences and published in a journal in the related field The authorship of the results paper and the conference abstracts will include the authors of this protocol together with other researchers who may contribute to the procedures or to the analysis of the data Any modifications of this protocol will require a formal amendment and they will be approved by the Ethics Committee of the Nove de Julho University The modifications will be properly reported and justified in the manuscript for the publication of the results The main results obtained will be sent to the participants by mailAcknowledgements The authors would like to thank the Nove de Julho University UNINOVE S£o Paulo Brazil for the availability of its laboratories the company Cosmedical for the development of the equipment for PBM and the SCS Medicina Diagn³stica Laboratory S£o Caetano do Sul Brazil for their partnership in the analyses of the laboratory testsContributors MM LR M DFTS and CP designed the study MM and LR M will conduct the experiments and will be making the data acquisitions DFTS and CP will perform data analysis and interpretation MM and LR M drafted the work while CP and DFTS revised it critically for important intellectual content All of the authors approved the final version of the manuscriptFunding The authors have not declared a specific grant for this research from any funding agency in the public commercial or not for profit sectorsCompeting interests None declaredPatient consent for publication ObtainedProvenance and peer review Not commissioned externally peer reviewed access This is an access article distributed in accordance with the Creative Commons Attribution Non Commercial CC BY NC license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial See a0http creativecommons licenses by nc ORCID iDsDaniela F¡tima Teixeira a0Silva http orcid Christiane a0Pavani http orcid REFERENCES Silva Figueiredo P Carla Inada A Marcelino G et a0al Fatty acids consumption the role metabolic aspects involved in obesity and its associated disorders Nutrients Landecho MF Tuero C Valent V et a0al Relevance of leptin and other adipokines in obesity associated cardiovascular risk Nutrients Kong Y Zhang S Wu R et a0al New insights into different adipokines in linking the pathophysiology of obesity and psoriasis Lipids Health Dis Jim©nez Flores P Jim©nez Cruz A Bacardi Gasc³n M Body image dissatisfaction in children and adolescents a systematic review Nutr Hosp Weinberger N A Kersting A Riedel Heller SG et a0al Body Dissatisfaction in individuals with obesity compared to normal weight Marreira a0M et a0al BMJ 202010e036684 101136bmj 2019036684 0cindividuals a systematic review and meta analysis Obes Facts Kouris A Platsidaki E Christodoulou C et a0al Patients self esteem before and after chemical peeling procedure J Cosmet Laser Ther Stundzaite Barsauskiene G Tutkuviene J Barkus A et a0al Facial perception self esteem and psychosocial well being in patients after nasal surgery due to trauma cancer and aesthetic needs cluster analysis of multiple interrelations Ann Hum Biol Ribeiro F Steiner D Quality of life before and after cosmetic procedures on the face a cross sectional study in a public service J Cosmet Dermatol Bensoussan J C Bolton MA Pi S et a0al Quality of life before and after cosmetic surgery CNS Spectr Appleton SE Ngan A Kent B et a0al Risk factors influencing transfusion rates in DIEP flap breast reconstruction Plast Reconstr Surg Lievain L Aktouf A Auquit Auckbur I et a0al [Abdominoplasty complications particularities of the post bariatric patients within a patients series] Ann Chir Plast Esthet Sterodimas A Boriani F Nicaretta B et a0al Revision Abdominoplasty with truncal Liposculpting a year experience Aesthetic Plast Surg Al Dujaili Z Karcher C Henry M et a0al Fat reduction complications and management J Am Acad Dermatol Krueger N Mai SV Luebberding S et a0al Cryolipolysis for noninvasive body contouring clinical efficacy and patient satisfaction Clin Cosmet Investig Dermatol Neira R Arroyave J Ramirez H et a0al Fat liquefaction effect of low level laser energy on adipose tissue Plast Reconstr Surg Karu T Mitochondrial mechanisms of photobiomodulation in context of new data about multiple roles of ATP Photomed Laser Surg Feng J Zhang Y Xing D Low power laser irradiation LPLI promotes VEGF expression and vascular endothelial cell proliferation through the activation of ERKSp1 pathway Cell Signal Croghan IT Hurt RT Schroeder DR et a0al Low level laser therapy for weight reduction a randomized pilot study Lasers Med Sci Thornfeldt CR Thaxton PM Horn
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the molecular heterogeneity of renal cell carcinoma rcc complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge this study investigates the role of thelncrna cdkn2bas1 and mir1413p interactions in the progression and metastasis of kidney cancer human renalcancer cell lines achn and caki1 normal rptec cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study an overexpression of cdkn2bas1 was observed in rcc compared to normal samplesin tcga and our inhouse sfvamc tissue cohorts reciprocally we observed reduced expression of mir141 in rcccompared to normal in the same cohorts cdkn2bas1 shares regulatory mir141 binding sites with ccnd1 andccnd2 genes direct interactions of cdkn2bas1mir141cyclin d1d2 were conï¬rmed by rna immunoprecipitationand luciferase reporter assays indicating that cdkn2bas1mir141cyclin d1d2 acts as a cerna network in rccfunctionally attenuation of cdkn2bas1 andor overexpression of mir141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model furtheroverexpression of cdkn2bas1 is positively correlated with poor overall survival of rcc patients expression of mir141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal rptec cells induced procancerous characteristics cdkn2bas1 attenuation or mir141 overexpression decreased ccnd1ccnd2 expressionresulting in reduced rac1ppxn that are involved in migration invasion and epithelialmesenchymal transition thisstudy for the ï¬rst time deciphered the role of cdkn2bas1mir141cyclin d axis in rcc and highlights this networkas a promising therapeutic target for the regulation of emt driven metastasis in rccintroductionrenal cell carcinoma rcc is one of the most commoncancers in the usa accounting for nearly deathsand new cases in surgery is the ï¬rst line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of morecorrespondence rajvir dahiya rdahiyaucsfedu1department of urology veterans affairs medical center san francisco anduniversity of california san francisco san francisco ca usa2department of surgery university of miami miller school of medicine miamifl usafull list of author information is available at the end of the these authors contributed equally pritha dasgupta priyanka kulkarniedited by e candithan however in approximately of localizedrcc cases recurrence occurs with distant metastasis2the obstinate nature of rcc to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence lack of sensitivity to both chemotherapytherapeuticoptions difï¬cult3 it is therefore of utmost importanceto improve our understanding of rcc pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive rcc6and immunotherapy makesemerging lines of evidence suggest that cancer aggressiveness is associated with epithelialmesenchymal transition emt7 it is a wellorchestrated process involved in the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40ofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8emt changes in cellular morphology and migratoryproperties are governed by numerous factors9 increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like ncadherbronectinvimentin and matrix metalloproteinasemmps and decreased expression of epithelial markerslike ecadherin αecatenin claudin etc10 are common emt phenomena often the progression of cancerthrough emt is significantly induced by the interaction ofcyclind with its binding partner cdk4 which act astranscriptional regulators controlling cell proliferationand migration14 it is well known that cyclind regulates the ratelimiting step in cell cycle progression fromg1 to s phase accumulating evidence also suggest thatabnormal cyclindcdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodreports show that noncoding rnas are involved in thefactors involved in emt18 micrornasregulation ofmirnas a naturally occurring class of small noncodingrna molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mrna degradation20 whereas long noncoding rnaslncrnas with more than nucleotides can also actas regulators for tumorsuppressive mirnas in differentcancers21 recently a class oflncrnas have beencategorized as competing endogenous rna cernawhich involves crosstalk among lncrnas mrnas andtheir shared mirnas thus a novel regulatory mechanismis hypothesized suggesting that lncrnas and mrnascommunicate with each other by competing for commonmirna response elements24in this context we describe the novel role of lncrnacdkn2bas1 and mir1413p mir141 in the regulation of cyclind to govern the metastatic progression ofrcc to our knowledge this is the ï¬rst report to directlydemonstrate that cdkn2bas1mir141 interaction is acrucial component in rcc progression and metastasisthrough the cyclindrac paxillin pathwaymaterials and methodscell lines and cell culturethe normal rptec atcc number crl4031 andrenalcancer achn atcc number crl1611and caki1 atcc number htb46 cell lines were purchased from the atcc manassas va these humanderived celllines were authenticated by dna shorttandem repeat analysis by atcc cell line experimentswere performed within months of their procurementresuscitation achn cells were cultured in mem mediaofï¬cial of the cell death differentiation associationcaki1 cells in and mccoy 5a medium and rptec cellsin dmemf12 medium atcc® ¢ all mediawere supplemented with fbs and 1x antibioticspenicillin and streptomycin cell lines were maintainedat °c and humidiï¬ed atmosphere of co2mirnasirna transfectionsto induce overexpression or knockdown cells were transiently transfected with either mirvana mirna mimic nmoll or antimir mirna inhibitor nmollthermo fisher scientiï¬c and nmoll of sirna sigmaaldrich using lipofectamine rnai max thermo fisherscientiï¬c according to the manufacturers protocol toverify transfection efï¬ciency mirvana mirna mimicnegative control mirna inhibitor control and sirnacontrol were used respectively in each transfection experiment at the same concentration all transfection experimentswere carried out for hclinical specimensformaldehydeï¬xedparafï¬nembedded ffpe tissue specimens from patients undergoing radical nephrectomy wereobtained from the san francisco veterans affairs medicalcenter sfvamc written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines irb approval no allpatient samples were pathologically conï¬rmed for clear cellrcc ccrcc and slides were reviewed by a boardcertiï¬edpathologist for the identiï¬cation of tumor foci and adjacentnormal tissue apart from sfvamc cohort tcgakirctcgakich tcgakirp icgc and geo cohorts forrcc from online databases were also used to check theexpression levelsrnamirna extraction and quantitative realtime pcrqrtpcrtotal rna was extracted from microdissected ffpetissues and cell lines using mirneasy ffpe and mirneasymini kits qiagen respectively in accordance to manufacturers instructions mature mirna and mrnas wereassayed by qrtpcr using quantstudio flexreal timepcr system applied biosystem using fast sybr®green master mixtaqman universal pcr master mixprobes and primers applied biosystems inc foster cityca usa following manufacturers protocol humangapdh and rnu48 were used as endogenous controlsand relative expression of rnamirna were calculatedusing comparative ct threshold cycle primer sequencesare provided in supplementary table t1dna methylation analysis insilico in cell lines and 5azacdr treatmentdna hypermethylation of the mir141 promoter innormal and rcc samples was ï¬rst conï¬rmed in the 0cdasgupta cell death and disease page of tcga database using wanderer software27 in order toconï¬rm the methylation status of the mir141 promoterin rcc cell lines we extracted dna from achn andcaki1 using dneasy tissue kit qiagen sodium bisulphite modiï¬cation was done using ez dna methylationgold kit zymo research orange ca usa followingthe manufacturers protocol bisulï¬tetreated dna wasanalyzed by methylationspeciï¬c quantitative polymerasechain reaction msqpcr with primer pairs speciï¬c formethylated and unmethylated regions of the mir141promoter msqpcr was performed as described earlier28 for each sample the percent of methylation wascalculated by the difference of ct in methylated samplectm and ct in unmethylated sample ctu the primers sequences are mentioned in supplementary table achn and caki1 cells were treated daily with μmoll5azadeoxycytidine 5azacdrfor h29 and total rna was isolated using a mirneasy minikit qiagen to check mir141 expressionsigmaaldrichcell viability clonability migratory invasion andapoptosis assayscell viability was measured at and h using acelltiter aqueous solution cell proliferation assay kitpromega madison wi following the manufacturersinstructions for colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed plates were then stained with giemsafollowed by crystal violet and colonies were countedculture inserts of 8µm pore size transwell costar wereused for migration and invasion assay inserts were coatedwith matrigel bd biosciences µgwell for invasionbrieï¬y h posttransfection cells were counted andplaced on inserts at à cellsml for migration and à for invasion in serumfree medium and wereallowed to migrateinvade for h at °c cellsmigrated or invaded through the pores were ï¬xed stainedwith crystal violet crystal violet was solubilizedwith methanol and quantiï¬ed at nm by a kineticmicroplate reader spectra max molecular devicesfacs analysis for apoptosis was done h posttransfection using annexin vfitc and 7aad kit beckmanin accordance with the manufacturerscoulter incinstructions cold pbs washed cells were resuspended in1x binding buffer and stained with annexin vfitc7aad viability dye after min of incubation at roomtemperature in the dark stained cells were analyzed usingbd facsverse bd pharmingendualluciferase reporter assaythe wild type wt and offtarget ot luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingofï¬cial of the cell death differentiation associationsites and corresponding nontarget mutant sites into thepmirglo reporter vector luciferase constructs µgwere cotransfected into achn and caki1 cells along with nmoll mir141 mimic or controlmir using transfection reagent jetprime polyplustransfection illkirchfrance luciferase activities were measured using thedualluciferase assay promega madison wi h posttransfection relative luciferase activity was calculated bynormalizing fireï¬y luciferase to renilla luminescencerna immunoprecipitation rip assaysimultaneous binding of mir141 to lncrna andmrna was conï¬rmed by rip assay an imprint rip kitwas used following the manufacturers protocol sigmaaldrich st louis mo usa igg control and ago2antibodies were used forimmunoprecipitation theimmunoprecipitated rna fraction was reverse transcribed to cdna using high capacity cdna reversetranscription kit thermo fisher fold enrichment oflncrna and mrna to ago2 with respect to igg wascalculated using quantitative rtpcrwestern blot and immunoï¬uorescence analysistotal protein extraction was performed as describedpreviously18 proteins were then separated by nupage bistris protein gels invitrogen and subsequentlytransferred onto nitrocellulose membraneresulting blots were blocked using odyssey blockingbuffer licor and subsequently probed with primaryand secondary antibodies blots were scanned using anodyssey infrared imaging system scan and quantiï¬cationwas carried out with the licor odyssey® scanner andsoftware licor biosciences the primary antibodiesused are listed in supplementary table for immunoï¬uorescencetransfected achn andcaki1 cells were ï¬xed in paraformaldehyde for minfollowed by blocking 1x pbs5 normal goat serum triton x100 for h at room temperature cellswere then incubated overnight in fold diluted primary antibody at °c cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoledapi for min cells were then mounted on a slideusing prolong gold antifade reagent images were captured using zeiss microscope model axio imagerd2transientlyrenal cancer xenograftswe studied the antitumorigenic effects of mir141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 male nude mice weekold n charles river lab were subcutaneously injected with à caki1 cells oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups ï¬ve in 0cdasgupta cell death and disease page of each synthetic mirna mir141 mimicmircon of μg was complexed with μl siportamine transfection reagent ambion in μl pbs and deliveredintratumorally in 3day intervals tumor volume wascalculated according to the formula x2 y2 where x yx width y length experiments were terminated days after the last treatment day tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups all animal care was in accordance withinstitutional guidelines iacuc approval no statistical analysisall quantiï¬ed data represents an average of at leastthree independent experiments or as indicated statisticalanalyses were performed using graphpad prism andmedcalc error bars represent ± standard error meansem the mannwhitney u test was used to assessthe difference between mirna expressions in tumornormal adjacent tissue significant differences betweenthe groups were determined using the students ttest alltests were performed either one tailed or two tailed andresults were considered statistically significant at p ¤ receiver operating curves roc were performed toevaluate the potential of mir141 to differentiate betweenmalignant and nonmalignant samples using medcalcsoftware showing area under curve auc and conï¬dence interval kaplanmeier analyses for overall survival with respectto mir141 methylation levels intcgakirc cohort were generated using softwareezrhttpsdoi101038bmt2012244 tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsresultslncrna cdkn2bas1 is oncogenic and is a direct target ofmir141initially we found cdkn2bas1 is an oncogeniclncrna in rcc based on tcga fig 1a icgc andgeo databases fig s1 tcga cohort also revealed thathigh cdkn2bas1 expression increases from lower gradeand stage to higher grade and stage fig 1b moreoverhigher expression is significantly p correlated tooverall survival fig 1c in agreement with these datacohorts significantly higher cdkn2bas1 expression wasalso seen in rcc cell lines achn caki1 as compared tonormal rptec fig 1d and sfvamc cohort fig 1epatient and tumor characteristics are summarized insupplementary table t3 roc analysis shows an auc of p ci fig 1f suggesting the diagnostic potential of cdkn2bas1 to discriminate between normal and tumor tissues we usedcomputational algorithms and identiï¬ed putative mir141binding sites in the cdkn2bas1 sequence fig 1g toofï¬cial of the cell death differentiation associationexamine potential mir141cdkn2bas1 interactionexperimentally we performed luciferase reporter assayboth achn and caki1 cells cotransfected with mir141and cdkn2bas1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmir141 directly interacts and regulates cdkn2bas1fig 1h thus all these data suggest that clinicallyimportant cdkn2bas1 is an oncogenic lncrna in rccand is a novel target of mir141cdkn2bas1 inhibition by sirna suppressestumorigenicity in rcctransient transfection of achn and caki1 cells withcdkn2bas1 sirnas for h showed significant reduction in cdkn2bas1 expression fig 2a cdkn2bas1knockdown in both cell lines significantly inhibited cellproliferation figs 2b s2a and clonogenic survivalfigs 2c s2b with a significant increase in apoptosis fig2d e decreased cell migrationinvasion figs 2f s2c dwith simultaneous changes in emt markers such as anincrease in epithelial markers αecatenin and claudinand decrease in mesenchymal markers vimentin andï¬bronectin were also observed figs 2g s3expression of mir141 and its clinical importance in renalcarcinomaand samples lowersince our results conï¬rmed cdkn2bas1 as a directtarget of mir141 we examined mir141 status andclinical importance in rcc expression of mir141 wassignificantly downregulated in rcc cell lines fig 3aand in tumor samples fig 3be compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors fig 3ce patientand tumor characteristics are summarized in supplementary table t3 roc analysis showed an auc of p ci fig 3f suggesting thatmir141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsepigenetic regulation of the mir141 locuswe identiï¬ed a genomic site rich in cpg island locatedupstream of the mir141 in chromosome 12p13 in thetcga cohort we observed hypermethylation of mir141promoter in tumor tissues as compared to normalfigs 3g s4a which is significantly associated with poorpatient survival fig 3h similarly rcc cell lines achnand caki1 also showed hypermethylation compared tonormal rptec cells fig 3i further we treated achnand caki1 cell lines with demethylating agent 5azacdrand observed decrease in methylation fig s4b withconcomitant increase in mir141 expression fig 3jindicating possible epigenetic regulation a significantdecrease in the expression of methylation regulatory 0cdasgupta cell death and disease page of fig lncrna cdkn2bas1 is oncogenic in renal cancer and is a direct target of mir141 a expression levels of cdkn2bas1 among kircnormal tumor kich normal tumor and kirp normal tumor patient samples in tcga cohort using wanderersoftware pvalue calculated by mannwhitney twotailed test b expression of cdkn2bas1 in tcgakirc cohort among different grades normal grade grade and stages normal stage iii and stage iiiiv c overall survival in tcgakirc cohort as performedby kaplanmeier analysis using ualcan software d relative expression levels of lncrna cdkn2bas1 in rcc cell lines achn and caki1 e expressioncdkn2bas1 in matched pairs of rcc tissue samples from sfvamc cohort pvalue calculated by mannwhitney twotailed test f receiver operatingcurve roc analysis on sfvamc cohort showing ability of lncrna cdkn2bas1 to differentiate between malignant and nonmalignant samples sfvamccohort g predicted binding sites of mir141 in cdkn2bas1 sequence h luciferase assays showing decreased reporter activity after cotransfection witheither wildtype wt offtarget ot cdkn2bas1 or luciferase control constructs ev with mirconmir141 in achn and caki1 cellsgenes such as dnmtl dnmt3a and dnmt3b werealso noted after 5azacdr treatment compared to control dmso in both achn and caki1 cell lines31mir141 overexpression phenocopies functional effectsobtained with cdkn2bas1 inhibition in vitro andsuppresses tumorigenicity and in vivowe sought to determine if cdkn2bas1 causes itsantitumorigenic effects through mir141 we checkedthe effect of mir141 overexpression in rcc cellstransient transfection of mir141 mimic in achn andcaki1 cells for h led to over expression of mir141compared to control mircon fig 4a also overexpression of mir141 significantly reduced cdkn2bas1 expression fig 4b indicating a reciprocal correlation between mir141 and cdkn2bas1 a significant decrease in cell proliferation over time fig 4cand marked decreasefig 4din clonogenicityofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of fig knockdown of lncrna cdkn2bas1 reduces tumorigenicity in renal cancer a relative expression of cdkn2bas1 in achn and caki1 cellstransfected with cdkn2bas1 sirnas b cell proliferation assessed by mts assay after knockdown of cdkn2bas1 in both cell lines with sirna2 cgraphical representation showing knockdown of cdkn2bas1 with sirna2 significantly decreased colony formation in achn and caki1 cellsd e achn and caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of cdkn2bas1f reduced migration and invasion in cdkn2bas1 sirna transfected cells compared to control treatment g changes in emt related proteins in bothachn and caki1after knockdown of cdkn2bas1compared to controls were also observed further westudied the therapeutic potential of mir141 in amouse xenograft model a significant decrease intumor growth was observed by intratumoral delivery ofmir141 mimic compared to control over the course ofexperiment average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received mir141 mimic fig 4e in additionmir141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both rcc celllines compared to controlfig 5a this proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyadpribose polymerase parp an increase in bax and adecrease in bcl2 at protein levels fig 5b a significantdecrease in migration fig 5c and invasion fig 5dwas also observed in both rcc cell lines with mir141overexpression we also examined emt markers aschange in migration and invasion are directly associated with emt our results showed an increase inepithelial markers αecatenin and claudin with concomitant decrease in mesenchymal markers ï¬bronectinand vimentin at both protein fig 5e and mrnaofï¬cial of the cell death differentiation associationfig s5 levels taken together overexpression of mir phenocopies the functional effects of cdkn2bas1 inhibition in vitro and tumor growth suppressioneffects in vivolike cdkn2bas1aremir141cdkn2bas1 interaction negatively regulatescyclind and its downstream effectors in rcccyclind1cyclind2alsooncogenic in rcc fig s6 and are direct targets of mir as discussed earlier lncrnas can act as cernas tocarry out their regulatory functions32 we observedthat cdkn2bas1 shared regulatory mir141 bindingsites with cyclind1cyclind2 fig 6a and therebysponges mir141 allowing cyclind1d2 to be expressedin tumors to determine potential mir141cdkn2bas1cyclind interaction experimentally we performedrip assay both achn and caki1 cells over expressingmir141 revealed significant enrichment of cyclind1cyclind2 and cdkn2bas1 with ago2 as compared toigg control fig 6b moreover decreased luciferaseactivity also conï¬rmed direct binding of mir141 tocyclind in mir141overexpressing achn andcaki1 cells compared to controls fig 6c we also found 0cdasgupta cell death and disease page of fig expression clinical signiï¬cance and epigenetic regulation of mir141 in renal cancer a mir141 expression levels in achn caki1 andrptec cells b expression levels of mir141 in kirctcga cohort normal and tumor c expression levels of mir141 in normal n nonmetastatic n metastatic n kirc patient samples in tcga cohort d expression levels of mir141 in kirctcga cohort amongdifferent grades normal grade grade and stages normal stage iii and stage iiiiv e relative mir expression in rcc tissue vs matched adjacent normal regions n among different grades normal grade grade andin different stages normal stage iii stage iiiiv as assessed by qrtpcr sfvamc cohort f receiver operating curve roc analysisshowing ability of mir141 to differentiate between malignant and nonmalignant samples g methylation for kirc patient samples in tcgakirccohort for probe cg02624246 h overall survival with tcgakirc methylation as performed by kaplanmeier analysis i methylation status of mir141promoter in rcc cell lines compared to nonmalignant rptec as assessed by msqpcr j expression of mir141 in 5azacdr treated and untreatedachn and caki1 cell lines results are representative of three independent experiments p value calculated by student t test bar mean ± standarderror mean semthat overexpression of mir141 or inhibition of cdkn2bas1 significantly decreased cyclind1d2 expression atboth the mrna fig 6d e and protein levels figs 6fhs8a b this effect was significantly attenuated by mir inhibitor fig s7 indicating that cyclind expressionis dependent on the interaction between mir141 andcdkn2bas1 we further observed a decrease in rac1 asmall gtpase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mrna levelsfigs s3 s5 and protein levels figs 6g ij s8cf inboth mir141 overexpressed and cdkn2bas1 inhibitedrcc cell lines which in turn are involved in regulatingcellular migrationinvasion cumulatively these resultsindicate that suppression of cdkn2bas1 by mir141inhibits renal cell proliferation invasion and migration byinhibiting cyclind rac1 and phosphorylation of paxillinofï¬cial of the cell death differentiation association 0cdasgupta cell death and disease page of fig mir141 overexpression mimics the knockdown effect of lncrna cdkn2bas1 in vitro and reduces tumorigenicity in vivo a relativeexpression of mir141 in achn and caki1 cells transfected with mir141 mimic and control b significant decrease in cdkn2bas1 expressioncompared to control in both cell lines overexpressed with mir141 c rcc cell proliferation after transfecting with mir141 mimic and control asassessed by mts assay d colony formation and its graphical representation in mir141 overexpressing achn and caki1 cells compared to controlse pictures of excised tumors are taken at the termination of experiment day graph represents tumor volume after intratumoral injection ofcontrol or mir141 mimic into established tumors injection was started at day and was followed for days each mouse in both groups mirconand mir1413pmimic received a total of eleven injections intermittently data represent the mean of each group and error bars are sem results arerepresentative of three independent experiments p value calculated by student t test bar mean ± standard error mean semattenuation of mir141 exerts tumorigenic attributes innormal rptec cellswe next determined whether attenuation of mir141induces tumorigenic characteristics in normal rpteccells by targeting cdkn2bas1 and cyclind transienttransfection of mir141 inhibitor indeed showed a significant decrease in mir141 expression fig 7a and anincrease in cdkn2bas1 expression fig 7b along withother procancerous phenotypes such as increased cellproliferation fig 7c colony formation fig 7d migration and invasion fig 7e as compared to controlsadditionally a significant increase in cyclind1 cyclind2 rac1 and paxillin pxn expressions were observed inmir141 inhibited rptec cells fig 7f a noticeableincrease in prometastatic ï¬bronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαecatenin genes were also observed in mir141 inhibited rptec cells compared to controls fig 7gdiscussionprior studies have shown the regulatory role of noncoding rnas in tumorigenesis especially in the emtofï¬cial of the cell death differentiation associationpathway leading to cancer aggressiveness cdkn2bas1also known as anril is located at chromosome 9p21cdkn2bas1 is reported to be upregulated in tumortissues and function as an oncogenic lncrna in pancreatic ovarian and laryngeal squamous cell carcinoma36 human mir141 is located at chromosome12p1331 and is transcribed from a mir200 familyclusterinterestingly expression of mir141 is controversial since it exhibits either oncogenic39 or tumorsuppressive roles42 in speciï¬c types of cancer theprime goal of the present study was to understand therole of cdkn2bas1mir141 interactions in regulatingrcc progression and metastasisin this study we identify cdkn2bas1 to be a crucialoncogenic lncrna that plays an important role in renalcarcinogenesis cdkn2bas1significantly overexpressed in rcc and the expression increases fromlower to higher grades and stages lncrna cdkn2bas1directly interacted with mir141 as it was found to be anovel target of mir141 in contrast to cdkn2bas1 weobserved significant attenuation of mir141 expression inrcc cell lines and tumor samples compared to normalis 0cdasgupta cell death and disease page of fig ectopic mir141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a apoptosis assessed by ï¬owcytometric analysis of annexinvfitc 7aadstained achn and caki1 cells transfected with mirconmir141mimic graph represents totalapoptosis early late b immunoblots showing apoptotic proteins in mirconmir141mimic treated achn and caki1 cells with gapdh asendogenous control c migration assay and d invasion assay as seen in pictures and graphical representation of both achn and caki1 cells after mir overexpression compared to control e immunoblot assay showing emt related proteins in mirconmir141mimic treated achn andcaki1 cells with βactin as endogenous control graphs are average of three independent experiments p value calculated by student t testbar mean ± standard error mean semcell line or matched normal samples as it is known thatextensive dna hypermethylation of cpg islands is highlycorrelated to activation of cancerspeciï¬c genes45 wechecked the methylation status of mir141 in normal andrcc tissues interestingly insilico analysis showed thepresence of cpg island in the promoter region of mir and we also found hypermethylation of mir141 intcga samples as compared to normal this hypermethylation is also found to be significantly associatedwith poor survival of patients similar results were alsoobserved in rcc cell lines compared to a normal rpteccell line functionally inhibition of cdkn2bas1 andoroverexpression of mir141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in rcc in vitro in vivodata show suppression of tumor growth by mir141overexpression conversely attenuation of mir141 innormal rptec cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionfrom a clinical point of view noncoding rnas signatures are powerful tools for early cancer diagnosisofï¬cial of the cell death differentiation associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 our results revealed thathigher expression of cdkn2bas1 is positively correlatedwith poor overall survival probability of rcc patientsindicating its prognostic capability cdkn2bas1 canalso discriminate normal from tumor samples showing itsdiagnostic potential similarly mir141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be a diagnostic biomarker for rcc collectivelythese resultshighlight the biomarker potential of cdkn2bas1mir expression in rcc although it needs to be conï¬rmedin a larger independent sample cohortinterestingly we found tha
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purpose pancreatic ductal adenocarcinomas exhibit a high degree of desmoplasia due to extensive extracellular matrix deposition encasement of mesenteric vessels by stroma in locally advanced pancreatic cancer lapc prevents surgical resection this study sought to determine if the addition of a monoclonal antibody to connective tissue growth factor pamrevlumab to neoadjuvant chemotherapy would be safe and lead to improved resectability in this surgically adverse patient populationmethods in this phase iii trial patients with lapc were randomised to gemcitabinenab paclitaxel plus arm a n24 or minus arm b n13 pamrevlumab those who completed six cycles of treatment were assessed for surgical eligibility by protocol defined criteria resection rates progression free and overall survival were evaluatedresults eighteen patients in arm a and seven in arm b completed six cycles of therapy with similar toxicity patterns in arms a and b carbohydrate antigen response as defined by ¥ decline from baseline occurred in and respectively sixteen per cent of patients were radiographically downstaged by national comprehensive cancer network criteria in arm a and in arm b positron emission tomography normalised in vs of patients in arm a vs arm b respectively and correlated with surgical exploration eligibility for surgical exploration was vs p00019 and resection was achieved in vs of patients in arm a vs arm b p01193 respectively postoperative complication rates were not different between armss neoadjuvant chemotherapy with pamrevlumab holds promise for enhancing resection rates in patients with lapc without added toxicity this combination merits evaluation in a larger patient cohortintroductionpancreatic cancer is currently the third leading cause of cancer death in the usa1 and by it will likely become the second leading cause of cancer related death after key questionswhat is already known about this subject º pamrevlumab is anti ctgf1 inhibitor highly safe when given to patients with pancreatic cancer and potentially adds to the activity of gemcitabine and erlotinib when given in metastatic diseasewhat does this study add º this study examines a the safety and activity of pamrevlumab when added to gemcitabine and nab paclitaxel in locally advanced pancreatic cancer b the impact of this regimen and surgical resection and postoperative safety c explores the utility of a novel study design for locally advanced pancreatic cancerhow might this impact clinical practice º this study has the potential to lead to practice changing activity in locally advanced pancreatic cancer via the eventual approval of pamrevlumab for use in this situation the promulgation of a new study design for locally advanced pancreatic cancer and increased potential for surgical resection and thus prolonged os curability in locally advanced pancreatic cancerlung cancer surpassing breast and colon cancer2 surgical resection is generally necessary for treatment with curative intent or to extend life expectancy3 however only of patients have disease amenable to upfront curative resection at the time of diagnosis4 approximately of patients are diagnosed with locally advanced disease5 determined surgically unresectable per national comprehensive cancer network nccn guidelines6 patients with locally advanced pancreatic cancer lapc have a prognosis similar to those with metastatic disease with a historical median overall survival os of picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access months with recent trials demonstrating median os of months7 recent single institution retrospective studies have reported the potential for resection of lapc with neoadjuvant therapy irrespective of imaging findings with promising results8 however these are limited by significant selection bias lack of strict radiographic classification and chemotherapy standardisation current prospective trials have documented resection rates of lapc in the range of to therefore novel approaches are needed to improve patient outcomesthe tumour biology inherent to pancreatic ductal adenocarcinoma pdac significantly contributes to the poor outcomes seen in this disease notably pdac exhibits a high degree of desmoplasia often in association with elevated connective tissue growth factor ctgf expression12 ctgf appears to play a central role in the biology of pancreatic cancer affecting both its cellular biology and its extracellular matrix composition this leads to many simultaneous biological effects that promote pancreatic cancer growth including increased cellular proliferation and differentiation increased cellular adherence and migration antiapoptosis vascular permeability angiogenesis and suppression of tumour immunological responses13 this stroma may also contribute to the radiographic imaging findings of mesenteric vessel involvement or encasement that is used to determine resectability of pancreatic tumours executing a pharmacological intervention on the pancreatic cancer stromal environment is therefore a major goal of the development of novel pancreatic cancer therapeuticspamrevlumab is a human monoclonal antibody that targets ctgf preclinical studies showed that ctgf overexpression is associated with both desmoplasia and gemcitabine resistance in the kpc pancreatic cancer mouse model14 when pamrevlumab was used in combination with gemcitabine sensitivity to gemcitabine was enhanced which correlated with inhibition of xiap an antiapoptotic protein15 when tested in patients with advanced pancreatic cancer stage iv and locally advanced stage iii treated with gemcitabine and erlotinib in a phase iii study n75 pamrevlumab displayed multiple favourable outcomes16we hypothesised that through inhibition of the downstream effects of ctgf overexpression on tissue adhesion and other mechanisms pamrevlumab may influence resectability of pdac tumours with this in mind this novel phase iii randomised multicentre trial was designed to explore the safety and efficacy of pamrevlumab in combination with gemcitabinenab paclitaxel in lapc with special emphasis on surgical eligibility and safetymethodsstudy designthis was a phase iii randomised trial of safety and efficacy in patients with lapc who received gemcitabine and nab paclitaxel with or without pamrevlumab as neoadjuvant therapy the randomisation was preplanned and blinded to the investigator the study was approved by individual institutional review boards at nine us institutions and conducted according to the declaration of helsinki the trial was registered at clinicaltrials gov as nct eligibilitykey protocol eligibility requirements included biopsy proven diagnosis of pdac radiographic staging consistent with locally advanced unresectable disease as defined nccn guidelines v2 clinical stage confirmed by diagnostic laparoscopy radiographically measurable disease per response evaluation criteria in solid tumors recist v11 eastern cooperative oncology group ecog performance status of or adequate haematological renal and hepatic function no prior therapy for pdac and no concomitant cancer diagnosis within the past yearsstudy schemaeligible patients were randomised to arm a or arm b to receive a total of six treatment cycles weeks of therapy figure patients in arm a received pamrevlumab mgkg by intravenous infusion on days and of each day cycle with an additional dose given on day in the first cycle patients in both arms a and b received gemcitabine mgm2 by intravenous infusion on days and of each day treatment cycle nab paclitaxel mgm2 by intravenous infusion on days and of each day cycle doses for gemcitabine and nab paclitaxel were modified for haematological and non haematological toxicity as per standard of care soc15 patients remained on therapy for six treatment cycles weeks unless they had disease progression an intolerable adverse event ae or toxicity withdrew consent or were withdrawn at the investigators discretion all patients were followed for drug toxicity until days after the last drug dose patients undergoing surgery were followed for days following hospital discharge for surgical complications ctgf levels were obtained prior to treatment from all patients plasma samples for pamrevlumab level determination were obtained from all patients receiving this drug after all protocol specified therapy was completed patients were followed for disease progression survival and additional oncological therapy postoperative complications including day readmissions and day mortality were notedresponse assessmentpatients were evaluated for response by the following measures carbohydrate antigen ca measured at baseline first day of each cycle and end of treatment eot recist v11 read based on full body ct imaging high resolution dual phase fine cut ct imaging at baseline and every weeks thereafter fluorodeoxyglucose fdg positron emission tomography pet imaging and nccn v2 resectability criteria at baseline and eotpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessfigure patient flow and surgery outcomes in arm a four of the eligible subjects had their surgeries cancelled 1portal vein thrombosis 3medical issues precluding surgery in arm a four eligible subjects underwent surgery but resection was not achieved 3metastatic disease discovered 1extensive sma encasement in arm b one eligible subject underwent surgery but resection was not achieved 1extensive vascular encasement sma superior mesenteric arterysurgical assessmentsubjects who finished six cycles of combination chemotherapy were evaluated for eligibility for surgical exploration per protocol pp defined criteria given that patients included in the trial were determined to be initially unresectable by radiographic imaging and nccn criteria objective criteria were developed to standardise attempts at surgical resectionpatients were deemed eligible for surgery if one or more of the following criteria were met reduction in plasma ca level by ¥ at eot compared with baseline reduction in fdg pet maximum standardised uptake value suvmax by ¥ at eot compared with baseline radiological tumour response per recist of partial response pr or complete response cr at eot or met the definition of resectable or borderline resectable per nccn guidelines subjects were classified as ineligible for surgical exploration if any of the following occurred development of distant metastases or local progression on ct scan tumour anatomy precluding vascular reconstruction unreconstructible local complications preventing surgery eg portal vein pvsplenic vein thrombosis pancreatitis or decline in performance status to a karnofsky score ¤ or picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668absolute contraindication to surgery from comorbidity eg recovery from myocardial infarction or uncontrolled diabetes the final decision regarding whether resection was to be performed was made by the treating surgeonendpointssafety endpoints included serious adverse events sae during neoadjuvant therapy and surgical complications postresection the efficacy endpoints included surgical eligibility r0 resection r0r1 resection median os progression free survival pfs and year survival rate all patients were followed and data analysis was stratified by pp population and intention to treat itt cohortstatistical considerationsthe comparison between selected clinical characteristics toxicity profiles and eligibility for surgical exploration or completed surgical resection was performed using the ϲ test exact cis for the point estimates as well as the treatment difference were obtained from the sas proc freq procedure with the exact option the two treatment arms were compared using the cochran mantel haentzel test controlling for baseline factors tnm stage ecog ca pet suvmax 0copen accesssuperior mesenteric artery sma involvement coeliac abutment and so on as prespecified in the protocol all cause mortality was used in determining os which was analysed by the kaplan meier method survival status was updated within month before the data cut off date data from patients who were alive at the cut off date were censored for survival analysis all statistical tests were performed at the significance level of α005 using two sided testsresultspatient characteristics and dispositionthirty seven patients were randomised to study treatment to arm a pamrevlumabgemcitabinenab paclitaxel and to arm b gemcitabinenab paclitaxel alone patient characteristics at baseline are summarised in table all patients enrolled were unresectable by nccn criteria patients had tumour arterial involvement sma encasement ° coeliac abutment table patient characteristicsbaseline demographics years years ¥ years median male femaleage group sex bmi kgm2 mean sd median min maxecog grade grade tnm stage t3 n0 m0 t3 n1 m0 t4 n0 m0 t4 n1 m0 t4 nx m0location of the tumour in the pancreas non resectability per nccn criterion head body tail median tumour size mm ° sma encasement any coeliac abutment inferior vena cava invasion or encasement unreconstructible smvportal occlusion aortic invasion and encasementarm agnppn24 arm bgnpn13 totaln37 to to to · ok as isnot mutually exclusivebmi body mass index ecog eastern cooperative oncology group g gemcitabine n number of subjects nccn national comprehensive cancer network np nab paclitaxel p pamrevlumab pv portal vein sma superior mesenteric artery smv superior mesenteric veinpicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accesswithout gastroduodenal artery involvement or aortic involvement inferior vena cava invasion and unreconstructible pvsuperior mesenteric vein smv occlusion a higher percentage of patients with sma encasement ° were randomised to arm a vs arm b patient disposition is summarised in figure twenty four patients in arm a received gemcitabinenab paclitaxel and pamrevlumab patients completed six treatment cycles six patients discontinued treatment early due to progressive disease three patients aes two patients or physician decision one patient thirteen patients in arm b received gemcitabinenab paclitaxel patients completed six treatment cycles six patients discontinued treatment early due to progressive disease two patients aes two patients or patientphysician decision two patientssafetysaes are summarised in table forty one per cent of patients had a treatment emergent sae arm a arm b no individual toxicity category occurred with frequency except systemic infection patients there was no demonstrable increase in any toxicity with the addition of pamrevlumab to gemcitabinenab paclitaxel chemotherapytable summary of treatment emergent serious adverse eventssystem organ classpreferred term ascites nausea pancreatitis vomiting device occlusion drug withdrawal syndrome feverno of patients with any treatment emergent saeblood and lymphatic disorders haemolytic uremic syndrome lymphadenopathycardiac disorders cardiac failure supraventricular tachycardiagastrointestinal disorders general disorders and administrative site conditions hepatobiliary disorders infections sepsis cellulitis urinary tract infectioninjury poisoning and procedural complications respiratory thoracic and mediastinal disorders skin and subcutaneous disorders cholangitis hyperbilirubinaemia craniocerebral injury pneumonitis pulmonary embolism rasharm an24n arm bn13n overalln37n · ok as ispicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen accessresponse to therapyin arm a had ¥ ca decline at eot response by recist pr ¥ decline in pet suvmax and were radiographically downstaged by nccn criteria during the treatment period the median ca decline was patients were non secretors seven out of patients had best objective recist response crpr some patients had exceptional responses defined as normalisation or ¥ decline of ca patients or normalisation pet suvmax in in arm b had ¥ ca decline at eot response by recist pr ¥ decline in pet suvmax and were radiographically downstaged by nccn criteria four out of patients had best objective recist response cr pr in arm b of patients had an exceptional ca response and had an exceptional pet response as defined by either ¥ normalized ca response normalized suv max andorradiographic downstaging post therapy completion surgical evaluationoverall of the total study patients were eligible for surgical exploration using protocol defined criteria arm a arm b p00019 resection was completed in of the patients arm a arm b p01193 details of the nine resected patients are shown in table in arm a of the patients were eligible for surgical exploration in the itt population and of the patients were eligible in the pp population patients who completed six cycles of treatment in arm a out of eligible patients ultimately underwent surgical exploration for resection five operations were cancelled four due to drug toxicitymedical comorbidity sepsis gallbladder perforation declined performance status and fever and one patient declined eight out of patients in arm a were resected r0 r1 the remaining four patients who were explored were not resected due to progression or unresectable disease intraoperatively in arm b of the patients were eligible for surgical exploration in the itt population and were eligible in the pp population of the two subjects found to be surgically eligible only one was resected as the other patient had local progressionpredictors of resectionhigh ca response ¥ decline andor normalisation was contributive to surgical eligibility vs p03 normalisation versus non normalisation of pet suvmax was predictive of surgical eligibility vs p0013 and successful resection vs p0002 combining these two criteria was highly predictive for surgical eligibility vs p0003 and completed vs p0002 resection all nine successful resections were identified by one or both of these criteria table summary of resected patientssitesubject idtreatmentarmresponse to treatmentnccnbaselinenccnend of treatmentresection statusaaaaaaaab unresectablecoeliacunresectablesma smvunresectablecoeliacunresectablecoeliacunresectablesmvunresectablesmaunresectablesma smv coeliacunresectablesmaunresectablecoeliacunresectablecoeliacunresectablesma smvunresectablecoeliacborderline resectableunresectablesmvunresectablesmaunresectablecoeliacunresectablesmaunresectablecoeliacr0r1r0r0r1r1r1r0r0protocol defined criteria ca decrease fdg pet suvmax decrease ¥ recist v11 response pr or cr nccn resectable or borderline resectable criteriaca carbohydrate antigen cr complete response fdg fluorodeoxyglucose nccn national comprehensive cancer network pet positron emission tomography pr partial response recist response evaluation criteria in solid tumors sma superior mesenteric artery smv superior mesenteric vein suvmax maximum standardised uptake valuepicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cconversely radiographic features of response did not correlate with operative potential neither recist response nor radiographic downstaging per nccn criteria statistically correlated with completed resectionsurgical complicationspostoperative complications were summarised according to the clavien dindo classification posthoc analysis ischaemic gastritis and ulceration and right lower lung lobe collapse were reported for one patient in arm a grade ii there was one episode of clinically significant pancreatic leak in each arm grade iiia no reoperations and no day or day surgical mortality were noted one patient in arm b had a delayed gastric perforation following a distal pancreatectomy with coeliac axis resection likely due to thermal injury and was treated non operatively grade iiib no wound complications or superficial site infections were noted in either group four out of patients and out of patients in arm a and b respectively were readmitted within days and the difference between treatment arms was not clinically or statistically significantsurvivalas of the data cut off date of evaluable patients were known to be alive with a median length of follow up at approximately months pfs was months ci to and months ci to in arm a and arm b respectively one year survival and median os were and months ci open accessto in arm a and and months ci nr in arm b the median os for all patients who were eligible for surgical exploration arm a arm b vs ineligible arm a arm b was months ci nr vs months ci to p00766 the median os for resected arm a arm b vs non resected patients arm a arm b was not reached ci nr vs months ci to p00141 figure discussionthe treatment of lapc with neoadjuvant therapy remains challenging and there is no established soc several high volume centres have reported their single centre experiences with varying neoadjuvant chemotherapy and chemoradiation strategies8 the combination of more active regimens delivered over an extended period and surgeons comfort with resecting and reconstructing major mesenteric vessels has led to an increase in resection rates a meta analysis of studies using folfirinox has demonstrated resection rates ranging from to in lapc17 one of the larger studies including patients with lapc reported a resection rate of that was more dependent on duration of therapy months than chemotherapy regimen folfirinox or gemcitabine based18 recently a single institution and single arm prospective study of neoadjuvant folfirinox and losartan with selective use of radiation in patients with lapc reported an r0 resection rate of figure overall survival resected vs non resected patientspicozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0copen access however the lack of randomisation makes it difficult to determine what aspect of therapy was responsible for this effect and only of resected patients needed any type of vascular reconstruction perhaps suggesting more favourable outcome than usually seen in locally advanced disease these retrospective studies contain significant interpretative challenges including selection bias treatment variables including radiation and the use of different definitions of lapcthe anti ctgf mechanism of action with respect to gemcitabine based therapy a recent large scale prospective trial of patients with lapc treated with induction gemcitabine with or without erlotinib followed by radiation only patients were able to undergo resection10 furthermore the addition of erlotinib to gemcitabine did not improve any downstaging capacity and there was no difference in survival with the addition of radiation more recently the la pact trial examining the role of induction gemcitabine nab paclitaxel found that only out of patients with lapc were able to undergo surgery following neoadjuvant therapy with combination gemcitabine and nab paclitaxel for six cycles by investigators choice11 last although folfirinox has been the most studied induction combination chemotherapy regimen in this population recent randomised data from european patients who received neoadjuvant folfirinox versus gemcitabinenab paclitaxel prior to resection20 showed no clear difference with respect to r0r1 to resection rate vs p0135 or os vs months p0268given for pamrevlumab its use in the neoadjuvant setting has the potential to impact tumour regression and modulate the desmoplastic niche and possibly affect tumour margins allowing for improved resection rates previous studies have looked at the ability of gemcitabinenab paclitaxel to reduce cancer associated fibroblasts resulting in a softening of tumours by endoscopic ultrasound elastography21 this stromal depletion also translated into a decrease of suv uptake on pet22 in the study reported herein we combined gemcitabine and nab paclitaxel with pamrevlumab to explore its effect in terms of therapeutic response the impact on eligibility for surgical exploration and improved resection rates in locally advanced patientsthe protocol specified therapeutic response criteria ca pet suvmax recist and nccn criteria were used as criteria to determine eligibility for surgical exploration in lapc this is a novel approach specific to the protocol and allows participating patients to be explored for resection when otherwise they may not qualify by current treatment standards nccn criteria for example by nccn conversion alone ie converted from unresectable to borderline resectable only of patients in arm a would have been eligible for surgical exploration however by protocol criteria of patients in arm a were eligible for surgical exploration a higher percentage of patients were eligible for surgical exploration by the above criteria in arm a vs arm b vs respectivelyoverall the rate of successful resections in the pamrevlumab treated group was higher than in the control group but this did not reach statistical significance most probably due to small sample size of the nine subjects that were successfully resected in this trial only one was converted by nccn criteria to borderline resectable prior to surgical exploration despite this phenomenon the data support the hypothesis that pamrevlumab a human monoclonal antibody with anti ctgf mechanism of action could alter tumour characteristics allowing resection in otherwise unresectable patients this hypothesis needs to be confirmed and patients should be stratified by coeliac andor sma involvementthe most common predictive factors for eligibility for surgical exploration and resection were ca decline and pet suv max response which are indicators of tumour response to treatment the combination of these two factors proved to be a highly sensitive objective readout for prediction of potential surgical success both the ability of ca response and the inability of radiographic response recist and nccn criteria of resect ability to predict surgical outcome has been observed by others3 and these observations deserve further examination in subsequent clinical trials in the mpact study both ca and pet response correlated to improved survival in metastatic patients treated with gemcitabine and nab paclitaxel23 recent surgical series of patients with borderline resectable and lapc have also corroborated their impact in the localised setting25 correlation of clinical response with plasma levels of endogenous ctgf and pamrevlumab exposure as shown in the prior study by picozzi et al16 may provide added prognostic and predictive insightwith regard to safety no major incremental toxicity in any category was noted with the addition of pamrevlumab to gemcitabinenab paclitaxel in addition a higher number of patients were able to complete six cycles of the three drug combination including pamrevlumab when compared with gemcitabinenab paclitaxel alone pamrevlumab is well tolerated and considered safe compared with the soc drugs for patients with pdac these observations represent a very favourable attribute when considering potential neoadjuvant chemotherapy in a patient population with the frequency of medical problems typically seen in lapc in addition there were no signals of increased surgical morbidity or wound healing problems with ctgf blockade by pamrevlumab in fact there were only two clinically significant pancreatic leaks one in each arm which is comparable to national outcome data from high volume pancreatic surgery centres similarly readmissions following resection were comparable between arms and reflected the complexity of this challenging patient populationfinally while survival data are not yet mature both patients who were eligible for surgery and those that picozzi a0v et a0al esmo open 20205e000668 101136esmoopen2019000668 0cwere ultimately resected had longer pfs and os highlighting the importance of surgical resection of the tumour therefore more investigation into newer agents targeting lapc and increased consideration of candidacy for surgery in those patients who do not progress on therapy or suffer toxicity should be of utmost importance to improve outcomes in this diseasein this is the first prospective randomised multicentre trial examining the role of neoadjuvant therapy in lapc with prespecified criteria for surgical exploration the use of pamrevlumab in combination with gemcitabine and nab paclitaxel showed a potential to enhance tumour response and increase resection rates further evaluation of this drug combination in the neoadjuvant treatment setting for lapc is warranted and a larger phase iii trial with resection and survival endpoints is ongoingcontributors fibrogen inc was the study sponsor that designed the study in consultation with the principal investigator vp and surgical co investigator fgr all authors except those of the sponsor contributed patients to the study fibrogen was responsible for data collection and analysis all authors reviewed the manuscript and signed off on its accuracyfunding the study was funded by fibrogen inc san francisco cadisclaimer the corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors an exclusive licence or non exclusive for government employees on a worldwide basis to the bmj publishing group ltd and its licensees to permit this article if accepted to be published in esmo open editions and any other bmjpgl products to exploit all subsidiary rights as set out in our licencecompeting interests mc mz sp ek and ec are employees of fibrogen and hold stock andor stock options in fibrogenpatient consent for publication not requiredprovenance and peer review not commissioned externally peer revieweddata availability statement data are available on reasonable request all data relevant to the study are included in the article or uploaded as supplementary information data will be available as presented in this manuscriptopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idewa a0carrier http orcid org references american cancer society cancer facts figures available httpswww cancer org content dam cancer org research cancer facts and statistics annual cancer facts and figures cancer facts and figures pdf rahib l smith bd aizenberg r et a0al projecting cancer incidence and deaths to the unexpected burden of thyroid liver and pancreas cancers in the united states cancer r
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this study hypothesizes that bromelain bl acts as radiosensitizer of tumor cells and that it protects normal cells from radiation effects in vitro and in vivo studies have been carried out to prove that assumption in vitro mtt cell proliferation assay has shown that the irradiated ehrlich ascites carcinoma eac cell line could be sensitized by bl pretreatment in vivo animals were randomly divided into groups group control pbs ip for days group ehrlich solid tumor est bearing mice group est Îradiation fractionated dose gy group est bl mgkg ip daily for days group est bl for days followed by Îirradiation gy the size and weight of tumors in gammairradiated est bearing mice treated with bl decreased significantly with a significant amelioration in the histopathological examination besides bl mitigated the effect of Îirradiation on the liver relative gene expression of poly adp ribose polymerase1 parp1 nuclear factor kappa activated b cells nfκb and peroxisome proliferatoractivated receptor α pparα and it restored liver function via amelioration of paraoxonase1 pon1 activity reactive oxygen species ros content lipid peroxidation lpo and serum aspartate transaminase ast alanine transaminase alt and albumin alb it is concluded that bl can be considered as a radiosensitizer and radioprotector suggesting a possible role in reducing radiation exposure dose during radiotherapykeywordsbromelain tumor Îradiation radiosensitizer radioprotectorsubmitted april revised july accepted july introductionradiotherapy has been used for a long time in treating cancer1 however from the clinical perspective radiotherapy provides inadequate success due to the radioresistance of many tumors as well as the high risk of recurrence and effects on normal cells may occur23 radioresistance occurs as the microenvironment of solid tumors is hypoxic compared with normal tissue4 in addition some tumors have either an intrinsic resistance to ionizing radiation or can attain this property through accumulation of genetic mutations causing an increased survival and proliferation5 thus strategies to improve radiation therapy could include increasing resistance of normal tissues to radiation andor increasing sensitivity of the tumor cells6radiosensitizing agents increase the sensitivity of tumor cells via enhancing the generation of reactive oxygen species ros increasing lipid peroxidation depletion of glutathione which leads to dna damage inhibition of dna repair inhibition of dna synthesis induction of cell cycle arrest induction of apoptosis and inhibition of proliferation7 numerous nutritive cancer chemopreventive compounds having antioxidant properties have been recognized to potentiate radiation therapyinduced cytotoxic 1drug radiation research department national centre for radiation research and technology egyptian atomic energy authority nasr city cairo egypt2biochemistry department alazhar university cairo egyptcorresponding authorhanan a fahmy drug radiation research department national centre for radiation research and technology atomic energy authority p o box nasr city cairo egypt email fahmyhananyahoocomcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies effects on cancer cells inversely decreasing its toxicity on normal adjacent tissues89 in this regard much research has aimed to develop numerous antioxidant drugs of both natural and synthetic origin tested in both in vitro and in vivo models and also human clinical trials to overcome injuries caused by ir exposure and to induce killing of cancer cells at the same time previous studies have reported that phytochemical soy isoflavones genistein daidzein and glycitein which exhibit anticarcinogenic properties through their antioxidant activities could be used as potent radiosensitizers to enhance the efficacy of radiotherapymediated suppression of the growth and metastatic ability of cancers1011 along parallel lines resveratrol and piperine which possess antitumor activity have been shown to augment ionizing radiation irinduced apoptosis and loss of mitochondrial membrane potential in murine colon carcinoma and melanoma cells via enhancing irinduced ros generation12 moreover pentoxifylline ptx a methylxanthine that possesses antioxidant properties is known for improving tumor tissue oxygenation in murine hypoxic tumors and inhibiting post radiation induced normal tissue injury in mice1314 consequently searching for a natural product possessing anticancer activity that increases radiosensitivity of tumor cells and radioresistance of normal cells may lead to a potential future drug in cancer therapyamong the natural products bromelain bl extract attracts interest due to its anticancer antioxidant as well as antiinflammatory effects1517 bl an extract from pineapple stem ananas comosus belongs to a group of protein digesting enzymes it is a mixture of diï¬erent thiol endopeptidases and other components like phosphatase glucosidase peroxidase cellulase escharase calcium and several protease inhibitors1819 the anticancer activity of bl has been examined in various types of gastrointestinal and breast cancers cell lines in in vivo models bl has shown antimetastatic effect and reduction in local tumor growth2023 it is also used for reducing the severity of such radiation therapy side effects as mucositis skin reactions and dysphagia in patients24 hence this study was aimed to evaluate the radiosensitizing and radioprotective effect of bl using in vivo and in vitro approachesmaterials and methodin vitro studiesmtt cell proliferation assay the growth and viability of ehrlich ascites carcinoma eac cell line were tested in vitro by 345dimethylthiazol2yl25diphenyltetrazolium bromide mtt assay according to freimoser and buch 2526 to verify the antitumor and radiosensitizing effect of bl two plates were designed for this study the first one contained eac cells maintained by serial subculturing at the national cancer institute egypt incubated for hour before irradiation irr gy alone and with different concentrations of bromelain bl in phosphate buffer saline pbs the second one contained eac cells serving as a control and eac with different concentrations of bl each test was seeded in triplicate into a plate at concentration of cellswell containing rpmi media with fbs nahco3 uml penicillin and µgml streptomycin and each plate was incubated for hours at °c in co2 and humidity atmosphere then μl mtt reagent bio basic inc canada was added over the cells in each well and the plate was incubated in the dark for to hours until a purple precipitate was seen and the absorbance was measured at nm the amount of color produced was directly proportional to the number of viable cells viable cell a samples a blanka control a blank the inhibitory concentration ic50 is the dose of a drug which reduces the viability to and was calculated using nonlinear regression analysisfree radical scavenging assay the antioxidant activity of bromelain was evaluated by 1diphenyl2picrylhydrazyl dpph radical assay and its scavenging power was compared with some antioxidants naringin polyphenolic antioxidant garlic oil and glutathione sulfur containing antioxidants about µl of samples mgml dissolved in dist water was added to µl of a solution of dpph g100 ml dissolved in vv methanol after minutes incubation at room temperature in the dark the absorbance was read at nm against a blank µl dist water µl dpphmethanol solution the experiments were done in triplicate according to the method of braca 27 glutathione mgml was used as a standard antioxidant the scavenging percentage of dpph was calculated according to the followscavengin ing equation where b was the absorbance of the blank and a was the absorbance of samples or standard ec50 is defined as concentration of sample that causes dpph loss there values were calculated using nonlinear regression analysisb ab\uf8ee\uf8ef\uf8f0\uf8f9\uf8fa \uf8fbin vivo studiesradiation processing whole body Îirradiation of mice was carried out using gamma cell40 137cesium manufactured by the atomic energy of canada limited ontario canada installed in the national center for radiation research and technology ncrrt cairo egypt the dose rate was gymin during the experimental period daily correction for humidity barometric pressure and temperature were madeanimals adult female swiss albino mice weighing to g obtained from the breeding unit of ncrrt cairo egypt all animal procedures were performed in accordance with the committee of scientific ethics at faculty of 0cmekkawy table sequences of primers for realtime quantitative pcrgeneparp1 nm0074152nfκb nc0000696pparα nc0000816βactin nc0000716forward primerreverse primer²ccatcgacgtcaactacga3²²caatggctacacaggacca3²²actccacctgcagagcaacca3²²gcgtggggacagccgcatctt3²²gtgcgtggtagcatgagtgt3²²cactgtcacctggaaccaga3²²tagatctcctgcagtagcggg3²²atcggcagaaggggcggaga3²pharmacy alazhar university egypt following the guidelines for animal use the animals were housed in colony cages micecage under proper environmental conditions that is hours darklight cycle good ventilation condition and temperature to humidity at the ncrrt animal house fed with standard diet pellets and provided with water ad libitum animals were left week for acclimatization on the lab environment before starting the experimenttumor transplantation the eac cell line was supplied by serial subculturing at the national cancer institute cairo university egypt it was implanted in each donor female swiss albino mice by ip injection of cells22 g b wt and allowed to multiply28 the ehrlich solid tumor est was obtained by the intramuscular inoculation of ml of viable eac in the right lower limb of each mouse29 mice with a palpable solid tumor diameter mm3 that was maintained within to days after inoculation were used in the studyanimal grouping animals were randomly divided into groups mice each group control not bearing tumor received pbs ip for days group ehrlich solid tumor est bearing mice received pbs ip for days group est Îirradiation gy fractionated doses starting days after tumor appearance mm3 and lasting for days group est bearing mice receiving freshly prepared bl dissolved in pbs mgkg ip daily for days according to pilot study starting once est becomes mm3 bl was purchased from merck kgaa co darmstadt germany group est bearing mice received bl as in group hours before Îirradiation as in group mice were anesthetized days after last irradiation dose using urethane mgkg30 blood samples were collected through cardiac puncture and divided into parts edta coated and plain vials at that time they were euthanized by cervical dislocation liver and tumor tissues were dissected out rinsed with icecold saline dried on a filter paper and weighed then homogenized in icecold pbs ph and stored at °c until used for subsequent biochemical analysisestimation of total body tumor and liver weights animals in each group were checked daily for any adverse clinical symptoms and deaths after to days post inoculation with eac body weights were recorded so body weight change could be estimated tumor and liver weights were measured during sample collection and then the tumor inhibitory ratio was calculated by the following formula inhibition ratio aba where a is the tumor weight average of the control and b is that of the treated group also relative liver weight was calculated as liver weighttotal body weight histopathological examination three tumors of each group were collected and fixed in neutral buffered formalin the specimens were dehydrated in ascending grades of ethyl alcohol cleared in xylene and embedded in paraffin wax four micron thick paraffin sections were mounted on clean slides stained with ehrlichs hematoxylineosin he31 and examined using an olympus microscope bx41 hamburg germany histopathological evaluation was done by assessment of necrosis and calculation of tumor area percentage using image analysis software image j 146a nih usa through the following equation of tumor area area of tumortotal area of the field molecular analyses the mrna levels of poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα genes and of the housekeeping gene βactin were measured by real time polymerase chain reaction rtpcr total rna was isolated from liver tissues using qiagen tissue extraction kit qiagen usa in accordance with the manufacturers instructions the extracted rna μg was used for cdna conversion using high capacity cdna reverse transcription kit fermentas usa and μl reaction volume sybr chemistry in applied biosystems thermal cycler usa to amplify pcr under the following conditions °c for denaturation then °c to °c for annealing using primers mentioned in table and °c for elongationresults were expressed using the comparative ct method for relative mrna quantification of target genes normalized to an endogenous reference βactin and a relevant control equal to ct ct is the difference between the mean ctsample and mean ctcontrol where ctsample is the difference between the mean ctsample and the mean ctβactin and ctcontrol is the difference between the mean ctcontrol and the mean ctβactin 0c integrative cancer therapies estimation of lipid peroxidation lpo reactive oxygen species ros and paraoxonase pon1 in liver homogenate liver lipid peroxidation was estimated by measurement of malondialdehyde mda formation using the thiobarbituric acid method of yoshioka 32 a modified technique of vrablic 33 was used to measure the generation of ros by the intracellular conversion of nitro blue tetrazolium nbt to formazan by the action of superoxide anion paraoxonase activity was estimated by using fluorometric assay enzchek® kit invitrogen uk for the anophosphatase activity of paraoxonase based on the hydrolysis of a fluorogenic anophosphate analog34hematological and biochemical analyses whole blood was immediately analyzed for complete blood count with platelet count using the fully automated analyzer abx cobas micros roche germany estimation of serum alanine aminotransferase alt aspartate aminotransferase ast and albumin alb assays follow the recommendations of the international federation of clinical chemistry ifcc but were optimized for performance and stability using the rochehitachi cobas c 311systemstatistical analysis the statistical analysis was performed using oneway analysis of variance anova and the groups were compared by tukeykramer test viability percentage at different concentrations and body weight change analyzed by twoway anova followed by bonferronis posttest graphs were sketched using graph pad prism isi® software usa version software data were presented as mean ± standard error se and p values considered significantresultsin vitro studieseffect of bromelain and gammairradiation blirr on tumor cell growth and viability the radiosensitizing effect of bl on eac cells was determined by performing mtt assay eac cells exposed to gy Îradiation showed high cell viability percentage reflecting a radioresistance of eac cell line while bl treatment showed in vitro cytotoxic activity with ic50 value of mgml however the maximum cytotoxic effect appeared when the eac cells were subjected to bl then Îradiation gy compared to control or irradiated group with ic50 mgml table effect of bromelain and some natural compounds as free radical scavengers the inhibitory percentage of each compound is shown in figure the ec50 value concentration of sample that causes dpph activity loss is a reliable way for estimation of the radical scavenging activity the ec50 value of glutathione referenced antioxidant is mgml while table cytotoxic activity of blirr against eac cell line bromelain concentration mgmleac bl mgmlic50 mgmlviability non irradiated eac irradiated eac48ab59ab60ab69a787a10ab158ab18ab27ab52ab ± ± each value indicates the mean of records statistical analysis carried out by twoway anova followed by bonferroni posttests a significant versus control ehrlich ascites carcinoma eac group where b significant versus irradiated eac group at p ic50 ± se values were calculated by using nonlinear regression analysisbromelain and garlic oil ec50 are almost equal and mgml respectively however the naringin phenolic antioxidant is the least potent one ec50 mgml in this comparisonin vivo studieseffect of bromelain and gammairradiation blirr on tumor weight and volume table shows a significant decrease in tumor weight in groups treated with bl andor Îirradiation as compared to the est nontreated group the more drastic decrease in the tumor weight ratio observed in the combined therapy group bl irr compared with the estirradiated group as well as est group indicates that combination therapy is more significantly effective than single agent therapy the photograph of est xenografts at the time of sacrifice shows the synergistic effect of bl and irr on tumor volume figure effect of bromelain and gammairradiation blirr on tumor histopathological features of est bearing mice histopathological examination of solid tumor sections revealed typical malignant features including sheets of malignant cells infiltrating adjacent muscular tissue the malignant cells show pleomorphism hyperchromatism and mitotic activity while the necrotic cells appear as nonviable homogenous structureless material with degenerated or karyorrhectic nuclei untreated est bearing group shows a deeply stained tumor cells arrow head and areas of necrosis arrow figure 3a also it displays intact cancer cells arrow and giant cells arrow figure 3b and c the estirradiated group shows muscle fibers invaded by deeply stained tumor cells arrow head and large areas of necrosis arrow figure 3d also displays a notable necrosis of cancer cells n figure 3e the bl treated group shows a 0cmekkawy figure dpph 1diphenyl2picrylhydrazyl reduction curve for glutathione bromelain naringin and garlic oil each value represents mean ± se all experiments were replicated timestable tumor weight and inhibition ratio of ehrlich solid tumor estbearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupsestest irrest blest bl irrtumor weight g ± ± 004a ± 005a ± 005abtumor inhibitory ratio ± 24a ± 14ab ± 204ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe values shown are mean ± se of data a significant versus est group where b significant versus estirradiated group at p figure a photograph of ehrlich solid tumor est xenografts at the time of scarification showing the effect of bromelain and gammairradation blirr on tumor volume e ehrlich solid tumor e ir ehrlich solid tumor irradiation e br ehrlich solid tumor bromelain e ir br ehrlich solid tumor bromelain irradiation 0c integrative cancer therapies figure photo micrograph of ehrlich solid tumor est xenografts in different animal groups est sections show the degree of tumorogenesis necrosis n regression of tumor by appearance of muscle fibers m and a b c est ehrlich solid tumor d e est irr ehrlich solid tumor irradiation f g est bl ehrlich solid tumor bromelain h i est bl irr ehrlich solid tumor bromelain irradiationwide area of necrosis arrow and n few groups of cancer cells arrow head and muscle fiber m figures 3f and 4g however combined treatment bl irr displays muscle fiber m significant regression of tumor or wide areas of necrotic cancer cells n and few groups of intact cancer cells arrow figure 3h and 3i the tumor area percentage per total tissue area could determine the degree of proliferation as seen in figure there is a great regression of tumor area in the group treated with bl alone or bl and irr compared with untreated est or estirradiated group indicating that combination therapy significantly more effective than single agent therapy 0cmekkawy bl irr shows nonsignificant group change additionally bl irr group significantly upregulated pparα expression compared with est and estirradiated groups indicating that bl might have a hepato as well as radioprotective effect figure effect of bromelain and gammairradiation blirr on the hepatic lipid peroxidation lpo level reactive oxygen species ros content and paraoxonase1 pon1 activity of ehrlich solid tumor est bearing mice lpo in liver tissues significantly increased in all est bearing groups compared to the control group except the combined treated group irr bl succeeded in returning mda lpo measured as mda malondialdehyde level to the normal level however liver ros significantly increased only in untreated and Îirradiated est bearing groups when compared to the control group while a significant decrease in liver ros showed in estirradiated mice treated with bl in comparison with both est untreated and estirradiated groups pon1 activity in liver homogenate was significantly decreased in est untreated and estirradiated groups when compared with the control group bl treated groups revealed significant increases in pon1 when compared with both est untreated and estirradiated groups showing that bl might have a hepato and radioprotective effect figure effect of bromelain and gammairradiation blirr on hematological measurements wbcs and plts were significantly elevated while hgb and hct significantly decreased in the untreated estbearing mice in comparison with control mice however Îirradiation resulted in a significant decrease in wbcs rbcs plt hgb and hct compared with the control mice treatment of the estbearing mice with bl shows a significant amelioration in wbcs plt and hct compared to est untreated mice combined treatment bl irr shows an enhancement in wbcs plt and hct compared to est untreated and gammairradiated est bearing mice table effect of bromelain and gammairradiation blirr on the serum alanine transaminase alt aspartate transaminase ast and albumin alb to investigate the cytoprotective effects of bl against irradiation the levels of serum alt ast and alb were measured figure it was found that alt and ast significantly increased and conversely alb significantly decreased in est untreated and estirradiated groups compared with the control group however estbearing mice treated with bl alone show nearly the same result of alt and alb as control values estbearing mice treated with bl in combination with irradiation initiated a significant decrease in ast and alt as compared with estirradiated group which may reflect a potential hepatic radioprotective effect of blfigure percentage of tumor areatotal tissue area of ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bl mgkg the values shown in the plotted area are mean of records from animals ± se significant versus est group where significant versus estirradiated group at p effect of bromelain and gammairradiation blirr on body weight change and relative liver weight regarding the day by day documented recording of body weight bwt illustrated in figure there is almost no change in bwt of bl treated group while it increases significantly in the untreated est group conversely estirradiated groups with or without bl treatment show a significant decrease in bwt when compared with the control group table relative liver weight was compared after normalization to mg body weight untreated estbearing group shows a significant increase in liver weight by hepatomegaly while nonsignificant changes were observed in bl treated groups compared to the normal group table effect of bromelain and gammairradiation blirr on the poly adpribose polymerase parp1 nuclear factor kappa b nfκb and peroxisome proliferatoractivated receptors pparα relative gene expression of ehrlich solid tumor est bearing mice to test the possibility that bl reduces radiation damage to the liver mrna gene expression of parp1 nfκb and pparα was measured in the liver homogenates of est bearing mice and compared to control pbs treated mice the results illustrated in figure show that irr causes significant increases in parp1 and nfκb expression compared to the control group however combined treatment bl irr shows a significant increase in parp1 and nfκb expression compared to control group and a significant attenuation compared to estirradiated groupmoreover all est bearing groups show significant decreases in hepatic pparα relative gene expression compared to the control group except the combined therapy 0c integrative cancer therapies figure effect of bromelain and gammairradiation blirr on body weight during experiment period each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationtable change in body weight and relative liver weight of control mice and ehrlich solid tumor est bearing mice treated with gammairradiation irr gy andor bromelain bl mgkggroupscontrolestest irrest blest bl irrbody weight change ± ± 101a ± 217ab ± 201b ± 341abrelative liver weight ± ± 026a ± 022a ± 026a ± 026ababbreviations est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationeach value represents the mean ± se a significant versus control group where b significant versus est group at p body weight changes percent are related to the initial weight of animalsdiscussionresistance of tumor cells to chemoradiotherapy as well as the damaging effects to nearby normal tissues remains a major obstacle to successful cancer management therefore the current study has been conducted to estimate the effect of bromelain bl as a tumor radiosensetizer and to show to what extent it can protect normal tissue from radiation hazardsradiosensitizers are compounds that when combined with radiation therapy achieve greater cytotoxicity they can be determined in vitro by the mtt assay2635 the present study has found that the radioresistant eac cells could be sensitized when incubated with bl before irradiation it was known previously that in vitro treatment with bl on mouse tumor cell lines resulted in inhibition of cell growth and invasion capacities3637 the anticancer property of bl has been mainly attributed to the protease component through digestion and diffusion in tumor cells38 it may also be due to the bl enhancement of p53 expression as well as another activator of apoptosis eg bax39 in addition it decreases the activity of cell survival regulators such as akt and erk it also deactivates aktdependent proapoptotic regulator foxo3a thus promoting apoptotic cell death in tumors40it is well known that during cancer and radiotherapy excessive energy is used from the host41 ultimately contributing to mechanisms that promote loss of weight as shown in the present study which also showed that bl could return body weight to a normal level by decreasing tumor weight and volume currently the combined therapy bl irr has been shown to be more effective than single agent therapy in reducing tumor volume and weight indicating that bl could possess a radiosensitizing effect in addition the combined therapy has revealed a drastic decrease in tumor area percentage wide areas of necrotic cancer cells and presence of muscle fiber in the histopathological examination compared with the control est and estirradiated groups this seems to be in agreement with other findings of the role of bl in reducing metastasis and local tumor growth2342 in chemically induced mouse skin papillomas topical application of bl reduced tumor formation tumor volume and caused apoptotic cell death39 bl is a hydrolytic enzymatic complex which shows an efficient digestion and diffusion in tumor cells through attacking the glycosidic linkages and hence denatures glycoproteins thus it protects against tumor growth37 another study has demonstrated the use of controlled proteolytic activity on tumor as a successful strategy to increase therapeutic efficacy43 0cmekkawy figure effect of bromelain and gammairradiation blirr on relative gene expression of liver a parp1 b nfκb and c pparα each value represents the mean of records ± se significant versus control group where significant versus est group and significant versus estirradiated group at p est ehrlich solid tumor est irr ehrlich solid tumor irradiation est bl ehrlich solid tumor bromelain est bl irr ehrlich solid tumor bromelain irradiationthe aim of the radiotherapy protocols is to achieve the maximum curative effect on tumor cells with minimal damaging effect on normal cells hence antioxidants and other nutrients which do not interfere with therapeutic modalities for cancer may enhance the killing property decrease side effects and protect normal tissue44for estimation of the antioxidant ability of bl dpph assay was conducted in vitro and the free radical inhibitory action of bl was compared with some antioxidant compounds it was found that bl has a powerful free radical scavenging power bl belongs to thiol proteases in which the catalytic nucleophile is sulfhydryl groups of cysteine residues which in turn accounts for its antioxidant activity45the involvement of ros mda and pon1 are important mechanisms that play a vital role during radiation toxicity the use of antioxidants is an important preventive to decrease the toxic and pathological effects associated with oxidative stress caused by radiation46 the attained results show a hepatic impairment on the third day from exposure to Îradiation elevation of lpo and ros levels and inhibition of pon1 activity compared to normal mice however treatment with bl revealed an amelioration in hepatic damage caused by irradiation these results were in accordance with liu 47 who described the effect of radiation induced ros generation which in turn might attack cell membrane phospholipids and circulating lipids and thus increases production of mda48 lpo acts as a sensitive biomarker for oxidative stress that occurs as part of the pathogenesis of irradiation49 bl has sulfhydryl groups consequently accounting for its antioxidant activity45 thus it could act as ros scavengermeasurement of pon1 postradiotherapy could be an effective clinical biomarker of hepatic and systemic oxidative stress and may be used as an index of the usefulness of radiotherapy50 it has been demonstrated to catalyze hydrolysis of lipid hydroperoxides and lactones51 pon1 protects serum hdl and ldl ps against lipid peroxidation52 in the present study the decreased activity of pon1 upon radiation exposure might be due to its super saturation of lipid hydroperoxides and lactones upon treatment with bl the activity of pon1 was restored near to the normal level hence the pon1
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" tumor associated macrophages tam constitute the most abundant immune cells in the tumor stroma initiating pro inflammatory m1 or immunosuppressive m2 responses depending on their polarization status advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patientsmethods to test the effects of radiotherapy on tam we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry we furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short course radiotherapy and compared findings to non pretreated rectal cancer using an immunostaining approach anotypic assays ota consisting of macrophages cancer associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophagesresults we demonstrate that short course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of tam towards an m1 like pro inflammatory phenotype in addition ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for t cell activation in ota we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles ev derived from irradiated tumor cells we identified high mobility group box in ev from irradiated tumor cells as a potential effector signal in that crosstalks our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro inflammation our data indicate that clinically applied short term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategiesintroductionsince the introduction of immune checkpoint blockade immunotherapy has become an attractive therapeutic option in cancer1 irradiation used as standard therapy in a number of solid malignancies induces immunogenic cell death icd by the release of damage associated pattern damp4 studies based on murine models indicated that irradiation induced dna damage of tumor cells elicited an antitumor immune response5 it was shown that alterations of the infiltrating immune cells by irradiation might be augmented when combined with immune modulating drugs6 a detailed understanding of the impact of radiotherapy on the immune system in humans should allow application of radiotherapy as a part of novel immunotherapeutic concepts however little is known about the detailed regulation of the immunogenic effect of irradiation in clinical settingsmacrophages are one of the most abundant immune cell subsets in tumor tissue9 and play a key role in the cancer microenvironment11 tumor associated macrophages tam are functionally diverse13 and display high plasticity upon immunological stimuli14 the concept of m1 and m2 macrophages was introduced to describe the heterogeneity of this cell subset16 m1 like macrophages possess the capacity to clear infections in support of a t helper type th1 immune response17 whereas m2 like macrophages respond in general to th2 cytokines and are strongly enriched in the tumor microenvironment18 the concept of m1m2 has been challenged and is seen as an oversimplified approach to the phenotype of macrophages but can be viewed as a linear scale on which m1 and m2 present two extremes19 tam recognize damp and respond by producing a variety of cytokines and growth factors to promote innate and stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access adaptive immunity9 in response to these signals macrophages are able to undergo reprogramming with enhanced antitumor features making them an attractive target for anticancer therapies22 there are conflicting results with respect to the effect of irradiation on the macrophage phenotype klug et al demonstrated in a murine model of breast cancer and human pancreatic cancer that low dose irradiation gy induced repolarization of m2 like macrophages to m1 like macrophages via induction of nitric oxide synthase inos24 moreover expression of inos correlated with vessel normalization and an influx of cd8 t cells suggesting a tumor ablative as well as pro inflammatory effect of repolarized macrophages upregulation of inos was also observed in murine prostate cancer which has been irradiated with up to gy25 similarly agonists of the toll like receptor further stimulated the m1 phenotype of macrophages and enhanced the antitumor effects of irradiation in a murine model of breast cancer26 other studies suggested that irradiation of tumors was associated with a more immunosuppressive phenotype of tam27 jones found that the depletion of macrophages by an anti csf antibody greatly increased tumor ablation upon irradiation gy in murine tumors generated from a colorectal and a pancreatic cell line obviously the effect of irradiation appears to depend on the model irradiation dose tissue as well as on the investigated time point after irradiation however despite the controversial reports an important role for tam in response to radiotherapy seems evidentmore recently extracellular vesicles ev have attracted attention in mediating signals to immune cells ev are rich in molecular cargos and are emerging as critical messengers in the cell to cell crosstalk they contain a variety of small signaling molecules which can be transferred to other cell types to modulate cell functions30 thus we hypothesized that ev might be involved in macrophages regulation in the tumor microenvironment in irradiated cells using a clinically relevant approach we show that short course irradiation increased the proportion of m1 like tam in human rectal cancer tissue primary short term cultures and anotypic tumor assays ota consisting of tumor cell lines macrophages and cancer associated fibroblasts allowed to further dissect irradiation induced changes in colorectal tam using irradiated tumor cell lines we demonstrate that ev are able to mediate irradiation induced repolarization of macrophagesmethodspatients and tissue materialpatients with clinical t3 rectal cancer patients characteristics online supplementary table received neoadjuvant hyperfractionated radiotherapy over the course of days within the frame of a controlled clinical study34 as a control group we used historical surgical tumor specimen of non pretreated rectal cancer lesions of patients from the same institution with no history of irradiation therapy or cytoablative treatment for immunofluorescence and immunohistochemical stainings surgical paraffin embedded specimen of the cancer lesions were cut in µm sections and mounted on slides for ex vivo cultures and subsequent flow cytometric stainings rectal cancer tissue was obtained from patients with histologically verified rectal cancer with no history of irradiation therapy or cytoablative treatment studies involving patient material were performed according to the declaration of helsinki and approved by the local ethics committee of the medical university of vienna for ota hct116 ccl147 and dld1 ccl221 were purchased from atcc peripheral blood mononuclear cells for macrophage differentiation were isolated from healthy blood donorsprimary ex vivo cultures of leucocytes and flow cytometrytissue samples of non irradiated rectal cancer were minced resuspended in rpmi medium with fetal bovine serum and plated in a mm petri dish irradiation protocol was applied as indicated below after hour of incubation °c co2 a single cell suspension from cancer tissue was prepared briefly tissue was digested with collagenase iv unitsml and dnase i mgml for hour min in rpmi supplemented with fetal bovine serum and hepes buffer solution at °c afterwards cell suspension was rinsed through a µm mesh and leucocytes were isolated by density gradient centrifugation using ficoll gradients for flow cytometry analysis cells were stained with fluorescence antibodies listed in the online supplementary table livedead discrimination was performed with fixable viability dye biolegend samples were acquired on a facsaria iii bd and analyzed with flowjo software v1061irradiation protocola theratron mds nordion radiotherapy unit with a cobalt60 source was used for γirradiation of ota ex vivo cultures of rectal cancer tissue monocyte derived macrophages and cancer cell lines tissue samples of rectal cancer were minced and cultivated petri dishes with cancer tissue were irradiated with à gy after hours of incubation a single cell suspension was prepared ota and cancer cell lines hct116 and dld1 were irradiated with à gy or à gy and subsequently cultivated over the course of hours monocyte derived macrophages were irradiated with gy or gy a negative control gy was always transported to the radiotherapy unit but was not exposed to γirradiationex vivo phagocytosis assaymononuclear cells isolated of rectal cancer lesions were spun down at g for min at °c for ex vivo escherichia coli e coli phagocytosis assay irradiated and non irradiated leucocytes were resuspended in µl phrodo red e coli bioparticles thermo fisher scientific as per the manufacturers instruction a same set of cells were resuspended in phosphate buffered saline pbs as controls after hours incubation at °c stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessleucocytes were washed and stained for flow cytometry tam cd11b cd14 hla dr viable cd45 cells that were pe high were considered to be phagocytosing for ex vivo tumor cell phagocytosis assay tam were isolated via fluorescence activated cell sorting facs cd11b cd14 hla dr viable cd45 cells from healthy colon mucosa or colorectal cancer lesions and incubated overnight with ev isolated from irradiated à gy and non irradiated gy dld1 cells evtam after washing dld1 were labeled with carboxyfluorescein diacetate succinimidyl ester cfse thermo fisher µm and cocultured with tam at an effector to target cell ratio of the proportion of cfse tam was assessed by flow cytometry after hours of incubation at °cat the air liquid interface in dmem supplemented with egm for seeding of tumor cells nylon discs with collagen cell cylinders were transferred into well plates five hundred microlitres of a tumor cell suspension with à cellsml hct116 or dld1 were added to each well after hours the tumor cells attached to the surface of the collagen gels and the nylon meshes with the tumor cell colonized collagen cell cylinders were put back onto the metal grids and incubated for days with media changes every days prior to further experiments for further assessment ota were embedded in optimal cutting temperature media snap frozen in liquid nitrogen and stored at °c until processing or fixed and embedded in paraffinimmunofluorescence and immunohistochemical stainingdirectly and indirectly labeled monoclonal antibodies as listed in online supplementary table and dapi as a nuclear marker were used an isotype was used as negative control in brief after incubation with the primary antibodies overnight at °c an appropriate secondary fluorescence labeled antibody was applied for min at room temperature followed by staining of dapi nuclear marker for immunohistochemical staining antibodies were mixed with bovine serum albumin in pbs and applied overnight to sections in a humid chamber at °c for visualization of the cells aec was used as chromogen sections were counter stained with hematoxylin for evaluation of staining results images of whole tissue sections one section per patient were acquired using a z1 axio observer microscope equipped with a ld plan neofluar à objective zeiss for leucocyte evaluation the tumor normal interface mm on tumor and normal zone was defined as regions of interests roi for γh2ax staining only tumor tissue was selected as roi roi were automatically quantified using tissuefaxstissuequest image analysis software tissuegnostics gmbhpreparation of otaota were set up as previously described35 briefly cancer associated fibroblasts caf were isolated and cultured from fresh samples of colon adenocarcinomas for the preparation of macrophages monocytes were isolated using the easysep direct human monocyte isolation kit stemcell technologies according to the manufacturers instructions à caf and monocytes per ota were mixed and pelleted by centrifugation at g for min for the collagen gel preparation all steps were performed on ice collagen solutions were prepared by mixing mgml of collagen i rat tail mgml becton dickinson and the fibroblastmonocyte suspension in dulbecco's modified eagle medium dmem supplemented with endothelial cell growth medium mv2 egm promocell a total of µl each of the collagen cell suspension were transferred into silicone gel casting devices after polymerization of the collagen solution the gels were lifted up on nylon mesh discs and transferred onto metal grids in well plates and cultured macrophage differentiationpbmc were obtained by ficoll plaque density gradient centrifugation pbmc were seeded at a concentration of Ã106well well plates in rpmi medium monocytes were isolated using the ability of monocytes to adhere to non tissue culture treated plastic culture dishes attached cells were cultivated in rpmi medium with glutamax thermo fisher scientific supplemented with ngml macrophage colony stimulating factor m csf thermo fisher scientific fetal bovine serum uml penicillin uml streptomycin and µgml fungizone in a humidified atmosphere at °c cells were cultivated for days with two medium changes to obtain m1 lpsifnγ polarized macrophages cells were stimulated with ngml lipopolysaccharide lps sigma aldrich and ngml interferonγ ifnγ thermo fisher scientific for hours m2 il4il13 polarized macrophages were generated using ngml interleukin il4 strathmann and ngml il13 biolegendev preparation and characterizationev were isolated by differential centrifugation hct116 and dld1 cells were cultured in mccoys 5a medium supplemented with exosome depleted fetal calf serum briefly cancer cell line culture medium was centrifuged at g for min to pellet cells supernatant was transferred to new falcon tubes and then centrifuged at g for min to pellet dead cells and apoptotic bodies rotanta 460rc hettich supernatant was transferred to the high speed centrifugation tubes and large ev were removed by ultracentrifugation at g for min sorvall evolution rc thermo fisher scientific after filtration of the supernatant through a µm syringe filter small ev were pelleted by ultracentrifugation at g for min rotor t1250 suspended in ice cold pbs and collected after another ultracentrifugation at g for min rotor type sorvall wx ultra thermo fisher scientific the ev pellet was resuspended in cold pbs for further use ev count was determined using a nanoparticle tracking analyzer zetaview particlemetrix results were analyzed with zetaview software ev were added to macrophages at a stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access concentration of Ãà cells we have submitted all relevant data of our experiments to the ev track knowledgebase ev track id ev20006537western blotcells were scraped from cell culture plates washed in ice cold pbs and lysed in à ripa buffer mm trishcl ph mm nacl np40 sodium deoxycholate sds containing à halt protease inhibitor cocktail thermo fisher scientific ev suspensions were mixed with à ripa buffer containing à halt protease inhibitor cocktail to obtain à concentration protein extracts were incubated at °c for min and centrifuged at g for min clear protein extract supernatants were quantified with the bca protein assay kit thermo fisher scientific one to five micrograms of protein were denatured with laemmli buffer loaded into each well of a acrylamide bisacrylamide gel containing sds and electrophoresed with magel for vhours proteins were transferred to a polyvinylidene fluoride membrane using the transblot turbo and the rta ready to assemble transfer kit bio rad after several hours of blocking pvdf membranes were incubated with primary antibodies online supplementary table at °c overnight after washing with tris buffered saline tween detergent membranes were incubated with the secondary antibody anti rabbit immunoglobulin g igg hrp linked antibody cell signaling technology or anti mouse igg hrp linked antibody cell signaling technology at room temperature for hour detection was done with clarity or clarity max western ecl substrate bio rad protein band intensities were quantified with imagequant tl ge healthcareelectron microscopyafter mounting cu mesh r22 holy carbon grids quantifoil with a tweezer into a leica gp leica microsystems grid plunger µl of ev sample solution were applied to grids front side and blotted for s grids were plunge frozen into liquid ethane for instant vitrification and transferred to a glacios cryo transmission microscope thermo fisher scientific equipped with a x feg images were recorded in low dose mode using the serialem software mastronarde with a falcon3 direct electron detector at magnifications of and with pixel sizes of and respectivelystatistical analysisstatistical analysis was performed using graphpad prism graphpad software statistical significance was determined by student's t test when comparing two groups the two way analysis of variance followed by tukey's multiple comparison test was used when comparing three or more groups significance was set at a p value of less than resultstam in rectal cancer polarize towards m1like phenotype upon irradiationwe first investigated the effect of irradiation on human tam in a primary ex vivo culture of rectal cancer specimen for this purpose minced tumor tissue was irradiated with gy and subsequently cultivated for hours before leucocyte isolation figure 1a the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer is shown in figure 1b tam as defined by cd14cd11bcd68hla dr accounted for over of all viable cd45 leucocytes in rectal cancer figure 1c with over cells per gram of tissue figure 1d there was no significant difference in percentage or absolute numbers of tam in non irradiated versus corresponding ex vivo irradiated tissue samples figure 1cd we then assessed the phenotype of treatment naïve and ex vivo irradiated tam using flow cytometry in naïve rectal cancer lesions tam were characterized by high expression of cd206 cd163 and cd64 and low levels of the chemokine receptor ccr7 indicating that the m2 like macrophage phenotype was present in untreated rectal cancer figure 1e this was also reflected in their cytokine pattern as more tam in untreated rectal cancer samples produced il10 il13 and il4 while few interferonγ ifnγ and tumor necrosis factor alpha tnfα producing tam were found in untreated samples in contrast ex vivo application of gy γirradiation to primary rectal cancer leucocytes resulted in a reduced presence of cd163 tam this phenotype correlated with enhanced levels of tnfα and inos tam as well as diminished detection of il10 and il13 tam hence low dose irradiation of rectal cancer tissue can polarize tam towards a pro inflammatory m1 like phenotype and may therefore directly contribute to antitumor activity by tnfα and inos productionlowdose irradiation reduces pd1 expression and enhances phagocytosis in tam to e colito further assess the distinct functional phenotype of tam on irradiation we investigated the ability of tam to phagocyte as an aspect of direct effector function and activation to model the phagocytic behavior of irradiated tam we incubated leucocytes derived from irradiated ex vivo tumor culture with ph sensitive pe labeled e coli particles in the phagosome the fluorescence of e coli particles increases as demonstrated in a representative example figure 2a phagocytosis by tam was significantly elevated upon irradiation with gy figure 2b demonstrating that the acquired shift towards m1 polarization due to irradiation was also functionally relevant as pd1 was previously shown to inhibit phagocytosis38 we next determined pd1 expression on irradiated ex vivo tumor culture derived tam we found variable pd1 expression on tam in non irradiated rectal cancer and observed a significant decrease of pd1 on tam derived from irradiated cultures figure 2c these stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0cchanges induced by irradiation can be instrumental for phagocytic activity of tamirradiation promotes antigen presentation and cytokines associated with th1 responseone of the hallmarks of m1 polarization of macrophages is the acquisition of antigen presenting features leading to efficient th1 response17 as a first step to determine whether irradiation may result in improved capability of tam to initiate immune responses we analyzed the expression of cd86 on tam derived from irradiated ex vivo tumor cultures irradiation correlated with a significant increase of cd86 on the cell surface of tam as compared with tam derived from non irradiated naïve cultures figure 2d we next investigated hla dr on non irradiated tam versus tam irradiated with gy we observed hla dr low expressing tam and high expressing tam clearly dividing them into two groups figure 2e in non irradiated samples more than of tam expressed high levels of hla dr while irradiation significantly increased the fraction of hla drhigh tam to over figure 2f we next investigated whether irradiation can induce the expression of il12 p70 and il23 p19 as markers for the initiation of adaptive immune responses whereas il23 p19 showed a tendency to be produced by tam on irradiation il12 p70 was significantly induced in irradiated tumor tissue compared with open accesstreatment naïve samples figure 2g together these results suggest that low dose irradiation influences tam polarization in rectal cancers by equipping the cells with an hla drhiil12hi il23hi cytokine profile this observation emphasizes that irradiated tam have a higher probability to participate in antitumor immune responses directly by phagocytosis and cytokine secretion as well as indirectly by exhibiting a broad armamentarium that potentially activates t cells as main players of antitumor adaptive immunityshortcourse irradiation of patients with rectal cancer increases the m1m2 ratio of tamto corroborate our ex vivo data with the in vivo situation on short course irradiation we examined tam polarization and function in rectal cancer patients treated by a routinely applied radiotherapy protocol we made use of a cohort of patients in clinical stage t3 rectal cancer who received neoadjuvant hyperfractionated short course radiotherapy two times gy per day over the course of days figure 3a surgery was performed days after radiotherapy in these patients as a control we used surgical specimen from treatment naïve clinical t3 rectal cancer patients importantly the indication for radiotherapy was not correlated with a more severe tumor progression compared with the treatment naïve cohort figure ex vivo γirradiation induces polarization of tumor associated macrophages a tissue samples of naïve rectal cancer were minced and irradiated after incubation a single cell suspension of cancer tissue was prepared b representative example of the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer c percentage of macrophages compared to total of viable cd45 cells in non irradiated and ex vivo irradiated rectal cancer lesions assessed by flow cytometry and presented as mean±sd n10 d mean numbers of macrophages in rectal cancer per gram in non irradiated and ex vivo irradiated rectal cancer presented as mean±sd n10 e expression of intracellular and extracellular markers in macrophages assessed by flow cytometry bars presented as mean percentage of indicated markers±sd of non irradiated and ex vivo irradiated macrophages n10 p005 p0001 p00001 by two tailed paired students t test gy gray tam tumor associated macrophages ifnγ interferonγ tnfα tumor necrosis factorα il interleukin inos inducible nitric oxide synthasestary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access irradiation increases phagocytosis and marker associated with antigen presentation and th1 response a figure representative histogram and flow cytometry plots indicating difference in pe fluorescence of non irradiated blue versus irradiated red tam in ex vivo phagocytosis assay total phagocytosis was analyzed by first gating on tam and then gating on pe cells b analysis of tam phagocytosis of phrodo e coli particles data presented as percentage of phagocytosis of non irradiated tam and corresponding irradiated tam gy n5 c percentage of tam positive for pd1 non irradiated versus ex vivo irradiated and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 d percentage of expression of cd86 in non irradiated versus ex vivo irradiated tam and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 e representative plots histogram and f expression of hla dr on tam of non irradiated and ex vivo irradiated rectal cancer data given as mean±sd n10 g expression of il12 p70 and il23 p19 data presented as mean percentage±sd of total tam n10 representative example of il12 p70 and il23 p19 expression on tam p005 p0001 by two tailed paired students t test th1 t helper type gy gray tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessfigure short course irradiation in patients modulates the immune infiltrate with induction of macrophage polarization a irradiation protocol of patients with neoadjuvant hyperfractionated à gy per day over the course of five days radiotherapy for clinical t3 rectal cancer surgery was performed the following week b quantitative in situ assessment of infiltrating macrophages cd68 t cells cd3cd56 nk cells cd56cd3 and nkt cells cd56cd3 in non irradiated n25 and irradiated rectal cancer n45 assessed by multicolor immunofluorescence staining data are given as absolute numbers of positive cells per mm2±sd c analysis of immunohistochemical staining for γh2ax as irradiation response in non irradiated n25 and irradiated tissue sections data are given as mean±sd d representative examples of immunohistochemical staining for γh2ax images below are magnified à e representative immunohistochemical staining of cd68 cells in non irradiated n25 and irradiated tissue sections n45 images below are magnified à f representative example of immunofluorescence multicolor staining of cd68 macrophages pe m1inos fitc m2 cd163 apc g quantitative in situ analysis of immunofluorescence staining of ratio of inos m1 to cd163 m2 tams cd68 macrophages presented as m1m2 in non irradiated n25 compared to irradiated sections n45 data are given as mean±sd h percentage of il10 cells to total cd68 cells data are presented as mean±sd i representative image of multicolor immunofluorescence staining of il10 in macrophages cd68 peil10 fitc of irradiated rectal cancer p0001 p00001 by two tailed unpaired students t test gy gray inos inducible nitric oxide synthase tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access since the stage in the tnm classification of malignant tumors and cancer differentiation of the two cohorts were comparable online supplementary table to assess the composition of the leucocytic infiltrate and the impact of radiotherapy we evaluated the distribution of macrophages t cells nk cells and nkt cells via immunofluorescence staining and automated image analysis irradiated tumor sections showed significantly less infiltration of cd68 cells which correlated with a tendency for a decrease of cd3 t cells while the amount of nk cells cd56 cd3 or nkt cd56 cd3 cells did not show significant differences figure 3b immunostaining of phosphorylated γh2ax was used to confirm radiotherapy induced dna damage in analyzed tumor specimen irradiated tumors revealed a significantly higher amount of γh2ax positive cells than non irradiated tissue figure 3c γh2ax foci were primarily located in the nuclei of tumor cells rather than in the cells of the tumor infiltrating microenvironment figure 3d in both patient cohorts cd68 cells accumulated in regions surrounding tumor cell clusters figure 3eto evaluate the in vivo effect of irradiation on macrophage polarization and to correlate results with ex vivo irradiated tumor specimens we determined the ratio of m1 like tam to m2 like tam by staining for inos and cd163 in cd68 cells irradiation was associated with higher numbers of inos expressing tam while cd163 tam were drastically reduced resulting in a significant increase in the m1m2 ratio figure 3fg similar to the ex vivo cultures we observed a sixfold decrease of il10 tam in tumors from irradiated patients as compared with treatment naive tumors figure 3hi these data are corroborating hallmarks of our ex vivo irradiation protocols with macrophages becoming active players within the tumor microenvironment upon γirradiationirradiation of anotypic cultures promote macrophage activation towards an m1like phenotypeto be able to further dissect the irradiation induced findings in patients tissue sections and ex vivo cultures we used anotypic cultures which were established with colorectal cancer cell lines hct116 or dld1 primary caf and peripheral blood derived macrophages online supplementary figure a in collagen i gels in contrast to primary cultures ota allowed culture periods of up to days to mimic the clinical protocol of short course irradiation ota containing either hct116 or dld1 were exposed to irradiation with two times gy or two times gy over the course of hours compared with non irradiated controls figure 4ab frozen tissue sections of ota were assessed for macrophage marker using immunostaining and automated image analysis the number of macrophages in ota was comparable and not significantly different among the distinct conditions online supplementary figure b non irradiated gy ota harbored macrophages resembling an m2 like phenotype of cd11bcd68 macrophages as indicated by high expression of cd163 figure 4cd low expression of ccr7 figure 4e and modest expression of il10 figure 4fg upon irradiation of ota cd163 and il10 were reduced whereas the expression of the pro inflammatory marker ccr7 was elevated thus the marker profile of ota correlated with those of primary ex vivo tumor cultures and surgical specimen following neoadjuvant irradiation these observa
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" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearmans correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturers instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearmans correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time à power time à power¼¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci pgml and pgml ci pgml respectively the median level of il p40 before the mwa treatment was pgml ci pgml there was a slight increase to pgml ci pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci pgml and increasedto pgml ci pgml days afterthe mwa treatment and to pgml ci pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci pgml there was a significant increase at h postmwa with a median level of pgml ci pgml the median level ofil1 before the mwa treatment was pgml ci pgml and a significantincrease wasnoted days after the mwa treatment pgml ci pgml the median level of il6before the mwa treatment was pgml ci pgml and significantly increased daysafter the mwa treatment pgml ci pgml the median level ofil8 before themwa treatment was pgml ci pgml and increased significantly to pgml ci pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci pgml and increasedsignificantly days after the mwa treatment pgml ci pgml the median level oftnfα before the mwa treatment was pgml ci pgml and increased significantlyto pgml ci pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of energy time à power time à power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional 10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci ci ci ci ci ci ci ci ci ci h postmwa pgml ci ci ¼ ci ci ci ci ci ci ci ci il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa ¼ 2fold vs premwa days postmwa pgml ci ci ci ci ci ¼ ci ¼ ci ¼ ci ¼ ci ¼ ci days postmwa pgml ci ci ci ci ci ci ci ci ci ci effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 its mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of in situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaenergyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearmans rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the energyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wascancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a cancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled abscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as differentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmeds work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors contributionsjz conceptualization data curation writingoriginal draft and writingreview and editing ql conceptualization and writingreview and editingmm conceptualization and writingreview and editing brconceptualization and writingreview and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writingreview and editing rl conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the six one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim
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"pd1pdl1 blockade therapy is a promising cancer treatment strategy which has revolutionized the treatmentlandscape of malignancies over the last decade pd1pdl1 blockade therapy has been trialed in a broad range ofmalignancies and achieved clinical success despite the potentially curelike survival benefit only a minority ofpatients are estimated to experience a positive response to pd1pdl1 blockade therapy and the primary oracquired resistance might eventually lead to cancer progression in patients with clinical responses accordingly theresistance to pd1pdl1 blockade remains a significant challenge hindering its further application to overcomethe limitation in therapy resistance substantial effort has been made to improve or develop novel antipd1pdl1based immunotherapy strategies with better clinical response and reduced immunemediated toxicity in thisreview we provide an overview on the resistance to pd1pdl1 blockade and briefly introduce the mechanismsunderlying therapy resistance moreover we summarize potential predictive factors for the resistance to pd1pdl1blockade furthermore we give an insight into the possible solutions to improve efficacy and clinical response inthe following research combined efforts of basic researchers and clinicians are required to address the limitation oftherapy resistancekeywords pd1pdl1 blockade cancer immunotherapy resistance immunotherapy is a validated and significant cancertreatment strategy which eliminates tumors by normalizing the antitumor immune responses [ ] over thelast decade cancer immunotherapy has revolutionizedthe treatment landscape of malignancies and achievedclinical success especially in immune checkpoint inhibitors correspondence 189whueducn lschrjjs163com jinyu sun and dengke zhang are cofirst authors4department of general surgery the first affiliated hospital of nanjingmedical university nanjing china2key laboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui chinafull list of author information is available at the end of the signalsandprogrammed death1 pd1 is a class of receptorexpressed on the t cell surface which could downregulate the immune system by abrogating t cellreceptorinducedantigenmediated t cell activation the interaction betweenpd1 and its ligand programmed deathligand pdl1 plays an essential role in maintaining selftoleranceand avoiding autoimmune diseases however pd1pdl1 could also prevent the activation of t cells in thetumor and thus result in immune resistance preventingpd1pdl1 blockade is a breakthrough in cancerimmunotherapy and it has been trialed in a broadrange of malignancies in the preclinical or clinicalincluding melanoma hodgkins lymphomastage breast cancer [ ] nonsmall celllung cancer as well as hepatocellular carcinomansclc the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun biomarker research page of [ ] despite the longterm potentially curelikeclinical benefits therapy resistance remains a significant challenge for the further application of pd1pdl1 blockade therapy only a minority of patientsin general are estimated to experience apositive response to pd1pdl1 blockade therapy[] and the primary or acquired resistance mighteventually lead to cancer progression in patients withclinical response [ ]in this review we provide an overview on the resistance to pd1pdl1 blockade and its underlying mechanisms moreover we summarize potential predictivefactors for the resistance to pd1pdl1 blockade furthermore we give an insight into the possible solutionsto improve efficacy and clinical response of pd1pdl1blockade therapyresistance to pd1pdl1 blockade therapycheckpoint inhibitors targeting pd1 or pdl1 coulddisturb the interaction between pd1 and pdl1 whichwould preserve antitumor properties of t cells withdraw immune escape and normalize their ability to induce tumor cell death currently pd1pdl1 blockadehas shown sustained survival benefits in multiple malignancies and is at the forefront of cancer immunotherapy howeverjust as tumor cells can avoid immuneevasion several cancers may evolve to resist pd1pdl1 blockade therapy clinical evidence indicated thateven for patients with tumors highly positive for pdl1more than of them might not respond to pd1pdl1 blockade due to tumor heterogeneity and manyother reasons clinical responses vary largely across different tumor entities the objective response rate was in melanoma in nsclc in head and neck carcinoma and in kidneycancer besides for most patients experiencing initial clinical response acquired resistance remains another problem which would lead to cancer progressionor relapse after a few years [ ]many studies have demonstrated that antipd1therapy can significantly improve survival outcomes forpatients with metastatic or unresectable melanoma however only a small number of patients approximately could achieve a complete response in arecent phase i trial of atezolizumab antipdl1 involving patients with metastatic melanoma the overall response rate was among efficacy evaluablepatients and the median response duration was months moreover in another study on the longtermoutcomes of melanoma patients receiving antipd1therapy complete responses were only observed in of patients after a median followup of months of patients were alive without additional melanoma therapy additionally in the retreatedpatients after disease progression the response was onlyobserved in retreated patients receiving singleagent pd1 blockade therapy and of patientsescalated to pd1 blockade plus ipilimumab therapy inthis cohort most complete responses were durable withthe treatment failure rate of at three years whilethe response to retreatment remained relatively infrequent with a response rate of for patients withsingleagent pd1 blockade therapy moreover in aphase ii study of pembrolizumab on patients withadvancedobjectiveresponse was observed in of patients with a diseasecontrol rate of after a median followup of months adrenocorticalcarcinomatheinterestingly the response rate of some malignanciesis relatively high in hematological malignancies for example for patients with relapsed or refractory classicalhodgkin lymphoma tislelizumab antipd1 achievedan objective response rate of and a completeresponse of in a phase ii singlearm multicenterstudy similarly the complete response rate of camrelizumab antipd1 was with a partial remission rate of mechanisms underlying the resistance to pd1pdl1 blockadesince therapy resistance remains a significant limitationof pd1pdl1 blockade in clinical practice interest isgrowing in understanding the mechanisms underlyingthe resistance the response to pd1pdl1 blockaderelies on a preexisting immune response and determinants of adaptive immunity currently multiple factorshave been discovered to be involved in the efficacy ofpd1pdl1 blockade therapy such as tumor immunogenicity t celltumormicroenvironment and so forthfunction pdl1 expressionthe lack of tumor antigensthe genetic alterations are centralin the oncogenicprocess which could lead to tumor immunogenicity andprovide an opportunity for cancer immunotherapy tumor immunogenicity is positively associated with theability of the t cell to recognize tumor cells which isessential for the antitumor effect of pd1pdl1 blockade however the lack of tumor antigen will significantlyimpede the recognition ability of t cells and eventuallyresult in the failure of immunotherapymicrosatellites are prone to dna replication errorswhich will usually be repaired in normal cells however in tumors with mismatch repair mmr deficiencythese errors will accumulate which eventually result in alarge number of mutations and lead to microsatellite instability msi importantly high msi positivelycontributes to increased neoantigen production greater 0csun biomarker research page of immunogenicity and a more robust immune response moreoverthe resultant high tumor mutationburden would contribute to tumor immunogenic andenhance the response to pd1pdl1 blockade therapy[ ]multiple studies have demonstrated that the tumormutation burden is positively correlated with neoantigenburden as well as response to immunotherapy [ ]for example in colorectal cancer with mmr deficiencywhich usually exhibits a high tumor mutation burdenantipd1 therapy showed a higher response rate andbetter survival outcome compared to other subtypeswith mmr proficiency [] yarchoan analyzed the objective response rates of pd1pdl1blockade therapy for the corresponding tumor mutationburden in various cancers and their results showed thatthe mutation burden was closely associated with the objective response rate moreover pancreatic cancer generally exhibits a lowermutation load compared with other solid tumors andtherefore pd1pdl1 blockade is usually ineffective forthose patients and fails to improve their survival outcomes nevertheless in pancreatic cancer patients harboring an mmr deficiency they appear to be responsiveto pd1pdl1 blockade therapy mmr deficiency significantly increases the somatic mutation rate whichcould be translated into neoantigens and recognized bythe immune system thus making these patients responsive to pd1pdl1 blockade therapy [ ] accordingly pembrolizumab has been approved for selectedcancer patients with mmr deficiencyt cell dysfunctioneffective pd1pdl1 blockade therapy relies on the tcell function and any disruption in the processes of tcell immune function will result in the failure of pd1pdl1 blockade therapy a recent review by ren has provided an indepth insight into the mechanisms underlying the t cell dysfunctionmediated resistance with a focus on t cell recognition activationdifferentiation infiltration depletion as well as chemotaxis identification byantigen presentation is a critical process for the tumorantigensinitial t cells beta2microglobulin b2m is a significant hla1 moleculewhose mutation will hinder tumor antigen presentationand result in therapy resistance [] zaretsky analyzed biopsy samples from patients with metastatic melanoma receiving pembrolizumab who exhibited disease progression after an initial tumor regressionand they found a truncating mutation in the b2m genein the following research gettinger identifiedacquired homozygous loss or downregulation of b2m inlung patients with resistance to pd1pdl1 blockadeto further explore the role of b2m in mediating resistance they knocked out the b2m gene in immunocompetent lung cancer mice by crispr technology and theloss of b2m resulted in the resistance to pd1pdl1blockade additionally b2m mutationinducedresistance primarily occurred in an environment ofactivated pd1 positive t cellinfiltration whichresistance to pd1pdl1 blockadesuggested thattherapy might be particularly common in patients withhigh pd1 positive t cell for example t cellmoreover t cell activation is another critical processfor pd1pdl1 blockade therapy after blocking pd1pdl1 tumor cells can still counteract the activity ofimmune checkpoints and activate additional inhibitorypathways via expression of other immune checkpointsand their ligands within the tumor immune microenvironmentimmunoglobulinmucin3 tim3 is another type of immune checkpointreceptor expressed on tumorinfiltrating lymphocytes inhuman head and neck squamous cell carcinoma tumorinfiltrating lymphocytes pd1 blockade was demonstrated to upregulate tim3 expression which inhibitedt cells activation and contributed to tim3mediatedescape from pd1 blockade in the tumor microenvironment via pi3kakt pathway pd1 or pdl1physiologicallyinteractions between pd1 and pdl1block t cell activation pathways related to the immuneresponse against specific antigens and the expression ofpd1 or pdl1 has gained importance as a significantplayerin regulating the response to pd1pdl1blockade therapy pd1 and pdl1 are upregulated inthe tumor immune microenvironment of various malignancies which is considered as a strategy to evadeimmunosurveillance and imposes a significant barrier ofthe antitumor immune response importantly pdl1 primarily exhibits two distinct expression patternson tumor cells or on tumorinfiltrating immune cellspdl1 expression on immune cells reflects the adaptiveregulation meditated by ifnγ which is accompanied byincreased effector t cells as well as tumorinfiltratinglymphocytes effector t cells differently the expressionof pdl1 on tumor cells is less prevalent and it indicates the epigenetically dysregulated pdl1 gene whichis correlated with reduced immune infiltration scleroticor desmoplastic stroma as well as mesenchymal molecular features multiple studies have revealed a significantly higherobjective response rate in tumor pdl1 positive patientsthan pdl1 negative subgroups together with an improved progressionfree and overall survival [ ]kowanetz observed that atezolizumab antipdl1 achieved an objective response rate of in 0csun biomarker research page of patients with high pdl1 levels on tumor cells alone andof in those with a high expression on immune cellsalone although these observations indicated that thefunctional importance of pdl1 expression in regulatingpd1pdl1 blockadeinduced t cellthemechanistic significance of pdl1 on tumor cells or immune cells remains vagueresponsenoncoding rnasa large amount of micrornas mirnas and some longnoncoding rnas lncrnas have emerged as players inregulating tumor immunity [] and resistance topd1pdl1 blockade therapy recently huber identified a panel of circulating mirnas mir146a mir155 mir125b mir let7e mir125a mir146b mir99b which wereassociated with phenotypic and functional features ofmyeloidderived suppressor cells mdscs in melanomapatients importantly mdscs are a subclass of immature myeloid cells pathologically associated with cancerand play an inhibitory role against antitumor t cell immunity the transcriptional analysis showed thatthese mirnas could facilitate the conversion of monocytes into mdscs by melanoma extracellular vesiclesand the expression level ofthese mirna was upregulated in circulating cd14 monocytes and tumorsamples which was associated with myeloid cell infiltration and could predict the resistance to pd1 blockadetherapy moreover hu revealed the role of oncogeniclncrna for kinase activation linka in losing antigenicity and evading immune checkpoints and demonstrated lncrnadependent antigenicity downregulationsuppression for patients withand intrinsic tumortriplenegative breast cancer and resistantto pd1blockade therapythey showed upregulated linkalevels and downregulated peptideloading complex components the analysis suggested that linka expressioncould attenuate protein kinase amediated phosphorylation of the e3 ubiquitinprotein ligase trim71 via facilitating the crosstalk between phosphatidylinositol []trisphosphate and inhibitory gproteincoupled receptor pathways consequently linka could contribute to the degradation of the antigen peptideloadingcomplex and upregulate intrinsic tumor suppressors gut microbiomethe gut microbiome is a complex system composed ofmore than trillion microanisms which has beendemonstrated to regulate the efficacy and toxicity ofcancer immunotherapy many studies have reported theinfluence of the gut microbiome on cancer immunotherapy and the therapeutic response of pd1pdl1blockade therapy can be improved or diminished via gutmicrobiome modulationin mice models with distinct microbiome a significantly different response to pd1pdl1 blockade therapy was observed for example melanoma mice with anincreased bifidobacterium species in the gut microbiomeexhibited an effective response to pd1 blockade therapy similarly antibiotic administration was reported toreduce the diversity and aggravate dysbiosis of the gutmicrobiome thus influencing the clinical response topd1pdl1 blockade in tumorbearing mice as well ascancer patients [] compared to patients withoutantibiotic treatment the oral antibiotic application couldsignificantly diminish the clinical benefit of pd1pdl1blockade therapy and decrease progressionfree survivaland overall survival therefore dysbiosis of the gut microbiome is considered as one of the putative mechanisms underlying poorresponse to pd1pdl1 blockade therapy and thedualdirectional modulation of the gut microbiome oncancer immunotherapy is increasingly revealed howeverit is still unclear how gut microbiome regulatestherapy response and whether a specific bacterial taxaor gut microbiome as a whole plays a primary role remains largely unclear further research is required toprovide a more indepth understanding of the underlying mechanismspredictive factors for pd1pdl1 blockadetherapydespite the clinical success achieved in pd1pdl1blockade across multiple cancers the knowledge concerning therapy selection criteria is relatively limitedconsidering the potential adverse events and high costof immune checkpoint inhibitor agents there is a substantial need to identify predictive factors to select patients likely to benefit from this therapy currently apartfrom the functional status of immune cells [] ortumor infiltrating lymphocytes multiple factorshave been identified to predict the response to pd1pdl1 blockade therapy such as pd1pdl1 expression antigen recognition gut microbiome and so forthtable pd1 or pdl1 expressioninhibiting the pd1 pathwaymediated immune suppression is the basis and premise of pd1pdl1 blockadetherapy accumulating research has suggested that pdl1 is a biomarker to predict therapeutic response to pd1pdl1 blockade across multiple tumor types forexample atezolizumab achieved overall survival benefitacross all pdl1 expression subgroups in nsclc patients while those with high pdl1 expression experienced a more substantial survival benefit currently 0csun biomarker research page of table predictive factors for pd1pdl1 blockade therapytumor typenonsmall cell lung canceragentatezolizumabmultiple cancerscolorectal cancerurothelial carcinomaurothelial carcinomaurothelial cancermelanomamelanomamelanomapembrolizumabnivolumabatezolizumabatezolizumabatezolizumabantipd1 therapyantipd1 therapyantipd1 therapymmr mismatch repair msi microsatellite instability tmb tumor mutation burdenpredictive factorpdl1pdl1mmr msitmbtmbtmbgut microbiomegut microbiomegut microbiomereference pdl1 testing is recommended as a predictive test fornsclc urothelial carcinoma or head andneck cancers and so forthott assessed the predictive value of pdl1expression in patients with advanced solid tumors receiving pembrolizumab and the analysis showed that tumors with higher pdl1 expression and tumor mutationburden were significantly associated with higher response rate and more prolonged progressionfree survival heat map analysis revealed a close correlationbetween pdl1 expression and a broader pattern ofcoregulated gene expression which involved cytokine recruitment of t cells t cell activation markers as well asantigen presentation also the regression metaanalysisdemonstrated that pdl1 expression level was positivelyassociated with objective response rate p aswell as progressionfree survival p moreover nct02853305 and nct02807636 evaluated the efficacy of pembrolizumab or atezolizumab asfirstline treatment and the current data showed reduced survival in patients with low expression of pdl1accordingly it is advised that pembrolizumab or atezolizumab should be used for adult patients with a relativelyhigh pdl1 expression pdl1 expression of ¥ foratezolizumab and a combined positive score of ¥ forpembrolizumab however the efficacy of pd1pdl1blockade therapy as firstline therapy for advancedurothelial carcinoma still remains unclear [ ]importantly pdl1 positive only is not a predictivefactor for the response to pd1pdl1 blockade sincemultiple factors are involved in the pd1pdl1 blockade therapy in a study on patients with metastaticmelanoma receiving pembrolizumab preexisting cd8t cells were demonstrated as a prerequisite for thetumor regression after pd1pdl1 blockade therapy besidesin advanced adrenocortical carcinomatumor pdl1 expression status was not associated withtherapy response additionally it was reported thatpdl1 expression on tumor cells was not associatedwith therapy response in resected head and necksquamous cell cancer additionalinvestigation isrequired to illustrate the mechanisms accounting for thedifferenceantigen recognitionantigen recognition plays a vital role in initiating theadaptive immune response while the lack of tumor antigens significantly impedes the response to pd1pdl1blockade therapycurrently the fda has approved pembrolizumab totreat unresectable solid tumors with high msi or mmrdeficiency in a study on recurrent or metastaticcolorectal cancer patients with mmr deficiency or highmsi nivolumab showed an objective response rate of and of the patients had a disease control rateof ¥ weeks which indicated that patients with highmmr deficiency or high msi might exhibit better responses to pd1pdl1 blockade therapy [ ] interestingly the responses of tumors with mmrdeficientare highly variable and approximately half are resistantto pd1pdl1 blockade therapy mandal revealed that msi and the resultant mutation load wereresponsible for the variable response to pd1 blockadetherapy in mmrdeficiency tumors and the responsedegree was significantly correlated with the degree ofinsertiondeletion mutation loadseveral studies have revealed the association betweentumor mutation burden and the response to pd1pdl1blockade therapy [ ] mariathasan examined samples from patients with metastatic urothelial cancer receiving atezolizumab treatment and identified highneoantigen and tumor mutation burden as major determinants of clinical outcome their results showed that thetumor mutation burden was closely correlated with the response in the excluded and inflamed phenotypes 0csun biomarker research page of gut microbiome compositionclinical experiments on the human gut microbiomehave identified several specific bacteria genres that playimportant roles in human immunity and can be used asprognostic biomarkers for clinical response to pd1pdl1 blockade therapy based on the gut microbiome analysis of melanomapatients receiving pd1 blockade gopalakrishnan found that patients with prolonged progressionfree survival showed a higher multiplicity of bacteriaand clostridiales ruminococcaceae and faecalibacterium were abundant in therapy responders moreovermatson evaluated the baseline stool samplesfrom patients with metastatic melanoma before pd1pdl1 blockade treatment and the results showed thatcommensal microbial composition was significantly associated with the clinical response bifidobacteriumlongum collinsella aerofaciensand enterococcusfaecium were more abundant in responders similarlyin patients with epithelial tumors routy revealed that akkermansiacea muciniphila and enterococcus hirae were significantly abundant in those withbetter clinical response progressionfree survival months all these results indicate that gut microbiomecomposition may be a potential determinant of therapyresponse and might be used as a predictive factor inthe following research more studies are needed to validate the predictive value of gut microbiome in largercohorts and explore their efficiency in the context ofvarious types of tumorsstrategies and it hasfuture perspectivesimmunotherapy is one of the most promising cancertreatmentrevolutionized thelandscape of cancer management over the last decadehowever together with the costly and timeconsumingtrialanderror approach the limited therapy responseremains a tricky problem which hinders the furtherapplication of pd1pdl1 blockade to overcome therapy resistance and potential adverse events substantialeffort has been made on developing novel antipd1pdl1 based immunotherapy strategies with better clinical response and limited immunemediated toxicityfigs tobetterclinicallikelyachievesystem issince the interaction between cancer and the immunecomplex and involves multiplefactors strategies in combination with multiple agentsareoutcomescompared with singleagent administration a largenumber ofcombinedtherapy is an effective therapeutic strategy againstcancers for example transforming growth factor βtgfβblocking agents concomitantly with combinedpd1pdl1 blockade combined provides a clinicallyrevealed thatstudies haveexperimentson mice withfeasible strategy to improve efficacy and reduce toxicity mariathasan revealed that metastaticurothelial cancer with upregulated tgfβ signalingbefore treatmentresponded poorly to pd1pdl1blockade therapy the tumors with dense collagenfibrils could trap t cells in the stromal compartmentthus preventing them from playing their functions inpreclinicalimmuneexcluded phenotype they demonstrated that the coadministration of pdl1 blockade and tgfβblockingagents could reduce tgfβ signaling facilitate t cellinfiltration and achieve active antitumor immunityand tumor regression similarly the combination ofpd1pdl1 blockade with tumor necrosisfactorinhibitor [ ] metformin antivegf agents or otherinhibitors egcxcr4 has been demonstrated as a clinicallyfeasible strategy with improved antitumor efficacyand reduced toxicityimmune checkpointinhibitor agentspd1pdl1 blockade usually acts on the whole hostimmune system instead ofsitespecifically targetingtumorspecific immune cells while nanomedicine technology provides a powerful tool to selectively deliverimmune checkpointto tumors orlymphoid ans using drugloaded nanops usually to nm in diameter recent studies suggest that the pd1pdl1 antibody could be conjugatedor modified on the surface of nanops which couldmaintain their stability enhance efficiency and minimizethe toxicity of pd1pdl1 blockade [ ] forexamplein gastric cancer cells the pdl1 blockadeconjugated nanops contributed to significantlyhigher cellular uptake and achieved more effective inhibition of pdl1 expression compared with the controlgroups moreover in patients with metastatic triplenegative breast cancer the coadministration of nabpaclitaxelatezolizumabprolonged progressionfree survival owing to thesuccess in previous research clinical trials on nanoimmunotherapysuch asnct03589339 and nct03684785 these clinical trialsshould provide substantial evidence for the combinationof nanomedicine and pd1pdl1 blockade in the nextfew yearscurrently underwayblockadepdl1plusarethe manipulation offurthermore accumulating evidence has demonstrated that gut microbiome significantly impacts theefficacy of cancer immunotherapy which in turn indithe gut microbiomecates thatcould latently affectthe response to pd1pdl1blockade therapy [] currently antibiotic applicationfecal microbiota transplantation fmt anddiet regulation are considered as practical approachesto manipulate gut microbiome for example fmtfrom patients with a positive response to germfree or 0csun biomarker research page of fig overview on the strategies to improve the resistance to pd1pdl1 blockade therapy multiple strategies have been proposed toimprove the resistance to pd1pdl1 blockade therapy including combined therapy nanoimmunotherapy gut microbiome manipulation andso forthin contrastantibiotictreated mice could improve tumor controlaugment t cell responses and ameliorate the antitumor effects of pd1 blockadethetransplantation from resistant patients did not resultin improvement similarly responses to pdl1blockade are distinctin mice with different commensal microbes and the positive response of micewith advantageous gut microbiome can be transplanted to mice with negative responses by fmt orcohousing conclusionsdespite the success across multiple types of cancersonly a minority of patients are estimated to exhibit apositive response to pd1pdl1 blockade therapy andthe primaryacquired resistance might eventually leadto progression in patients with clinical responses thelimitation in clinical response impairs the efficacy andhinders its further application since the understandingof the mechanisms underlying therapy resistance remains vague only a few therapeutic options areavailable for those patients currently illustrating thedeterminants of response or resistance is significant toaccelerate improving survival outcomes and developingimproved treatment options for cancer patients tobetter realize the therapeutic potential of pd1pdl1blockade therapyit is essential to identify predictivebiomarkers for therapy response develop novel therapeutic strategies and improve therapeutic strategies incombination with other agents in the following research combined efforts of basic researchers and clinicians are required to address the pd1pdl1 blockadetherapy resistanceabbreviationspd1 programmed death1 pdl1 programmed deathligand nsclc nonsmall cell lung cancer mmr mismatch repair msi microsatelliteinstability b2m beta2microglobulin tim3 t cell immunoglobulin mucin3mirnas micrornas lncrnas long noncoding rnas mdscs myeloidderived suppressor cells tgfβ transforming growth factor β fmt fecalmicrobiota transplantationacknowledgmentsnot applicableauthors contributionsjys dk z mx and xz wrote original draft preparation sq w jsj and xjprovided critical revision all authors read and approved the final manuscriptfundingthis study was supported by national key research and developmentprojects intergovernmental cooperation in science and technology of chinano 2018yfe0126900 to jiansong ji the key research and developmentproject of zhejiang province no 2018c03024 to jiansong ji the nationalnatural science foundation of china to xl 0csun biomarker research page of availability of data and materialsnot applicableethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1the first college of clinical medicine the first affiliated hospital of nanjingmedical university nanjing medical university nanjing china 2keylaboratory of imaging diagnosis and minimally invasive interventionresearch lishui hospital of zhejiang university the fifth affiliated hospitalof wenzhou medical university clinical medicine of center hospital of lishuicollege lishui china 3college of medicine lishui college lishui china 4department of general surgery the first affiliated hospitalof nanjing medical university nanjing china 5department of radiologyaffiliated lishui hospital of zhejiang university lishui chinareceived april accepted august referenceshellmann md pazares l bernabe caro r zurawski b kim sw carcerenycosta e nivolumab plus ipilimumab in advanced nonsmallcell lungcancer n engl j med niglio sa jia r ji j ruder s patel vg martini a programmed death1or programmed death ligand1 blockade in patients with platinumresistant metastatic urothelial cancer a systematic review and metaanalysis eur urol sun jy lu xj cancer immunotherapy current applications and challengescancer lett andrews lp yano h vignali daa inhibitory receptors and ligands beyondpd1 pdl1 and ctla4 breakthroughs or backups nat immunol prestipino a zeiser r clinical implications of tumorintrinsic mechanismsregulating pdl1 sci transl med betof warner a palmer js shoushtari an goldman da panageas ks hayessa et
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" bridge to surgery bts using a selfexpandable metallic stent sems for the treatment of obstructivecolorectal cancer improves the patients quality of life this study aimed to examine prognostic factors ofobstructive colorectal cancermethods we analyzed stage iiiii resectable colon cancer cases cur a retrospectively registered between january and december overall patients with cur a obstructive colorectal cancer were evaluated ofthem underwent emergency surgery es group and of them after bts with sems placement bts group wecompared surgical results and prognoses between the two groupsresults a total of patients underwent endoscopic sems placement which technical success of andmorbidity rate of primary anastomosis rates were in es and in bts p postoperativecomplication in es and in bts p pathological findings of lymphatic invasion in es and in bts p venous invasion were in es and in bts p and recurrence of in esand in bts the 3year overall survival was significantly different between two groups es 868bts bts is worse than es logrank test p venous invasion independently predicted worsened recurrencefreeand overall survivals the vascular invasiveness was correlated with tumor progression after sems placement and thesurvival rate was lower in bts sems potentially worsens prognostic outcomes in stage iiiii obstructive colorectalcancerkeywords bowel obstruction colorectal cancer selfexpandable metallic stent colorectal cancer crc remains the leading cause ofcancerrelated deaths worldwide because several patientsare initially diagnosed during advanced stages correspondence ohtakmedkindaiacjp1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japanfull list of author information is available at the end of the approximately of patients with crc were diagnosed with acute colonic obstruction [] severe malignancy with bowel obstruction needs urgent surgicalintervention which includes primary lesion resectionand stoma creation leading to increased morbidity andmortality and a potential failure to achieve completeoncological resection [ ]an endoscopic procedure with selfexpandable metallic stent sems is an acceptable bridge to surgery bts the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc surgery page of treatment for acute colonic obstruction [] preoperative sems placement provides an opportunity to perform medical resuscitation comorbidity optimizationbowel preparation tumor staging and observation ofproximal lesions the procedure prevents highriskemergency surgeries and increase oncological resectionand primary anastomosis rates [ ] after the inclusion of colonic sems placement as bts in the coverageof the national health insurance in japan several physicians joined the colonic stent safety procedure researchgroup and developed skills to provide safe treatmentthe largest multicenter prospective study demonstratedthe feasibility and safety of sems placement as bts inpatients with malignant colorectal obstruction the oncological safety and minimalinvasiveness ofthis procedure have confirmed that sems placement asa bridge to elective surgery is not recommended as astandard treatment for symptomatic leftsided malignantcolonic obstruction [ ] several studies reportedthat prognostic factors of malignant colonic obstructionin sems placement had oncological disadvantages compared with those in emergency surgery es [ ] incontrast several trials showed that sems placement as abridge to elective surgery did not improve the survivalrates [] how sems placement worsens prognosticoutcomes remains unclear [ ]this study aimed to evaluate the induction of curativesurgery in patients with malignant colorectal obstructionafter a sems placement and its longterm results andprognostic factors postoperatively compared to patientswithout sems placement we demonstrated prognosticfactors and overall survival os and recurrencefreesurvival rfs rates for curative surgery after a semsplacementmethodspatientsmedical records of patients who underwent primarycolorectal resection at higashiosaka city medical centerbetween january and december werereviewed all participants provided written informedconsent oralintake and symptoms before and aftersems placement were assessed in table using thecolorectal obstruction scoring system cross from to we recruited patients with all class oftable the colorectal obstruction scoring system crosspatients symptom and their condition of an oral intakesolid meal low residue and full diet without symptomcrosscross as stent insertion candidate from we excluded patients with cross and based on updatedstent insertion guideline malignant colorectal obstruction was diagnosed through clinical examinationcross radiography and computed tomography surgery was performed using three approaches es comprised laparotomylymph node dissection as possibleand primary anastomosis on the same day between and bts after sems placement comprised standbylaparoscopy d3 lymph node dissection and primaryanastomosis since january overall patientswith stage iiiii cur a obstructive colorectal cancerwere evaluated of them underwent emergency surgery as es group and of them after bts with semsplacement as bts group we compared surgical resultsand prognoses between the two groupssems devices and the procedurepatients were endoscopically treated with placement ofan uncovered wallflex enteral colonic stent boston scientific corporation natick ma usa or nitis enteralcolonic uncovered stent taewoong inc gimpo southkorea placements were performed as presented in thepreintroduction publicity announcement placement details were mentioned on the website as a brief guideline obstruction structures were determined using aguide wire and a contrast tube was inserted into theproximal colorectal lumen obstructions were measuredusing contrast agents and then the endoscopist determined the number size and type of stent pathologicalbiopsies were recommended after sems locations andintraluminal or extraluminal marking using an endoscopic clip were recommended via visual recognition ofthe endoscopist dilatation of the colonic obstructionbefore sems placement was generally not allowedhistological findingsparaffinembedded specimens were obtained from a cohort of patients diagnosed by the union for international cancer control stage iiiiisurvival definitionsos was defined as the duration from surgery to anydeath or last followup diagnosis of recurrence was calculated based on recist according to the chemotherapy criteria rfs was defined as the durationfrom surgery to any recurrence includes local recurrenceor distant metastasissolid meal low residue and full diet with symptomliquid or enteral nutrient intakeno oral intakerequiring continuous decompressionstatistical analysisstudents ttest and wilcoxon test for continuous variables and the Ï2 and fishers exact tests for categoricalvariables were conducted survival curves were generated using the kaplanmeier method and compared 0cohta bmc surgery page of table baseline characteristics and outcomes of endoscopicsems placementtable comparison of baseline characteristics in patientsundergoing emergency surgery and bridge to surgerybts n es n pmalefemalemedian range genderagelocationmalefemalemedian rangececumascendingtransversedescendingsigmoidrectumlength of obstructionmedian range cmtechnical successprocedurestentingmorbiditymortalityclinical successthrough the scopethrough the wirewall flex cmnitis cmoverall cda iiicda vcrossbts n a claviendindo classificationusing a logrank test univariate and multivariate survival analyses were performed using the cox proportional hazards regression model all statistical analysesused jmp version sas institute cary nc or statistical scripting language r httpwwwrprojectpvalues of ¤ twosided were considered statistically significant this prognostic study complied withthe reporting recommendationsfor tumor markerprognostic studies resultsa total of patients underwent endoscopic semsplacement which was technically safe for malignantcolorectal obstruction with the technical success rate of the clinical success rate was and the patientssymptoms and oral intake dramatically improved afterthe sems placement shown in table a total of patients were reviewed and patients underwentes and bts respectively as shown in table baselineclinical characteristics were balanced between the twogroups moreover cases of patients underwent es on the same day as in open surgery the median waiting period for surgery was days for bts thegenderagelocationtype of operationcecumascendingtransversedescendingsigmoidrectumstandbyemergency duration tooperationmedian range dayssurgical procedurelaparotomy laparoscopy timemedian range minblood lossmedian range ml before afterstoma creation morbidity30day complication cda iii anastomosticleakagehospital stayaclaviendindo classificationmedian range days primary anastomosis ratios were in es and in bts p postoperative complication rateswere in es and in bts p postoperative hospital stay was shorter in bts days comp patients withpared to esobstructive crc showed significantinpostoperative complication rate and hospital stay withsems placement operative procedures were dramatically changed and the primary anastomosis rate improved after the sems placementimprovement daysthe pathological tissue type accounted for of differentiated types shown in table tumordepth was similar between the two groups lymphatic vesselinvasion ratios were in es and in bts p and venous invasion ratioswere in es and in bts p recurrence rates were cases in es and cases in bts nodenegative patients stage iimorewhereas nodepositive patients stage iii more frequently had liver metastasis in the kaplanmeier survival analysis in fig 1a the 3year rfs waslung metastasisfrequentlyhad 0cohta bmc surgery page of table comparison of pathological characteristics of emergency surgery and bridge to surgerypt factortotal lymph nodespn factorhistologicallymphatic invasionvenous invasionsurgical clearancet4b t4a t3median rangen0 n1 n2 n3tub1 tub2 othersly v cur a b csignificantly different between the two groups es bts which was significantly low inbts than that in es logrank test p the3year os rate was also significantly different between thetwo groups es bts p shown in fig 1b the relationship between lymph node metastasis and sems placementwas also evaluated the pathological nodenegativestage ii 3year rfs rate was not different betweenthe two groups es bts as shown infig 2athe pathological nodepositivestage iii 3year rfs rate was different between thetwo groups es bts as shown in fig 2bthe stage ii 3year os rate was not different between thetwo groups es bts as shown in fig 2cwhereas stage iii 3year os rate was different between thetwo groups es bts as shown in fig 2dthese results suggestthat vascular invasiveness andpathological nodepositive status were correlated withtumor progression after sems placementthein contrastthuses n bts n p survival rate was affected by poor prognosis in the btsgroupresults of adjusted multiple cox proportional hazard regression for rfs and os in all stages and stage iii diseaseare presented in table after adjusting for possible confounders venous invasion and bts independently predicted poor rfs in all stages and venous invasionindependently predicted poor rfs in stage iii disease venous invasion and bts were also significantly associatedwith os in stage iii diseasediscussionacute colonic obstruction requires emergent surgicalintervention a mandatory conventional treatment skillemergent surgicalis associated with highmorbidity mortality and stoma creation rates affectingthe quality of life of patients malignant colorectal obstruction is not only an intestinal obstruction but alsoan advanced stage crc their prognosis was poorerthan that in patients with nonocclusive disease becausetreatmentfig kaplanmeier survival curves in patients undergoing emergency surgery vs bridge to surgery a recurrencefree survival b overall survival 0cohta bmc surgery page of fig kaplanmeier survival curves in patients undergoing emergency surgery vs bridge to surgery a rfs nodenegative patients b rfs nodepositive patients c os nodenegative patients d os nodepositive patientsof highly invasiveness and distant metastasis [ ]chen revealed that the prognosis in patients withperforation associated with obstruction was poor early intervention in the clinical setting before the colonic perforation has been established endoscopic placement of colonic stents improves the high decompressioneffect and reduces clinical symptoms high postoperative complication rates were correlatedwith poor prognosis in patients with cancer in severalans [] reducing complication rates can improve the prognosis our results showed high clinicalsuccess rate after sems placement and high primarysurgicalinterventions howeveranastomosis rate stentrelated complications requiredemergentthe stentplacement is safe and feasible in this study moreoverthe laparoscopic rate was high and postoperative complication rate was clinical results including shortterm outcomesin bts after sems were verifiedthrough a metaanalysis [ ]the prognosis was poor in patients with stent perforation and increased local recurrence rate after the colonic stent placementthe longtermprognosis in patients with colorectal obstruction afterbts was not different compared with that in patients however 0cohta bmc surgery page of table multivariate analysis of recurrencefree survival at all stages and stage iiipvaluevariablesrecurrence free suvivalall stages hazard ratio ± sd cistage iii hazard ratio ± sd cipvaluees vs semspt factor t3t4pn factor n0n1n2verous invasion v01v2lymphatic invasion ly01ly2hr ± hr ± hr ± hr ± hr ± overall suvivales vs semspt factor t3t4pn factor n0n1n2verous invasion v01v2lymphatic invasion ly01ly2hr ± hr ± hr ± hr ± hr ± without obstruction [] according to the europeansociety of gastrointestinal endoscopy clinical guidelinethat considers the risk of perforation due to colorectalstents only limited uses are allowed therefore colorectalstent placement is not a standard treatment []the prognostic outcomes of bts in this study were significantly worse than those of es particularly in lymphnodepositive patientslymphatic and venous invasionseemed to be a significant prognostic factor althoughreduced postoperative complication rate would improve the prognosis our results were contradictoryafter the stent replacement these results suggestedthat stent placement leads to poor prognosis a concern that colonic stents may be associated with adverse effects of mechanical expansion also exists mechanical expansion may be associated with thegrowth of solid tumors particularly lymphatic andvenous invasion [ ]we found that recurrence and os were associatedwith high vascular invasion after a colonic stent placement venous invasion was an independent factor for recurrence and prognosis the ck20 mrna level anepithelial marker is significantly increased in peripheralblood serum suggesting stent deployment into the vasculature alliteratively ki67 level associated withcellular proliferation and p27 gene assisting cell cycleprogression were measured using specimens obtainedbefore and after sems insertion next the ki67 leveldecreased in the specimen after an sems placementcompared with that before and cell proliferation wassuppressed the prognostic nutritionalindex andserum albumin levels were significantly decreased afterstenting suggesting its disadvantage as bts theduration from stent placement to surgery was daysoncological and nutritional factors might change in theblood and contribute to poor prognosis during the waiting period mechanical expansion of the replacement hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± should be minimized to prevent perforation and molecular cytological factors to improve the materials expansion and establishment of new mechanism are necessaryin colorectal obstruction [ ]thisisafirstretrospectivethese findings should be considered in light of severallimitationsnonrandomized small sample sized study from a single institution thereby the heterogeneity of the surgical strategy may have affected the prognostic factors secondalthough validated endoscopic procedures were validated stent devices used in this study had differentlengths types and thickness and obtained from differentvendors lastly we performed stent placement in the patients with cross and who are not indicated forstent insertion until to investigate the oncological longterm prognosis ofcolonic sems placement as a bridge to elective surgerylarge sample size and prospective randomized controlledstudies are warranted to develop a treatment strategy forcrc with obstructionvascular invasiveness was correlated with tumor progression after a sems placement and os and rfs rateswere lower in bts sems placement potentially worsensprognostic outcomes in stage iiiii malignant colorectalobstructionabbreviationsbts bridge to surgery crc colorectal cancer cross colorectal obstructionscoring system es emergency surgery esge european society ofgastrointestinal endoscopy jsccr japanese society for cancer of the colonand rectum recist response evaluation criteria in solid tumors version os overall survival rfs recurrencefree survival sems selfexpandablemetallic stent wses world society of emergency surgeryacknowledgementsthe authors thank for contribution as endoscopic technical adviser kenkonishi md phd from department of surgery hyogo prefecturenishinomiya hospital 0cohta bmc surgery page of authors contributionsall authors have read and approved the manuscript ko protocolproject development data collection and management andmanuscript writingediting mi protocolproject developmentmanagement and manuscript writingediting mu protocolprojectdevelopment and data collection and management jm se jm and ikdata collection and management yt and sn data collection ki andtn data analysis and manuscript writingediting mt and ty dataanalysis and managementfundingauthors have no grant support and no financial relationship for this studyavailability of data and materialsthe datasets used and analyzed during this study are available from thecorresponding author upon reasonable requestethics approval and consent to participateall procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional researchcommittee and with the helsinki declaration and its later amendmentsor with comparable ethical standards all participants or their guardians haveprovided their written informed consent and that the study protocol wasapproved by higashiosaka city medical center ethical committee on humanresearch assignment number consent for publicationno applicablecompeting intereststhe authors declare that they have no conflict of interest to discloseauthor details1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japan 3thoracic surgeryhigashiosaka city medical center osaka japan 4digestive surgery kawasakimedical school okayama japan 5gastroenterology higashiosaka citymedical center osaka japanreceived june accepted august referencesjemal a bray f center mm ferlay j ward e forman d global cancerstatistics ca cancer j clin pubmed pmid epub eng winner m mooney sj hershman dl feingold dl allendorf jd wright jd incidence and predictors of bowel obstruction in elderly patients withstage iv colon cancer a populationbased cohort study jama surg pubmed pmid pubmed central pmcid pmc45 epub engjullumstro e wibe a lydersen s edna th colon cancer incidencepresentation treatment and outcomes over years color dis pubmed pmid epub engcheynel n cortet m lepage c benoit l faivre j bouvier am trends infrequency and management of obstructing colorectal cancers in a welldefined population dis colon rectum pubmed pmid epub engcuffy m abir f audisio ra longo we colorectal cancer presenting assurgical emergencies surg oncol pubmed pmid epub eng mcardle cs hole dj emergency presentation of colorectal cancer isassociated with poor 5year survival br j surg pubmedpmid epub eng mainar a tejero e maynar m ferral h castanedazuniga w colorectalobstruction treatment with metallic stents radiology pubmed pmid epub engzhang y shi j shi b song cy xie wf chen yx comparison of efficacybetween uncovered and covered selfexpanding metallic stents inmalignant large bowel obstruction a systematic review and metaanalysiscolor dis 2012147e367 pubmed pmid epub engtilney hs lovegrove re purkayastha s sains ps westonpetrides gk darziaw comparison of colonic stenting and open surgery for malignantlarge bowel obstruction surg endosc pubmed pmid epub engsaito s yoshida s isayama h matsuzawa t kuwai t maetani i aprospective multicenter study on selfexpandable metallic stents as a bridgeto surgery for malignant colorectal obstruction in japan efficacy and safetyin patients surg endosc pubmed pmid epub engsaida y sumiyama y nagao j uramatsu m longterm prognosis ofpreoperative bridge to surgery expandable metallic stent insertion forobstructive colorectal cancer comparison with emergency operation discolon rectum suppls44 pubmed pmid epub engtomita m saito s makimoto s yoshida s isayama h yamada t selfexpandable metallic stenting as a bridge to surgery for malignant colorectalobstruction pooled analysis of patients from two prospectivemulticenter series surg endosc pubmed pmid pubmed central pmcid pmc6342866 epub eng chen hs sheenchen sm obstruction and perforation in colorectaladenocarcinoma an analysis of prognosis and current trends surgery pubmed pmid epub eng huang x lv b zhang s meng l preoperative colonic stents versusemergency surgery for acute leftsided malignant colonic obstruction ametaanalysis j gastrointest surg pubmed pmid epub engkim hj huh jw kang ws kim ch lim sw joo ye oncologic safetyof stent as bridge to surgery compared to emergency radical surgery forleftsided colorectal cancer obstruction surg endosc pubmed pmid epub engshigeta k baba h yamafuji k kaneda h katsura h kubochi k outcomesfor patients with obstructing colorectal cancers treated with onestagesurgery using transanal drainage tubes j gastrointest surg pubmed pmid epub engvan hooft je bemelman wa oldenburg b marinelli aw lutke holzik mfgrubben mj colonic stenting versus emergency surgery for acute leftsided malignant colonic obstruction a multicentre randomised trial lancetoncol pubmed pmid epub engvan hooft je fockens p marinelli aw timmer r van berkel am bossuyt pm early closure of a multicenter randomized clinical trial of endoscopicstenting versus surgery for stage iv leftsided colorectal cancer endoscopy pubmed pmid epub engsloothaak da van den berg mw dijkgraaf mg fockens p tanis pj vanhooft je oncological outcome of malignant colonic obstruction inthe dutch stentin trial br j surg pubmed pmid epub eng gorissen kj tuynman jb fryer e wang l uberoi r jones om localrecurrence after stenting for obstructing leftsided colonic cancer br j surg pubmed pmid epub eng ormando vm palma r fugazza a repici a colonic stents for malignantbowel obstruction current status and future prospects expert rev meddevices pubmed pmid epub englauro a binetti m vaccari s cervellera m tonini v obstructing leftsidedcolonic cancer is endoscopic stenting a bridge to surgery or a bridge tonowhere digestive diseases and sciences pubmed pmid epub engschwartz lh litière s de vries e ford r gwyther s mandrekar s recist 11update and clarification from the recist committee europeanjournal of cancer oxford england p pubmed pmid pubmed central pmcid pmc5737828 epub eng mcshane lm altman dg sauerbrei w taube se gion m clark gm reportingrecommendations for tumour marker prognostic studies remark eur jcancer pubmed pmid epub eng hashiguchi y muro k saito y ito y ajioka y hamaguchi t japanesesociety for cancer of the colon and rectum jsccr guidelines for thetreatment of colorectal cancer int j clin oncol pubmed pmid epub eng cortet m grimault a cheynel n lepage c bouvier am faivre j patterns ofrecurrence of obstructing colon cancers after surgery for cure a population 0cohta bmc surgery page of based study color dis pubmed pmid epub eng nojiri t maeda h takeuchi y funakoshi y kimura t maekura r predictive value of btype natriuretic peptide for postoperative atrialfibrillation following pulmonary resection for lung cancer eur jcardiothorac surg pubmed pmid epub engpubmed pmid pubmed central pmcid pmc4128744 epub engkaragiannis gs poutahidis t erdman se kirsch r riddell rh diamandis epcancerassociated fibroblasts drive the progression of metastasis throughboth paracrine and mechanical pressure on cancer tissue mol cancer res pubmed pmid pubmed central pmcidpmc4399759 epub eng nojiri t inoue m yamamoto k maeda h takeuchi y funakoshi y b maruthachalam k lash ge shenton bk han af tumour celldissemination following endoscopic stent insertion br j surg pubmed pmid epub eng matsuda a miyashita m matsumoto s sakurazawa n kawano y yamahatsuk colonic stentinduced mechanical compression may suppresscancer cell proliferation in malignant large bowel obstruction surg endosc pubmed pmid epub eng haraguchi n ikeda m miyake m yamada t sakakibara y mita e colonic stenting as a bridge to surgery for obstructive colorectal canceradvantages and disadvantages surg today pubmedpmid epub eng zhu z li b liao w lv n chen y shu x novel predictive nomogram foridentifying difficult guidewire insertion in patients with malignant colorectalobstruction and sphincterotomeassisted guidewire insertion for improvingthe success rate of selfexpandable metal stent insertion front oncol pubmed pmid pubmed central pmcid pmc7237730epub eng miyasako y kuwai t ishaq s tao k konishi h miura r newlydeveloped selfexpandable nitis md colonic metal stent for malignantcolonic obstruction world j gastrointest surg pubmedpmid pubmed central pmcid pmc7215972 epub engpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationstype natriuretic peptide as a predictor of postoperative cardiopulmonarycomplications in elderly patients undergoing pulmonary resection for lungcancer ann thorac surg pubmed pmid epub eng cowie mr struthers ad wood da coats aj thompson sg poolewilsonpa value of natriuretic peptides in assessment of patients withpossible new heart failure in primary care lancet pubmed pmid epub eng cuthbertson bh card g croal bl mcneilly j hillis gs the utility of btypenatriuretic peptide in predicting postoperative cardiac events and mortalityin patients undergoing major emergency noncardiac surgery anaesthesia pubmed pmid epub engsabbagh c browet f diouf m cosse c brehant o bartoli e isstenting as a bridge to surgery an oncologically safe strategy for themanagement of acute leftsided malignant colonic obstruction acomparative study with a propensity score analysis ann surg pubmed pmid epub engtung kl cheung hy ng lw chung cc li mk endolaparoscopic approachversus conventional open surgery in the treatment of obstructing leftsidedcolon cancer longterm followup of a randomized trial asian j endoscsurg pubmed pmid epub eng gianotti l tamini n nespoli l rota m bolzonaro e frego r aprospective evaluation of shortterm and longterm results from colonicstenting for palliation or as a bridge to elective operation versus immediatesurgery for largebowel obstruction surg endosc pubmed pmid epub eng yang sy park yy han yd cho ms hur h min bs oncologicoutcomes of selfexpandable metallic stent as a bridge to surgery andsafety and feasibility of minimally invasive surgery for acute malignantcolonic obstruction ann surg oncol pubmed pmid epub engvan hooft je van halsema ee vanbiervliet g beetstan rg dewitt jmdonnellan f selfexpandable metal stents for obstructing colonic andextracolonic cancer european society of gastrointestinal endoscopy esgeclinical guideline endoscopy pubmed pmid epub eng ansaloni l andersson re bazzoli f catena f cennamo v di saverio s guidelenines in the management of obstructing cancer of the leftcolon consensus conference of the world society of emergency surgerywses and peritoneum and surgery pns society world j emerg surg pubmed pmid pubmed central pmcid pmc3022691epub eng arezzo a balague c targarona e bhi f giraudo g ghezzo l colonic stenting as a bridge to surgery versus emergency surgery formalignant colonic obstruction results of a multicentre randomisedcontrolled trial esco trial surg endosc pubmedpmid epub eng amelung fj burghgraef ta tanis pj van hooft je ter b f siersema pd critical appraisal of oncological safety of stent as bridge to surgery inleftsided obstructing colon cancer a systematic review and metaanalysiscrit rev oncol hematol pubmed pmid epub eng domingo s puertolas s graciavilla l puertolas ja mechanical comparativeanalysis of stents for colorectal obstruction minim invasive ther alliedtechnol pubmed pmid epub engsuzuki n saunders bp thomasgibson s akle c marshall m halligan scolorectal stenting for malignant and benign disease outcomes incolorectal stenting dis colon rectum pubmed pmid epub eng voutouri c mpekris f papageis p odysseos ad stylianopoulos t roleof constitutive behavior and tumorhost mechanical interactions in thestate of stress and growth of solid tumors plos one 201498e104717 0c"
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"Early detection of capecitabineresistance could largely increase overall survival of colorectal cancerCRC patients Previous studies suggested examination of immune cells in peripheral blood would help to predictefficacy of chemotherapyMethods We examined the immunological characteristics of peripheral blood in CRC patients with capecitabinetreatment We analyzed the relationships between the abnormal immune cell population in capecitabineresistancepatients and major clinical features Furthermore RNA sequencing analyses of cell surface marker expression andthe correlations with other major immune cell populations were performed using this population to explore thepossible function of these cellsResults The expression level of CD16 on neutrophils was downregulated in capecitabineresistant CRC patientsPatients with CD16lowneutrophils after capecitabine therapy had adverse clinical features Whats important thechange of CD16 expression level on neutrophils appeared much earlier than CT scan RNA sequencing revealedthat CD16lowneutrophils in capecitabineresistant patients had lower expression level of neutrophilrelated genescompared to CD16neutrophils in capecitabinesensitive patients suggesting this CD16lowpopulation might beimmature neutrophils Furthermore the expression level of CD16 on neutrophils in patients with capecitabinetreatment was positively correlated with the number of antitumor immune cell subsets such as CD8T cell CD4Tcell NK cell and monocyteConclusions Our findings indicated that CD16 expression on neutrophils in peripheral blood was a goodprognostic marker for predicting efficacy of capecitabine in CRC patientsKeywords CD16 Neutrophils Capecitabineresistance Colorectal cancer Correspondence drzhongming1966163com gaoweiqiangsjtueducnyanzhsjtueducnYu Lu Yizhou Huang and Lei Huang share first authorship2Department of Gastrointestinal Surgery Renji Hospital School of MedicineShanghai Jiaotong University Shanghai China1State Key Laboratory of Oncogenes and Related Genes RenjiMed X StemCell Research Center Renji Hospital School of Medicine Shanghai JiaotongUniversity Shanghai ChinaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLu BMC Immunology Page of BackgroundColorectal cancer CRC is one of the leading cause ofdeath worldwide More than million patients are diagnosed with CRC every year [] Whats more this lifethreaten disease kills nearly million people annually []In north America and Europe the morbidity and mortalityremain at high level [] despite developments of cancerscreening and endoscopy [ ] In China CRC becomesthe 5th most diagnosed cancer and 5th most deadly cancer[] Nearly million new cases are diagnosed andabout million people die from the disease every year []Postoperative adjuvant chemotherapy is firstline treatment for CRC patients [ ] Capecitabine a carbamatederivative of fluoropyrimidine is the backbone of CRCchemotherapy [ ] Asthe oral prodrug of fluorouracil 5FU it is widely used for postoperative adjuvant chemotherapy due to its long stable durationlower toxicity and convenient dosing compared to infusional 5FU [ ] However this chemotherapeutic drughas only modest efficacy the response rates of 5FU foradvanced CRC is only for single treatment and for combined chemotherapy [ ] The chemoresistance is recognized as a principal obstacle for cancer therapy [] leading to tumor recurrence or metastasisespecially liver and lung metastasis and cause over ofCRC mortality [] Intense researches on the mechanisms underlying the resistance revealed that changes oftumor cells themselves cause resistance although thesefindings are mainly restricted to tumor specimen examinewhich is not that suitable for posttreatment surveillanceWhats more CT computed tomography scan and colonoscopy are insensitive to micro metastasis despite theirgoodrecurrenceCapecitabineresistant patients could only be diagnosedwith cancer recurrence by CT scan or colonoscopy about years after capecitabine therapy [] when tumorsare big enough to be discovered Thus good prognosticmarkers are indispensable for predicting capecitabineresistance in the early stage after capecitabine therapydetection ofaccuracytheforCancer cells and their microenvironment could interactwith each other Immune cells could dynamically reflectcancer status and display multifaceted functions in cancerdevelopment [] Myeloid cells including monocytesmacrophages granulocytes neutrophils eosinophils basophils and mast cells play critical roles in cancer progression [] Myeloidderived suppressor cells MDSCs aheterogeneous population of myeloid cells remain at different stages of differentiation are immature counterparts ofmyeloid cells in cancer MDSCs acquire immunosuppressive features and mainly inhibit lymphocytes including Tcells and NK cells [] Recent studies report that chemotherapeutic agents like 5FU could interact with myeloid cells and influence antitumor efficacy []Vincent J reported that 5FU selectively inducedMDSC apoptotic cell death and increase IFNÎ productionby tumorspecific CD8T cells [] Other researchersshowed that activation of NLRP3 inflammasome and increased amount of HSP70 exosomes on MDSC by 5FUlead to MDSC activation [ ] Yuan Y found thattumorassociated macrophages secret IL6 to induce 5FUchemoresistance []ImportantlyIn this study we discovered that the expression ofCD16 on CD11bmyeloid cells was dramatically decreased in capecitabineresistant CRC patients after capecitabine adjuvanttherapy The expression level ofCD16 was closely related to poor prognosis after capecitabine therapythe downregulation ofCD16 on CD11bmyeloid cells appeared as early as month after capecitabine therapy in patients who werediagnosed with capecitabineresistance by CT scansabout years after the treatment The cutoff value ofCD16 expression would be helpful for the prediction of capecitabine chemoresistance Further analysisdemonstrated that these CD11bCD16lowmyeloid cellswere mainly immature neutrophils and expression levelof CD16 on neutrophils had a positive relationship withfrequencies of antitumor immune cell populations suchas CD8T cells and NK cellsResultsCD16 expression levels on CD11bmyeloid cells inperipheral blood of capecitabineresistant CRC patientsare different from capecitabinesensitive CRC patientsafter capecitabine therapyTo explore if myeloid cells in peripheral blood could predict the treatment efficacy of capecitabine we chose CRC patients with capecitabine adjuvant treatment whoseimmune cells populations in peripheral blood were examined by flow cytometry before and about months afterthe treatment Patients were divided into capecitabinesensitive and capecitabineresistant groups based on thediagnosis of recurrence by CT scan in about years aftercapecitabine treatment Table Additional file Fig S1ENo significant change was observed in major myeloid cellsubsets such as monocytes CD11bCD14CD15 neutrophils CD11bCD15CD14 or CD11bCD66bCD14and MDSCsbetweencapecitabinesensitive patients and capecitabineresistantpatients Additional file S1A B C and D But we foundthat the frequency of CD11bCD16myeloid cells was decreased in capecitabineresistant patients after capecitabinetreatment compared to that before the treatment Fig 1aWhats important a dramatic lower expression level ofCD16incapecitabineresistant patients compared to that of drugsensitive patients Patient and patient are representative patientsgroup andcapecitabineresistant group respectively Fig 1b TheCD11bHLADR\\lowCD33from capecitabinesensitiveon CD11bmyeloidcells wasobserved 0cLu BMC Immunology Page of Table Baseline characteristics of CRC patients in Fig GroupNumber of PatientsAgeSexTNM StageLocationCEA ngmlCA199 ngml Diagnosis of Recurrence AfterCapecitabinesensitiveCapecitabineresistantMMMMMFFFMFMFMMMFMMFFFMFMFMMMMFMMMFFMIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIRectumRectumColonColonRectumRectumRectumColonRectumColonColonColonRectumColonRectumRectumRectumRectumColonColonRectumRectumRectumRectumColonRectumRectumColonColonRectumRectumRectumRectumRectumRectumColonCapecitabine TreatmentNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYesYesYesYesYesYesYesYesYesYesdiagnosis of capecitabine resistance was determined by CTscan Additional file Fig S1E However when we analyzed these CD11bCD16myeloid cells in healthy donorsHDs and CRC patients before capecitabine therapy wefound no difference between these two cohorts Additionalfile Fig S1F and G This indicated that change of CD16expression on CD11bCD16myeloid cells was particular inCRC patients who were resistant to capecitabine therapyDecreased CD16 expression is correlated with poorpathological features in CRC patients after capecitabinetherapyTo determine whether the expression level of CD16 onCD11b myeloid cells is related to treatment efficacy of capecitabine we collected peripheral venous blood of CRCpatients months after capecitabine treatment and divided these patients into two groups CD16 group and 0cLu BMC Immunology Page of Fig CD16 expression of peripheral blood myeloid cells were differential in CRC patients after capecitabine therapy Peripheral venous bloodfrom CRC patients received singleagent oral capecitabine adjuvant therapy was collected before the therapy and months after the therapyand analyzed for myeloid cellrelated markers Attention Blood were collected months after capecitabine treatment unless particularlynoted a Frequencies of CD11bCD16myeloid cells were compared before and after capecitabine therapy in capecitabinesensitive andcapecitabineresistant patients n in sensitive group and n in resistant group respectively b Representative images of CD16 expressionon CD11bmyeloid cells before and after capecitabine therapy in two CRC patients from capecitabinesensitive group or capecitabine resistantgroup respectively Diagnosis of drugresistance was proved by CT scan during the followup in Fig S1e Mean ± SEM P005 by t tests aCD16low group Firstly Kmean clustering algorithm wasused to determineto divideCD11bCD16myeloid cells into CD11bCD16highcells andthe boundaryvalueCD11bCD16lowcells based on mean fluorescent intensityMFI of CD16 on CD11bCD16myeloid cells in peripheralblood after capecitabine therapy Additional file Fig S2A 0cLu BMC Immunology Page of ROCanalysisThe boundary value of CD16 MFIfor division ofCD11bCD16high cells and CD11bCD16low cells was à Next we analyzed frequency of CD11bCD16high cellsin peripheral blood after capecitabine therapy Additional file Fig S2B and determined the cutoff value for CD16 expression on CD11bmyeloid cells by receiver operating characteristicand Youden Index valuesAdditional file Fig S2C and S2D The cutoff value was Patients of CD16 group or CD16low group were determined if their frequencies of CD11bCD16highcells werehigher or lower than the cutoff value Additional file FigS2B S2C and S2D Then we assessed correlations betweenthe expression level of CD16 and CRC clinicopathologicalcharacteristics by Ï2 test The data revealed that patients inCD16low group had more cancer recurrence P and high level of carcinoembryonic antigen CEA P as well as carbohydrate antigen CA199 P compared to patients in CD16 group Table There were CRC patients developing recurrenttumor in CD16low group whereas only cases were observed in CD16 group Among CRC patientswith high CEA level patients belonged toCD16low group while only patients wereCD16 And patients with high CA199level were found in CD16low group compared with cases in that of CD16 However no significant difference was observed between these twogroups on age gendertumor sizeand Tumor Node Metastasis TNM stage Table tumor locationTo further confirm these results we divided CRCpatients after capecitabine treatment into two groupsbased on the level of CEA or CA199 and compared theexpression level of CD16 on CD11bCD16myeloid cellsbetween CEAhigh CEA ng and CEAlow CEA ¤ Table Relationship between CD16 expression on CD11bmyeloid cells after capecitabine therapy and clinicopathologiccharacteristicsCharacteristicsCD16low after therapy n nAll patients n nCD16 after therapy n nAge years¥GenderMaleFemaleTumor locationRectumColonTumor Size¥ cm cmCEA level after therapy¤ ngml ngmlCA199 level after therapy¤ ngml ngmlTNM stage AJCCStage IIStage IIILocation of recurrenceLocoregionalliver lungliverlungperitoneumPvalue 0cLu BMC Immunology Page of ng groups or between CA199high CA199 ngand CA199low CA199 ¤ ng groups The boundaryvalue of CEA and CA199 were decided by clinical guidelines The results showed that the expression level ofCD16 was dramatically decreased in either CEAhigh orCA199high groups compared to CEAlow or CA199low groups Fig 2a and b suggesting that the decreasedexpression level of CD16 on CD11bmyeloid cells aftercapecitabine treatment was related to the poor pathological features In conclusion low level of CD16 expression was related to poor pathological features such astumor recurrence CEA and CA199in CRC patientswith capecitabine therapyCD16 serves as a prognostic marker for CRC patientsreceived capecitabine adjuvant chemotherapyTo further explore the prognostic significance of CD16expression on CD11bmyeloid cells in predicting thetreatment efficacy of capecitabine chemotherapy wecompared the differences of overall survival OS anddisease free survival DFS between CD16 group andCD16low group The survival curves revealed that therewere significant association between the expression levelof CD16 and OS P 00006Fig 3a or DFS P 00023Fig 3b suggesting that low expression level ofCD16 was associated with shorter survival Next weused univariate analysis to further elucidate the significance of CD16 expression in predicting prognosis ofCRC patients receiving capecitabine The result demonP HR strated that CD16 expression level was prognostic factor for OS Table Whatsimportant Cox multivariate analysis also demonstratedthat expression level of CD16 P HR wasindependent predictors of OS Table Thesestillresults demonstrated that the expression level of CD16on CD11bmyeloid cells may serve as a good prognosticmarker for overall survival in CRC patients with capecitabine adjuvant chemotherapy[] Next we wondered ifDownregulation of CD16 expression on CD11bmyeloidcells appears earlier than diagnosis of capecitabine byimaging testsAs we know adjuvant chemotherapy remains the firstline therapy for CRC patients Capecitabine the oralprodrug of 5fluorouracil is one of the primary drugsfor the treatment A number of CRC patients becomeinsensitive to the therapy and suffer from cancer recurrence In clinic capecitabineresistance is mainlydiagnosed by cancer recurrence discovered throughcolonoscopy or CT scan in about years after capecitabine treatmentthechange of CD16 expression level on CD11bmyeloidcells appeared earlier than CTshowed recurrence Weselected CRC patients with capecitabine treatmentwhose blood samples were examined before and aftercapecitabine treatment Table The results showedin patients in capecitabineresistant groupthefrequency of CD11bCD16myeloid cells was decreased months after treatment compared to thatbeforecapecitabineresistance was diagnosed by CT scan about yearsafter the treatmentfile Fig S1E Whats important in a resistant patient decreased expression level of CD16 was found as earlyas month after capecitabine treatment Fig 4a Thefrequency of CD11bCD16high cell population waslargely lower than the cutoff value Neverthelesstumor monthsTable and Additional1a whiletreatmentFigafterthecapecitabinetherapyFig CD16 expression of CD11bCD16myeloid cells related to pathological features of CRC patients with capecitabine therapy CRC patientsreceiving capecitabine therapy were divided into different groups according to their CEA or CA199 level n in CEAhigh CEA ng groupand n in CEAlow CEA ¤ ng group n in CA199high CA199 ng group and n in CA199low CA199 ¤ ng group CD16MFI of CD11bCD16myeloid cells in CRC patients acquired from flow cytometry analysis was compared between different groups Mean ± SEMP001 P0001 by t tests a b 0cLu BMC Immunology Page of Fig CD16 high expression on CD11bmyeloid cells was good prognostic marker for CRC patients survival KaplanMeier analysis of overallsurvival OS and disease free survival DFS was performed in CD16 group and CD16low group p values were calculated by logrank test n in CD16 group and n in CD16low grouprecurrence was found in the liver from CT scan Fig 4bThese data suggested that downregulation of CD16on CD11bmyeloid cells served as a more sensitiveexamine than CT in CRC patientstreated withcapecitabineCD11bCD16lowmyeloid cells are mainly immatureneutrophils after capecitabine therapyTo further characterize the population of CD11bCD16lowmyeloid cells we isolated CD11bCD16myeloid cells fromcapecitabinesensitive patients and CD11bCD16myeloidcells from capecitabineresistant patients after capecitabinetherapy Fig 5a The data from flow cytometry revealed thatthese two populations were mainly neutrophils provedby their CD15 and CD66b expression Additional file Fig S3A To further verify these CD11bCD16myeloid cells and CD11bCD16myeloid cells were bothneutrophils we sorted these cells from capecitabineresistant patients and capecitabinesensitive patientsrespectively Characteristics ofthese patients werelisted in Additionalfile Table S1 We comparedour data of RNA sequencing with published data ofneutrophils from Jiang K [] using gene set enrichment analysis GSEA The results revealed thatin gene sets of neutrophil signature the expressionpattern of these cells was similar to that of the neutrophils provided by other group Additionalfile Fig S3B Additionalfile Table S2 Neverthelessthe decline of CD15 and CD66b expression combinewith the elevation of hematopoietic progenitorrelatedmarkers especially CD33 and CD117 suggested thatthese CD11bCD16myeloid cellsin capecitabineresistant patients became more immature after thetherapy compared with CD11bCD16myeloid cells fromcapecitabinesensitive patients Fig 5b The data of RNA sesomequencing also revealed declined expression ofTable Univariate and multivariate analyses for survival in CRC patients after capecitabine therapyPrognosticparameterUnivariate analysisHRCD16 expressionGenderAgeTumor locationTumor sizeCEACA199TNMRecurrenceHR Hazard ratio CI Confident interval95CIp valueMultivariate analysisHR95CIp value 0cLu BMC Immunology Page of Fig Analysis of CD16 expression was more sensitive than CT scan after capecitabine therapy a Peripheral venous blood from CRC patientsreceiving singleagent oral capecitabine adjuvant therapy was collected at different time before capecitabine therapy month and years afterthe therapy Frequencies of CD11bCD16highmyeloid cells were analyzed by flow cytometry b CT scan was performed during followup afteradjuvant chemotherapy in same patients as that of a respectively Sensitive patient normal operation site with no recurrence Resistant patientresectable metachronous liver metastases red arrowsand ATP wereneutrophilrelated genes in CD11bCD16myeloid cells fromcapecitabineresistant patients after capecitabine therapywhich implied immature status of these neutrophils Fig 5cIn addition active metabolism of nitrogen species purinenucleosidetheseCD11bCD16myeloid cells which are tightly related toimmunosuppressive role of MDSC [ ] Fig 5d To verify the immunosuppressive role of these CD11bCD16myeloid cells we sorted peripheral blood CD11bCD16myeloidcellsandCD11bCD16myeloid cellsfrom capecitabinesensitiveCRC patients or HDs and autologous T cells as well Aftercoculture T cells with these myeloid cells in the presence offrom capecitabineresistant CRC patientsinalsofoundleukocyte activators proliferation of T cell was significantlydeclined in resistant CRC patients group compared withsingle T cell group HD group and sensitive CRC patientsgroup Fig 5e ThetheseCD11bCD16myeloid cells in capecitabineresistant patientsmight exert immature cell status and play immunosuppressive role like MDSCsuggested thatresultsThe low expression level of CD16 on neutrophils isrelated to protumor status in CRC patients aftercapecitabine therapyAs we know immature myeloid cells are usually MDSCswhich could exert powerfulimmunosuppressive role 0cLu BMC Immunology Page of Fig CD11bCD16myeloid cells became immature neutrophils after therapy in capecitabineresistant patients a Peripheral venous blood fromcapecitabineresistant and capecitabinesensitive CRC patients was collected after the treatment in months CD11bCD16myeloid cells insensitive patients and that of CD11bCD16 in resistant patients were sorted for further analysis in b c and d b Expression of myeloidassociated and hematopoietic progenitorassociated markers on CD11bCD16myeloid cells in sensitive patients and on CD11bCD16myeloidcells in resistant patients was analyzed by flow cytometry c Peripheral blood CD11bCD16myeloid cells in sensitive patients andCD11bCD16myeloid cells in resistant patients were sorted and analyzed by RNA sequencing Expression of neutrophilrelated and monocyterelated genes derived from the results of RNA sequencing was shown in the heatmap d GO enrichment terms of differentially expressed MDSCrelated immunosuppressive biological processes derived from RNA sequencing e Autologous T cells were cultured alone cocultured withperipheral blood CD11bCD16myeloid cells from HDs and sensitive CRC patients or CD11bCD16myeloid cells from resistant CRC patientsfor h respectively Proliferation of T cells were analyzed by flow cytometry after incubation n for each group CD16N HD CD11bCD16myeloid cells from HDs CD16N CRC S CD11bCD16myeloid cells from sensitive CRC patients CD16N CRC R CD11bCD16myeloid cells from resistant CRC patients Mean ± SEM P005 P001 by t tests epatientscapecitabinesensitiveespecially in inhibiting T cells and NK cells [ ]As our results showed that CD11bCD16myeloid cellsfromandCD11bCD16myeloid cells from capecitabineresistantpatients were mainly neutrophils we tried to find out therelationship between the expression level of CD16 on neutrophils and other major immune cell subsets We collected peripheral venous blood from colorectal cancerpatients months after capecitabine therapy and analyzed frequencies of immune cells by flow cytometry Therelationships between expression level of CD16 on neutrophils and frequencies ofimmune cell subsets wereanalyzed by Pearsons correlation test The results showedthat CD16 expression was positively related to CD8T cellCD4T cell monocyte and NK cell frequencies Fig 6a bc and d but not that of cDC and pDC in patients aftercapecitabine therapy Fig 6e and f suggesting thatCD16lowneutrophils might have immunosuppressive activity as MDSCsDiscussionOver the past few decades numerous researchers haveattempted to improve the efficacy of capecitabine adjuvant therapy to ameliorate prognosis of CRC patients 0cLu BMC Immunology Page of Fig CD16 low expression on neutrophils predicted protumor immune status in CRC patients with capecitabine therapy Peripheral venousblood from CRC patients received singleagent oral capecitabine adjuvant therapy was collected months after the therapy and analyzed fordifferent immune cell subsets by flow cytometry CD16 MFI of peripheral blood neutrophils was calculated by flow cytometry analysis and thecorrelations between CD16 MFI of neutrophils and frequencies of CD8 T cells a CD4 T cells b monocytes c NK cells d cDCs e and pDCsf among total peripheral blood leukocytes were analyzed by Pearsons correlation testHoweverit remains one of the principal obstacle forcancer therapy at present In this study we demonstrated that the expression level of CD16 was downregulated in capecitabineresistant patients and lower expression level of CD16 on neutrophils in peripheralblood was correlated with poor prognosis in CRC patients with capecitabine adjuvant therapy Importantlydownregulation of CD16 was observed as early as month after capecitabine treatment which was moresensitive than CT scan indicating its great value in clinical application We determined the cutoff value ofCD16 expression on neutrophils for the prediction of capecitabine chemoresistance which would behelpful for clinical application and further researchesAnalyzationincapecitabineresistant patients revealed their immaturestatus and the expression of CD16 on neutrophils waspositively correlated with frequencies of antitumor immune cell populationsCD16lowneutrophilstheseofrecurrence which is vitalTo this day coloscopy and CT scan are still themain examines to supervise CRC progression and discoverfor capecitabineresistance diagnosis Unfortunately these two methodscould only provide evidence untiltumors are bigenough to be discovered patients wont have enoughtime to adjustthe treatment CEA and CA199 arewidely used to CRC surveillance as well especiallyCEA [] However CEA and CA199 cannot predictcancer progression so precisely and the false positivelead to anxiety and excessiveor negative results willtherapy Whats more some clinicaltrial also suggested that combining CEA and CT got no advantagecompared with single examine [] In this study ourresults showed that CD16 expression could serve as agood prognostic marker for poor CRC progressionafter capecitabine therapy Analyzation of CD16 expression hasthe downregulation of CD16 expression on neutrophils couldbe observed atcapecitabineresistance after the treatment Fig Previous studieshave demonstrated that CRC patients had primary resistance to 5FU single treatment[ ]thus the marker is essential for the drugselection inthese patients Second this marker is quite accuratefor predicting capecitabineresistance after the therapy In our study we collected totally CRC patients with capecitabinetheexpression level of CD16 on neutrophils Among patients who werecapecitabineresistance patients were observed to have downadvantages Firstto examinediagnosedtherapyasgreattheearlystage of 0cLu BMC Immunology Page of regulation of CD16 in months after capecitabinetreatment Table Third the examination of CD16expression only takes about ml peripheral bloodand it is noninvasive and has nearly no effect on patients healthCapecitabine the oral form of 5FU which is widelyused in CRC therapy has only modest efficacy due tothe chemoresistance Great efforts have been taken tofind out the mechanism Previous studies mainly concentrated on tumor cells themselves such as expressionof specific genes or generation of particular tumor cells[ ] In this research we worked on the correlationbetween changes on immune system and capecitabinechemoresistance and illustrated the conversion fromneutrophilsto immunosuppressive PMNMDSClikeneutrophils in these capecitabine insensitive patients byRNA sequencing and flow cytometry Our conclusioncould also be supported by other studies that 5FUcould promote MDSC protumor function The study byBruchard M found that 5FU could activate NLRP3inflammasome in MDSC and promote tumor growth[] Gobbo J also discovered that 5FU facilitatedproduction of tumorderived HSP70 exosomes whichfavored MDSC activation [] Thus prevention ofMDSC function after capecitabine or 5FU therapyholds great promise for improving drug efficacyreceptorResearchers have revealed that CD16myeloid cellswere tightly related to CRC development[ ]Giulio S found that CD16myeloid cell infiltration in CRC tumor tissue represented favorable prognosis [] and by using in vitro studies these studiesalso demonstrated that colon cancer infiltrate neutrophils enhance the responsiveness of CD8 T cells byTcelltriggering [] Our work differedfrom theirs in some ways Firstly our study focusedon CRC patients who received capecitabine adjuvanttreatment after surgery while Giulio Spagnoli groupfocused on all CRC patients and some healthy donorsSecondly biopsies from different positions were analyzed Peripheral blood was used in our study whileGiulio Spagnoli group mainly focused on tumor biopsies Exceptthese differences some of our resultswere also consistent with studies from Giulio Spagnoligroup Firstly both our data and Giulio Spagnoligroups data found that phenotype of peripheral bloodCD11bCD16myeloid cells had no difference betweenhealthy donors and CRC patients without capecitabinetherapy Fig S1F and G Secondly our work indicated that CD16 highpositive expression after capecitabine therapy predicted sensitivity to the therapyand good prognosis These results were consistentwith the work from Giulio Spagnoli groupthatCD16myeloid cells related to good prognosis of CRCpatientsMDSCs are a heterogeneous population of myeloidcells stay at different stages of differentiation PMNMDSCs are a great part of MDSCs that could be considered as counterparts of immature granulocytes chieflyimmature neutrophils []In this study we founddownregulation of CD16 expression on myeloid cells incapecitabineinsensitive CRC patients after capecitabinetreatment These CD16lowmyeloid cells after the therapy were mainly immature neutrophils CD16 is a lowaffinity FcÎ receptor which could activate antibodydependent process like phagocytosis in neutrophils andother phagocytes [] It is expressed on neutrophilsduring the maturation Researchers also revealed thatCD16 is typically associated with PMN activation andphagocytosis and its expression will change in differentmaturation status [ ] MDSCs could exert protumor roles mainly through inhibition of effective Tcells and NK cells [ ] Our study demonstrated thatlow expression of CD16 on neutrophils after the therapywas related to decreased frequencies of antitumor immune cells like CD8T cells and NK cells suggestingthatthey may have immunosuppressive activity asMDSCs The mechanism underlying the changes induced by capecitabine would be investigated further andit could be a good target to compete against capecitabinechemoresistanceConclusionsIn conclusion CD16 seems to be a promising target forCRC progression surveillance after capecitabine therapyStudies of CD16 expression on neutrophils may light thepath for not only predicting prognosis but also solvingcapecitabine resistance in CRC patientsMethodsPatients and peripheral bloodPeripheral venous blood of CRC patients in Departmentof Gastrointestinal Surgery Renji Hospital ShanghaiChina from January to December was gottenbefore capecitabine adjuvant treatment and at differenttime after the treatment as indicated in figure legendPeripheral venous blood of healthy donors was gotten inRenji Hospital The pathological information of patients was retrieved from the Pathology Department ofRenji Hospital These peripheral blood was used for flowcytometric analysis All the patients were provided withwritten informed consent before enrolment and thestudy was approved by the Research Ethics Committeeof Shanghai Jiao Tong University School of MedicineRenji Hospital Approval No Renji [] N013 Noneof patients had received radiotherapy or chemotherapybefore surgery All patients were followedup until deathor until the final followup May 0cLu e
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"Product size was 203 bp. Southern blot analysis Genomic DNA from ESCs and ans was extracted with the Gentra Puregene Tissue Kit (Qiagen). Genetic chimerism in various tissues was determined by Southern blotting of EcoRV digested DNA hybridized to the Trp53 5? XbaI probe which is a 700 bp genomic XbaI fragment subcloned in pBSK and labeled by PCR (Jonkers et al 2001). Intensity of bands was quantified with ImageJ software (version 1.43u) by generating a profile plot and measuring the surface area of individual peaks. Targeting with the Col1a1-frt vector was monitored by Southern blotting of EcoRI digested DNA hybridized to the Col1a1 3? probe which is an 842 bp genomic PstI-XbaI fragment. Flp-in of frt-invCAG-Luc and frt-invEF1-Luc was monitored by Southern blotting of BglII digested DNA hybridized to the same Col1a1 3? probe. Ad5-Cre virus administration Mice were treated with cyclosporine A (Novartis) orally in the drinking water 1 week prior to adenovirus administration and 23 weeks following infection. Viral Ad5-CMV-Cre ps (1 109; Gene Transfer Vector Core University of Iowa) were injected intratracheally into KrasLSL-G12D mice and Rb1F/F ;Trp53F/F mice and intrathoracically into Nf2F/F ;Trp53F/F ;Cdkn2a*/* mice. Immunohistochemistry of tumors Mice were monitored biweekly for development of tumors and general health status. Mice were sacrificed when signs of discomfort became evident. Tissues were collected for pathological examination. Formalin fixed and paraffin embedded material was sectioned H&E stained an analyzed microscopically by a dedicated mouse pathologist (by JYC). Array comparative genome hybridization Genomic DNA from ESCs was extracted with the Gentra Puregene Tissue Kit (Qiagen). DNA (500 ng) was labeled with either Cy5 or Cy3 using the NimbleGen Dual-Color DNA Labeling Kit (NimbleGen). Labeled DNA was hybridized on the mouse array comparative genome hybridization (aCGH) 12 135 K whole-genome tiling array (NimbleGen) using the MAUI hybridization station. Targeted ESC clones were analyzed against the parental ESC clones and the Flp-in ESC clones against the targeted ESC clones. Data was analyzed using the NimbleScan (Roche) and Nexus 6.0 (BioDiscovery) software. Data are available at the MIAMExpress database (http://www.ebi.ac.uk/miamexpress) under accession number E-MEXP-3998. The paper explained Problem The ever-growing list of potential cancer genes and drug targets associated with specific cancers demand validation in relevant in vivo models. Genetically Engineered Mouse Models (GEMMs) have proven valuable for the distinction of driver mutations from less relevant modifiers or bystanders though the inherent complexity of these models and time spent introducing additional modifications has hampered their general use. Results We have developed an approach for fast and flexible adaption of existing mouse models. Three elements are central to this system; (i) The efficient derivation of authentic Embryonic Stem Cells (ESCs) from established GEMMs (ii) the routine introduction of transgenes of choice in these GEMM-ESCs by Flp recombinase-mediated integration and (iii) the direct use of the chimeric animals in tumor cohorts. By applying stringent quality controls the GEMM-ESC approach proofs to be a reliable and effective method to speed up cancer gene assessment and target validation as exemplified for MycL1 in a model for Small Cell Lung Cancer. Impact The GEMM-ESC approach speeds up the generation/modification of mouse models while minimizing the breeding efforts. Experimental cohorts of mice are generated on-demand and ready-to-use. This reduces the number of mice needed per experiment and is therefore in compliance with one of the 3R's of animal testing. Furthermore the establishment of an archive of ESCs from various validated mouse models will allow easy distribution and gives researchers worldwide the opportunity to evaluate their favorite genes in the most suitable model. DNA copy number analysis Genomic DNA from tumors was extracted with the Gentra Puregene Tissue Kit. Real time PCR was performed on genomic DNA using SYBRGreen in the StepOnePlus real time PCR system (Applied Biosystems) with primers specific for MycL1 (Dooley et al 2011) and related to at least two reference genes in the same sample either Actin GapdH Tfrc or Tert. Primer sequences are provided in supplementary Table S5. Some samples were analyzed by low-coverage sequencing. Nexus 6.0 software was used to process Illumina Hiseq2000 generated signal intensity. A reference samples was created by combining three ESC clones invCag-Luc;Rb1F/F ;Trp53F/F clones 1.5_1B1_6 and _ 9 and Col1a1-frt;Rb1F/F ;Trp53F/F clone 1.5_1B1_r4. The FASST2 segmentation and default Illumina setting with Gain 0.4 and Loss ?0.4 were used to identify regions of CNV for each sample. In vivo bioluminescence imaging of tumors In vivo bioluminescence imaging was performed and quantified as described by Hsieh et al (2005) on a cryogenically cooled IVIS system (Xenogen Corp. CA USA) using LivingImaging acquisition and analysis software (Xenogen). Statistical analysis Survival analysis was performed by comparing two survival curves using the Log-rank (Mantel-Cox) test within the Prism 6 software. The comparison of the incidence of MycL1 copy number gains between three groups was performed using the Fisher's Exact Test. The cut-off for MycL1 amplified tumors was more than four copy numbers present in the tumor. We are grateful to J. Nichols and A. Smith (Welcome Trust Centre for Stem Cell Research Cambridge UK) for providing training in ESC derivation and culture. We thank R. Jaenisch (Whitehead Institute for Biomedical Research Cambridge MA USA) and J. Gribnau (Erasmus University Rotterdam the Netherlands) for Flp-in plasmids. We thank F. van der Ah S. Kautschitsch B. Siteur H. van der Gulden L. Henneman for technical assistance; M. Snoek and T. Meehan for the official MGI nomenclature of ESC clones; the personnel of the animal facility for their excellent animal husbandry; and the Genomics Core Facility for their help with aCGH. This work was supported by the Dutch Cancer Society (KWF) by a National Roadmap grant for Large-Scale Research facilities provided by the Netherlands anization for Scientific Research (NWO) and by the EuroSyStem and EurocanPlatform projects as part of the European Union's seventh framework programme. Author contributions IJH conceived and designed experiments performed project coordination data analysis and wrote the paper. RBA performed ESC derivations and injections. CP designed vectors and performed embryonic stem cell manipulation. MC and NP performed health monitoring of mice viral intubations and luciferase imaging. MCK performed Mycl1 copy number analysis. JYS analyzed tumors. HdV established primary mesothelioma cultures. JB and KS provided cohorts for conventional mesothelioma and lung tumor groups respectively. EMM acquired technology. PK JJ and AB conceived and designed experiments. JJ and AB supervised the work. "
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"gut microbiota composition influences the balance between human health and disease increasing evidencesuggests the involvement of microbial factors in regulating cancer development progression and therapeuticresponse distinct microbial species have been implicated in modulating gut environment and architecture thataffects cancer therapy outcomes while some microbial species offer enhanced cancer therapy response othersdiminish cancer treatment efficacy in addition use of antibiotics often to minimize infection risks in cancer causesintestinal dysbiosis and proves detrimental in this review we discuss the role of gut microbiota in cancerdevelopment and therapy we also provide insights into future strategies to manipulate the microbiome and gutepithelial barrier to augment therapeutic responses while minimizing toxicity or infection riskskeywords intestinal dysbiosis cancer development cancer therapy microbial therapy human intestinal microbiota is essentialfor microbialhomeostasis regulation of metabolism and immune tolerance intestinal dysbiosis occurs when there are alteredratios of healthy microbial flora along with changes in theirdiversity and density such changes may lower mucus layerthickness reduce antimicrobial defense and disrupt theepithelial tightjunction barriers to allow increased translocation of intestinal bacteria and bacterial products intothe systemic circulation and trigger inflammation andimmune responses circulating bacterial products such asendotoxin genotoxin and trimethylamine oxide have beenimplicated in many human disorders including metabolicsyndrome cardiovascular complications atherosclerosisand thrombosis and various neoplastic conditions intestinal dysbiosis may also affect adaptive immunity by correspondence seahhlimyahoocom1division of hematology and oncology suny downstate health sciencesuniversity clarkson avenue room b5495 brooklyn new york usa2division of hematology and oncology department of medicine new yorkmedical college valhalla new york usamodulating the functions of t lymphocytes and promotingtumor immune escapewhile increased translocation of intestinal luminal content is associated with carcinogenesis and poor therapeuticresponse the causeeffect relationship is often bidirectionalin this review we will discuss the role of gut microbes inmodulating tumor immunity intestinal permeability andcancer development next we will highlight the effects ofintestinal dysbiosis and increased permeability in cancertherapy finally we will explore the options to improve guthealth to enhance the efficacy of cancer therapyintestinal immunity and permeabilitythe intestinal architecture and microbiota regulateinnate and adaptive immunity disruption of the architecture andor microbiota affects these functions therelationships between the different players in the intestinal microenvironment is summarized in fig the composition of microbes in the gut dictatesmucus layer thickness and production of antimicrobialsignals in germfree mice mucus layer and effector t the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cdutta and lim biomarker research page of fig interplay between different factors involved in gut immunity and permeability a the intestinal epithelial cells containing paneth cellsgoblet cells enterocytes and enteroendocrine cells coordinate with intraepithelial lymphocytes to generate a functional immune responsepaneth cells secrete antimicrobial peptides and goblet cells produce mucus to cover the epithelial layer this mucus layer prevents adhesion ofmicrobes to the epithelial cells lamina propria situated under the mucus layer contains peyers patches and immune cells including antigenpresenting cells apcs like dendritic cells dcs t cells and b cells pattern recognition receptors prrs such as tolllike receptors tlrs onepithelial cells interact with microbederived pathogenassociatedmolecular patterns pamps such as lipopolysaccharide lps to activatemyd88dependent signaling dcs travel to mesenteric lymph nodes mln and promote the differentiation of na¯ve t cells to regulatory t tregcells that migrate to other sites treg cells secrete il10 to elicit an antiinflammatory response b dysbiosis decreases mucus layer thickness andshortchain fatty acid scfas production this affects the secretion of antimicrobial peptides and allows microbes to come in close proximity tothe epithelial cells reduction in scfas influences gut barrier dysfunction as a result the gut luminal content also translocated and spreadedthrough the systemic circulation to trigger local and systemic immune responses in addition to pamps damps released from damaged intestinalepithelium interact with prrs to facilitate expression of macrophages and maturation of dcs mature dcs promote the differentiation of na¯ve tcells to effector t cells such as t helper cells th1 th2 th17 th1 release tnfα and ifnÎ and th17 secrete il17 to recruit polymorphonuclearneutrophils pmns these cytokines create a proinflammatory condition 0cdutta and lim biomarker research page of cells are absent [ ] microbes secrete shortchain fattyacids scfas such as propionate and butyrate thatprevent microbial binding to the epithelial cells and helpmaintain barrier function and immune homeostasisbutyrate promotes tightjunction formation [ ] andactivates peroxisome proliferatoractivated receptor gammapparÎto enhance epithelial oxygen consumptionresulting in reduced emanation of oxygen from the mucosalsurface it helps in maintaining an anaerobic condition inthe gut lumen needed for colonization of obligate anaerobes this intestinal microenvironment determines thecomposition of resident bacterial species for example onlyclostridium lactobacillus and enterococcus are enrichedon the epithelial surface and in the mucus layer whereasbacteroides bifidobacterium streptococcus enterobacteriaceae enterococcus clostridium and lactobacillus are allpredominant in the intestinal lumen dysbiosis increases inflammatory signals that shiftthe metabolism of enterocytes epithelial hypoxia iseliminated and increased oxygenation results in therelease of more oxygen from the mucosal surfacesince only facultative anaerobes can respire oxygendysbiosisinduced shift in epithelial oxygenation altersgut microbial community from obligate to facultativeanaerobes intestinal pathogens such as proteobacteria produce genotoxins like colibactin and cytolethaldistending toxin cdt to induce inflammation andhost deoxyribonucleic acid dna damage that initiates tumor formation dysbiosis also decreasesmucus layer thickness reduces scfa production anddamages mucosal barrier allowing pathogenassociatedmolecular patterns pamps to interact with pattern recognition receptors prrs and activate tolllike receptortlr 24myeloid differentiation primary response protein myd88 signaling pathways in addition changes inmicrobial composition and density triggers epithelial releaseof damageassociated molecular patterns damps such asextracellular adenosine triphosphate atp cytoplasmiccalreticulin high mobility group box hmgb1 proteinsendogenous nucleic acids and intracellular proteins tointeract with prrs prr engagementtriggers a proinflammatory condition that causes tissue damage and localinflammation microbiotadriven tlr immune signalinghas been implicated in cancer formation and modificationof treatment efficacy [] for example cpg oligodeoxynucleotides that mimic bacterial dna acts as a pamp totrigger a tlr9dependent tlr4 activation and tumor necrosis factor tnfα production by tumorinfiltratingmyeloidderived cells mice bearing el4 lymphomamc38 colon carcinoma and b16 melanoma when treatedwith cpg oligodeoxynucleotides show reduced tumrowth and enhanced survival rate the beneficial effects ofcpg oligodeoxynucleotides were positively associated withthe abundance of alistipes shaii in the gut effects of intestinal microbiota on cancerdevelopmentintestinal microbes can influence local and distantcarcinogenesis through infection and microbial productsor by modulating tumor immunosurveillance this isaccomplished via altering the balance between the rateof cell proliferation and apoptosis triggering chronicinflammation andor immunosuppression or changingthe metabolism of the products produced by host andmicrobes in this section we will discuss how intestinaldysbiosisrelated permeability may contribute to tumorigenesis in different anscolorectal cancerfusobacterium nucleatum a gramnegative mucosaadherent anaerobic bacteria has been implicated in theinitiation and progression of colorectal cancer crc[ ] fada an adhesion molecule on f nucleatumbinds to host ecadherin to enter epithelial cells this activates the wntβcatenin pathway leading toan increased secretion of inflammatory cytokines including il6 il8 and tnfα and upregulation of nuclearfactor kappa light chain enhancer of activated b cellsnfκb that facilitates crc development in addition itattracts myeloidderived suppressor cells and the autotransporter protein fap2 interacts with the human inhibitoryreceptor t cellimmunoreceptor with ig and itimdomains tigit to create a tumor immunosuppressivemicroenvironment f nucleatum may also induce chemoresistance by modulating the tlr4myd88 signalingpathway following 5fluoruracil treatment in crc patients an increased abundance of f nucleatum along with clostridium difficile and species ofstreptococcus campylobacter and leptotrichia has beendemonstrated in tumor tissue and fecal materials []f nucleatummediated colorectal carcinogenicity occursdownstream of apc introduction of f nucleatum resultedin rapid onset of colonic tumors in mice deficient in onecopy of adenoma polyposis coli apc apcmin gene both intestinal dysbiosis and loss of apc disruptepithelial tightjunctions and mucus layer [ ] andallow increased infiltration of f nucleatum and other nonresidential microbes to drive crc development the roleof defective gut barrier in crc has been confirmed inmucin 2knockout muc2 mice in which the lack ofgastrointestinal mucin resulted in spontaneous crc development therefore dysbiosisinduced gut permeabilitymay play an important role in tissue enrichment of fnucleatum and increased risks for crchepatobiliary cancerthe liver is chronically exposed to intestinal microbiotaand its products via the portal vein intestinal dysbiosisand increased permeability enhance translocation of gut 0cdutta and lim biomarker research page of microbiota to trigger inflammation and chronic liver disease that predisposes patients to the development of hepatocellular cancer alteration in bile acid metabolism due tochanges in clostridium spp suppress anticancer immunity in mice eradication of grampositive bacteria by oralvancomycin inhibits secondary bile acid conversion resulting in the upregulation of chemokine cxc motif ligandcxcl16 in liver sinusoidal endothelial cells cxcr16recruits natural killer t nkt cells in the tumor microenvironment and kill tumor cells in a cd1ddependentmanner in addition gut microbiotaderived lipopolysaccharides lps promote tumor progression in liver cancerby activating the tlr4 signaling in a study involving cholangiocarcinoma patients bile ducttissues haddistinct dominance of dietziaceae pseudomonadaceae andoxalobacteraceae members pancreatic cancergut microbiota influences the development of pancreaticcancer through activating tlr4 signaling the stromain pancreatic tumor harbors an abundance of microbiotaespecially bifidobacterium pseudolongum compared tonormal pancreas this helps in creating an immunosuppressive environment by differentially activating distincttlrs in monocytes pancreatic adenocarcinoma has anenrichment of proteobacteria synergistetes and euryarchaeota longer survival is observed in patients with amore diverse intratumor microbial composition primarilyof sachharopolyspora pseudoxanthomonas streptomycesand bacillus clausii tumoral colonization with mycoplasma hyorhinis and gammaproteobacteria is associatedwith gemcitabine resistance antibiotics diminishmyeloidderived suppressor cells and increase antitumorm1 macrophages to promote th1 differentiation of cd4t cells and cd8 t cell activation in the tumor cotreatment of gemcitabine with ciprofloxacin abrogatedgammaproteobacteriainduced chemotherapy resistance the efficacy of immune checkpoint inhibitors icistherapy is also enhanced by antibiotics lung cancerwhile local microbiota is important there are reportsthat gut microbiome may also contribute to lung cancerdevelopment lung cancer patients demonstrated an abundance in intestinal enterococcus and depletion in bifidobacterium and actinobacteria they are also enrichedwith veillonella bacteroides and fusobacterium depletedof dialister enterobacter escherichiashigella fecalibacterium and kluyvera in nonsmall celllung cancernsclc patients butyrate producers such as faecalibacterium prausnitzii clostridium leptum clostridial cluster iruminococcus spp clostridial cluster xiva and roseburiaspp were significantly reduced since butyrate isessential for preserving mucosal homeostasis reduction ofintestinal butyrate producers may imply a compromised intestinal barrier in these patientshematologic malignanciesdysbiosisinduced intestinal permeability affects mucosaassociated lymphoid tissue malt and plays a significantrole in hematologic malignancies composition of intestinalmicrobiota is responsible for maintaining the pool of bonemarrow myeloid cells preleukemic myeloproliferationis driven by microbial signals in teneleven translocation2tet2deficient mice [ ] these mice show increasedinfiltration of inflammatory cells disrupted mucosal barrierand increased translocation of bacteria [ ] it wassuggested that dysfunction of small intestinal barrier andleakage of microbes can occur due to tet2 mutation in occurrence of tet2hematopoietic compartmentmutation intestinal dysbiosis and leaky gut is common inleukemia and lymphomaacute myeloid leukemia aml and acute lymphoblasticleukemia all patients have a compromised intestinalbarrier [] fecal microbiota in all patients showedlower microbial diversity they were enriched in enterococcaceae porphyromonadaceae and bacteroidetes mainlyb fragilis and depleted in blautia erysipelotrichiales lachnospiraceae and clostridiales members [ ] abundanceof staphylococcaceae and streptococcaceae have also beenreported in pediatric all and adult aml [ ]helicobacter pylori is associated to malt lymphoma and chamydophila psittacito ocular maltlymphoma while borrelia burgdorferi was linked tocutaneous bcell nonhodgkin lymphoma two studiesdid not find significant risk of borrelia burgdorferi in thedevelopment of nonhodgkin lymphoma [ ] abundance of proteobacteria is a predictor for neutropenicfever and enrichments of enterococcaceae and streptococcaceae are strong predictors of infectious complications inall similarly higher gut microbiota diversity inmultiple myeloma is associated with reduced risk fordisease relapse all patients with infectious complications have an abundance of brevundimonas diminuta andagrobacterium tumefaciens whereas faecalibacteriumprausnitzii producer of scfas is completely absent similar findings have been reported in nonhodgkinlymphoma with infectious complications effects of intestinal microbiota on cancer therapythe efficacy of cancer treatment is in parts dependenton normal immune function since gut microbiota playsa crucial role in modulating immune response it is notsurprising that dysbiosis affects treatment outcomesprophylactic antibiotics are commonly used for cancerpatients undergoingallogeneichematopoietic stem cell transplantation allohsct toreduce the risk of neutropeniaassociated infectionchemotherapyand 0cdutta and lim biomarker research page of however antibiotic use causes intestinal dysbiosis thatresults in negative outcomes including poor treatmentresponse and toxicity and the development of clostridium difficile infection cdi in addition to antibioticsopioid analgesics for cancer pain management may alsotrigger dysbiosis opioid analgesics impair intestinal motility and promote bacterial overgrowth resulting in dysbiosisand gut permeability intestinal dysbiosis induces mucosal injury and triggers the release of damps damps have a dual andbidirectional effect on cancer although damps exertimmunosurveillance and immunemediated cell death toeliminate tumor cells and protect against cancer development chronic inflammation induced by damps maypromote tumor initiation damps released by apoptoticcells from cancer therapy may also induce chemoresistance and promote metastasis for example tlr78expressed on tumor cells may bind damps loxoribinefor tlr7 and poly u for tlr8 and promote chemoresistance through the activation of nfκb and the upregulation of bcl2 damps may also activate tlr9on human breast prostate and lung cancer cells to trigger tumor invasion and metastasis [ ] given theclinical significance of dysbiosismediated mucosal injuryand permeability in cancer we will in this section discusshow the treatment outcome by various cancer therapymay be affected by intestinal microflora and permeabilitychemotherapy and radiation therapyintestinal microbial composition and mucosal barrier function influence chemotherapeutic outcome and the effect isbidirectional while dysbiosis can exacerbate chemotherapy drug toxicity and reduce its efficacy chemotherapy canitself cause dysbiosis although prevalence of certain intestinal microbes in the gastrointestinal tract offer beneficialeffects others contribute to chemoresistance and drug toxicity this multiplepathway effect is best covered by timer mechanisms translocation of microbes immunomodulation metabolism and enzymatic effects on drugsand reduced microbial diversity these mechanistic effectsalter chemotherapy efficacy and toxicity and risks forinfections for example translocation of microbes due tochemotherapy induceddysbiosis and disruption of mucosal barrier can increase the risk of infection howevercertain chemotherapy drugs such as cyclophosphamideand doxorubicin damage intestinal barrier for the translocation of commensal bacteria into secondary lymphnodes to elicit antitumor immune response vancomycin prophylaxis inhibits antitumor effects of cyclophosphamide in fibrosarcoma inoculated mice irinotecanused for crc treatment is transformed into its active formsn38 by tissue carboxylesterase it is detoxified in theliver by host udpglucuronosyltransferases into inactiveglucuronide sn38g and excreted into the gut via bileducts in the gut bacterial βglucuronidases reconvertssn38g into active sn38 which causes severe intestinaltoxicity and diarrhea streptomycin inhibits irinotecanabsorption and reduces epithelial carboxylesterase activityand diarrhea ciprofloxacin inhibit βglucuronidases and low dose amoxapine βglucuronidases inhibitorsuppress irinotecanassociated diarrhea in rats table provides a selection of chemotherapeutic agents affectingand affected by intestinal microbial composition andpermeabilitylocal pelvic irradiation damages intestinal epitheliumand barrier integrity and produce reactive oxygen speciesirradiation increase alistipes and decrease prevotella inmice in gynecologic cancer patients receiving pelvicradiotherapy firmicutes and fusobacterium were significantly decreased in addition to reduced diversitysignificant enrichment of clostridium iv roseburia andphascolarctobacterium was associated with radiationenteropathy in pelvic cancer patients the effects oftotal body irradiation which is a preparative regimen forallohsct that causes dysbiosis and gastrointestinaltoxicity is discussed in more details in the allohsctsection belowimmunotherapycancer cells often create an immunosuppressive microenvironment to mediate tumor immune escape this immune escape mechanism may be reversed by icis directedat cytotoxic t lymphocyteassociated antigen ctla4programmed death receptor pd1 or pd1 ligandspdl1 since intestinal microbes influence local and systemic antitumor immune reaction by modulating prrspamps and dampsintestinal dysbiosis may impacttreatment outcome figure illustrates how the potentialmechanisms ofthe antitumor immune responses aredownregulated by intestinal dysbiosis the effects of intestinal microbiome on responses to icis have been discussed previously [ ] broadspectrum antibioticsbefore during or after icis therapy alter intestinal microbiome and resulted in lower tumor response rate inferiorprogressionfree survival and reduced overall survival responses to inhibition of ctla4 by ipilimumab inmouse models of mca205 sarcoma ret melanomaand mc38 colon carcinoma were inferior in germfreeor in broadspectrum antibiotic treated mice poorresponses were associated with decrease in intestinalbacteroides thetaiotaomicron bacteroides uniformis andburkholderia cepacia and increase in clostridiales suchdysbiosis was also associated with mucosal damage andcolitis oral feeding with either bacteroides thetaiotaomicron or bacteroides fragilis individually or with acombination of bacteroides fragilis and burkholderiacepacia restored the antitumor effects of ctla4 blockade through augmentation of th1 responses in tumor 0cdutta and lim biomarker research page of table selection of chemotherapeutic agents and the bidirectional effects between the chemotherapy and intestinal microbiotachemotherapy drugcisplatintoxicity infectioncdi ototoxicity effects on gut changes in microbiotadamages mucosal barrier by impairing dnareplication of rapidly proliferating epithelialcells facilitates translocation of gut bacteriacommensal gut bacteria influencesgenotoxicity by inducing reactive oxygenspecies ros production and recruitment ofpathogenic th17 cells in the tumormicroenvironment independently ofimmunity elicited by immunogenic celldeath microbial interventionantibiotics against grampositive bacteriaabrogate antitumor chemotoxicityincrease tumor size and decrease survivalratecisplatin alone show better responsecompared to a combined treatment ofcisplatin and antibiotics in mice with lungcancer the combination treatmentincreased tumor size and decreasedsurvival ratelactobacillus acidophilus restores antitumorefficacy following antibiotic treatment[ ]restoration of gut microbiota andepithelial integrity by fmt andtreatment with dmethionine [ ]prevent infections and ototoxicity withoutaffecting tumor chemotoxicityfmt increases a muciniphilaabundance and reduces cipn oral butyrate supplementation improvesgut barrier by reducing inflammation andmucositis antibiotics reduce mucositis and cytokineproduction but also diminish antitumorefficacy and promote chemotherapyresistance antibiotics against grampositive bacteriareduce th17 responses and subsequentdevelopment of cyclophosphamideresistancereestablishment of e hirae alone restoresantitumor activity e hirae decreases tumorinfiltrating tregsbarnesiella intestinihominis accumulates inthe colon and increases the number ofintratumoral ifnÎproducing Îδt cellse hirae and b intestinihominissynergistically stimulate local and systemicimmunity to improve anticancer effects nod1nod2 mice having abundant bintestinihominis demonstrate increased Îδtcells in tumor beds and enhancedcyclophosphamide efficacy paclitaxel5fluoruracilincreases gut permeability as indicated by5fold elevation in circulating lpsbindingprotein and systemic inflammation reduces abundance of roseburiaporphyromonadaceae and akkermanisamuciniphila [ ]reduces clostridium spp and increasesmembers of proteobacteria mainlyenterobacteriaceae damages mucosal barrierchemotherapyinducedperipheral neuropathicpain cipn cdi [ ]mucositis along the entiregastrointestinal tract cdi [ ]cyclophosphamidecdi triggers disruption of gut barrier by alteringbacterial compositiongrampositive bacteria such asenterococcus hirae lactobacillus johnsoniiand l murinus translocate from gut intomesenteric lymph nodes and spleen this enhances immune responses by theproduction of interferon gamma ifnÎ andactivation of th17 cellsdraining lymph nodes and promotion of maturation oftheintratumoral dendritic cells dcscombination treatment of bacteroidesandburkholderia cepacia prevented intestinal damage andrefractory colitisin additionfragilisfecal microbiota analysis of melanoma patients beforeand after ipilimumab treatment showed a change in therelative proportions ofenterotypeclusters cluster a was dominated by prevotella spwhereas clusters b and c by different bacteroides sppfecal microbiota transplantation fmt from patientsinto tumorbearing germfree mice showed that onlyfecal material from cluster c resulted in colonizationthree dominantwith bacteroides thetaiotaomicron or bacteroides fragilisand enhanced ipilimumab response in another study ofipilimumab in mice vancomycin treatment resulted in amore severe manifestation of colitis whereas oraladministration of bifidobacterium ameliorated the sideeffects similarly melanoma patients with increasedabundance of bacteroidaceae rikenellaceae and barnesiellaceae members responded better to ctla4 antibodies however a different study in ipilimumabtreated melanoma patients found that bacteroides spp were associatedwith decreased response whereas faecalibacterium andother firmicutes members improved clinical outcome 0cdutta and lim biomarker research page of fig potential antitumor immune mechanisms induced by intestinal dysbiosis a in the presence of intact mucosal barrier and signals fromcommensal microbiota effector t cell activation is modulated by t cell receptor tcr ligation with major histocompatibility complex mhc classi and costimulation of cd80cd86 and cd28 binding of cytotoxic t lymphocyteassociated antigen ctla4 receptor to antictla4 antibodyon treg impairs its effector tcell inhibitory function it also downregulates ctla4 expression on apc ligation of repressive receptorprogrammed death receptor pd1 and its ligand pdl1 to antipd1 and antipdl1 antibodies respectively activate effector tcell proliferationand function activated effector t cells interact with tumor cells and release cytokines to induce tumor cell death b signals from unfavorablemicrobes due to dysbiosis upregulates ctla4 pd1 and pdl1 expression to inhibit tcell activation ctla4 on treg binds to cd80cd86 onantigen presenting cell apc cd80cd86 on apc also disengages from cd28 and binds to ctla4 on effector t cells pdl1 the ligand of pd1is expressed on antigen presenting cell apc and tumor cells pd1 on effector t cells ligates to pdl1 on apc and tumor cells these activitiesinhibit effector tcell activation reduces immune checkpoint inhibitor ici efficacy and causes tumor escape 0cdutta and lim biomarker research page of patients with higher abundance of faecalibacteriumand improved response to ctla4 antibodies showedhigher incidence of enterocolitis and lower level of treg inperipheral blood thus the beneficial effects of specificand isolated gut microbes may depend on the commensalassociation with other microbial species and may differ between humans and micepd1 blockade may also be modulated by intestinalmicrobiota melanoma patients who responded to pd1blockade had increased abundance of enterococcus faeciumcollinsella aerofaciens bifidobacterium adolescentis klebsiella pneumoniae veillonella parvula parabacteroidesmerdae lactobacillus sp and bifidobacterium longumwhereas in nonresponders the intestinal microbiome wasenriched in ruminococcus obeum and roseburia intestinalis another study found higher abundance of faecalibacterium species in responders and enrichment with bacteroides thetaiotaomicron escherichia coli and anaerotruncuscolihominis in nonresponders clinically nonsmallcell lung cancer nsclc and renal cell carcinoma rccpatients experienced increased resistance to pd1 blockadeafter antibiotic treatment these patients had shorterprogressionfree survival as well as overall survival in thisstudy response to pd1 blockade correlated with higherfecal abundance of akkermansia muciniphila fmt fromresponders to germfree or antibiotictreated mice improved the outcome of pd1 blockade administration ofa muciniphila after fmt from nonresponders restoredresponsesimilarly intestinal microbiota may influence the outcome of chimeric antigen receptor t cell car t therapypatients with complete response to cd19 car ttherapyexhibited enrichment of oscillospiraceae ruminococcacaeae and lachnospiraceae in their intestinal microbiomewhereas patients who did not attain a complete responseshowed increased abundance of peptostreptococcaceae effectiveallogenic hematopoietic stem cell transplantationalthough allohsct issomein treatinghematological malignancies the immunosuppressive agentsbroadspectrum antibiotics and chemoradiation used withthe transplant often induce intestinal dysbiosis gut permeability and impaired systemic immune response highermicrobiota diversity is associated with longterm survivaland lower diversity in gut microflora is associated with reduced overall survival and higher transplantrelated mortalityfollowing allohsct [ ] severe infections that occurdue to intestinal dysbiosis such as cdi and vancomycinresistant enterococci vre infections are also associatedwith higher treatmentrelated mortality [] allohsctdisrupts the equilibrium of bacterial composition in feceswith a dominance of enterococcus streptococcus and proteobacteria members [ ] and reduces beneficial bacteriasuch as faecalibacterium and ruminococcus higherabundance of blautia was found to be associated with improved overall survival moreover allohsct patientswith reduced risk of relapse had an enrichment of eubacterium limosum ofgraftversushost diseaseone of the major complications of allohsct is thedevelopmentgvhdoccurrence of cdi during allohsct increases the riskof gvhd besides the loss of overall microbial diversityreduction in beneficial faecalibacterium blautia lactobacillus and ruminococcus and increased abundance ofenterococcus and clostridiales was observed in gvhd[ ] patients without gvhd had increasedabundance of parabacteroides and bacteroides in theirpretransplant feces in a preclinical study reducedgvhd and improved overall survival was observed afterthe administration of the probiotics lactobacillus rhamnosus gg alone or in combination with ciprofloxacindue to the preservation of gut mucosal integrity in therecipient mice restoration of normalintestinalmicrobiome by fmt has been found to benefit patientswith steroidrefractory gvhd [ ] multipleclinical trials are currently ongoing to investigate howmanipulation of gut microbiota using dietary intervention and fmt might reduce the risk of gvhdmanipulation of intestinal microbiome and barrierto improve outcome of cancer therapeuticsifintestinal dysbiosis and its associated increased gutpermeability are associated with cancer development andtherapyrelated complications and treatment outcomes itfollows that intervention of the intestinal microbiomeandor gut barrier may alter cancer outcome in thissection we will explore three broad approaches fig that might be investigated nonselective modificationof intestinal microbiome using fmt semiselectivemodification of intestinal microbiome using antibioticsand biologic modification of intestinal barrier we willdiscuss the challenges and obstacles each of the approaches may encounternonselective modification of intestinal microbiome usingfmtmodification of the intestinal microbiome is theoreticallybest accomplished by fmt unmanipulated fmt will notonly replete the dysbiotic intesti
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"as metastasis is a major cause of death in cancer patients new antimetastatic strategies are needed to improvecancer therapy outcomes numerous pathways have been shown to contribute to migration and invasion ofmalignant tumors aspartate hydroxylase asph is a key player in the malignant transformation of solid tumorsby enhancing cell proliferation migration and invasion asph also promotes tumor growth by stimulation ofangiogenesis and immunosuppression these effects are mainly achieved via the activation of notch and srcsignaling pathways asph expression is upregulated by growth factors and hypoxia in different human tumors andits inactivation may have broad clinical impact therefore small molecule inhibitors of asph enzymatic activity havebeen developed and their antimetastatic effect confirmed in preclinical mouse models asph can also be targetedby monoclonal antibodies and has also been used as a tumorassociated antigen to induce both cluster ofdifferentiation cd and cd4 t cells in mice the pan3011 vaccine against asph has already been tested in aphase clinical trial in patients with prostate cancer in summary asph is a promising target for antitumor andantimetastatic therapy based on inactivation of catalytic activity andor immunotherapykeywords asph small molecule inhibitor metastasis immunotherapy cancer is a multifactorial disease with an approximate million fatalities in worldwide it is the secondleading cause of death the complex modifications inthe genome affected by the interactions between hostand environment lead to cancer development and progression despite advancements in characterizing themolecular mechanisms of oncogenesis tumor progression and metastasis delayed cancer detection limitedsurgical options therapeutic resistance and tumor recurrence are serious obstacles in decreasing the prevalence and fatality rate of cancer since metastasis is theprimary cause of deaths from cancer the design oftherapeutictarget mechanisms oftumorcell migration and invasiveness is essential in thisapproachesthat correspondence smahelmnaturcunicz1department of genetics and microbiology faculty of science charlesuniversity biocev vestec czech republicfull list of author information is available at the end of the regard a growing number of investigations of signalingpathways involving products of oncogenes and tumorsuppressor genes in human carcinomas has helped toelucidate the mechanisms underlying malignant transformation of cells and facilitated the development ofnew and more efficient therapeutic methodsaspartate hydroxylase asph has been identified asone of the cell surface proteins associated with malignant transformation of tumor cells [ ] asph belongsamong the most important biological targets to controlmigration and invasion of tumor cells as its overexpression has been observed in of human solid tumors [] the overexpressed asph is transportedfrom the endoplasmic reticulum to the plasma membrane which results in exposure of the cterminal regionto the extracellular environment where it is accessible toantibody binding recently molecular targeted therapyhas been developed against this target using small molecule inhibitors smi that can inhibit the catalytic site the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ckanwal of experimental clinical cancer research page of in the cterminal region moreover as antigenic epitopes that reside on the asph protein can efficientlystimulate cluster of differentiation cd and cd8 tcell responses unique to tumor cells harboring asphthis enzyme can be used as a tumor associated antigentaa in immunotherapy [ ]thedioxygenasesstructure of the asph gene and isoformsasph is a type ii transmembrane protein of approxito the family of αmately kda that belongsketoglutaratedependenthydroxylated products of asph hydroxylation were firstdetected in blood coagulation proteins [] asphwas initially identified in the bovine liver as an enzymeresponsible for catalyzing the hydroxylation of aspartyland asparaginyl residues in calcium binding epidermalgrowth factor cbegflike domains of various proteins fig thereafter the human asph gene wascloned and characterized this gene spanning base pairs long region of genomic dna and containing exons is located at the position q123 of thehuman chromosome the asph sequence is highlyconserved in mammalian evolution the sequence of thehuman protein is from about identical to the sequences of rat and mouse analogs and the catalytic siteis quite conserved among proteins of these three species the whole asph protein consists of five domainsan nterminal cytoplasmic a universal transmembranea positively charged luminal a calcium binding and a cterminal catalytic domain tissue specific transcription is directed from two putative promoters p1 and p2which differ in their regulation sequences [ ] whilethe transcription from the p1 promoter was observed inmost human tissues the p2 promoter is activated by thecalciumdependent transcription factor myocyte enhancer factor mef2 particularly in muscle tissues the asph gene undergoes extensive alternative splicingresulting in four protein isoforms ie asph humbugjunctate and junctin [ ] these proteins vary in thecterminal region which affects their function [ ]the two longest asph transcript variantsthat aretranscribed from the p1 and p2 promoters and differ inthe length of the ²untranslated region encode the fulllength asph protein this protein contains the catalyticcterminal domain that catalyzes the posttranslationalhydroxylation in the cbegflike domains of numerousproteins supplementary fig including receptors receptor ligands and extracellular adhesion moleculesthat influence cell motility and invasiveness [ ] thetruncated isoforms humbug junctate and junctin sharethe nterminal part with the asph protein but lackcatalytic function they are involved in calcium homeostasis humbug has a potential role in cell adhesionand calcium flux and similar to asph its overexpressionhas been correlated with aggressive tumorcell behavior reticulummembranebound protein that is known for its functionin the regulation of the intracellular ca2 concentrationjunctin is a structural membrane protein and as an integral part of the complex consisting of the ryanodine receptor calsequestrin and triadin influences calciumrelease from the sarcoplasmic reticulum [ ]sarcoendoplasmicjunctateisalocalization in cells tissue distribution andexpression regulationasph is predominantly a cellsurface protein that isalso localized in the endoplasmic and sarcoplasmicreticulum furthermore a recent study identifiedmitochondriallocalization of asph in hepatocellularcarcinoma hcc in that study asph overexpressioncorrelated with an instability of mitochondrial dna andmitochondrial dysfunction that may lead to more aggressive pathological outcomes in hcc asph is abundantly expressed in proliferating placental trophoblastic cells [ ] and in decidua and endometrial glands and has a potential role in placentalimplantation and fetal growth on the contrary theasph expression in normal adult tissues is relativelylow or negligible however asph expression is inappropriately activated during oncogenesis when asph is required for generation of malignant and metastaticphenotypes the elevated expression of asph at bothfig asph catalytic reaction aspartyl and asparaginyl residues in cbegflike domains are hydroxylated 0ckanwal of experimental clinical cancer research page of transcription and translation levels has been shown in awide range of transformed cell lines as well as humancarcinoma tissues including hepatocellular pancreaticcolon prostate lung breast ovarian and cervical carcinoma cholangiocarcinoma neuroblastoma and gastriccancer table the first study that demonstrated thesignificantly higher expression of both asph mrnaand protein in hcc and cholangiocarcinoma relative totheir normal adjacent tissue counterparts was by lavaissiere subsequently they verified the role of upregulated asph protein production and its enzymaticfunction in the malignant transformation on biliary epithelium the nih3 t3 cell line and animal models the level of asph also correlated with cell motility andinvasiveness in in vitro experiments [ ] in thestudy by maeda the overexpression of theasph protein was in accordance with worse clinical andhistopathological characteristics of the intrahepatic cholangiocarcinomas and prognosis of patients similar findings were obtained in other studies for hepatocellular[ ] nonsmall cell lung and colon carcinomas and glioblastoma multiforme recentlytheprognostic significance of 2oxoglutaratedependent oxygenase expression was demonstrated by analysis of expression profile datasets of tumor samples and nontumor samples asph has been identified asone of the genes which upregulated expression couldserve for risk stratification of patients with cancertypes in glioblastoma the prognostic significance ofasph was suggested by profiling of alternative mrnasplicing asph gene expression is upregulated via wntcatenin and insulininsulinlike growth factor igf1insulin receptor substrate irs1 signaling [ ]through extracellular signalregulated kinaseerkmitogenactivated protein kinase mapk andphosphatidylinositol3kinaseprotein kinase b pi3kakt pathways fig for review see ref insulinigf1irs1 signaling affects cell growth and survival andcan be involved in oncogenesis in various human tumorstable summary of the studies which have identified the elevated asph expression in human tumor tissuesstudypositive cases of studied samples nntumor tissuesdetectionmethodihcantibody recognized region ofasph proteinfb50 ab nterminusfb50 ab nterminusfb50 ab nterminusfb50 ab nterminusfb50 ab nterminus or 15c7 abcatalytic domainfb50 ab nterminusmab g3 hybridomapolyclonalfb50 mab nterminusihcihcihcihcrtqpcrihcrtqpcrihcihcihcrtqpcrihcfb50 ab nterminuslavaissiere hepatocellularcholangiocarcinomabreastcolonpalumbo pancreatic adenocarcinomasepe primitive neuroectodermalmedulloblastoma neuroblastomamaeda cantarini cholangiocarcinomahepatocellularmonte hepatocellularyang wang dong tang lin types of tumor tissuesahepatocellularpancreatic cancerhepatocellularbreast 75fold higher level of mrnacompared to normal tissue 7fold higher level of mrnacompared to normal tissuepancreatic ductal adenocarcinomaogawa aliver kidney breast cervical ovarian fallopian tube laryngeal lung thyroid pancreatic thymic prostate bladder esophagus gastric gall bladder colon andrectum cancer and cholangiocarcinomafb50 ab nterminusihc 0ckanwal of experimental clinical cancer research page of fig regulation of asph expression and asph involvement in signaling pathways the expression of the asph protein can be regulated atseveral levels the asph gene can be amplified in tumor cells and its transcription activated by inigf1 and wnt catenin pathways or inducedby hypoxia at the posttranscriptional level mir200a and mir135a can downregulate asph expression stability of the asph protein can bereduced by phosphorylation with gsk3 conversely asph can enhance gsk3 activity by inhibition of its phosphorylation with akt and p38kinases asph also supports cell proliferation epithelialmesenchymal transition migration invasion and angiogenesis and consequently tumrowth and metastasis by hydroxylation of the notch receptor and ligands ex jag and interaction with prb vimentin and adams finallyinactivation of nk cells by asph has been demonstrated green arrow activation signal red bar inhibitory signal the catenindependent wnt pathway regulatescell proliferation motility and differentiation and is oneof the most frequently modified pathways in humanmalignancies upon aberrant activation of wnt signalingcatenin is accumulated in the cytoplasm and subsequently translocated to the nucleus where an interaction between catenin and tcellfactorlymphoidenhancerbinding factor tcflef proteins forms atranscriptional regulatory complex which enhances theexpression of wnt target genes including irs1 asph was proposed as a common link between wntcatenin and insulinigf1irs1 pathways and downstream signaling the regulation of asph gene expression in tumorsmight also be affected by a copy number variation inthe study by kadota the asph gene locus hasbeen identified as one of the dna regions with focalamplification in primary breast cancer in colorectal cancer asph gain or amplification was found in ofsamples next a suppressant role of the micrornamir200a in posttranscription regulation of the asphexpression in hepatoma cells has been found mir200a belongs to mir200 family which plays significantrole in preventing cancer initiation and metastasis forreview see ref similarly mir135a has beenshown to suppress asph in endometrial cancer moreover consistent with the protein sequence analysis that recognized numerous prospective phosphorylation sites of glycogen synthase kinase3 gsk3casein kinase ck2 protein kinase a pka and protein kinase c pkc on asph several studies demonstrated that phosphorylation can regulate the asphprotein expression [ ] inhibition of the gsk3activity did not modify mrna expression but increased 0ckanwal of experimental clinical cancer research page of the asph protein level direct phosphorylation ofasph by gsk3 probably decreases asph stability andthus reduces cell mobility asph protein expressionwas also increased by inhibitors of pka pkc and ck2 mutational analysis of potential sites of phosphorylation demonstrated complex and nonuniform effects ofasph phosphorylation on protein expression enzymaticactivity and subcellular localization [ ] thereforeasph phosphorylation probably regulates the functionof this protein by various mechanismsasph expression can also be regulated by hypoxia andoxidative stress in human neuronal cells this effect wasmediated by hypoxia inducible factor alpha hif1αthat is stabilized under hypoxiaoxidative stress whenthe prolyl hydroxylase domain phd proteins and factorinhibiting hif fih are inactivated consequently thehif1 heterodimer made up of subunits hif1α andhif1 functions as a transcription factor likely enhancing asph expression by binding to hypoxiaresponsiveelements in hypoxic regions of glioblastoma bothhif1α and asph were highly expressed particularly inmore aggressive mesenchymal subtype of glioblastomasuggesting a possible involvement of asph in mesenchymal transition brewitz showed reducedasph hydroxylation activity at low oxygen concentrations and suggested an asph role in oxygen hypoxiasensing asph upregulation induced by hypoxia couldcompensate for reduced enzymatic activity moreover a recent study reported an oxidative stress state ofthe castrationresistant prostate cancer cells upon asphoverexpression which was reversed by silencing asphexpression or generating hypoxic conditions resulting inimpaired cell proliferation and invasion asph protein interactions and signaling pathwaysthe asph hydroxylation consensus sequence is confined within cbegflike domains that are found in proteins of diverse function including notch receptors andligands clotting factors structural proteins of the extracellular matrix and ligands of the tyro3axl family ofreceptor tyrosine kinases the notch signaling cascade is a remarkably conservedpathway notch proteins notch1 notch4 are singlepasscell surface receptors that mediate communication betweencells and their expression is crucial for proper embryonicdevelopment notch signaling mainly results in cell differentiation but also plays a significant role in proliferationapoptosis and the maintenance and selfrenewal of stemcells dysregulation of the notch pathway is directly linkedto cancer vascular disorders and congenital defects in mammals notch signaling activated by binding ofone of two families of canonical notch ligandsjaggedjag1 and jag2 and delta like dll1 dll3 and dll4leads to the generation of the cleaved notch intracellulardomain nicd fragment and its nuclear translocation inthe nucleus the nicd fragment interacts with the dnabinding complex csl cbf1rbpjκ suh lag1 thiscomplex is then converted from a repressor into an activator leading to increased transcription of target genes suchas hes family bhlh transcription factor hes1 heswith yrpw motif hey1 cd44 epithelial cell adhesionmolecule epcam cmyc protooncogene matrix metallopeptidase mmp29 cyclin d1 cyclooxygenase vascular endothelial growth factor vegf and proliferatingcell nuclear antigen pcna [ ]upregulation of asph results in enzymatic modification of the cbegflike repeats in the notch receptorextracellular domain and its ligands which promotes thereceptor interaction with the ligands and the activationof notch signaling [ ] furthermore the interactionof asph with a disintegrin and metallopeptidase domainadam stabilizes this complex and enhances thes2 cleavage ofthe notch receptors and subsequentnicd fragment release the activation of the targetgenes in malignant cells increases cell proliferation migration and invasion through the epithelialtomesenchymal transition emt that is probably upregulated by the interaction of asph with vimentin consequentlythis activation supports tumor growthand metastasis the asphnotch axis also stimulatesthe release of exosomes that transfer proteins involvedin invasion metastasis metabolism and immunosuppression [ ]the src kinase pathway is another important pathwayin malignant cell transformation that regulates a complex signaling network promoting angiogenesis invadopodia formation and maturation and metastasis asph has been identified as an src pathway activatoroverexpressed asph directly interacts with adam12 and strengthens the src activation by these proteinswhich promotes mmpmediated extracellular matrixdegradation and tumor invasiveness asph can also contribute to malignant phenotype ofcells by interaction with other proteins iwagami revealed the interaction of asph with gsk3 that prevents gsk3 inactivation by phosphorylation with upstream kinases this mechanism was confirmed ina castrationresistant prostate cancer model gsk3 is a multifunctional kinase that is involved in variousprocesses including glycogen metabolism cell divisionand cell fate determination some types of tumors aresensitive to gsk3 inhibitors recently huang elucidated a direct binding of asph with retinoblastoma protein prb leading to prb phosphorylation they also showed that this effect was mediated byincreased binding of cyclindependent kinase cdk cdk4 and cyclins d1 and e with prb and wasdependentasasph enzymaticonactivity 0ckanwal of experimental clinical cancer research page of phosphorylation of prb inactivates its tumorsuppressorfunction asph can contribute to the progression of cellcycle via interaction with prbeffect of asph on an immune systemtumor generation and progression are influenced bycancer immunoediting that involves immunosurveillanceand escape from a host immune system in theseprocesses various mechanisms of both innate and adaptive immunity are included immune cells that infiltrate developing tumors are initially antitumorigenicbut in tumor microenvironment they can be modifiedinto cells with protumorigenic properties as potential targets of asph hydroxylation are alsoexpressed on immune cells this enzyme could affect thefunction of immune system particularly in tumor microenvironment when asph is overexpressed on cancercells indeed such effect was demonstrated for humannatural killer nk cells by using recombinant asphwhich reduced viability and cytotoxicity of these cells viaenhancing caspase signaling and decreasing the surfaceexpression of activating receptorsrespectively antibodies against asph inhibited these effectsinteraction of asph with other immune cells has notbeen studied however we suppose possible influence ofasph on different tumorinfiltrating cells this assumption comes from the involvement of notch signaling indifferentiation and function of various immune cells fibroblasts mesenchymal cells and endothelial cells forinstance notch activation contributed to stimulation ofproinflammatoryantitumorigenic m1 polarization inboth bone marrowderived primary macrophages and tumorassociated macrophages whennotch signaling was abrogated protumorigenic m2polarization was induced even by stimulators of m1polarization mir125a has been identified as adownstream mediator of notch signaling in macrophages similarly the notch pathway plays an important role in differentiation of other types of myeloidcells and probably all subsets of cd4 and cd8 t cells different notch receptors and their interactionwith different ligands contribute to these processes moreover noncanonical notch signaling is implicatedin regulation of immune cells while activation ofnotch signaling in some cells eg t helper cells cytotoxic cd8 t cells and m1 macrophages supports induction ofincluding antitumorimmunity in other cells particularly regulatory t cellsit leads to immunosuppression thus immunostimulatory effect of notch signaling is often inhibited intumor microenvironment to enable the tumor cells toescape from the host immunity therapeutics affecting notch signaling in malignant diseases are being developed and tested in clinical trials but their effects onimmune reactionsimmune reactions and possible combination with immunotherapy have not been properly studiedasph as a therapeutic targetoncogenic abilities of asph have been experimentallydemonstrated using tumor cell lines and mouse and ratmodels of different types of human tumors with asphoverexpression including cholangiocarcinoma [ ] hepatocellular carcinoma [ ]neuroblastoma pancreatic cancer [ ] glioma breast carcinoma castrationresistant prostatecancer and colorectal cancer in studies analyzingasph function various approaches were utilized to revealsignaling pathways affected by asph particularly asphexpression was diminished by using small interfering rnas[ ] short hairpin rnas [ ] or thecrisprcas9 system [ ] the importance of asphenzymatic activity in these processes was shown by the sitedirected mutagenesis [ ] or treatment by smis [ ] in vitro assays showed asph involvement in cell proliferation migration and invasion cellularalterations included emtinhibition of apoptosis andstemness acquisition tumor growth and invasivenesscould further be supported by asphinduced extracellularmatrix degradation angiogenesis and transendothelial migration notch and src signaling are probably major pathways influenced by asph fig and contributing toincreased aggressiveness of tumor cells that was verified inin vivo models thus these studies also demonstrated thatasph is a suitable target for cancer treatment especially bysmis or immunotherapysmall molecule inhibitorssmis of asph fig have been developed and used totest the role of asph in a wide range of cancer modelsincluding subcutaneous orthotopic and patient derivedxenograft in vivo models [ ] a small orallybioavailable inhibitor has severalintrinsic advantagesover immunotherapy approaches not only can these inhibitors inhibit the catalytic activity of asph unlike conventional antibodies that simply bind to the protein butthey can also penetrate into the cell and inhibit asphcatalytic activity in the endoplasmic reticulum differentcancers have different asph expression patterns andwhile surface expression is quite common in pancreaticcancer and hepatocellular carcinoma intracellular overexpression patterns have also been observed the first asph smis published were the tetronimidesmoi500 and moi1100 tetronimides were originallysynthesized in by dahn and are redoxactivemimics of ascorbic acid and 2oxoglutarate moi500 isa mixed inhibitor that inhibits both asph and the fatmass and obesity protein fto and is not onlyorally bioavailable but also can penetrate the blood 0ckanwal of experimental clinical cancer research page of fig small molecule inhibitors of asphthereand kinasesbrain barrier moi1100 is a more potent inhibitor ofasph and is also more selective despite investigation against a wide range ofirondependent dioxygenasesare no other knownenzymatic targets for moi1100 enhanced activity wasobserved by replacing the chlorine with a trifluoromethylgroup as in moi1151 and even a greater improvement in in vivo activity was found by replacing the trifluoromethyl group with a carboxymethyl group as inmoi1182 although it is not yet clear if the nature ofthis enhancement is due to increased inhibitory activityorenhanced solubility parameters moi1182 isreported to have the ability to suppress invasive activityat a concentration of nm smis of asph have acharacteristic in vitro concentration dependent profilewhere the activity of the smi plateaus at value around viability emphasizing the noncytotoxic properties of this class of inhibitorsnatural products and inhibitors of other enzymes thathave been repurposed as asph inhibitors have alsorecently been reported in the patent literature includingbosutinibcepharanthinecn2019101414327 and guaianolides related to nortrilobolidecn2019104575886cn2019101414219cn2019101414187 0ckanwal of experimental clinical cancer research page of sesquiterpene with complex anticancerbosutinib is a wellknown inhibitor of bcrabl and srctyrosine kinases approved for the treatment of chronic myelogenous leukemia cepharanthine is a natural productactivityincluding ampactivated protein kinase ampk activationand nuclear factor kappa b nfκb inhibition nortrilobolide and related compounds are reported to be potentcytotoxic agents with subnanomolar sarcoendoplasmicreticulum calcium atpase serca inhibition recently a family of potent pyridine dicarboxylates have alsobeen published utilizing a mass spectrometrybasedinhibition assay these compounds are related toknown irondependent dioxygenase inhibitors 23pyridinedicarboxylate 24pyridine dicarboxylate and 26pyridinedicarboxylate the synthesized pyridine dicarboxylateswere assayed for activity against a range of other enzymesto include phd2 fih and lysinespecific demethylase 4ekdm4e in addition to asph with varying degrees of selectivity however while cellbased activities have not beenevaluated the dicarboxylate nature of the compounds maybe useful for cell surface asph inhibitors that may nothave cell penetrating activity immunity as a target of humoralimmunotherapyasph can be used not only as a target of the inhibitors inactivating its enzymatic activity but also as a target of immune reactions leading to destruction of tumor cells andtumor growth suppression since asph is cell surface displayed on tumor cells it represents a tumorassociatedantigen that can be targeted by both cellmediated andhumoralimmunityasph on the surface of cancer cells can be bound by antibodies that mediate antibodydependent cellular cytotoxicity adcc complement dependent cytotoxicity cdcor antibodydependent cellular phagocytosis adcp when the asph antigen is processed in tumor cells orantigen presenting cells antigenic peptides are presentedon these cells by human leukocyte antigen hla class ior class ii molecules and recognized by cd8 or cd4 tlymphocytes respectively that can be stimulated byimmunization breaking tolerance to selfantigens induction of asphspecific cd4 and cd8 t cells wasexamined in blood samples of hcc patients using synthetic peptides derived from asph after prediction of hlaclass i and hla class iirestricted epitopes it has beenfound that asph is a highly immunogenic protein that activates both types of analyzed t cells thus efficientantitumor reactions could be stimulated by immunizationthe first vaccine against asph was based on matureddendritic cells dc loaded with the asph protein andtested in an orthotopic rat model of intrahepatic cholangiocarcinoma this study showed that vaccinationstimulated cytotoxicity against cancer cells in an in vitroassay and decreased tumor growth and metastasis bothcd8 and cd4 cells contributed to an antitumor effectinduced in a mouse model of hcc by immunizationwith asphloaded dcs the next antiasph vaccine was based on a bacteriophage lambda display system the viral capsid proteingpd was fused with the n or cterminus of asph andimmunogenicity ofthese nanopforming constructs was verified in two mouse tumor models the vaccine pan3011 containing these constructs hasalready been examined in a phase clinical trial in patients with biochemically relapsed prostate cancer this study demonstrated safety and immunogenicity of pan3011 and indicated an antitumor effect interms of the reduction of prostate specific antigen psaor psa doubling time asphspecific immune responseswere mediated by both antibodies and t lymphocytesas asph is a type ii transmembrane protein its cterminus carrying the enzymatic domain is exposed outside cells and can be bound by antibodies that can be usedfor diagnostic and therapeutic purposes development ofasphspecific antibodies has been described in several s [] the human igg1 pan622 recognizesthe catalytic domain of asph this antibody is not directly cytotoxic for tumor cells but is internalized and candeliver cytotoxic moieties into cells in the subsequent study with a mouse model of metastatic breast cancerandradioimmunotherapy with promising results mouseigg1 monoclonal antibody binding to the cterminalasph domain mediated adcc by human nk cells recently a secondgeneration antibody approach hasbeen disclosed the prepared antibody binds to the extreme cterminus of asph us that is involved in specific substrate recognition thereforethis antibody has direct asph inhibitory activity anddoes not require any radioisotope or cytotoxic payloadfor potential therapeutic activitypan622 wasbioimagingusedforconclusionsasph is an important enzyme in malignant transformationof cells it stimulates tumor cell proliferation migration andinvasion but it can also affect other cells in tumor microenvironment two main pathways notch and srcthrough which asph promotes the tumor growth havebeen identified it has also been shown that asph expression is induced by some growth factors and hypoxia and isregulated at various levels the overexpression of asphand its downstream targets has been detected in numeroushuman malignancies since asph is not expressed in appreciable level in normal adult tissues and the catalytic domain is localized on the cell surface it has been proposedas one of the most exciting potential therapeutic targetsfig small inhibitory molecules orally bioavailable havebeen developed and successfully tested in several cancer 0ckanwal of experimental clinical cancer research page of fig asph as a therapeutic target asph expression is upregulated by growth factors and hypoxia its enzymatic activity can be inhibited bysmis or monoclonal antibodies which results in reduction of cell proliferation angiogenesis immunosuppression and cell migration and invasionconsequently tumor growth and metastasis are also reducedmodels but they have not yet advanced into clinical trialsadditionally as asph was identified as a tumorassociatedantigen immunotherapy approaches vaccines and monoclonal antibodies were tested with promising results in preclinical experiments and results of pha
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in the uk the death toll from severe covid19 is among the highest worldwide1 severe covid19 is characterised by respiratory failure with socalled cytokine storm occurring in some patient subsets2 pathological correlates are required to understand the pathophysiology of covid19 autopsybased histopathological analysis is crucial in this respect in anticipation of the covid19 pandemic our group produced national guidelines for autopsy performance in suspected covid19 cases3covid19 is caused by infection with severe acute respiratory syndrome coronavirus sarscov245 although sarscov2 and its predecessor sarscov causing severe acute respiratory syndrome [sars] are toll similar on a molecular and clinical level covid19 has a lower death rate for covid19 vs for sars and a substantially higher death deaths worldwide from covid19 as of aug vs from sars than sars due to a higher basic reproduction number1 the postmortem findings in patients with sarscov infection included diffuse alveolar damage dad splenic and nodal lymphocyte depletion haemophagocytosis renal acute tubular injury cerebral oedema microthrombosis and adrenalitis with necrosis with intracellular sarscov detected in the lungs kidney brain and haematological ans6 various autopsy series on covid19 have begun to emerge in the literature7 here we document the major pathological lancet microbe published online august 101016 s2666524720301154department of cellular pathology northwest london pathology b hanley mbbch prof k n naresh md c roufosse phd j weir frcpath prof r goldin md p viola md m osborn frcpath and department of hepatology p manousou phd imperial college london nhs trust london uk centre for haematology b hanley prof k n naresh and centre for inflammatory diseases c roufosse department of immunology and inflammation department of infectious disease prof g s cooke frcp a abdolrasouli phd o c swann mres l baillon bsc r penn msc prof w s barclay phd and department of metabolism prof m thursz md faculty of medicine imperial college london london uk department of histopathology royal brompton and harefield nhs foundation trust and national heart and lung institute imperial college london london uk prof a g nicholson dm renal and transplant centre hammersmith hospital imperial college healthcare nhs london uk r corbett phd department of neuropathology kings college hospital london uk prof s alsarraj frcpath death investigation committee royal college of pathologists london uk m osborn and nightingale nhs hospital london uk m osborn correspondence to dr brian hanley department of cellular pathology northwest london pathology charing cross hospital campus london w6 8na uk bhanleyimperialacukwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cresearch in contextevidence before this studycovid19 is a new disease and comprehensive descriptions of the histopathological findings at autopsy are scarce we reviewed the literature available on covid19 autopsy findings up to and including may for this we searched pubmed and google scholar databases with no language restrictions using the search terms covid19 sarscov2 histology autopsy and postmortemadded value of this studyour series focused on providing a comprehensive description of the histopathological findings in patients with severe fatal covid19 and correlating these findings with data on viral tropism the most prominent findings included diffuse alveolar damage thrombosis haemophagocytosis and immune cell depletion several novel autopsy findings in patients with covid19 were also described including pancreatitis pericarditis adrenal microinfarction secondary disseminated mucormycosis and brain microglial activationimplications of all the available evidenceour study supports the existing clinical and autopsy literature that identified diffuse alveolar damage thrombosis immune cell depletion and macrophage activation as the most prominent pathological features in covid19 other factors including acute kidney injury pancreatitis pericarditis secondary fungal infections and preexisting liver disease require further investigation the presence of ongoing viral replication in late stage covid19 supports the continued use of antiviral therapy even at a point in illness when immunopathology is dominantsee online for appendixfindings of ten postmortem examinations done on patients with clinically confirmed covid19methodspatient selectionfor this study eligible patients were older than years with premortem sarscov2 infection and covid19 listed clinically as the direct cause of death under part on the medical certificate of cause of death [mccd] consent was obtained for all included patients according to the human tissue authority codes of practice by a member of the trust core postmortem consent team consent rate was · ten of patients exclusion criteria included extended postmortem interval before autopsy days and patients with covid19 contributing but not directly leading to death under part of the mccd patients were from imperial college national health service nhs trust nine patients london uk and royal brompton harefield foundation nhs trust one patient london uk premortem sarscov2 infection was identified using the coronavirus typing multiplextandem pcr highplex system aus diagnostics chesham uk ethical approval for this project was provided by the imperial college healthcare tissue bank r20012autopsy proceduresfull autopsies were done on nine patients pm1 and one patient underwent percutaneous biopsy sampling heart lungs pancreas kidneys and liver using percutaneous biopsy under ultrasound guidance pm10 full postmortem examinations included standard sampling and were done according to royal college of pathologists guidelines3 eight different regions of the brain were sampled for each full neuropathological examination all tissue samples were fixed in formalin for a minimum of h before embedding histochemical stains and immunohistochemistry were applied according to local protocols appendix p ans were reviewed by subspecialist pathologists in lung agn and pv haem ato pathology and immune pathology knn liver rg gastrointestinal mo neuropathology sas and renal pathology cr integrated interpretation was done by a subspecialty autopsy pathologist bh and mo all cases were reviewed independently by at least two pathologistspcr proceduresfresh tissue for quantitative rtpcr qrtpcr analysis was processed within the biosafety level facilities at st marys hospital london uk approved by the uk health and safety executive and in accordance with local rules at imperial college london total rna was obtained from fresh tissue samples by use of trizolchloroform extraction followed by precipitation and purification using the rneasy kit qiagen hilden germany qrtpcr against e gene rdrp and subgenomic rna was done as described elsewhere1617 in patient pm5 total fungal genomic dna was extracted from four to five ribbon slices of a formalinfixed paraffinembedded lung tissue block purified dna was amplified with pcr for panfungal and mucoralesspecific targetsstatistical analysisall data was analysed using spss software version and expressed using median iqr and percentagerole of the funding sourcethe funder of the study had no role in study design data collection data analysis data interpretation or writing of the the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publicationresultsbetween march and april ten patients were included in the study the median age at death was years wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ciqr seven of ten patients were men three were women and most patients were white or asian nine hypertension four patients and chronic obstructive pulmonary disease three were the most common contributing factors to death according to mccd all ten patients developed fever and had at least two respiratory symptoms or signs cough shortness of breath reduced oxygen saturations or pleuritic chest pain during their early presentation of eight patients assessed for inflammatory markers all had elevated inflammatory markers these features were either apparent upon presentation to hospital eight of ten patients or developed in an inpatient two patients pm8 and pm9 most patients died within weeks of symptom onset seven patients and were not intubated or ventilated six patients four patients were intubated during their presentation pm2 for days pm5 for days pm6 for less than day and pm7 for days the median bodymass index bmi was in the obese range · iqr ·· and more patients were obese according to bmi at post mortem five of nine than indicated on the mccd one of ten the median interval between death and postmortem examination was days iqr ·· although the limited post mortem had a shorter interval less than h after death detailed clinical case vignettes are available in the appendix p and clinical data are summarised also in the appendix p all patients had dad six showed purely exudative phase dad and four showed a mixture of exudative and anising dad appendix p figure three of four patients with anisingphase dad had spent a substantial period on a ventilator days days and days florid acute bronchopneumonia and ventilatorassociated pneumonia were not noted in this series although mild interstitial neutrophilic inflammation three of ten patients and patchy acute bronchopneumonia three patients were observed interstitial macrophages were prominent macrophages were accompanied by scattered plasma cells mild or moderate lymphocyte inflammation was present in all ten patients although focal lymphocyte cuffing of small vessels was noted in six patients we noted that lymphocytes in the lung were predominantly cd4positive t cells cd56positive natural killer cells were rarely found occasionally a patient had small aggregates of small b cells chronic bronchiolitis was seen in most patients nine of ten no granulomas or viral inclusion were seen invasive mucormycosis was noted in one patient pm5 figure and confirmed with mucoralesspecific pcr the mucormycosis was vasculocentric and disseminated involving the hilar lymph nodes heart brain and kidney in the same patientmacroscopic two of nine patients and microscopic eight of nine pulmonary thromboemboli were frequent observations appendix p figure both fibrinrich and plateletrich thrombi were identified in smallsized and mediumsized vessels and within the capillaries in alveolar septa figure external examination findings of deep venous thrombosis were not noted very focal lymphocytic vasculitis was identified in one patientthrombotic features were universal in this cohort and all nine patients who underwent a full autopsy had at least one microthrombosis or macrothrombosis in a major an one of nine patients had a macroscopic acute coronary thrombosis in the right coronary artery whereas five patients had thrombi in the microcirculation of the heart on histological analysis coronary artery disease was negligible or mild in most patients seven of nine acute myocardial ischaemic damage h old was noted in the patient with an acute coronary artery thrombus figure 2a pm1 a mottled myocardium and subendocardial contraction band necrosis was noted in a acebdf 03m µm µmfigure pulmonary pathological findings in patients with covid19a macroscopic subpleural petechial haemorrhage in a 24yearold man pm6 b hyaline membranes indicative of exudative phase diffuse alveolar damage in a 79yearold woman pm9 at 20x magnification c cd61 immunohistochemical staining indicating plateletrich microthrombosis in alveolar capillaries pm6 d squamous metaplasia in a 61yearold man pm1 with exudative phase diffuse alveolar damage at à magnification e interstitial multinucleated giant cells in a 79yearold man pm7 with anising phase diffuse alveolar damage at à magnification the top right insert is of multinucleated giant cells showing positive cd68 staining indicative of macrophage lineage the bottom left insert shows absence of staining for cytokeratins f periodic acid schiff staining indicating wide irregular aseptate and ribbonlike hyphae with openangle branching and a vasculocentric pattern indicative of mucormycosis in a 22yearold man pm5 the insert is a grocott silver stain highlighting mucormycosis at 20x magnificationwwwthelancetcommicrobe published online august 101016s2666524720301154 s 0csecond patient pm2 whether the contraction band necrosis was related to ischaemia or inotropic medication received in the intensive care unit is uncertain appendix p pm1 and pm2 were the two patients with the highest active viral load detected in the heart a single patient had a right atrial thrombus pericarditis was acegbdfh µm µm 03m µmfigure thrombotic features identified at autopsy in patients with covid19a macroscopic right coronary artery thrombosis arrow in a 61yearold man pm1 with exudative phase diffuse alveolar damage b macroscopic pulmonary thromboembolism arrow in a 97yearold man pm8 c thrombus in the lung of a 79yearold woman pm9 on haematoxylin and eosin staining at 20x magnification the insert shows cd61 immunohistochemistry indicating moderate staining for platelets d plateletrich thrombus in the mediumsized vessels surrounding the heart in a 61yearold man pm1 the insert shows strong cd61 staining for platelets periodic acid schiff staining showing a glomerular microaneurysm arrow e and microthrombi within glomerular capillary loops arrow f at 40x magnification indicative of thrombotic microangiopathy in a 97yearold man pm8 macroscopic splenic g and hepatic h infarction in a 22year old man pm5identified in two patients one patient showed florid fibrinous pericarditis containing fungal hyphae pm5 while the other showed only microscopic acute pericarditis appendix p figure the median heart weight was high g and four of nine patients had left ventricular hypertrophy nonbacterial thrombotic marantic endocarditis was noted in one patient pm5 with no known history or autopsy findings consistent with malignancy or chronic disorder associated with nonbacterial thrombotic marantic endocarditis appendix p figure pm5 had disseminated mucormycosis and numerous other thrombotic features appendix p cardiac amyloidosis and right atrial thrombosis were identified in one of ten patients pm8 appendix p lymphocyte depletion involving specific compartments and increased phagocytosis were prominent findings appendix p figure increased phagocytosis of other cells was identified in the sinusoidal macrophages of the red pulp of the spleen in four of seven patients sinus histiocytes of hilar lymph nodes in three of six and bone marrow four of eight phagocytosis was identified in at least one of these ans in six of nine patients bone marrow haemophagocytosis was prominent in two patients pm4 and pm8 and focal in two patients pm7 and pm9 depletion of periarteriolar tcell sheaths within the white pulp was observed figure red pulp was generally congested showing reduced numbers of cd8positive t cells plasma cells were variably prominent and sinusoidal histiocytes showed phagocytosis of red blood cells and other cells to varying extents both igmpositive and iggpositive plasma cells were identified and they were polytypic for lightchain expression figure lymph nodes showed preservation of follicles and relative depletion of paracortical areas medullary areas showed prominence of plasma cells and histiocytes were prominent in the sinuses bone marrow samples showed reactive changes with trilineage hyperplasia and prominence of plasma cells and histiocytes were a common finding a necrotising granuloma was noted in a single hilar lymph node in one patient and acidfast bacilli were noted on ziehl neelson staining appendix p all spleen and lymphoid material examined with immuno histochemistry were negative for epsteinbarr virus and cytomegaloviruspancreatitis was noted in two of eight patients pm5 was a 22yearold man with frank necrotichaemorrhagic pancreatitis and secondary mucor mycosis figure no fungal hyphae were noted in the pancreas pm8 was a 97yearold man who showed no substantial macroscopic pancreatitis although micro scopic acute inflammation within the pancreas and periadrenal fat necrosis was noted figure a third of patients three of nine showed patchy areas of infarcttype adrenocortical necrosis with one patient showing anising microthrombi in adrenal vessels figure no wwwthelancetcommicrobe published online august 101016s2666524720301154s 0cadrenalitis was noted two of nine patients showed chronic inflammation in the thyroid with follicular epithelial cell disruption however the significance of this finding is uncertainmedian combined kidney weight was within normal range at g iqr salient renal pathology findings were acute tubular injury in all nine patients underlying moderate cortical scarring of uncertain cause in one patient glomerular microaneurysm and thrombi in one patient figure and rare thrombi in interlobular arteries in four patients pm6 24yearold man of arterial intimal thickening than expected for that age appendix p we observed no evidence of focal and segmental glomerulosclerosis diabetic glomerulopathy or glomerulonephritisdegree had a higher large droplet fatty change was seen in most patients seven of eight cirrhosis or bridging hepatic fibrosis were noted in three patients no liver thrombosis was identified histologically but one patient showed macroscopic liver infarction figure the median liver weight was g iqr and three of nine patients showed hepatomegaly liver weighing g two patients pm4 and pm7 showed marked autolysis and were not included in analysismoderate to intense microglial activation was the most prominent pathological feature in the cns five of five patients mild tcell infiltration was noted around blood vessels and capillaries in all five patients but b cells were absent we found ischaemic changes of variable extent in the neurons of the cortex and in the white matter detected by bapp β amyloid precursor protein stain however no necrosis of brain tissue or extensive infiltration of inflammatory cells in brain parenchyma or meninges was observed on histological examination although one of nine patients showed macroscopic haemorrhagic transformation in a large recent cerebral infarction in the distribution of the middle cerebral arterytissues from five patients were analysed for presence of viral genomes against e gene and indications of viral replication against subgenomic rna transcripts by qrtpcr viral rna was present in respiratory tract samples including lung of all five cases analysed in addition two of three patients had detectable viral rna in the nasal epithelium and four of five patients in the trachea evidence of viral genomes outside the respiratory tract was found for all five patients but the distribution and viral loads varied case by case figure 5a viral genomes were also detected using a different qrtpcr targeted at rdrp gene and patterns were consistent between the two sets of primers data not shown a third primer set that detected subgenomic rna indicated virus replication in all tissues examined with variation between patients in levels and distribution figure 5bace µmbdf µm µmfigure other notable autopsy findings in patients with covid19a contained aortic dissection green arrow and fibrinous pericarditis red arrow in a 22yearold man pm5 insert is a haematoxylin and eosin stain image of the pericardium showing fibrinous pericarditis 10x magnification b adrenocortical microinfarcts in a 79yearold woman pm9 with reendothelialising thrombus in small adrenal vessels highlighted by cd34 insert bottom left and haematoxylin and eosin insert top right marantic endocarditis c highlight with haematoxylin and eosin staining bottom left at 10x magnification and necrotising haemorrhagic pancreatitis d in a 22yearold man pm5 with covid19 and a secondary fungal lung infection e periodic acid schiff staining showing a granular cast arrow indicative of acute tubular injury in a 24yearold man pm6 20x magnification f microscopic acute pancreatitis on haematoxylin and eosin staining in a 97yearold man pm8 20x magnificationdiscussionin this series we have described the major pathological findings identified at autopsy in ten patients who died of severe covid19 the most consistent findings were dad thrombosis haemophagocytosis and immune cell depletion although unexpected pathologies that are probably related to sarscov2 infection were also identifieddad was the most consistent and prominent feature in our series and others78 the specific phase of dad probably represents the degree and chronicity of the offending insult sarscov2 infection in relation to the time of death this is similar to previous coronavirus epidemics6 the conclusion by copin and colleagues7 that covid19related lung injury is not diffuse alveolar damage might relate to their sampling strategy and wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cadbc µm µm µm µm µmef µm µm µmfigure pathological findings in haematological ans in patients with covid19tcell depletion in the spleen of a 79yearold woman pm9 with covid19 haematoxylin and eosin staining of the spleen at 10x magnification a cd20 staining of spleen indicating presence of b cells b 10x magnification with the insert showing the same region at higher power 20x magnification and cd3 staining of spleen indicating depletion of t cells c 10x magnification with the insert showing the same region at higher power 20x magnification bone marrow phagocytosis in a 97yearold man pm8 with covid19 haematoxylin and eosin staining of a well preserved bone marrow with an arrow indicating presence of phagocytosis d 40x magnification and cd68pgm1 staining of bone marrow indicating presence of phagocytosis 20x [e] and 40x [f] magnificationchronicity five patients had spent approximately weeks on a ventilator barton and colleagues8 described prominent acute bronchopneumonia as the major finding in one of two patients although the authors acknowledge that this was probably affected by aspiration in their patient with muscular dystrophy reports of lung histology in early covid19 also suggest a degree of lymphocytic pneumonia although dad is probably superimposed on this over time in the majority of fatal cases7 pulmonary macrophage infiltration and multinucleated giant cell reactions are prominent similar to other series8 definite evidence of in covid19 will require quantitative analysis comparing tissues from covid19 patients with dad associated with other conditions and unaffected tissues several cases of invasive pulmonary aspergillosis have been reported in patients with severe covid19 pneumonia18 to our knowledge this is the first description of histologically proven mucormycosis in patients with covid19 and suggests that other human fungal pathogens including members of mucoromycotina can complicate covid19associated infectionslymphocyte depletion tissuerelated numerous clinical features including raised serum ddimer concentrations raised procalcitonin concentrations and imaging findings suggest thrombosis is prominent in patients with covid192 thrombotic features were universal among patients who underwent full autopsies all nine patients had thrombi in at least one major an and have been noted to be prominent in other covid19 autopsy series15 in a retrospective study of autopsies in patients with acute respiratory distress syndrome and dad of various causes only showed thrombi within the small vessels of the lung despite sampling of every lobe of the lung19 another study used postmortem angiography and identified thrombi in nearly all cases of acute respiratory distress syndrome from various causes20 whether thrombosis in covid19 is more common than in other causes of dad remains uncertain however our data support thrombosis as being a striking feature in these patients a study suggested endotheliitis as a prominent feature in patients with severe covid1910 but this was not a prominent feature in our patients importantly limited post mortem or postmortem crosssectional imaging are likely to underrepresent the true extent of thrombosis particularly microthrombosis and its impact on patient death the extent of cardiomegaly fibrointimal thickening of renal blood vessels and obesity in our series supports a contribution of hypertension beyond that noted clinically only four patients had hypertension documented on the mccda raised cytokine profile has been documented in a subset of patients with severe covid192 consistent with this haematological ans in our series showed prominent phagocytosis in several patients which has not been documented in previous series21 of the four patients with bone marrow haemophagocytosis one patient pm7 showed mild transaminitis hyperbilirubinaemia elevated serum ferritin concentrations and fever of ·°c however most clinical data were insufficient to assess the presence of haemophagocytic lympho histiocytosis wwwthelancetcommicrobe published online august 101016s2666524720301154s 0ca substantial feature in covid19 is lymphocyte depletion and this is supported in our series by the spleen and lymph node findings when compared with those with mild disease patients with severe covid19 tend to have a higher neutrophil to lymphocyte ratio and higher cd4positive to cd8positive tcell ratio22 additionally a negative correlation exists between peripheral blood lymphocyte count and viral copy number22 we have corroborated this evidence by documenting a low number of t cells especially cd8positive t cells and foxp3positive regulatory t cells in the spleen and lymph nodes in severe fatal covid19 notably normal plasma cell both igm and igg positive response was present in haematological ans in most patientsthe extent to which anspecific pathologies relate to direct viral replication or consequent immunological and cardiovascular complications is of clinical relevance we report here evidence of viral genomic rna outside the respiratory tract this finding is in agreement with several previous studies that have identified viral genomes by qrtpcr in postmortem tissues including the colon14 spleen14 liver1423 skin24 heart23 and brain25 we also report detection of subgenomic rna a product that is only produced in actively infected cells a report identified low viral load in the brain of three of patients with covid19 but could not detect the virus in subsequent immunohistochemistry and concluded that the viral genomes might have been present in the blood25 although we cannot exclude that the rnas detected in our series were similarly carried to the site of sampling in blood the distribution of rna in different tissues varied widely between postmortem casespm3 and pm4 appear to have died earlier in the disease course days after symptom onset and had higher viral loads in the respiratory tract than other patients whereas pm3 and pm4 died after long stays in intensive care units and had either lower overall viral rna pm5 or higher viral rna outside the respiratory tract pm2 pm1 and pm2 were the only patients in whom we detected viral rna and subgenomic rna in the heart and are the only two patients with evidence of acute myocardial injury moreover unlike the previously mentioned study where virus detected in the brain was times less than that detected in the lung the number of viral genomes detected in external tissues in our series was frequently of similar or even higher levels than that found in the respiratory tree one study has detected sarscov2 infection of the endothelium in the vasculature of the skin and lung by immunofluorescent staining for viral antigens24 it is not obvious what determines spread and tropism of sarscov2 outside the respiratory tract several studies have reported ace2 expression levels that were higher in some ans than in others26 the sites of highest expression did not correlate with areas of most severe pathological involvement in our series we should note that cellular receptor status is one of many things that determines the degree of anr latot g 03 rep seipocenege larivpm1pm2pm3pm4pm5abtcbone marrowbrainheartileumkidneyliverspleentonguetrachealungnasal epitheliumfigure tissue tropism of sarscov2 in postmortem samplesfresh tissues were collected from a subset of postmortem examinations and viral load quantified by use of qrtpcr targeting the viral e gene a detection of viral rna was verified by use of qrtpcr against the viral polymerase gene data not shown tissues were additionally tested for subgenomic viral rna transcripts b dotted lines indicate the limit of detection as ascertained by negative control data are included for a 61yearold man pm1 a 64yearold man pm2 a 69yearold woman pm3 a 78yearold man pm4 and a 22yearold man pm5 qrtpcrquantitative rtpcr sarscov2severe acute respiratory syndrome coronavirus pathological involvement other aspects are likely to play a role such as route of transmission acid lability in the stomach coreceptors expression of proteases required to activate entry of virus into the cell eg tmprss2 possibly other yet unidentified host factors and invivo routes of dissemination the evidence of ongoing replication late in disease supports the use of antiviral therapy even at a point in illness when immunopathology is dominantpancreatic pathology was a major unexpected pathology in this series which had not been previously reported in covid19 autopsies or large clinical covid19 series2 a 22yearold man had haemorrhagic necrotic pancreatitis pm5 although this patient also had disseminated mucormycosis he had evidence of viral persistence in the lung and no fungal hyphae were identified in the pancreas on histological sections the other patient with wwwthelancetcommicrobe published online august 101016s2666524720301154 s 0cpancreatic pathology had microscopic evidence of pancreatitis that could be missed at autopsy without adequate sampling the pancreas is a classic site of unexpected pathology identified at autopsy27 serum amylase is not part of the routine care bundle for covid19 in our trust whether the pancreatitis was related to sarscov2 iatrogenic comorbidities or secondary infection is not clear in our study most cases had evidence of hepatic steatosis which is consistent with clinical findings that obesity is a risk factor for poor outcome in covid19 and liver cirrhosis or bridging fibrosis was prominent in this cohort28 from these data nothing suggests direct viral inflammation of the liverinfection or other causes the interval between time of death and autopsy impaired histological interpretation in many ans and affected antigen preservation postmortem endothelial stripping did affect the endothelial interpretation in our study however a subs
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"Mareks disease MD is a chicken neoplastic disease which brings huge economic losses to theglobal poultry industry The wild type p53 a tumor suppressor gene plays a key role in blocking cell cyclepromoting apoptosis and maintaining the stability of the genome However the mutant p53 losses its tumorinhibitory role and become an oncogene when a mutation has happenedResults The mutation rate of p53 was in the experimentally and naturally infected chickens The mutationsincluded pointmutations and deletions and mostly located in the DNAbinding domain The mutated p53 wasexpressed in various tumor tissues in an infected chicken The mutant P53 proteins were notably accumulated inthe cytoplasm due to the loss in the function of nuclear localization Unlike the study on human cancer theconcentrations of P53 in the serums of MD infected chicken were significantly lower than the control groupConclusions The p53 mutations were apparent in the development of MD P53 and P53 antibody level in serumcould be a useful marker in the diagnosis and surveillance of MDKeywords Mareks disease p53 P53 antibodyBackgroundMareks disease MD is a lymphoproliferative neoplasticdisease caused by the chicken Mareks disease virusMDV or named Gallid alphaherpesvirus The infection caused by this virus may lead to lymphocyte proliferation tumor formation immunosuppression paralysisand mononuclear cell infiltration in peripheral nervesgonads and immune ans [ ] As one of the mosthighly contagious tumor diseases in chickens the data ofOIE [] reported by about half of the world has shownthat this disease accounts for the loss of up to billionUS dollars annually in the global poultry industry [] Correspondence liusidsdaueducn1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong ChinaFull list of author information is available at the end of the Described often as the guardian of the genome [] andthe cellular gatekeeper [] p53 is the most relevantand important tumor suppressor gene with the highestmutation frequency in human and animal tumor diseases [] The chicken p53 gene has a fulllength reading frame and untranslated regions and a polyadenylation signal which encodes amino acidsThese amino acids share a homology to the aminoacids of human P53 []p53 is divided into the wild type and the mutant typeThe wild type is a normal tumor suppressor gene whilethe mutant p53 is an oncogene transformed from atumor suppressor gene due to spatial conformationalchange As a result the mutant P53 protein losses itsability in regulating cell growth apoptosis and DNA repair [] which often a prerequisite for tumorigenesis and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhang BMC Veterinary Research Page of disease progression [] The proportion of p53 mutations in tumor tissue varies between []The wild type P53 has a very short halflife while themutant P53 protein is prolonged This abnormality ofP53 can lead to the accumulation of P53 antibody inserum as well as the P53 protein in tumor tissue []Several studies have demonstrated that the P53 antibodyplays a predictive role in tumorigenesis and its manifestation in serum is an early event in the development ofmalignant tumors in humans [] The level of P53antibody in serum correlates significantly with commonclinical neoplastic diseases but it has been barely detectable in the serum of healthy subjects [] This correlation may also exist in chicken and currently there is aknowledge gap concerning the role of mutant p53 inMareks disease in chicken Therefore this study aimedto investigate the role of p53 as a tumor marker inassisting the clinical diagnosis and prognosis of MDResultsHistopathological and immunohistochemical analysis ofp53 expressionHistopathological examination revealed the evidence ofliver cells undergoing necrosis in the infected SPFchicken Such liver tissues demonstrated focal infiltration and hyperplasia of lymphoid tumor cells Fig 1aIn addition the lymphoid follicle in the bursa of Fabricius was atrophied diffuse infiltration and proliferationof lymphoid tumor cells between the follicles were observed Fig 1b In the livers of the clinically infectedchickens the cytoplasm of the lymphoid tumor cellsunderwent multifocal proliferation which was stainedbrown by the immunohistochemicalFig 2aPositive staining was also detected in the tumor cells inIHCthe spleen and the bursa of Fabricius tissues Fig 2b and cIn contrast no positive stained cells were observed in thecontrol group Fig 2dMutations in the p53The mutation rate of p53 was in the infectedpoultry There were two types of p53 mutations deletions and point mutations detected which was consistent with the results reported in a previous study []Among the mutated p53 genes the base sites withhigh mutation frequency were and There was no mutation found in the control groupMost of the mutations were located in the core domainand the Cterminal domain The mutation analyses wereshown in Table P53 antigen and P53 antibody levels for MDThe concentrations of the P53 antigen of the clinicaland experimental MDVinfected group were significantlylower than the control group However the P53 antibody levels in the experimental infection group were significantly higher than the control group These analyseswere shown in Fig DiscussionThe human P53 is widely acknowledged as an intranuclear phosphorylated protein The wide type P53 normally exists in the nucleus for an extremely short period[] On the other hand the mutated P53 has a prolonged halflife to h as it is not digested quicklyand therefore accumulates inside tumor cells [] Thisallows the detection of mutant P53 protein via IHC forFig Histopathological observation of diseased chickens infected with MDV a Different sizes and shapes of focal infiltration and hyperplasia oflymphoid tumor cells were observed in the liver tissues HE b Diffuse infiltration and proliferation of lymphoid tumor cells between thefollicles were observed in the bursa of Fabricius HE 0cZhang BMC Veterinary Research Page of Fig Immunohistochemical staining of p53 in infected chickens a Liver lymphoid tumor cells with cytoplasm expression HE b Spleen lymphoid tumor cells with cytoplasm expression HE c Bursa of Fabricius lymphoid tumor cells with cytoplasm expression HE d Liver Negative controls were incubated with PBS HE which IHC has now become an important modality forthe detection of various tumor biomarkers P53 proteinrepresents an effective substitute marker for TP53 mutation status [] but at present P53 IHC is mainly applied for tumor diseases in humans P53 IHC allows theassessment of the stage and grade of cancers with only afew studies reported in poultry tumor diseasesIn this study the immunohistochemical analysis revealed that P53 was present in the liver spleen and thebursa of Fabricius of infected chicken with MDV Interestingly P53 was notably expressed in the cytoplasmThe potential explanation of this phenomenon could bethat the mutated P53 protein lost its function in nuclearlocalizationtheaccumulatedinandeventuallycytoplasm Furthermore p53 has been regarded as thehotspots given that p53 mutations have been frequently detected in a variety of tumors [] In thisstudy several types of p53 mutations were demonstratedin natural and experimental infections of MDV The fivemost frequent mutation sites were and The altered codons of mutant p53 comparedwith the wildtype were ACGACA GCAGCC CGCCGG GCCGCA and ACCACA but these were all synonymous mutationsIn our study a short form of p53 transcript was detected in a clinical case The deleted sequence was located at the bp in the reading frame of thereference sequence resulting in missense mutations fromTable Primers used toamplify chicken p53 cDNAPrimerp531Nucleotide sequenceGTGGCCGTCTATAAGAAATCAGA3AAAAAGGGGGCGTGGTCAGT3p532Annealing temperature Size of fragments amplifiedlocation bp 0cZhang BMC Veterinary Research Page of Fig Levels of p53 antigen and antibody in the serum of study groups The Yaxis represented the concentrations of antigen or antibody andthe Xaxis represented the different groups of samples Each group consisted of seven samples Statistical significance was designated as p or p the position to the termination The short form ofp53 transcript was also found in the cases infected byavian leukosis virus [] A series of missense mutationsfrom the position to the termination due to a base deletion was found in an experimentally infected chicken Inaddition this study found that the p53 was mutated in of poultry oncology with the mutation region concentrated mainly in the DBD The DBD allows the specificrecognition of target sequences [] Once the p53 is deleted or point mutations in this region occursit mayaffect the formation of tetramers which in turn leads tothe conversion of wild type P53 into a mutant type resulting in the loss of normal functionThe conformation of mutant P53 extends its halflifeto several hours in humans The accumulated mutantP53 then acts as a target antigen which elicits an autoimmune response [] However in our study the levelsof serum P53 antigen in clinical and experimentalMDVinfected groups were significantly lower than thecontrol group contrary to the findings in human cancerresearch Only the serum P53 antibody concentrationsof the experimentalinfected group were significantlyhigher than the control group This might be that P53as a tumor suppressor was largely consumed in response to the occurrence of MD Moreover tumorigenesis promoted mutation of p53 and further reduced P53concentrationConclusionsOur study revealed p53 was mutated and expressed inMDV infection which suggested that these mutationswere playing an important role in the development ofMD The mutant p53 was expressed in the tumor cellsof various tissues of the infected chicken Mutant P53protein lost its nuclear localization function and transferred from the nucleus to the cytoplasm Unlike studiesin human cancer the concentration of P53 was significantly lower in the natural and experimental MDV infectionnovelinnovations for the diagnosis and monitoring of MD inpoultryresults maygroup OurprovideMethodsNatural infection of MDV in chickensSeven 160dayold egglaying hens were obtained from alocal flock These hens were confirmed to have acquiredMDV infection naturally with the absence of other common viral diseases by PCR detection Their serums werecollected for the detection of the P53 antigen and antibody Their tissue samples including liver spleen pancreas and bursa of Fabricius were collected and fixedwith formaldehyde for h before histopathologicaland immunohistochemical studiesExperimental infection of chickens with MDVThe number of experimental animal was referred to thenumber of clinical samples Twenty 1dayold SPF chickens were obtained from the Shandong Academy of Agricultural Sciences Poultry Institute SPF Chicken ResearchCenter Jinan China They were randomly divided intotwo equal groups as an infection group and a controlgroup The two groups of chickens were raised separately in isolators with filtered air under positive pressureOn the first day of the experiment each of the chickenin the infection group was inoculated intraabdominallyia with plaqueforming units PFU of vvMDV 0cZhang BMC Veterinary Research Page of GX0101 which normally cause tumors at the tenthweek The chickens in the control group were inoculatedwith PBS In the tenth week serums were collected fromseven chickens in each group The experiment endedafter ten weeks and all chickens were put into a euthanasia box Thirty percent of carbon dioxide CO2 by volume was infused into the euthanasia box per minuteuntil all chickens lost their consciousness Then theCO2 flow rate was increased to for one minuteand the euthanasia box was kept airtight for ten minutesWhen all chickens were confirmed dead they were dissected and their livers and spleens were collected forhistopathology and immunohistochemistry studyHistopathology and ImmunohistochemistryThe fixed liver spleen pancreas and bursa of Fabriciuswere dehydrated waxed and cutinto µm slicesfollowed by Haematoxylin and eosin HE staining for ahistopathology examination In IHC processing tissueswere cut into sections of µm thickness and mountedon microslides treated with polyLlysine The sections were deparaffinized in xylene and rehydrated in agraded series of ethanol solutions into PBS then werepretreated in citrate buffer molL pH °Cfor antigen retrieval by microwaving and cooled at roomtemperature for min [] Endogenous alkaline peroxidase was quenched with hydrogen peroxide solutionin methanol for min [] Nonspecific antibody binding sites were obliterated by incubating the sections with fetal bovine serum for min at room temperatureFollowing this the antiP53 polyclonal antibody BOSTER China as a primary antibody was diluted into and immersed overnight at °C in a black humidchamber The secondary antibodies were HRP horseradish peroxidaseconjugated goatIgGCWBIO China Immunoreactivity was then visualizedwith diaminobenzidine DAB staining The sectionswere counterstained with hematoxylin and mountedNegative controls were incubated with PBS instead ofthe primary antibody in the immunohistochemicalanalysisantimouseTotal RNA isolation and reverse transcriptionThe total RNA ofliver and spleen tissue samples mgtissue were isolated by using TRIzol reagentTakara Japan according to the manufacturers instructions Extracted total RNA 1ug was reverse transcribedto cDNA with PrimeScript¢ RT Reagent Kit RocheSwitzerland According to the published complementaryDNA cDNA sequence of the chicken p53 GeneBankaccession number nm205264 specific primers were designed to amplify the DNAbinding domain DBD ofp53 as shown in Table Using PCR Polymerase ChainReaction techniques p53 cDNA genes sequences wereamplified The PCR reaction was performed by using athermal cycler Takara Japan under the following conditions for min followed by cycles of for s for s for s and a final Table Mutations of p53 genes in MDSample InfoNCMutation analysisBase mutation sitesNDMD CMD CMD CMD CMD CMD CMD EMD EMD EMD EMD EMD E TC AC C G AG AC gA CG CA AC CG CA CG CA delete TC TC AC CG CG CA delete CA CA TG AC CG CA gA CA CA AG AC CG CA CT gA AC CG CA gA gT CT CG CA gANC was SPF chicken without diseases E was experimental chicken C was clinical chickenAmino acid mutationsSite from toNDMutation area Y A E G R HNDND Stop StopNDND N S T I G DND R L V M E Y H YCore domainCterminal domainCore domainCore domainCore domainCore domainCterminal domainCore domainC terminal domain 0cZhang BMC Veterinary Research Page of for min extension cycle DNA templates that were acquired from the MDV positive chickens were amplifiedusing primer pair p5312 The SPF chickens wereregarded as negative controls DNA fragments were successfully amplified with sizes of bp The target fragment was gel extracted and connected to the pEASYT1vector Then the recombinant plasmid was transformedinto bacteria and sequencing analysis was made Finallythe sequencing results were compared with the wild typep53 cDNA sequences reported previouslyEnzymelinked immunosorbent assay ELISA for P53 andP53 antibodyThe P53 antigen and antibody levels of chicken weremonitored by ELISA Mlbio China In order to eliminate subjective interference the assessors were blinded tothe background of the samples The detection rangeswere pgml for P53 antigen and pgmlfor P53 antibody Samples were diluted five time with aspecial diluent and all processes were implemented inaccordance with the instructions The absorbance ODof each sample was finally measured at a wavelength of nm The sample concentration was calculated depending on the standard curveStatistical analysisStatistical analyses were conducted with GraphPadPrism Version San Diego CA USA The differences in the levels of P53 antigen and antibody were antwotailed Students Ttestalyzed by usingStatistical significance was designated as p or p theAbbreviationscDNA Complementary DNA DAB Diaminobenzidine DBD DNAbindingdomain ELISA Enzymelinked immunosorbent assay HE Haematoxylin andeosin IHC Immunohistochemical HRP Horse radish peroxidasePCR Polymerase chain reaction PFU Plaque forming unitsAcknowledgementsNot applicableAuthors contributionsHXZ carried out laboratory work wrote the manuscript and performed thedata analyses MDL designed the experiment wrote the manuscript andperformed the data analyses HZ SLC YL SNJ and YNS carried out laboratorywork SDL conceived and supervised this work revised manuscript All authorsread and approved the final manuscriptFundingThe work was supported by grants from the National natural sciencefoundation project NO The funding body was solely involved infunding and had no role in the design of the study the collection analysisand interpretation of the data or in writing the manuscriptEthics approval and consent to participateThe sample were collected and handled in accordance with the goodanimal practices required by the Animal Ethics Procedures and Guidelines ofthe Peoples Republic of China Informed consent to participate wasobtained from the chicken farmer owner All animal protocols andprocedures were performed according to the Chinese Regulations ofLaboratory Animals and were approved by the Animal Ethics Committee ofShandong Agricultural UniversityConsent for publicationNot applicableAvailability of data and materialsThe data supporting the findings are included in the manuscriptCompeting interestsThe authors declare that they have no competing interestsAuthor details1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong China 2China AnimalHealth and Epidemiology Center Nanjing Road QingdaoShandong ChinaReceived January Accepted August ReferencesJarosinski KW Tischer BK Trapp S Mareks disease virus lytic replicationoncogenesis and control Expert Rev Vaccines Reddy MS Book Review Mareks Disease An Evolving Problem Vet PatholBoodhoo N Gurung A Sharif S 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Mailo D Nardi H Corte MM Vincent E Barutta E Lizarraga GLizarraga P Montero H Gentili R Serological levels of mutated p53 proteinare highly detected at early stages in breast cancer patients Exp Ther MedSabapathy K Lane DP Understanding p53 functions through p53antibodies J Mol Cell Biol Kunizaki M Fukuda A Wakata K Tominaga T Nonaka T Miyazaki TMatsumoto K Sumida Y Hidaka S Yasutake T Sawai T Hamamoto RNanashima A Nagayasu T Clinical Significance of Serum p53 Antibody inthe Early Detection and Poor Prognosis of Gastric Cancer Anticancer Res Yue Q Yulong G Liting Q Shuai Y Delong L Yubao L Lili J Sidang LXiaomei W Mutations in and Expression of the Tumor Suppressor Gene p53in EggType Chickens Infected With Subgroup J Avian Leukosis Virus VetPathol Bykov VJN Eriksson SE Bianchi J Wiman KG Targeting mutant p53 forefficient cancer therapy Nat Rev Cancer Yemelyanova A Vang R Kshirsagar M Lu D Marks MA Shih IeM Kurman RJImmunohistochemical staining patterns of p53 can serve as a surrogatemarker for TP53 mutations in ovarian carcinoma an immunohistochemicaland nucleotide sequencing analysis Mod Pathol 0cZhang BMC Veterinary Research Page of Takagi M Ohashi K Morimura T Sugimoto C Onuma M The presence ofthe p53 transcripts with truncated reading frames in Mareks diseasetumorderived cell lines Leuk Res Arlt C Ihling CH Sinz A Structure of fulllength p53 tumor suppressorprobed by chemical crosslinking and mass spectrometry Proteomics Thierry S Analysis of p53 Gene Alterations in Cancer A Critical View Years of p53 Research Stiasny A Freier CP Kuhn C Schulze S Mayr D Alexiou C Janko C Wiest IDannecker C Jeschke U Kost BP The involvement of E6 p53 p16 MDM2and Gal3 in the clinical outcome of patients with cervical cancer OncolLett Shen FX Ma GP Cheng AC Wang MS Li CF Sun KF Chang H Zhu DK JiaRY Chen XY Sun T Development and application of an indirectimmunohistochemical method for the detection of duck plague virusvaccine antigens in paraffin sections and localization in the vaccinatedduckling tissues Poult Sci Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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Bone metastasis classification using wholebody images from prostate cancer patientsbased on convolutional neural networksapplicationNikolaos Papandrianos1 Elpiniki PapageiouID23 Athanasios Anagnostis34Konstantinos Papageiou4 General Department University of Thessaly Lamia Greece Faculty of Technology Dept of EnergySystems University of Thessaly Geopolis Campus Larisa Greece Institute for Bioeconomy and Agritechnology Center for Research and Technology Hellas Greece Department of Computer Science andTelecommunications University of Thessaly Lamia Greece npapandrianosuthgrAbstractBone metastasis is one of the most frequent diseases in prostate cancer scintigraphy imaging is particularly important for the clinical diagnosis of bone metastasis Up to date minimalresearch has been conducted regarding the application of machine learning with emphasison modern efficient convolutional neural networks CNNs algorithms for the diagnosis ofprostate cancer metastasis from bone scintigraphy images The advantageous and outstanding capabilities of deep learning machine learnings groundbreaking technologicaladvancement have not yet been fully investigated regarding their application in computeraided diagnosis systems in the field of medical image analysis such as the problem of bonemetastasis classification in wholebody scans In particular CNNs are gaining great attention due to their ability to recognize complex visual patterns in the same way as human perception operates Considering all these new enhancements in the field of deep learning aset of simpler faster and more accurate CNN architectures designed for classification ofmetastatic prostate cancer in bones is explored This research study has a twofold goal tocreate and also demonstrate a set of simple but robust CNN models for automatic classification of wholebody scans in two categories malignant bone metastasis or healthy usingsolely the scans at the input level Through a meticulous exploration of CNN hyperparameter selection and finetuning the best architecture is selected with respect to classificationaccuracy Thus a CNN model with improved classification capabilities for bone metastasisdiagnosis is produced using bone scans from prostate cancer patients The achieved classification testing accuracy is whereas the average sensitivity is approximately Finally the bestperforming CNN method is compared to other popular and wellknown CNN architectures used for medical imaging like VGG16 ResNet50 GoogleNetand MobileNet The classification results show that the proposed CNNbased approach outperforms the popular CNN methods in nuclear medicine for metastatic prostate cancer diagnosis in bonesa1111111111a1111111111a1111111111a1111111111a1111111111 ACCESSCitation Papandrianos N Papageiou EAnagnostis A Papageiou K Bonemetastasis classification using whole body imagesfrom prostate cancer patients based onconvolutional neural networks application PLoSONE e0237213 101371journalpone0237213Editor Jeonghwan Gwak Korea NationalUniversity of Transportation REPUBLIC OF KOREAReceived November Accepted July Published August Copyright Papandrianos This is an access distributed under the terms ofthe Creative Commons Attribution License whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are creditedData Availability Statement The data which areanonymized bone scintigraphy images without anyfurther information are available only after requestfor research purposes Data availability Thedataset from Diagnostic Medical CenterDiagnosticoIatriki AE was used under thelicense of the Board Committee Director of theDiagnostic Medical Center DiagnosticoIatriki AE Dr Vasilios Parafestas for the current studyand is not publicly available The dataset may beavailable only under request from the Director ofPLOS ONE 101371journalpone0237213 August PLOS ONE 0cthe Diagnostic Center vparafestasyahoogr andthe relevant Doctor of Nuclear Medicine DrNikolaos Papandrianos npapandrianosuthgrnikpapandrgmailcomFunding The authors received no specificfunding for this workCompeting interests The authors have declaredthat no competing interests existBone metastasis classification using convolutional neural networks IntroductionMost common tumors such as those of the breast lung and prostate frequently metastasize tothe bone tissue and so the skeleton seems to be a site with the most significant tumor burdenin cancer patients with advanced disease Statistical analysis results have shown that of allbone metastases originate from the breast in women and from the prostate in men Theremaining emanates from thyroid lung and kidney cancers [] In the case of metastaticprostate cancer diagnosis has a significant impact on the quality of patients life [] In mostmen the metastatic prostate cancer mainly sites on the bones of the axial skeleton causingsevere lesions that can cause pain debility andor functional impairment [] As this type ofcancer has great avidity for bone and could cause painful and untreatable effects an early diagnosis is crucial for the patient Reviews on clinical evidences and diagnostic assessments ofbone metastases in men with prostate cancer can be found in []The implementation of a properly selected diagnostic imaging can reveal the number ofmetastatic foci in the skeletal system [ ] Rapid diagnosis of bone metastases can be achievedusing modern imaging techniques such as scintigraphy Positron Emission TomographyPET and wholebody Magnetic Resonance Imaging MRIThe primary imaging method in the diagnosis of metastases that offers the highest sensitivity among all imaging methods is Bone Scintigraphy BS [] Through the depictionof the entire skeleton in one medical examination nuclear doctor is able to detect bone abnormalities in areas where intensive radionuclide activity is present However low specificityseems to be the main drawback of this method as it cannot tell whether the causes of boneturnovers are different than those of metastatic origin leukaemia healing fracture etc Atthe same time PET has been recognized as an efficient method for detecting cancer cellsbased on recent technological advancements in medical imaging PET and Computed Tomography CT combination can produce highresolution images []Although PET and PETCT are the most efficient screening techniques for bone metastasisBS remains the most common imaging procedure in nuclear medicine [ ] [] Asreported in European Association of Nuclear Medicine EANM guidelines [] BS is particularly important for clinical diagnosis of metastatic cancer both in men and women At presentwhen other imaging or examination methods are unable to provide a reliable diagnosis BSimaging becomes the proper modality for making a final diagnosis of bone metastasis []To address the considerable problem of bone metastasis diagnosis artificial intelligentmethods for medical image analysis implemented with deep learning algorithms have beenadequately investigated In this direction a recent survey reveals the entire penetration of deeplearning techniques into the field of medical image analysis detection segmentation classification retrieval image generation and enhancement registration and successful application ofdeep learning to medical imaging tasks are thoroughly examined []Implementation of deep learning in medical imaging is mainly conducted by ConvolutionalNeural Networks CNNs [ ] a relatively new and powerful way to learn useful representations of images and other structured data Before the application of CNNs these featurestypically had to be created by less powerful machine learning models or even handcraftedWith the introduction of CNNs such features could be learned directly from the provideddata since they include certain preferences in their structure that make them powerful deeplearning models for image analysis [ ] Typical CNNs have a similar structure withArtificial Neural Networks ANN and consist of one or more filters ie convolutional layers followed by aggregationpooling layers in order to extract features for classification tasks[] Gradient descent and backpropagation are both used as learning algorithms the sameway they are used in a standard ANN Their main difference lies in the fact that CNNs havePLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkslayers of convolutions along with pooling layers in the beginning of their architecture Thefinal outputs are computed via fully connected layers located at the end of the network architecture []In recent years CNNs have gained wider recognition in medical image analysis domain aswell as in vision systems [ ] Due to their enormous popularity several applications ofCNNs were investigated in the field of medical image analysis Two recent review studies []and [] gather all the important and most interesting applications of deep learningThe application of CNNs in medical imaging ranges from plain radiograph CT MRI andmicroscopy images to clinical photos dermatology capsule endoscopy and visual recognition[] In addition a CNN was investigated in [] that regards automatic detection of tuberculosis on chest radiographs while in [] a brain tumor segmentation in magnetic resonanceimages was made possible with the use of a CNN More examples of CNNs successful application in medical domain include automated cardiac diagnosis [] detection of lesions and prediction of treatment response by PET [ ] as well as dynamic contrast agentenhancedcomputed tomography where CNN showed high diagnostic performance in the differentiation of liver masses [] Furthermore CNNs have shown outstanding performance in radiology and molecular imaging []Some models with major impact in the context of deep learning and medical image processing were introduced in several s the Unet model for biomedical data semanticsegmentation [] the GoogLeNet model introducing the inception module [] the ResNetmodel introducing the residual learning building block for extremely deep convolutional networks [] and also Deeplab which deals with the inclusion of many convolutional layersatrous for semantic segmentation of images in deep convolutional neural networks []In medical image analysis the most widely used CNN methods are the following i AlexNet [] This network has a quite deep architecture similar to GoogLeNet by YannLeCun [] incorporates more filters per layer and includes stacked convolutional layersIt attaches ReLU activations after every convolutional and fullyconnected layer ii ZFNet [] Being a rather slight modification of AlexNet this network won the ILSVRCcompetition iii VGGNet16 [ ] It consists of convolutional layers having avery uniform architecture similar to AlexNet iv GoogleNet [] It is a convolutional neuralnetwork with a standard stacked convolutional layer having one or more fully connected layers called inception modules able to extract various levels of features on the same time vResNet [] It is another efficient CNN architecture that introduced the identityshortcut connection to solve the notorious problem of the vanishing gradients of the deepnetworks vi DenseNet [] Being another important CNN architecture DenseNetoffers the main advantage of alleviating the gradient vanishment problem with the direct connection of all the layers Related work in nuclear medical imaging for metastatic prostate cancerdiagnosis in bonesReviewing the relevant literature for diagnosis of bone metastasis using bone scintigraphyscans the authors notice that only a couple of previous works have been adequately conductedfor metastatic prostate cancer classification using CNNs while the others are devoted to ANNsand their application in ComputerAided Diagnosis CAD These works have investigated theuse of Bone Scan Index BSI which was introduced to assess the bone scanning process andestimate the extent of bone metastasis [ ] Specifically it serves as a clinical quantitativeand reproducible parameter that can measure metastatic prostate cancer bone involvement[] The software developed for the BSIbased ANN approach was EXINI bone EXINIPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksDiagnostics AB Lund Sweden and afterwards a revised version of this software calledBONENAVI FUJIFILM Toyama Chemical Co Ltd Tokyo Japan was engineered using alarge number of Japanese multicenter training databases []The first of the reported studies was devoted to the development of a classification algorithm based on CNNs for bone scintigraphy image analysis [] It was carried out as a masterthesis in Lund University and is focused mainly on classification problems without considering any identification and segmentation tasks The used dataset was provided by Exini Diagnostics AB in the form of image patches of already found hotspots The process in whichhotspots were segmented cropped and collected from bone scans was implemented using asoftware developed at Exini BSI was calculated for wholebody bone scans by segmenting theentire skeleton from the background in both the anterior and posterior views Due to timeframe restrictions only the hotspots found in the spine have been used to train the CNN sincethey were considered to be the easiest to classify A shape model based on a mean shape of several normal wholebody scans was fitted to the skeleton using an image analysis algorithmcalled Morphon registration The outcomes of the aforementioned thesis [] have shown thatthe calculated accuracy of the validation set was whereas the calculated accuracy of thetesting set was The second study explored CNNs for classification of prostate cancer metastases usingbone scan images [] The tasks of this master project appeared to have a significant potentialon classifying bone scan images obtained by Exini Diagnostics AB too including BSI The twotasks were defined as i classifying anterior posterior pose and ii classifying metastatic nonmetastatic hotspots The outcome of this study is that the trained models produce highlyaccurate results in both tasks and they outperform other methods for all tested body regions inthe case of metastatic nonmetastatic hotspots classification The evaluation indicator of thearea under Receiver Operating Characteristic ROC score was equal to which is significantly higher than the respective ROC of obtained by methods reported in the literature for the same test setThe remaining research that concerns the same imaging modality BS is devoted to theintroduction of CAD systems with the use of ANN and other Machine Learning ML methods for bone metastasis detection in bone scintigraphy images Sadik were the first todevelop an automated CAD system as a clinical quality assurance tool for the interpretation ofbone scans [] This bone scan CAD software was trained to interpret bone scans usingtraining databases that consist of bone scans from European patients who have the desiredimage interpretation metastatic disease or not The results showed a sensitivity of at aspecificity of These works result in certain outcomes that refer to the development of atotally automated computerassisted diagnosis system that can identify metastases after examining bone scans applying multilayer perceptron ANN techniques involving a small databaseof wholebody bone scans patients The highest sensitivity that was achieved from all thestudies and accomplished during this thesis was approximately []Horikoshi compared the diagnostic accuracy of two CAD systems one based on aEuropean and another on a Japanese training database in a group of bone scans from Japanesepatients [] The Japanese CAD software showed a higher specificity and accuracy comparedto the European Comparing the sensitivities the Japanese CAD software achieved whereas the European CAD software reached []In another study conducted by Tokuda the diagnostic capability of a completely automated CAD system which detects metastases in the images of bone scans by focusing on twodifferent patterns was investigated [] The first pattern was devoted to the detection ofmetastases per region the second one detects metastases per patient The investigated systemwas called BONENAVI version The produced results have shown that the new CADPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networkssystem is able to decrease the number of false positive findings which depends on the primarylesion of cancerIn Aslantas proposed CADBOSS as a fully automated diagnosis system forbone metastases detections using wholebody images [] The proposed CAD system combines an active contour segmentation algorithm for hotspots detection an advanced methodof image gridding to extract certain characteristics of metastatic regions as well as an ANNclassifier for identifying possible metastases The calculated accuracy sensitivity and specificity of CADBOSS were and respectively outperforming other state of theart CAD systemsAdditionally ML methods have been exploited and applied in CAD systems for bonemetastasis detection in bone scintigraphy images A parallelepiped classification method wasspecifically deployed in [] to assist physicians in bone metastases detection of cancer Decision Trees DT and Support Vector Machines SVM were exploited for predicting skeletalrelated events in cancer patients with bone metastases achieving higher accuracies with asmaller number of variables than the number of variables used in Linear Regression LR MLtechniques can be also used to build accurate models to predict skeletalrelated events in cancer patients with bone metastasis providing an overall classification accuracy of ± []As far as PET and PETCT imaging techniques in nuclear medicine are concerned thereare some recent and prominent studies that apply the advantageous features of CNNs In []deep learning has been applied for classification of benign and malignant bone lesions in [F]NaF PETCT images The authors in this work followed the VGG19 architecture for theirnetwork by employing à convolutional layers followed by fully connected layers and asoftmax layer as final activation The ImageNet database of natural images was further used topretrain the networks weights In this way the network first is trained on general image features and later is tuned using the lesion images and the physicians scores Taking a closer lookat the results it can be concluded that networks performance was improved when it wastrained to differentiate between definitely benign score and definitely malignantscore lesions The values of prediction metrics were and concerningthe accuracy sensitivity specificity and positive predictive value respectivelyAlso a CNNbased system was examined in a recent retrospective study which included sequential patients who underwent wholebody FDG PETCT [] The main purpose ofthe study was to detect malignant findings in FDG PETCT examinations while a neural network model equivalent to ResNet24 was built Additionally GradCAM was employed toidentify the part of the image on which the neural network used the largest information Thefindings of the study showed that GradCAM reasonably highlighted the area of malignantuptake allowing physicians to make a diagnosis The same research team recently developed a CNNbased system that predicts the location of malignant uptake and furtherevaluated predictions accuracy [] A network model with configuration equivalent toResNet24 was used to classify wholebody FDG PET imagesIn the research work [] a simple CNNbased system that predicts patient sex from FDGPETCT images was proposed Specifically consecutive patients have participated in thestudy and underwent wholebody FDG PETCT The CNN system was used for classifyingthese patients by sex Another CNNbased diagnosis system for wholebody FDG PETCT wasdeveloped in [] that predicts whether physicians further diagnosis is required or not Athorough analysis of the results shows that the accuracy considering images of patients presenting malignant uptake and images of equivocal was ± and ± respectivelyThe task of segmentation with the use of deep learning models in skeletal scintigraphyimages has been discussed in more research studies For example in [] the authors followedPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksdifferent approaches to convert convolutional neural networks designed for classificationtasks into powerful pixelwise predictors Moreover in [] a deeplearning based segmentation method was developed using prostatespecific membrane antigen PSMA PET imagesand showed significant promise towards automated delineation and quantification of prostatecancer lesions However this research domain that involves bone scintigraphy segmentationwith the inclusion of advanced deep neural networks has not been yet well establishedAlthough bone scintigraphy is extremely important for the diagnosis of metastatic cancer itis clear that a small number of research works have been carried out which presented onlysome preliminary results while the advantageous and outstanding capabilities of CNNs havenot been fully investigated Reviewing the relevant literature it appears that CNNs have notbeen sufficiently applied for the diagnosis of prostate cancer metastasis from whole bodyimages Aim and contribution of this research workCNN is an efficient deep learning network architecture that has recently found great applicability in the medical domain It has shown excellent performance on medical image applications including bone scintigraphy and nuclear medical imaging and can offer a positiveimpact on diagnosis tasksNowadays the main challenge in bone scintigraphy as being one of the most sensitiveimaging methods in nuclear medicine is to build an algorithm that automatically identifieswhether a patient is suffering from bone metastasis or not based on patients whole bodyscans It is of utmost importance that the algorithm needs to be extremely accurate due to thefact that patients lives could be at stake Deep learning algorithms whose potential lies in thefact that they can improve the accuracy of cancer screening have been recently investigatedfor nuclear medical imaging analysis Recent studies in BS and PET have shown that a deeplearningbased system can perform as well as nuclear physicians do in standalone mode andimprove physicians performance in support mode Even though BS is extremely importantfor the diagnosis of metastatic cancer there is currently no research paper regarding the diagnosis of prostate cancer metastasis from whole body scan images that applies robust and moreaccurate deep CNNsThis research study investigates the application of a deep learning CNN to classify bonemetastasis using whole body images of men who were initially diagnosed with prostate cancerThe proposed method employs different CNNbased architectures with data normalizationdata augmentation and shuffling in the preprocessing phase In the training phase the backpropagation technique has been used for updating the weights as part of the optimization process Finally the network architecture is finetuned and the configuration that offers the bestperformance is selected to train the CNN modelThe scope of the current work entails the following two main components First this studyintroduces the CNN method into the diagnosis of bone metastasis disease based on wholebody images In the second phase the paper deals with the improvement of the existing CNNmethod in terms of both network architecture and hyperparameter optimization Thedeployed process seemingly improves the diagnostic effect of the deep learning method making it more efficient compared to other benchmark and wellknown CNN methods such asResNet50 VGG16 GoogleNET Xception and MobileNet [ ]The innovations and contributions of this paper are well summarized as follows¢ The development and demonstration of a simple fast robust CNNbased classification toolfor the identification of bone metastasis in prostate cancer patients from wholebody scansPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networks¢ The rigorous CNN hyperparameter exploration for determining the most appropriatearchitecture for an enhanced classification performance¢ A comparative experimental analysis utilizing popular image classification CNN architectures like ResNet50 VGG16 GoogleNET Xception and MobileNetThis paper is structured in the following fashion Section presents the material and methods related to this research study Section presents the proposed solution based on CNNmethod for bone metastasis diagnosis for prostate cancer patients using whole body imagesSection shows the results of exploration analysis of CNNs in Red Green and Blue RGBmode for different parameters and configurations thus providing the best CNN model forthis case study Furthermore all the performed experiments with representative results aregathered in section whereas section provides a thorough discussion on analysis of resultsSection concludes the paper and outlines future steps Materials and methods Patients and imagesA retrospective review of consecutive whole body scintigraphy images from differentmale patients who visited Nuclear Medicine Department of Diagnostic Medical Center Diagnostico AE in Larisa Greece from June to June was performed The selection criterion was prostate cancer patients who had undergone wholebody scintigraphy because ofsuspected bone metastatic diseaseDue to the fact that whole body scan images contain some artifacts and other nonrelatedto bone uptake such as urine contamination and medical accessories ie urinary catheters[] as well as the frequent visible site of radiopharmaceutical injection [] a preprocessingapproach was accomplished to remove these artifacts and nonosseous uptake from the original images This preprocessing method was accomplished by a nuclear medicine physicianbefore the use of the dataset in the proposed classification approachThe initial dataset of images contained not only bone metastasis presence and absencepatients cases suffering from prostate cancer but also degenerative lesions [] Due to thisfact as well as aiming to cope with a twoclass classification problem in this study a preselection process concerning images of healthy and malignant patients was accomplished In specific out of consecutive wholebody scintigraphy images of men from differentpatients were selected and diagnosed accordingly by a nuclear medicine specialist with years of experience in bone scan interpretation Out of bone scan images bone scansconcern male patients with bone metastasis and male patients without bone metastasis Anuclear medicine physician classified all the patient cases into categories metastasis absentand metastasis present which was used as a gold standard see Fig The metastatic imageswere confirmed by further examinations performed by CTMRI Wholebody scintigraphy Bone scansA Siemens gamma camera Symbia S series SPECT System by dedicated workstation and software Syngo VE32B with two heads and low energy highresolution collimators was used forpatients scanning The speed of scanning was cmmin with no pixel zooming Two types ofradionuclide were used for bone scintigraphy 99mTcHDP TechneScan1 and 99TcMDPPoltechMDP 5mg Whole body scintigraphy was acquired approximately hours after intravenous injection of MBq of radiopharmaceutical agent depending on the patientbody type The common intravenous injection was MBq of radiopharmaceutical agentPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Image samples in the dataset Label a Metastasis is present or b absent101371journalpone0237213g001In total planar bone scan images from patients with known prostate cancer werereviewed retrospectively The wholebody field was used to record anterior and posteriorviews digitally with resolution à pixels Images represent counts of detected gammadecays in each spatial unit with 16bit grayscale depthThe image data acquired were originally in DICOM files a commonly used protocol forstorage and communication in hospitals These image data were extracted from these DICOMfiles to create new images in JPEGformat instead A novel dataset of bone scintigraphyimages containing men patients suffering from prostate cancer with metastasis present andmetastasis absent two distinct classes of healthy and malignant cases was prepared for experimentation This dataset consisting of wholebody scans is available for research use afterrequestThis study was approved by the Board Committee Director of the Diagnostic Medical Center DiagnosticoIatriki AE and the requirement to obtain informed consent was waived bythe Director of the Diagnostic Center due to its retrospective nature All procedures in thisstudy were in accordance with the Declaration of Helsinki MethodologyThe problem of classifying bone metastasis images is a complex procedure and so effectivemachine learning methods need to be exploited to cope with this diagnosis task Deep learningmethods such as CNNs are applied in order to train a classifier to distinguish images of prostate cancer patients with bone metastasis and metastasis absent on healthy patients The effective CNN method for bone metastasis classification proposed in this paper includes threeprocessing steps data preprocessing for the collected scan data normalization a trainingphase for CNN learning and validation and testing which includes the evaluation of the classification results as illustrated in Fig The proposed methodology is thoroughly presented inthe following sections Data preprocessing Step Load images to RGB The original images were saved inRGB mode All images are stored in a respective folder before loaded into the computer memory for CNN training In each image a suitable prefix was defined according to the patientscategory for example malignant_ and healthy_ Next a small script was set up in order toPLOS ONE 101371journalpone0237213 August PLOS ONE 0cBone metastasis classification using convolutional neural networksFig Flowchart of the proposed CNN methodology101371journalpone0237213g002assign a numerical value as label to each image according to its prefix In our case the value was assigned for malignant_ prefixes whereas the value for healthy_ prefixesStep Data normalization It is common to follow a normalization process feature scalingin most machine learning algorithms [] The minmax normalization processnormalizes thedataset values within the to ensure that all feature data are in the same scale for trainingand testing The data normalization process also assists the convergence of the backpropagation algorithmStep Data shuffle To avoid or eliminate unbiased sampling in machine learning anappropriate shuffling method is needed to be defined More specifically a randomnumbergenerator is used to reorder the images An image sample chosen randomly is meant to be animpartial representation of the total images An unbiased random sample is important formachine learning to provide reliable conclusions In this study Pythons randomshufflemethod is used for dataset shufflingStep Data augmentation Data augmentation is used as a method to artificially increasethe diversity of training data by a large margin by manipulating the existing data instead ofcreating new Data augmentation techniques such as cropping padding and horizontal flipping are commonly used to train large neural networks [] In this research the number ofimages used for learning processes was followed by an image augmentation processing such asrotation enlargementreduction range and flip Note that the original images used for the testwere not subjected to such an augmentation processStep Data split The dataset was split into three sections a training portion a validationportion that would allow the training process to improve and a testing holdout portionwhich is part of the dataset that is completely hidden from the training process The first datasplit takes place by removing of the total dataset and saving for later use as testing Theremaining of the dataset is then split again into an ratio where the small po
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"Ovarian cancer is the second most common gynecologic cancer with high mortality rate andgenerally diagnosed in advanced stages The 5year diseasefree survival is below MicroRNAs subset of thenoncoding RNA molecules regulate the translation in post transcriptional level by binding to specific mRNAs topromote or degrade the target oncogenes or tumor suppressor genes Abnormal expression of miRNAs were foundin numerous human cancer including ovarian cancer Investigating the miRNAs derived from the peripheral bloodsamples can be used as a marker in the diagnose treatment and prognosis of ovarian cancer We aimed to findbiological markers for early diagnosis of ovarian cancer by investigating BRCA1 gene mutation carrier monozygoticdiscordant twins and their high risk healthy family individuals miRNAsMethods The study was conducted on monozygotic twins discordant for ovarian cancer and the liquid biopsyexploration of miRNAs was performed on mononuclear cells that were isolated from the peripheral blood samplesThe miRNA expression profile changes in the study were found by using microarray analysis miRNA isolationprocedure performed from the lymphocyte in accordance with the kit protocol The presence and quality of theisolated miRNAs screened by electrophoresis Raw data logarithmic analysis was studied by identifying thethreshold normalization correlation mean and median values Target proteins were detected for each miRNA byusing different algorithmsResults After the comparison of monozygotic discordant twins for epithelial ovarian carcinoma upregulation of the miRNAs miR6131 miR1305 miR1973p miR3651 and downregulation of miRNAs miR3135b miR4430 miR664b5p miR7663p were found statically significantContinued on next page Correspondence hy2188istanbuledutrDepartment of Cancer Genetics Istanbul Faculty of Medicine OncologyInstitute Istanbul University Istanbul Turkey The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cTuncer Journal of Ovarian Research Page of Continued from previous pageConclusions The detected miRNAs out of miRNAs might be used in the clinic as new biological indicatorsin the diagnosis and follow up of epithelial ovarian cancer with complementary studies The miRNA expressionprofiles were identified to be statistically significant in the evaluation of ovarian cancer etiology BRCA1 mutationstatus and ovarian cancer risk in accordance with the obtained dataThere is a need for validation of the miRNAs which were particularly detected between monozygotic twins and itsassociation with ovarian cancer was emphasized in our study in wider cohorts including ovarian cancer patientsand healthy individualsKeywords Monozygotic twins miRNA expression profiles BRCA1 and BRCA2 BiomarkersBackgroundOvarian cancer is a significant cause of mortality in gynecologic cancers and one ofthe leading cause ofcancerassociated mortality [] In Turkey ovarian cancer is the 7th most common type of cancer in women inaccordance with worldwide Globocan datas showthat each year more than women are diagnosedwith ovarian cancer OC worldwide and approximately women die from it The data of Globocan for Turkey shows that annually women are diagnosed with ovarian cancer and women die fromthis malignancy The 5year survival rate was given as These data revealed that ovarian cancer is an important reason of gynecologic cancer associated mortality rate [] The epithelial ovarian cancersEOCoriginating from the ovarian surface epithelium constitutes approximately of ovarian malignancy [] Themajority of EOC patients are diagnosed in advanced stages Stage III and IV and year freesurvivalrate is below [] The standard treatment for newlydiagnosed ovarian cancer is the combination of cytoreductive surgery and platinbased chemotherapy Significant advances in radical surgery and chemotherapystrategies have improved clinical outcomes but unfortunately no progress has been made with relapse andtreatment resistance [] Ninety percent of ovarian cancer occurs sporadically in the population whereas hereditary type appears of ovarian cancer patientsBRCA1 and BRCA2 genes are the most common breastovarian cancer syndrome associated genes Both BRCA1and BRCA2 have roles in the control of the genomic stability cell cycle and apoptosis The mutations occurringin these genes result with the inability of DNA repairand therefore results in the accumulation of the mutations in the cell The rate of the breast cancer susceptibility of women with BRCA1 gene mutation until theage of years was and the rate of ovarian cancersusceptibility rate is breast cancer susceptibilitywomen with BRCA2 gene mutation until the age of years is and ovarian cancer development risk is [] Twin studies became important on genetics by theendandcentury Geneticnineteenthofthethe differentiated genesepidemiologic studies with monozygotic twins were accepted as highly useful investigation models in the pastdecades and have been used recently [] When a similarity for a disease or a quantitative feature betweenmonozygotic and dizygotic twins is compared variationsare excluded according to studies conducted in thepopulation and thereforeit is easier to identify andmake etiological differences visible via twin studies Because the affected siblings and dizygotic twins share theapproximately ofthephenotypic differences between twins are known to beassociated with the genetic variation In addition diversity may be revealed with a very limited patient population Therefore the results of the twin studies can beapplied to the population and can make valuable contributions to the genetic studies Monozygotic twins aregenetically similar and generally expected to be compatible for congenital malformations chromosomal abnormalities and Mendelian disorders There are numerousstudies conducted via discordant monozygotic twins revealing the genetic contribution [] Therefore investigating the genetic variability in monozygotic twins ishighly important and the majority of the human geneticsassociated research focus on finding genetic variability indiscordant monozygotic twins Phenotypically discordantmonozygotic twins are used as the model systems inidentification of the variable in understanding the pathogenesis of a disease The most striking study is the oneconducted with monozygotic twins in Canada and evidencing that multiple sclerosis MS was a genetic disease [] MicroRNAs are one of the subset of the noncoding RNAs generally consisting of single strand in nucleotide length not transformed to protein havingroles in post transcriptional regulation or suppression oftranslation of the target mRNAs [ ] The regulatoryroles of miRNAs were demonstrated to occur in tumorigenesis cell differentiation proliferation and apoptosis[] miRNA genes are known to locate in thechromosomal breaks This DNA breaks cause chromosomal abnormalities frequently associated with cancersusceptibility and tumor development [] The noninvasive biologicalindicators have been used for the 0cTuncer Journal of Ovarian Research Page of treatment resistance of ovarian cancer The most common of this indicators are the cancer antigen125 CA and cancer antigen153 CA153 These biological indicators can be used in the followup of thetreatment response in the diagnosed patients but cannotbe used in the early diagnosis and in differentiation ofthe malignant disease [] Therefore there is a need forspecial therapeutic agents customized for patients thatmay be used target specific therapies and in the earlydiagnosis of the ovarian cancer in identification of theefficacy of therapy and in the follow up period Thusstudies investigating the target molecules and biologicalindicators are required that will enable the early diagnosis and in the development of the better therapy optionsDifferentially synthesize miRNAs such as miR miR141 miR125b miR2223p or let7 family has beenshown in studies with ovarian cancer patients [] However the use of these miRNAs as a biomarker in ovariancancer is not yet available In order to clearly define therole of miRNAs in the pathogenesis of ovarian cancerwe planned to investigate the BRCA mutant monozygotic twins with the same genetic profile but with discordant for ovarian malignant transformation In thisstudy miRNAs which are thought to have the potential biological indicator role were studied from bloodsamples of both discordant monozygotic twins andBRCA wild type healthy siblingsMethodsPatients recruitmentThe peripheral blood lymphocytes of monozygotic twinsdiscordant for ovarian cancer and healthy individuals inthe same family were used in the study The patient diagnosed with ovarian cancer and all family members applied to the Cancer Genetics Clinic of OncologyInstitute of Istanbul University for BRCA breast cancersusceptibility gene testing were examined for BRCAgene mutation All family members in the study consisted of highrisk individuals with Hereditary Breast andOvarian Cancer HBOC syndrome and the people included in the study were given as BR codes according topatient file number The monozygotic ovarian cancer patient healthy monozygotic twin healthy sisters and niece were found to have BRCA1 gene mutation c5266dupC pGln1756Profs74 rs397507247 on exon Thepatients brother and daughter were found negative forBRCA1BRCA2 gene mutations In this study lymphocyte cells separated from peripheral blood belonging tototal of cases including younger age ovarian cancer patient and healthy monozygotic twin a patients daughter elder sisters a younger sister a nephew and a brotherwere examined by miRNA microarray method Thepedigree of the family included in the study and theirhierarchical cluster analaysis via Euclidean method isshown on Fig The study was approved by the Ethics Committee ofthe Istanbul Faculty of Medicine Following InstitutionalEthics Committee approval informed consents were obtained from all participants before enrollment into thestudy Ethics Committee Approval Number atstoredthey wereLymphocyte and miRNA isolationFicoll SigmaAldrich Darmstadt Germany density gradient was used to separate white blood cells mononuclear cells from other blood components miRNAisolation procedure was performed from the lymphocytein accordance with the kit protocol using the miRNeasyMini Kit Qiagen cat NoID The proceduresteps in accordance with the protocol are as follows μL QIAzol solution was included on the cells storedin nitrogen tank Cell fractionation was enabled by mixturing using the vortex For complete nucleoproteinfractionation °C roomtemperature for min By adding μL chloroformthey were shacked and mixed on hand The tubes wereincubated for min at °C room temperature thenwere centrifuged for min at °C and g Thesupernatant formed after centrifugation was transferredto collection tube using a pipette and was mixed usingvortex by inclusion of μL ethanol The supernatant formed after the ethanol centrifuging was transferred to collection tube was removed with a pipettewas mixed with vortex by including μL ethanol Seven hundred microliters was taken from the obtained mixture and was transferred to the RNeasyMiniElute spin colon placed on mL collection tubeThe tubes were centrifuged for s at g at °Croom temperature Seven hundred microliters RWT buffer was added to spin colons and the colons werewashed by centrifuging at g for sThe centrifuging procedure was repeated by including μL RPE buffer twice consecutively to colons The colons placed into clean tubes with mL were dried bycentrifuging min in maximum speed The colonsplaced in mL sterile tubes were included μL distilled water by centrifuging at g in min and themiRNAs were collectThe quality control of the miRNAsThe presence and quality of the isolated miRNAs werescreened by electrophoresis at V on Agarose gelThen the purity and concentrations of the miRNAswere measured on Thermo Scientific NanoDrop spectrophotometer NanoDrop Technologies Wilmington DE USA device The miRNA purity for each persondevicemeasurement result were obtained with the comparisonaccordance withthe NanoDropin 0cTuncer Journal of Ovarian Research Page of Fig The pedigree of the family included in the study and their hierarchical cluster analysis Legend The pedigree of the family and using thecorrelations between samples plotted a dendrogram for sample grouped by Hierarchical clustering Euclidean distance Complete Linkage BR Healthy Brother BRCA1 negative nonBRCA1 mutation carrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR Healthy Monozygotic twin BRCA1 positive BRCA1 mutation carrier BR Monozygotic twindiagnosed with ovarian cancer BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrierof the measurements at spectrophotometrically at nm and nm wave lengths The measurement rates at nm wave lengths is a sign of quality of the purity of the samples therefore the samples in the idealvalue interval of and for RNA measurementswere included in the study The purity of miRNAs wereevaluated using a Bioanalyser device BioanalyserAgilent Technologies Santa Clara CA USA AgilentRNA Nano Kit Agilent Technologies Santa ClaraCA USA for confirming whether the miRNAs were appropriate and in adequate level for microarray analysisThe evaluated sample concentrations and results wereanalyzed The samples with RNA concentrations between ngμL and rRNA rate over and RNA integrity number values between and were evaluated asthe appropriate samples for array study 0cTuncer Journal of Ovarian Research Page of Microarray trial protocolMicroarray protocol was performed by preparing theSpikein solution sample marking hybridization sampledephosphorylation sample denaturation sample ligationhybridization of the samples slide loading preparationof the hybridization unit and elution and scanning ofslides The slide scanning procedure was performedusing the Agilent Microarray Scanner Agilent Microarray Scanner with Surescan High Resolution Technology Agilent Technologies Santa Clara CA USAdevice The scanning procedure of the slides were performed on SurePrint G3 Human miRNA MicroarrayRelease 8x60K Agilent Inc Santa Clara CA platform and using the Agilent Technologies G2600D scanning protocol The analysis of the TIFF Tagged ImageFile Format extensioned files obtained after scanningprocedure was performed using the Agilent Feature Extraction v11011 programThe success levels of stages developed in all experiment process with this analysis program the quality ofthe levels the process were monitored and evaluatedThen Bioinformatic Analysis procedure was performedData analysisRaw data logarithmic analysis was studied by identifyingthe threshold normalization correlation mean and median values Then the miRNAs demonstrating differentexpression profile among the samples were filteredUsing the Fold change rates and independent twosample T test the possible difference between the compared groups were evaluated All evaluations were performed to enable the cutoff values as the foldchangerates FC ¥ and pvalue Hierarchical clusteranalysis was performed using the Euclidean method Fig and Complete Linkage cluster method The control ofthe experimental errors and the detection of the erroneous finding rate were identified using the HochbergmethodBioinformatic analysisTarget proteins were detected for each miRNA by usingtwo algorithms Targetscan71 httpswwwtargetscanvert_71 and MirdbV5 httpsmirdbmiRDBThe targeted genes thought for each miRNA wereconfirmed by also both algorithms and the miRNAtarget relations were also experimentally confirmed mirTarbase70httpsmirtarbasembcnctuedutwphpindexphp databaseComparison groupsIn the study miRNA analysis was performed at the genome level withwithout mutation in cases withwithoutovarian cancer The miRNA data was evaluated by comparing different groups in order to investigate the effectof BRCA mutation in ovarian malignancy developmentand determine the miRNAs that can be important in theovarian cancer pathogenesis In Group the monozygotic twins discordant for ovarian cancer were comparedin order to find the effects of miRNAs in the formationof ovarian cancer In Group the family members withBRCA1 mutation were compared with family memberswithout BRCA1 mutation to identify the changes ofmiRNAs expression levels according to BRCA positivityIn Group the monozygotic ovarian cancer patient withBRCA1 mutation carrier and the other healthy familymembers with mutation carrier were compared for investigate the effects of both ovarian cancer developmentand BRCA positivity on miRNAs expression level InGroup all family members were compared with ovarian cancer monozygotic twin in order to find the miRNAs that might be important in the predisposition ofovarian cancer The comparison groups also showed inTable ResultsWe identified differentially expressed comparisonof miRNAs between the groups The raw data obtainedafter experimental studies were filtered before the comparisons between the groups The upregulated or downregulated miRNAs expression levels more than foldFC and smaller than the p value p wereconsidered in evaluation and the comparisons betweenthe groups were performed based on these values Allthese comparisons were evaluated for ovarian cancer etiology BRCA1 mutation carriage and the ovarian cancerrisk Hierarchical cluster analysis of the expression of miRNAs represents sharp separations of upregulatedyellow from downregulated blue in Fig miRNAs total of miRNAs were found statistically different after the comparison of phenotypicallydiscordant monozygotic twin siblings The miRNAsmiR1273 g3p miR1305 miR1973p miR3651 miR and miR92a3p expressions were found to haveupregulated and the other miRNAs let7i 5p miR125a5p miR15b5p miR22 3p miR3135b miRTable Comparison groups and cases in the groupsCaseGroup BR1639ControlBR1447Group BR1639BR1447BR1547BR2030BR1546BR1861BR1850BR2028Group BR1639Group BR1639BR1447BR1447BR1547BR2030BR1546BR1861BR1547BR2030BR1546BR1861BR1850BR2028 0cTuncer Journal of Ovarian Research Page of Fig Hierarchical cluster analysis of the expression of miRNAs Legend BR Healthy Brother BRCA1 negative nonBRCA1 mutationcarrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR HealthyMonozygotic twin BRCA1 positive BR Monozygotic twin diagnosed with ovarian cancer BRCA1 positive BR Healthy Sister BRCA1positive BR Healthy Sister BRCA1 positive BR Healthy Sister BRCA1 positive The miRNAs that may be effective in the etiology ofovarian cancer were identified after the comparison of monozygotic twins who were phenotypically discordant for ovarian cancer diagnosis ingroup 320d miR3423p miR4430 miR451a miR664b5pand miR7663p expressions were found to have downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are given in Table and Fig Different miRNAs level were compared between group in order to determine the effect of BRCA1 gene mutation Group was consisted after the comparison of theBRCA1 gene mutation carrier family members and individuals not carrying BRCA12 gene mutation accordingto miRNAs expression profiles After the comparisonsdownregulated and upregulated miRNAsrelated toBRCA1 gene mutation carrier were determined The expression of a total of miRNAs including miR4449miR46533p miR4865p miR5739 miR6165 andmiR 8743p associated with the BRCA1 gene mutationcarrying were upregulated and the expression of a totalof miRNAs including miR1263p miR320a miR320b miR320c miR320d miR320e miR3243p miR miR miR4428 miR4516 miR4741 miR miR564 miR6089 miR68695p miR68915pmiR71075p and miR78473p were found downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are shown in Table and Fig AftercomparisonDifferent miRNA levels were compared between group in order to determine the relation with ovarian cancerdevelopment and BRCA positivity Group consists ofcomparison of miRNAs of BRCA1 positive ovarian cancer patient with all other BRCA1 positive healthy individualsanddownregulated miRNAs related to mutation carriage inBRCA1 gene and epithelial ovarian cancer etiology weredetermined The expression of miRNAs includingmiR1260a miR1260b miR165p miR175p miR181b5p miR 26b5p miR4281 miR4286 miR5100miR68403p miR71145p miR7975 and miR7977were found to have upregulated and the expression of miRNAs including miR12255p miR1423p miR 26a5p miR miR29a3p miR30d5p miR3196upregulated 0cTuncer Journal of Ovarian Research Page of Table Ovarian cancer etiology related upregulated and downregulated miRNAs and target proteins in monozygotic twinsmiRNAsSequence of miRNAmiRNAStatusTarget genesFold change FCvaluesACCACUGCACUCCAGCCUGAGUpregulatedZNF138 TMEM239 BMP3UUUUCAACUCUAAUGGGAGAGAUpregulatedPTPN4 PRKAA1 PAPD7FRAT2 DEPDC1 FBXO41UUCACCACCUUCUCCACCCAGCUpregulatedCD82 PMAIP1 MTHFD1 CHECK1 AGO1 CASP10CAUAGCCCGGUCGCUGGUACAUGA UpregulatedGGCUGGUCAGAUGGGAGUGUpregulatedRACGAP1 OLA1 TEX261PTGS1 NFIC ZNF200PAGR1 IGF2BP1 CACNG8UAUUGCACUUGUCCCGGCCUGUUGAGGUAGUAGUUUGUGCUGUUUCCCUGAGACCCUUUAACCUGUGA Downregulated ERBB3 CDKN1A TP53 ERBB2 EGFR STAT3MYCSTAT3 PTEN ATM NOTCH2 CDH1 NFKB1UpregulatedDownregulated TLR4 BMP4 EIF2C1 NEUROG1 SOCS1 IGF1VEGFAmiR1273 g3pmiR1305miR1973pmiR3651miR6131miR92a3plet7i5pmiR125a5pmiR15b5pUAGCAGCACAUCAUGGUUUACAmiR223pAAGCUGCCAGUUGAAGAACUGUDownregulated BCL2 VEGFA CCND1 CCNE1 CDK1 CDK4 CDK6 E2F3MAPK1Downregulated CDKN1A WNT1 ERBB3 MYCBP HMGB1 E2F2 PTENPOTED SOD2miR3135bmiR320dmiR3423pmiR4430miR451amiR664b5pGGCUGGAGCGAGUGCAGUGGUGAAAAGCUGGGUUGAGAGGAUCUCACACAGAAAUCGCACCCGUAGGCUGGAGUGAGCGGAGAAACCGUUACCAUUACUGAGUUUGGGCUAAGGGAGAUGAUUGGGUADownregulated BIRC5 ABL2 MAPK1 MYCNDownregulated DCTN5 SYNCRIP FBXO28Downregulated GEMIN4 DNMT1 ID4 SREBF1SREBF2 BMP7Downregulated ZNF485ABL2 MAPK1 MSH5 PTENDownregulated CPNE3 RAB5A IL6R AKT1 MMP2Downregulated CD55MSN RHOBTB3 PLAG1miR7663pACUCCAGCCCCACAGCCUCAGCDownregulated COX1 MAPK1 NF2 RAD51 STK4 STK24 VEGFCFig The upregulated and downregulated miRNAs and foldchanges associated with ovarian cancer etiology in monozygotic twins 0cTuncer Journal of Ovarian Research Page of Table BRCA1 gene mutation positivity related upregulated and downregulated miRNAs and target molecules An additional tablefile shows this in more detail [see Additional file ]miRNAsSequence of miRNATarget genesmiRNAStatusFold changeFC valuesmiR4449miR46533pmiR4865pmiR5739miR6165miR8743pmiR1263pmiR320amiR320bmiR320cmiR320dmiR320emiR3243pmiR3656miR4284miR4428miR miR4741miR484miR564miR miR6869 5pmiR68915pmiR71075pmiR7847 3pCGUCCCGGGGCUGCGCGAGGCAUpregulatedZFHX3UGGAGUUAAGGGUUGCUUGGAGAUpregulatedATG2A CREBL2 MAT2A FRS2 TMED4 UBN2UCCUGUACUGAGCUGCCCCGAGGCGGAGAGAGAAUGGGGAGCCAGCAGGAGGUGAGGGGAGCUGCCCUGGCCCGAGGGACCGAUpregulatedUpregulatedUpregulatedUpregulatedOLFM4CD40ARHGAP5 IGF1R DOCK3 CADM1DLX6CD207CHIC1PPL2A PLXDC1PER1TFAP2AFADS1 AMER1LUZP1 COX6B1HDAC1 AQP3 STAT3 CDK9UCGUACCGUGAGUAAUAAUGCGDownregulatedTOM1 CRK VEGFA SOX2 TWF1 PITPNC1 IGFBP2 KRASAAAAGCUGGGUUGAGAGGGCGADownregulatedMCL1 BANP ITGB3 BMI1 NRP1 NFATC3 TRPC5AAAAGCUGGGUUGAGAGGGCAADownregulatedCDK6DCTN5SYNCRIPARF1 BCL9L ZNF600AAAAGCUGGGUUGAGAGGGUAAAAGCUGGGUUGAGAGGAAAAGCUGGGUUGAGAAGGACUGCCCCAGGUGCUGCUGGGGCGGGUGCGGGGGUGGDownregulatedDownregulatedSYNCRIPFBXO28SMARCC NPM3DCTN5 SYNCRIP FBXO28DCTN5 NPM3 ZNF275 DDX19A NCAPD2 TXNL1DownregulatedDownregulated WNT9B CREBBP DVL2 WNT2BDownregulatedMRPL12 LSP1 MNT PRDM2ZNF770 CECR1GGGCUCACAUCACCCCAUDownregulatedBCL2L11RBBP5HNRNPA1 ZNF264 TRIB3 CRTAPCAAGGAGACGGGAACAUGGAGCDownregulatedMSL1MAPRE3MYH14CASP2 CCND2 CDK14TP63GGGAGAAGGGUCGGGGCDownregulatedSTAT3M6PRGPR137CCCND2 CCNT1 CDKN1A SCOC TP53CGGGCUGUCCGGAGGGGUCGGCUDownregulatedDDX39BMAPK1 ZBTB39 HMGA1UCAGGCUCAGUCCCCUCCCGAUDownregulatedFIS1 PAGR1 ZEB1 SLC11A2SMAD2 ANAPC7 TBRG1AGGCACGGUGUCAGCAGGCDownregulatedGID4 CNBP E2F3 RCAN3 AKT2 APPL1 SLC1A2 GPR155GGAGGCCGGGGUGGGGCGGGGCGGDownregulatedNKX2 TPT1 KCTD5 BBX SGCD CDH7 CCNB1GUGAGUAGUGGCGCGCGGCGGCDownregulatedTUBB2AMAPK1NRBF2 WEE1HMGA2 MAPK1 STAG2UAAGGAGGGGGAUGAGGGGDownregulatedCHD4 CD207 DDX6 CHRDL1CCND2 TP63UCGGCCUGGGGAGGAGGAAGGGDownregulatedVAV3 CASP16 CCND1 CASP16 MAPK14CGUGGAGGACGAGGAGGAGGCDownregulatedHAVCR1 POTED DNAJC10 SOD2 M6PR CDK19miR3423p miR3665 miR3960 miR4466 miR4530miR46873p miR47875p miR4943p miR50015pmiR50065p miR5787 miR6068 miR6087 miR miR6090 miR6124 miR6125 miR638 miR65105p miR68005p miR7704 miR8063 and miR were found to have downregulated After bioinformatic analysis a total of upregulated and downregulated statistically significant miRNAs and the targetmolecules are given in Table and Fig For identifying the ovarian cancer predisposition allfamily members were compared with ovarian cancermonozygotic twins in group The upregulated ordownregulated miRNAs in association with the epithelialovarian cancer risk were identified after the comparisonThe expression of the miRNAs consisting of let7a5plet7b5p miR181a5p miR1973p miR215pmiR2233p miR23a3p miR27a3p miR36533pmiR4255p miR572 miR5745p miR6127 and miR were detected to be upregulated The expression ofthe miRNAs consisting of let7i5p miR 125a5pmiR15b5p miR1505p miR22 3p miR3283pmiR4430 miR451a miR46975p miR664b5p andmiR7663p were detected to have downregulated Atotal of upregulated and downregulated statisticallysignificant miRNAs and the target molecules are givenin Table and Fig DiscussionWomen are diagnosed with ovarian cancer at an advanced stage due to limited number of biologicalmarkers for ovarian cancer patients Although existingovarian cancer biomarkers cancer antigen125 and cancer antigen153 CA125 CA15 are sensitive in thefollowup of diagnosed gynecological cancers they have 0cTuncer Journal of Ovarian Research Page of Fig The upregulated and downregulated miRNAs and foldchanges associated with BRCA1 gene mutation positivityofearlysensitivityin the diagnosislessstagegynecological cancers and separation of malignant tumorformations from benign formations [] Therefore tounderstand the underlying mechanisms of ovarian cancer and to explore targeting drugs and to improve newtreatment protocols for ovarian malignancy revealingsignificant genetic changes is necessary The genetic andepidemiologic studies conducted on monozygotic twinsare known to provide accurate and direct informationabout the gene and environment interaction with thedisease occurrence mechanism [] The changes in genesthat result in the occurrence of tumors such as miRNAexpression level among the monozygotic twins providesinformation on the etiology of disease and may have arole as a biological indicator in identifying the early stagedisease and in the follow up of the prognosis We aimedto identify the noninvasive biological markers that maybe used in the early diagnosis of ovarian cancer throughinvestigating the miRNAs in the peripheral blood ofmonozygotic twin siblings discordant for ovarian cancerwith the miRNA molecules of the other healthy members in the family Thus that may cause less bias thanthe controls to be selected from the population Ninetynine different miRNA molecules presented in the studywere detected after the comparison of monozygotic twinsiblings who were discordant for ovarian cancer andwith the other healthy individuals Seventeen differentmiRNAs were found that could be used for detectingearly diagnosis and prognosis of ovarian cancer betweenthe monozygotic twin siblings who were discordant forovarian cancer in our study The association between out of miRNAs and ovarian carcinoma is beingreported for the first time in this study Due to the highnumber of newly detected miRNAs in our study the discussion and comparison were only made between thecandidate miRNAs Although miR1973p miR1305miR6131 miR3651 miR3135b miR4430 miR664b5p and miR7663p have not been shown to be associated with ovarian cancer in literature but limited number of studies have suggested the association with othercancersWang found the elevated level of miR1973pinthe same way as we do The upregulated miR1973p expression level was shown to promote the cellular invasion and metastasis in bladder cancer in that studyResearchers reported that LINC00312 gene was responsible for invasion and metastasis mechanisms and thisgene inhibited the cellular migration and invasion bysuppressing the miR1973p expression Similar resultswere detected in thyroid cancer in the study of Liu et al[ ] Jin reported that increased expressionlevel of miR1305 caused pluripotent stem cells to accelerate the cell cycle G1S transfer in addition to causingthe cellular differentiation with the increased miR1305expression [] The expression levels ofreducedmiRNA125a5p and let7i5p found in the scope of ourstudy have been shown to parallel with other studies inthe literature Langhe suggested thatlet7i5pmight be described as a diagnostic indicator in ovariancancer [] The miRNA125a5p expression was upregulated to inhibit the cancer proliferation and migrationin the in vitro study of Qin in human cervical carcinomas [] and miR125a5p upregulated expressionlevel was demonstrated to inhibit the cervical cancer 0cTuncer Journal of Ovarian Research Page of Table BRCA1 mutation carriage and epithelial ovarian cancer etiology related upregulated and downregulated miRNAs targetmolecules An additional table file shows this in more detail [see Additional file ]miRNAsSequence of miRNAmiRNAStatusTarget genesAUCCCACCUCUGCCACCAAUCCCACCACUGCCACCAUUAGCAGCACGUAAAUAUUGGCGUpregulatedUpregulatedUpregulatedCAAAGUGCUUACAGUGCAGGUAGUpregulatedPSAT1UNC13A RPS27 BRD7SFRP1 DKK2 SMAD4 PSAT1UNC13A RPS27CCNE1 ARL2 BCL2 HMGA1 CDK6 CCND1VEGFA RECK PRDM4TGFBR2 PTEN CDKN1A BCL2L11E2F1TP53STAT3AACAUUCAUUGCUGUCGGUGGGUUpregulatedTCL1A TIMP3 PLAG1 BCL2RNF2VSNL1 ATMFold changeFC valuesmiR1260amiR1260bmiR16 5pmiR175pmiR181b5pmiR26b5pmiR4281miR4286miR5100UUCAAGUAAUUCAGGAUAGGUUpregulatedGGGUCCCGGGGAGGGGGGACCCCACUCCUGGUACCUpregulatedUpregulatedUUCAGAUCCCAGCGGUGCCUCUUpregulatedPTGS2 EPHA2 CHORDC1 EZH2CCNE1ABCA1 GATA4NCDN CDKN1A BCL3LDLR ZNF354B NSD1 RABGAP1TAOK1 MKNK2COX10 DEK KCNN3 RAB11FIP1DYNLT1 NOTCH2SLFN12L CTC1 GXYLT2GDE1FADS1PER1 ATG9AmiR68403pGCCCAGGACUUUGUGCGGGGUGUpregulatedmiR71145pUCUGUGGAGUGGGGUGCCUGUUpregulatedM6PR HNRNPUL1 SHMT1 ZNF529ACVR2B PAICS TAF8miR7975miR7977AUCCUAGUCACGGCACCAUUCCCAGCCAACGCACCAUpregulatedUpregulatedmiR12255pGUGGGUACGGCCCAGUGGGGGGDownregulatedKBTBD8GULP1 CASZ1 RAD51HSPA1B ZNF703 TMEM185BSF3B3COX6B1 CCDC9 CDH7ORC4 ODF2L MTRNR2L7PSMG2 MTRNR2L3miR1423pmiR26a5pmiR2861miR29a3pmiR30d5pmiR3196miR 3423pmiR3665miR3960miR4466miR4530miR46873pmiR47875pmiR4943pmiR50015pmiR50065pmiR miR6068miR6087miR6088miR6090miR6124UGUAGUGUUUCCUACUUUAUGGADownregulatedARNTLTGFBR1 RAC1 ROCK2 CCNT2 TAB2 PTPN23UUCAAGUAAUCCAGGAUAGGCUDownregulatedEZH2RB1ADAM17 HMGA2CCND2 CPEB3 DNMT3BGGGGCCUGGCGGUGGGCGGUAGCACCAUCUGAAAUCGGUUAUGUAAACAUCCCCGAC
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" pharmacology and toxicology laboratory csirinstitute of himalayan bioresource technology preproof 0c preproofinfection f0b7 systemic oxidative stress and inflammation are significant outcomes of sarscov2 highlights f0b7 activated gsk3 following sarscov2 infection provoke the oxidative stress and inflammation in the host f0b7 gsk3 phosphorylates nucleocapsid protein of sarscov2 and helps in disease progression f0b7 inhibition of gsk3 can be a suitable target in curbing of covid19 pandemic 0cwith the host defence mechanism by the help of gsk3 protein the virally infected cells show the coronavirus disease covid19 outbreak caused by severe acute respiratory syndrome coronavirus sarscov2 had turned out to be highly pathogenic and transmittable researchers throughout the globe are still struggling to understand this strain's aggressiveness in search of putative therapies for its control crosstalk between oxidative stress and systemic inflammation seems to support the progression of the infection glycogen synthase kinase3 gsk3 is a conserved serinethreonine kinase that mainly participates in cell proliferation development stress and inflammation in humans nucleocapsid protein of sarscov2 is an important structural protein responsible for viral replication and interferes activated gsk3 protein that degrades the nuclear factor erythroid 2related factor nrf2 protein resulting in excessive oxidative stress activated gsk3 also modulates crebdna activity phosphorylates nfκb and degrades catenin thus provokes systemic inflammation preproofinteraction between these two pathophysiological events oxidative stress and inflammation enhance mucous secretion coagulation cascade and hypoxia which ultimately leads to multiple ans failure resulting in the death of the infected patient the present review aims to highlight the pathogenic role of gsk3 in viral replication initiation of oxidative stress and inflammation during sarscov2 infection the review also summarizes the potential gsk3 pathway modulators as putative therapeutic interventions in combating the covid19 keywords covid19 gsk3 nfκb nucleocapsid protein oxidative stress sarscovpandemic list of abbreviations ace2 angiotensinconverting enzyme ad alzheimers disease adp adenosine diphosphate aiibb3 glycoprotein iibiiia ards acute respiratory distress syndrome 0care antioxidant response elements asc apoptosisassociated specklike protein containing a card atp adenosine triphosphate balf bronchoalveolar lavage bzip basic leucine zipper cats catalase cbp creb binding protein covid19 coronavirus disease creb camp response elementbinding protein cul3 cullin gpx glutathione peroxidase gsh intracellular glutathione gsk3 glycogen synthase kinase3 damp death associated molecular pattern gcsf granulocyte colony stimulating factor hcv hepatitis c virus hdac3 histone deacetylase ho1 heme oxygenase1 ifnΠinterferongamma preproofnfκb nuclear factorκb nlrp3 nodlike receptors protein mcp1 monocyte chemoattractant peptide mip1α macrophage inflammatory protein 1α myd88 myeloid differentiation primary response nadph nicotinamide adenine dinucleotide phosphate hydrogen ikk ikb kinase il6 interleukin iraks interleukin il 1rassociated kinase iκb inhibitor of kappa b keap1 kelchlike ech associated protein licl lithium chloride nlrp3 nucleotidebinding domain nodlike receptor protein nox nadph oxidase nprotein nucleocapsid protein nrf2 nuclear factor erythroid 2related factor 0cntd nterminal domain o superoxide anion o2 oxygen molecule oxpls oxidized phospholipids pamp pathogen associated molecular pattern par proteaseactivated receptors pd parkinsons disease pedv porcine epidemic diarrhea virus ros reactive oxygen species sarscov2 severe acute respiratory syndrome coronavirus tak1 transforming growth factor tgfactivated kinase tf tissue factor tirap tirdomaincontaining adaptor protein sgmrna sub genomic messenger rna sods superoxide dismutase ppr pattern recognition receptor psgl pselectin glycoprotein ligand1 rigi retinoic acidinducible gene i preproofvwf von willebrand factor xo xanthine oxidase xor xanthine oxidoreductase tlr3 toll like receptor3 tnf tumor necrosis factor tnfr tumor necrosis factor receptor tnfα tumour necrosis factoralpha traf6 tumour necrosis factor receptor associated factor trs transcription regulating sequence introduction in late december wuhan china got attention worldwide after getting several patients diagnosed with pneumonia following a viral infection on 11th february the pathogenic strain of the virus was taxonomically designated as severe respiratory syndrome coronavirus sarscov2 by the international committee on taxonomy of viruses ictv the 0cassociated diseased condition was termed covid19 by the world health anization who the who announced sarscov2 virus infection a pandemic as it infected nearly million persons and engulfed more than worldwide sarscov2 is a member of coronaviruses consisting of kb singlestranded positivesense rna as genetic material it shows genetic similarity between another human coronavirus ie sarscov while similarity with bat coronavirus ratg1 and shares a high similarity index with pangolin coronavirus respiratory droplets are the primary source of viral transmission either through nasopharyngeal or oral route dry cough and high fever are the sarscov virusassociated respiratory disease replication within the host cell in disease progression the present review provides an inthe infected cells however in the case of sarscov2 infection aggressive inflammation significant symptoms observed in patients within days following viral infection the disease pathophysiology of covid19 also shows a close resemblance with previous reported and oxidative stress help in viral replication and damage the airway epithelium cell that results in acute respiratory distress syndrome ards which makes the condition worst glycogen synthase kinase3 gsk3 is a serinethreonine evolutionary conserved central molecule that the majority of respiratory viral infections are associated with the recruitment of immune cells the release of proinflammatory cytokines oxidative stress and finally phagocytosis of mainly participates in cell proliferation migration development apoptosis and immune regulation acquired and innate activation of gsk3 is associated with suppression of host immunity and inhibition of antioxidant response it is also supporting viral genome preproofdepth knowledge of oxidative stress inflammation and viral replication related to gsk3 during sarscov2 infection further the review highlights the gsk3 pathway modulators' gsk3 is a versatile serinethreonine kinase that regulates glycogen metabolism it consists of two isoforms gsk3α and gsk3 encoded by two separate genes both the isoforms share sequence similarity between kinase domains despite they never compensate for each other's' loss of function gsk3 has two prime functional domains a substratebinding domain which acquires substrates to gsk3 while the other kinase domain is responsible for phosphorylation of the substrate the nterminal region of gsk3 contains atp binding domain whereas the cterminal region consists of a large conserved activation loop responsible for the enzyme's full activation activation of gsk3 depends on the siteputative role as therapeutic interventions in combating the covid19 pandemic gsk3 structure 0cspecific phosphorylation that is controlled by various kinases gsk3 prefers prephosphorylate substrate by recognizing consequence sequences stxxxphosphost on substrate gsk3 is also involved in wntcatenin and sonic hedgehog cell signalling pathways mediating in cell proliferation differentiation maturation and cell adhesion transcription factors cjun creb stat3 cebpα nfat myc nfκb and p53 are the major substrate of gsk3 that can manipulate the expression of several other genes impaired activity of gsk3 has recognized in several clinical conditions such as metabolic disorders cancers alzheimer's disease ad parkinson's disease pd bipolar disorders and various other neurodegenerative diseases sarscov2 infection and inflammation covid19 patients' systemic cytokine profile shows a close resemblance with cytokine release syndrome characterized by macrophage activation an elevated level of cytokines like tumour necrosis factoralpha tnfα interleukin6 il6 and interferongamma ifnΠfurther elevated levels of these cytokines trigger ards characterized by a low level of oxygen in the severity of symptoms and death in sarscov2 infected patients depends on the viral infection and is greatly affected by the aggressive behaviour of the host immune system blood and difficulty in breathing leading to the death of the infected patients previous data on sarscov demonstrated that the virus predominately affects the endothelium cells of preproofin counterdefence the virus encodes numerous immunesuppressive proteins that help employs the same host receptor angiotensinconverting enzyme ace2 for infection like sarscov indicating that both the viruses target the same set of cells for infection the as an antagonist of interferon signalling interruptions in interferon signalling happened at various stages preventing the recognition of viral rna through pattern recognition receptor expression of the ace2 receptor is reduced in the lungs following sarscov infection disrupting the reninangiotensin system that affects fluidelectrolyte balance blood pressure it to evade from host immune response and helps in replication similarly to counter such problem sarscov2 evolves with numerous structural and nonstructural proteins that act the airway alveoli vascular system and macrophages in the pulmonary an sarscov2 increases the vascular permeability and inflammation in the airway ppr inhibiting the synthesis of type i interferon protein via interrupting the tolllike receptor1 tlr1 and retinoic acidinducible gene i rigi signalling disturbing stat signalling and initiating the host mrna degradation and interrupting host translation machinery fig1 0cat the time of replication cytopathic viruses including sarscov2 show a massive death and injury of the infected epithelial and endothelial cells triggering the excessive release of cytokines and chemokines in addition to this inflammationinduced cell deathpyroptosis also observed in sarscov2 patients that further provoke the systemic inflammatory response pyroptosis signalling proceeds via nodlike receptors protein nlrp3 present on the cell membrane activate caspase1 through asc apoptosisassociated specklike protein containing a caspase recruitment domain adaptor protein activated caspase1 further triggers the synthesis of proinflammatory cytokines such as il1 and il6 fig1 these cytokines further attract the other immune cells mostly tlymphocytes and monocytes at the site of infection bronchoalveolar lavage balf fluid from the sever lymphocyte and immune cells' requirement at the site of infection in most of the patients these recruited cells clear the infection recedes the inflammatory response and leads to recovery however some patients show cytokine storms because of an imbalance in the population of monocytederived fcn1 macrophage in addition to these responses sever cases of sarscov2 infection also disclose a significant expansion in the population of proinflammatory monocytes cd14 and cd16 in the peripheral blood as compared to mild covid19 patients showed ccl2 and ccl7 chemokines which require the recruitment of ccr2 monocytes further balf analysis also revealed a highly inflammatory around of sarscov2 infected patients show lymphopeniainfiltration of preproofsevere hospitalized covid19 patients' blood plasma exhibits a higher level of alleviation in the t cell population which is more noticeable in severe cases the level of helper t cell cd4 cytotoxic t cell cd8 and regulatory t cell were below the average level in severe cases of covid19 as compared to mild cases cd8 t cells directly attack and kill the virusinfected cells while cd4 participates in the production of cytokines to recruit other immune cells at the same time regulatory t cell maintains the normal immune homeostasis along with inhibition of proliferation the proinflammatory activity of maximum immune cascade that further inflames the lungs sarscov2 infected patients also show cases lymphocytes natural killer cells and bcells fig1 granulocyte colonystimulating factor gcsf il2 il6 il10 monocyte chemoattractant peptide mcp1 macrophage inflammatory protein 1α mip1α and tnfα the blood plasma of the infected patients shows a significantly higher level of il6 in severe cases compared to mild or nonsevere cases which further contributes to macrophage activation syndrome pulmonary infiltrationbased assessment in ards patients also revealed that a 0cmore significant portion of lung injury is associated with a higher level of il6 in peripheral blood all of this evidences suggest that sarscov2 infection is responsible for dysregulation of the host immune system with the abnormal synthesis of cytokines chemokines and a decrease in the level of lymphocytes that ultimately leads to cytokine storm responsible of multian failure role of nuclear factorκb in disease progression nuclear factorκb nfκb is the leading player that responds immediately following the a pathogenic stimulus provoked by a bacteria or a virus invasion exposure of mitogen proinflammatory cytokines growth factors and stress activates ikb kinase ikk which relb and crel are grouped in firstclass characterized by the presence of transactivation domain while nfkb1 p50 and nfkb2 p52 belongs to the second group that is devoid of transcriptionalmodulation activity so both the classes of proteins need to be heterodimerized with each other to perform their functions under normal physiological conditions rela and p50 the heterodimer's predominant form is inactivated in the cytoplasm by ikb protein pathogen's invasion by promoting inflammation controlling cell proliferation and survival nfκb is a heterodimeric transcription factor that belongs to the rel protein family there are 05rel proteins present in mammalian cells that further divided into two classes rela p65 preproofmembranelike tolllike receptor tlr pathogen associated molecular pattern pamp and death associated molecular pattern damp are inflammatory stimulating molecules suggested that the nucleocapsid protein of sarscov directly interacts with nfκb translocate it to the nucleus and finally upregulates il6 gene expression ample of shreds of evidence is there that shows sarscov directly or indirectly activates nfκb protein excessive cytokine release especially il6 plays a crucial role in sarscov2 infection and further progression of pathogenic conditions nfκb is a transcription factor that controls the expression of proinflammatory genes responsible for the cytokine storm a study following infection nfκb also activated by receptors present on the cell surface further phosphorylates and degrades ikb protein via ubiquitination process released by virusinfected cells which act as ligands for tlr subsequently activating nfκb protein via myd88dependent pathway oxidative stress is another important factor responsible for cytokine storm generation via crosstalk between nuclear factor erythroid related factor nrf2 and nfκb pathway nfκb suggested as a negative regulator of nrf2 driven genes either by recruiting histone deacetylase hdac3 which promote local histone hypoacetylation or deprive the cbp creb binding protein fig1 0c sarscov2 infection and oxidative stress oxygen is a crucial molecule in the aerobic system to maintain normal life processes under normal cellular conditions the oxygen molecule utilized to generate chemical energy in the form of atp in a very tight and controlled manner the oxygen molecule combustion generates a small number of reactive oxygen species ros which utilized for usual cell signalling cascades ros are oxygen molecules with an unpaired electron that behaves as free radicals and reactive metabolites several ros forms were discovered so far such as peroxidase oxygenfree radicals nitrogen oxide and singlet oxygen molecules generally ros associated cellular damage is processed via sophisticated antioxidant machinery involving both enzymatic catalase cats superoxide dismutase sods and glutathione peroxidase gpx and nonenzymatic glutathione and nicotinamide adenine dinucleotide phosphate mitochondrial dna get degraded under the influence of oxidative stress subsequently hydrogen [nadph] mechanisms in normal physiological conditions the antioxidant systems can work simultaneously to combat the exceeded levels of ros however in a pathological state ros overwhelmed the antioxidant mechanism and generated oxidative stress in cells all the crucial cellular components such as proteins lipids nuclear and the available literature of clinical and preclinical experiments proposed that oxidative preproofensures the clearance of the virus but due to imbalanced host immune system they also start to release excessive cytokines that further aggravate to cytokine storm the recruited phagocytic cell participates in ros generation along with inflammatory response nicotinamide adenine dinucleotide phosphate oxidases nadph oxidase and xanthine cov2 infection activates the host airway epithelium and alveolar macrophage further releasing cytokines to attract another immune cell from the blood neutrophils and monocyte that further differentiate into macrophage at the site of injury recruitment of these cells burst is another prompting factor for mortality following sarscov infection sarstriggering the process of cell death oxidase xo are the two wellknown enzymes responsible for oxidative stress in respiratory viral infections nadph oxidase nox is an evolutionary conserved membranebounded enzyme complex that catalyzes the molecular oxygen into superoxide humans nadph oxidase family consists of members nox15 duox1 and duox2 its cterminal region comprises nadph binding site flavin adenine dinucleotide binding domain while the nterminal region consists of transmembrane α helical domains with four conserved hemebinding sites nox2 is predominantly expressed in the recruited 0cphagocytes neutrophils and macrophages at the viral infection site and contributes to oxidative stress a study reported that alveolar macrophage depended oxidative stress is responsible for acute lung injury progression following h5n1 viral infection in mice mostly via oxidized phospholipid and superoxide however the same pathological events reduced following the suppression of p47phox a regulatory subunit of nox2 in a study influenza a virusinfected nox2y mice showed reduced oxidative stress improved alveoli epithelium condition less production of superoxide and reduced airway inflammation compared to wild type mice fig inflammation xor is converted into xo by oxidation of cysteine amino acid or calciumin superoxide synthesis via nox2 enzyme complex xanthine oxidase xo is another dependent proteolysis xo shows more affinity toward molecular oxygen resulting in the transfer of a univalent and divalent electron to oxygen that further generates superoxide and ros generating enzyme that participates in oxidative stress following respiratory viral infection in the mammalian system this enzyme is existing in interchangeable form between hydrogen peroxide respectively fig2 in vitro rhino viruss infection in primary bronchial and a549 respiratory epithelial cell lines decreased the intracellular glutathione xo to xanthine oxidoreductase xor xor is predominantly distributed in healthy tissues and reduces nad to nadh by utilizing electron form substrate while during similarly ex vivo influenza a virusinfected alveolar macrophage exhibited an increase preproofdecreased superoxide generation thus revealed that xo also participates in oxidative stress during infection in vivo analysis also revealed that xo is the main contributor to and serum analysis however allopurinol and chemical modified superoxide dismutase decreased the oxidative stress and mortality rate this evidences revealed that xo also superoxide synthesis during a respiratory viral infection mouse infected with influenza viral showed a higher mortality rate which found to be associated with xo and superoxide in balf gsh level leading to oxidative stress via enhanced superoxide production serine protease inhibitor or xo inhibitor oxypurinol treatment enhanced the intracellular levels of gsh and participates in the viral associated disease progression via oxidative stress a part of these activated phagocyte releases prooxidant mediators such as tnf and il1 which further enhances the oxidative stress in host cells during viral infection tnf binds with the complex ii of the mitochondrial respiratory chain hampering oxidative phosphorylation via restricting electrons transport as a result the electron transport chain becomes leakier and lastly it enhances superoxide production tnf also helps in detachment of nfκb protein from ikb complex resulting in suppression of antioxidant gene expression via binding to their 0c1keap1 binding domain keap1 is a cystine rich and cytoplasmic protein whose npromoter region following translocation from the cytoplasm to the nucleus fig during stress condition neutrophils also release lactoferrin along with lysosomal protein under the influence of il1 which further binds to iron and start to accumulate in the reticuloendothelial system when an ironbinding threshold reached superoxide ions combine with free iron to generate hydroxyl radicals via fenton reaction and enhances oxidative stress nrf2 a key regulator of antioxidant genes nrf2 is the main transcription factor that plays an important role to overcome oxidative stress it is a basic leucine zipper bzip protein that belongs to the cap n collar family of transcription factors nrf2 consist of highly conserved functional domain termed as neh nrf2ech homologies neh1 is a leucine zipper domain through which nrf2 interact with other transcription factors whereas neh2 is the kelchlike ech associated protein however during stress conditions nrf2 detached from the keap1 protein translocate to the terminal domain binds with cul3dependent e3 ubiquitin ligase complex while cterminal domain binds with nrf2 protein under normal physiological conditions keap1 protein nucleus heterodimerize with small musculoaponeurotic fibrosarcoma mafs proteins and finally initiate or supress the transcription of genes that consists of electrophile response elements ere or antioxidant response elements are in their promoters nrf2 regulates preproofbecause of its highly vascular nature and indirect contact with environmental oxidant which had already proven in numerous of respiratory disease it was found that lungspecific nrf2 conditional knockout rodents showed pulmonary protective behaviour in respiratory disorders more than genes expression belonging to oxidative stress inflammation autophagy metabolism and excretion the pulmonary system is more exposed to oxidative stress ubiquitinates the nrf2 resulting in its proteasomal degradation infection systemic oxidative stress and inflammation linked thrombus formation in sarscovabnormal coagulation a higher level of ddimers and low platelet count are the signs of poor prognosis and significant reasons for multiple an failure and death in severe cases of covid19 microthrombus had reported in the lungs the heart the kidneys and the brain of covid19 patients cytokine storm induces aberrant coagulation by expressing the tissue factor tf pathway tf is a member of cytokine receptor 0csuperfamily and type i integral membrane glycoprotein which is highly abundant in the vasculature subendothelium especially in the brain lungs gut skin as well as in the monocytes in response to proinflammatory cytokines especially il6 the expression of tf is upregulated in the monocytes and the perivascular cells resulting in tf exposure to circulation the exposed portion of tf forms a complex with circulating factor vii thus enhance its catalytic activity that further activates downstream circulating factors such as ix and x activated factor x participates in the transformation of prothrombin into thrombin that finally leads to the formation of blood clots by conversion of fibrinogen into fibrin fig main consequences of the cytokine storm that also provokes thrombin production via proteasediphosphate adp thromboxane a2 translocate cell adhesion molecule pselectin and cd40 ligand on the surface of platelet along with activation of the integrin aiibb3 receptor the released thromboxane a2 and adp trigger activation of neighbouring platelets via activated receptors par signalling pathway par is a unique gprotein coupled cell surface receptor that carries its ligand and remains inactive until unmasked by proteolytic cleavage by the tffactorviia complex thrombin mediated par activated platelets undergo a morphological transformation release platelet activators such as serotonin adenosine thromboxane receptor and p2y12 respectively activated aiibb3 on platelets' surface binds with von willebrand factor vwf and fibrinogen that contributes to platelet aggregation platelet activation different proinflammatory events and fibrin clot formation are the preproofalong with cytokine storm oxidized phospholipids oxpls also participate in the recognition receptors in an experimental model of acute lung injury oxpls triggered cytokine storm release via tlr4triftraf6nfκb pathway il6 further promoted tf platelets during viral infection adherent leukocyteplatelets interaction provides positive feedback to amplify the overall inflammatory response and procoagulation events these activated endothelium cells following par signalling also exposes cell adhesion molecules eselectin pselectin icam1 and vcam1 and expresses monocyte chemo coagulation cascade via the tlr4 receptors present on the monocytes and endothelium cells attractant proteini that facilitates recruitment adhesion and migration of leukocytes and events are prothrombotic which further contributes to blood clot formation fig oxpls concerned as pams patterns that are recognized by numerous conserved patternexpression on monocytes and activated the endothelium cell to express monocyte adherent protein for their requirement which finally participated in inflammatory events thrombotic complications can be reduced in preexisting metabolic and cardiovascular disorders in covid19 patients by interfering with the oxpls activated monocyte or 0cwhich consists of domains including the nterminal domain ntddomain cterminal domain ctddomain and linker region lkrdomain nterminal domain enriched with endothelial cell additionally during inflammation the natural anticoagulant pathways such as tf pathway inhibitors or antithrombin are nearly diminished subsequently facilitating coagulation cascade gsk3 and sarscov2 infection the virus has to undergo many complex processes that are tightly regulated to infect a host cell it begins with viral genomic rna entry into the host cytosol transcription and finally budding off as viral progeny these viral progenies are similar to their parent in morphology and function that consists of structural proteins spike s protein envelop e protein matrix m protein and nucleocapsid n protein the n protein of severe acute respiratory syndrome coronavirus is the most abundant protein existing in an infected host cell among all be responsible for nuclear localization signal the cterminal domain of the n protein is also responsible for protein dimerization both the domains of n protein ie domain and protein sequencing also revealed that n protein is highly conserved among the species preproofinterestingly to perform such activity nprotein should recognize the viral genomic nucleocapsid protection of viral genome timely replication and proper transmission is the capsid's primary function nprotein also inhibits host cell proliferation and cytokines by rna associate with it and finally oligomerize by selfassociation to form capsid or terminal domain mapped between and amino acids is enriched with lysin thought to arginine motif responsible for the multimerization of the nprotein and predicted as a hot spot region for phosphorylation in brief nprotein divided into three main domains that play diverse functions during different stages of the virus life cycle nprotein is a type of capsid protein whose primary function is to pack the virus's genomic rna into the protective positive charge amino acids which is responsible for binding with viral rna whereas cother proteins covering domain are linked to each other through linker region domain that consists of serineinteracting with elongation factors 1α resulting in a halt of translation mechanism moreover the nprotein of sarscov also hijacks the host innate immune system for the progress of new viral progeny and associated disease development posttranslational phosphorylation of the virus nprotein is essential for their activity which results in an increased affinity of nprotein toward virus rna rather than nonviral rna gsk3 found 0cto be an essential kinase responsible for the phosphorylation of nprotein on the serine residue in linkerregion fig sarscov infected veroe6 cells showed an reduction in viral titer and cytopathic effects following the treatment of gsk3 inhibitor kenpaullone and lithium chloride thus suggested phosphorylation by this kinase be strongly linked with the viral replication several subgenomic mrnas synthesized due to discontinued transcription mechanisms during the coronavirus replication which encodes major structural proteins transcription regulating sequence trs responsible for the discontinuous transcription process exists in front of each gene body trs and after the leader sequence leader trs templateswitching events happen via base pairing between the body trs and leader trs to synthesize the discontinuous minusstranded rnas discontinuous nested plusstrand this discontinuous transcription mechanism tightly controlled for the successful compilation participate in discontinues to a continuous process of virus replication moreover of the virus life cycle among all the structural proteins nprotein tightly regulates the discontinued transcription mechanism as the synthesis of subgenomic mrna is reduced subgenomic mrna transcribed from the previously generated minusstranded rnas phosphorylation of nprotein at the serinearginine motif also inhibits the tra
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" gastric neoplasms containing neuroendocrine carcinoma nec components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure nec and mixed tumors containing neccomponents we aimed to investigate whether there is a distinct difference in overall survival os between gastricneoplasms containing nec components and gastric adenocarcinomamethods surgically resected gastric neoplasms containing nec components n and gastricadenocarcinomas n from january to december at peking university cancer hospital wereretrospectively analysed patients were categorized into a surgical group and a neoadjuvant group and adjustedusing propensity score matching in the two groups gastric neoplasms containing nec components were dividedinto pure nec and mixed tumors with less than ghminen between and ghminen andmore than ghminen neuroendocrine carcinoma components os was compared between thesegroups and the gastric adenocarcinoma groupresults the os of gastric neoplasms containing neuroendocrine nec components was poorer than that of gastricadenocarcinomas in the surgical group regardless of whether the percentage of neuroendocrine cancercomponents was less than between and more than or cox multivariable regressionanalysis suggested that tumor category neoplasms containing nec components or gastric adenocarcinoma wasan independent risk factor for prognosis interestingly among patients receiving neoadjuvant therapy thedifference was not significantcontinued on next page correspondence buzhaodecjcrcn jijiafuhscpkueducn jiahui chen anqiang wang and ke ji contributed equally to this workdepartment of gastrointestinal surgery key laboratory of carcinogenesisand translational research ministry of education peking university cancerhospital institute no fucheng road haidian district beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchen bmc cancer page of continued from previous pages gastric neoplasms containing any proportion of nec components had poorer overall survival thangastric adenocarcinoma in patients treated with surgery directly indicating that these neoplasms are moremalignant than gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference inoverall survival was not significant which was in sharp contrast with the results of the surgery group suggestingthat neoadjuvant therapy may have a good effect in the treatment of these neoplasmskeywords neuroendocrine carcinoma gastric adenocarcinoma overall survival gastric neoplasms containing neuroendocrine carcinomanec components are a heterogeneous subgroup ofgastric cancer with highly aggressive behavior and poorprognosis and include pure necs and mixed tumorscontaining nec components every yearthere areapproximately million new cases of gastric cancerworldwide and gastric neoplasms containing nec components account for approximately of thesecases [ ] given the low incidence there is little comprehensive basic and clinical research to systematicallyguide the treatment of these gastric neoplasms makingthe prognosis of these tumors unsatisfactory []according to the world health anizationwho digestive neuroendocrine tumor classificationneuroendocrine neoplasm nen can be divided intothree categories based on ki67 levels and mitotic counts hpf grade g1 ki67 mitoses grade g2 ki67 mitoses grade g3ki67 mitoses meanwhile the americanjoint committee on cancer ajcc defines highly differentiated nen as a neuroendocrine tumor net and thepoorly differentiated nen as a neuroendocrine carcinoma nec based on the degree of tumor cell differentiation generally g1 g2 and rare welldifferentiated g3nens belong to the nets while poorly differentiatedg3 nens belong to necs[ ] gastric mixedneuroendocrinenonneuroendocrineneoplasm gminen is a special type of gastric nen that is definedas containing more than of both neuroendocrineand nonneuroendocrine components accountingfor approximately of all gnens and of gastricneuroendocrine carcinomas gnecs [] for thosemixed tumors with less than or more than neuroendocrine carcinoma components there is no uniform definition consideringthe heterogeneity ofminen and the malignancy degree of the different components in the tumor la rosa [ ] proposeddividing minen into three categories highgradeintermediategrade and lowgrade highgrade minenconsists of nec and carcinomaadenoma intermediategrade mimen consists of net and carcinoma and lowgrade minen consists of net and adenoma thereforein this study gastric highgrade mixed neuroendocrinenonneuroendocrine neoplasm ghminen was defined as gastric cancer containing more than of bothneuroendocrineadenocarcinomacomponentscarcinomaandgenerally the prognosis of mixed tumors is largely determined by the most malignant component kim found that gnec has shorter progressionfree survival pfs than gastric adenocarcinoma huang found that the prognosis of patients with more than of neuroendocrine cancer components is significantly poorer than that of patients with less than components all of these studies provide evidence thattumors containing neuroendocrine cancer componentsmay contribute to a worse prognosis therefore wehypothesized that a mixed tumor containing neuroendocrine carcinoma components would have a worse prognosis than pure adenocarcinoma alone we sought tofind studies on the overall survival os comparison between ghminen and gastric adenocarcinoma butfailed thus we think that a study of the comparison ofthe os of ghminen and gastric adenocarcinoma willprovide a valuable supplement to current research on ghminen to overcome the bias caused by the differences between the covariates in the comparison we usedpropensity score matching psm to match importantfactors such as age gender tumor location tumor sizepathological staging and adjuvant chemotherapy between the two groups making the research results morereliablemethodspatient selectionwe retrospectively collected patients diagnosed withgastric nens and underwent radical resection at pekinguniversity cancer hospital beijing from january to december the inclusion criteria were as follows pathologically confirmed pure nec or tumorcontaining nec components no other tumors werediagnosed before the operation complete clinicopathological information and survival information thatcould be obtained through followup patients diagnosedwith cm1 or ct4b before surgery or died from perioperative complications were excluded from the study 0cchen bmc cancer page of patients with gastric adenocarcinomas undergoing radical surgery were randomly selected for psm analysesperformed the chisquared test and mannwhitney utest were used to further verify the matching resultsfollowupwe followed the patients at least twice a year serumtumor markers test gastroscope and computed tomography ct scans were used to reexamine patients aftersurgery depending on the patients status magneticresonance imaging mri and positron emission tomography computed tomography petct were alsoconsidered for patients who cannot regularly visit ourcenter for postoperative examination we use telephonefollowup to obtain survival informationdiagnosis and classificationwe reevaluated the diagnosis and classification of ghminen mixed tumors with less than or morethan neuroendocrine carcinoma components werealso included in this study which were defined as ghminen and ghminenrespectively atumor consisting of nec is defined as pure necall neuroendocrine tumors were identified diagnosedand classified by two independent pathologists in accordance with the who classification of tumors neuroendocrine components were identified byhistological features and immunohistochemical specificity marks such as synaptophysin syn chromogranina cga and neuro cell adhesion molecule cd56 orncam the tumor staging described in the study wasbased on the ajcc 8th edition tnm staging guidelines all possible disagreements were discussed in ourstudy groupdefinition of variables and groupsin this study patients were divided into a surgical groupand a neoadjuvant group based on whether they had received neoadjuvant therapy before surgery patients inthe surgery group were assessed by the ptnm stagingsystem while patients in the neoadjuvanttreatmentgroup were assessed by the yptnm staging system osrefers to the time from surgery to the last followup thetime of death or the end ofloss offollowup or other cause of deathfollowup egpropensity score matchingto accurately compare the prognosis of ghminenand gastric adenocarcinoma we employed psm to balance the differences between the two groups psm wasperformed through the pamatching plugin in spss software logistic regression models were used toestimate propensity scores based on gender age tumorlocation tumor size and pathological staging given a caliper width nearest neighbor matching wasstatistical analysisall statistical analyses were performed using spss statisticalsoftware ibm united states the chisquared test and mannwhitney u test were used forstatistical analysis of categorical variables and continuous variables respectively kaplanmeier method wasused for the comparison of os the logrank test wasused to compare survival rates multivariable cox proportional hazards models were used to identify predictors of survival outcome p was regarded as thethreshold of significanceresultspatient selection and psm resultsbetween and among the patients treated atthe gastrointestinal cancer center of peking universitycancer hospital a total of patients with gastric neoplasms containing nec components met the inclusioncriteria for the study including cases of pure necand cases of mixedtype of these patients a total of patients received neoadjuvant therapy nec ghminen ghminen ghminen while the remaining patients receivedsurgery directly nec ghminen ghminen ghminen there were aninsufficient number of patients in group ghminen group to conduct effective statistical analysisso we combined the ghminen group with thenec group for further analysis we also randomly selected patients with gastric adenocarcinoma whounderwent radical surgery among them patientsreceived neoadjuvant therapy and the remaining patients were treated with surgery directly fig immunohistochemical specificity markers were utilizedto identify the neuroendocrine components fig 2asyn was expressed in almost all neoplasms containingnec components while the positive rates ofcga and cd56 were much lower and respectively no significant difference in the positiverate of syn and cga was observed between pure nec ghminen ghminen and ghminenfig 2b c only the positive rate of cd56 was found tobe higher in the pure nec group than that in the ghminen group fig 2dtherefore priorto os comparison psm wasperformed to ensure that there were no significant differences in patient gender age tumor location tumorsize pathological staging and adjuvant chemotherapybetween the two groups 0cchen bmc cancer page of fig flow chart of patient enrolmentcomparison of os between all patients with neccomponents and patients with gastric adenocarcinoma inthe surgical group and neoadjuvant groupbefore psm we compared the survival curves between all patients with nec components and patientswith gastric adenocarcinoma by the kaplanmeiermethod fig apparently patients with nec components had a poorer os than those with gastricadenocarcinoma fig 3a p in the surgicalgroup in contrast no significant difference was observed between the patientsreceiving neoadjuvanttherapy fig 3b p according to the proportion of nec components patients were classifiedinto pure nec ghminen ghminenand ghminen the os was also comparedbetween patients with adenocarcinomaand thesegroups and the results were similar to the overallcomparison fig 3c dfig illustrations of immunohistochemical staining patterns in gastric neoplasms containing nec components a an overview of the expressionof syn cga and cd56 in tumors containing nec components b syn expression in different nec component groups c cga expression indifferent nec component groups d cd56 expression in different nec component groups cd56 neuro cell adhesion molecule cgachromogranin a nec neuroendocrine carcinoma syn synaptophysin pvalue 0cchen bmc cancer page of fig see legend on next page 0cchen bmc cancer page of see figure on previous pagefig comparison of os between gastric neoplasms containing nec components and gastric adenocarcinoma a os comparison betweengastric neoplasms containing nec components and gastric adenocarcinoma before psm in the surgical group b os comparison between gastricneoplasms containing nec components and gastric adenocarcinoma before psm in the neoadjuvant group c os comparison between differentnec content groups pure nec ghminen ghminen and ghminen and gastric adenocarcinoma before psm in the surgicalgroup d os comparison between the different nec content groups and gastric adenocarcinoma before psm in the neoadjuvant group e oscomparison for patients in the surgical group after psm f os comparison for patients in the neoadjuvant group after psm nec neuroendocrinecarcinoma os overall survival psm propensity score matchingbefore psm significant differences between the baseline characteristics were observed in the surgical groupand the neoadjuvant group table table to balance the clinicopathological differences between the twogroups psm was performed to ensure that there wereno significant differences in patient gender age tumorlocation tumor size pathological staging and adjuvantchemotherapy between the two groups the detailedclinicopathological characteristics before and after psmare shown in table and table as a result patients with nec components and patients with gastric adenocarcinoma were matchedin the surgical group table patients with nec components also had a poorer os than those with gastricadenocarcinoma fig 3e p multivariable analysis showed that adjuvant therapy tumor category andtnm stage werefactorstable independent prognosticto investigate whether neoadjuvant therapy had an effect on os patients with nec components and patients with gastric adenocarcinoma were matched inthe neoadjuvant group table interestingly kaplanmeier analysis showed that among patients receivingneoadjuvant therapy there was still no significant difference in os between the two groups fig 3f p comparison of os between patients with differentproportions of nec components and patients with gastricadenocarcinomato investigate whether the level of nec componentshad an effect on os in the surgical group ghminen ghminen pure nec and pure nec plus ghminen were compared with gastric adenocarcinoma after psm the results showed that even thegroup with the lowest proportion of nec componentsthe ghminen group had a poorer os thanadenocarcinoma fig 4a p as expected theghminen pure nec and pure nec plus ghminen groups each with relatively high proportionsof nec components had worse os than the gastricadenocarcinoma group fig 4bd p detailed clinical information after matching isshown in additional file tables s1s4psm was also performed in the neoadjuvant group incontrast to the results of the surgery group in the purenec group containing the highest proportion ofnec componentstill no significantdifference in os from gastric adenocarcinoma fig5d the other three groups with lower nec contentwere also notfrom gastricadenocarcinoma in terms of os fig 5ac detailedclinicopathologicaland afterpsm are shown in additional file tables s5s8characteristics beforethere wassignificantly differentdiscussionamong gastric neuroendocrine neoplasms the tumorcontaining nec components is a special type includingpure nec and mixed tumor containing nec components the incidence of these tumors is extremely lowbut they are more invasive and have a poorer prognosisthan welldifferentiated gnens [ ]received neoadjuvantin previous study kim found that in patientschemotherapywho had notprogressionfree survivalpfs of pure gnec waspoorer than that of gastric adenocarcinoma while thepfs of mixedtype tumors was not significantly differentin kimsfrom that of gastric adenocarcinoma study the mixed type was defined as net mixed withgastric cancer rather than nec net is much less malignant than nec [ ] this may be the reason whythere was no significant difference in os between mixedtype and gastric adenocarcinomas in addition mixed tumors with less than or more than of nec components were not included in that study which webelieve was a deficit of the study pfs is an important indicator for evaluating prognosis in many cases it can reflect the trend of os based on kims research resultswe regarded tumors containing nec components as awhole and found that the os of these tumors was poorerthan that of adenocarcinoma in the surgical group inthe comparison of os between mixed tumors with different proportions of nec components and gastricadenocarcinoma the results for pure nec cases wassimilar to kims while the os of mixed tumors was alsopoorer than that of gastric adenocarcinoma whether theproportion of neuroendocrine cancer components wasless than between and or more than which was not mentioned in kims study cox multivariable regression analysis showed thattumor categoryneoplasm with nec component or adenocarcinoma 0cchen bmc cancer page of table comparison of clinicopathological characteristics before and after psm in surgical grouppatient characteristicsunmatched comparisonpatients with neccomponents n p valuematched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm¥ cmtype of gastrectomytotal gastrectomydistal gastrectomy ± ± proximal gastrectomy surgical procedureopenlaparoscopict staget1t2t3t4n stagen0n1n2n3m stagem0m1ptnm stageiiiiiiiv gastricadenocarcinoman ± ± ± ± p value gastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer table comparison of clinicopathological characteristics before and after psm in neoadjuvant groupmatched comparisonpatient characteristicsunmatched comparisonpatients with neccomponents n age year mean ± sdgender malefemalebmi mean ± sdadjuvant therapyyesnotumor locationupper thirdmiddle thirdlower thirdentiretumor size cm¥ cmtype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedureopenlaparoscopict staget0t1t2t3t4n stagen0n1n2n3m stagem0m1yptnm stageiiiiiiiv ± ± gastricadenocarcinoman ± ± p valuepatients with neccomponents n ± ± page of p valuegastricadenocarcinoman ± ± bmi body mass index minen mixed neuroendocrinenonneuroendocrine neoplasm nec neuroendocrine carcinoma psm propensity score matchingpatients with nec components nec high grade minen high grade minen and high grade minen 0cchen bmc cancer page of table univariate and multivariate analyses of survival after psm in surgical grouppatient characteristicsunivariate analysishr cimultivariate analysishr cip valueage yeargendermale vs femalebmiadjuvant therapyyes vs notumor size¥ cm vs cmtumor categorycarcinoma with nec component vsgastric adenocarcinoma vstype of gastrectomytotal gastrectomydistal gastrectomyproximal gastrectomysurgical procedurelaparoscopic vs opentnm stageiiiiiiivp value tumor size and tnm staging were independent risk factors for prognosis this suggests that the prognosis ofgastric neoplasms with nec components is substantiallydifferent from that of gastric adenocarcinoma and evena small percentage of nec components can alsoimpair prognosis which challenges the current cutoffvalue of the proportion of each component that must theoretically be greater than was set in andsince who has also adopted this standard to define minen this largely avoids the overdiagnosisof minen in tumors with only focal neuroendocrinemarker expression and no corresponding morphologicalchanges in additionit also prevents clinicians fromdealing with these rare neoplasms too often withoutguidelines nevertheless it is now being questionedby an increasing number of scholars the componentsin mixed tumors are not evenly distributed for large tumorsthe randomness of biopsy and postoperativepathological sampling causes the proportion of eachcomponent to fluctuate greatly making it difficult to describe the proportion of each component precisely park compared the os between tumors with morethan nec components and gastric adenocarcinomawith or without less than nec and they found thattumors with an nec composition of more than hada worse prognosis this suggests that even a small proportion of malignant components can affect prognosis while in parks study for unknown reasons the authors did not compare the prognosis of mixed tumorswith nec components less than with gastricadenocarcinomas directly nor did they compare allneccontaining tumors as a whole with gastric adenocarcinoma which we believe was a deficit of the studyin our study we regarded tumors containing neccomponents as a whole and found that the os of thesetumors was poorer than that of adenocarcinoma in thesurgical group in addition we also found that the os ofmixed tumors with less than between and more than nec components or pure nec wasworse than that of gastric adenocarcinoma analysis ofimmunohistochemical markers show that there was nosignificant difference in the positive rate of syn and cgabetween different nec content groups only the positiverate of cd56 was found to be higher in the pure necgroup than that in the ghminen group therole of cd56 in the diagnosis of nec is still controversial however syn and cga are two wellrecognized 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec and gastric adenocarcinoma in the surgical group aoverall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparison between ghminen andgastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastric adenocarcinoma d overall survivalcomparison between pure nec alone and gastric adenocarcinomamarkers therefore from the results of immunohistochemistry we believed that there was no significantlydifference in tumors containing nec componentsstudies on the molecular mechanism of pathogenesisshow that nec components and adenocarcinoma components have similar genomic abnormalities similarlosses of heterozygosity loh and mutations at multiple loci and key oncogenes such as tp53 apc and rbgenes all these results imply that the two componentsin the mixed tumor may have a common origin and acquire biphenotypic differentiation during carcinogenesis[] moreoverin the who definition of mixedneuroendocrine and nonneuroendocrine neoplasms ofother ans ie lung and thyroid no minimumpercentage for either ingredient is established thereforewe believe that mixed tumors containing nec components are actually of the same origin have similar biological characteristics and are differentfrom gastricadenocarcinoma we propose considering mixed tumorscontaining nec components as a whole rather than defining them based on the definition for both tumorcomponents which has not been raised by other studiespreviously many studies have confirmed the efficacyof neoadjuvant chemotherapy in gastric adenocarcinoma[ ] in a retrospective study involving patientsma found that neoadjuvant chemotherapy improves the survival of patients with nec and hminenof the stomach van der veen reported that 0cchen bmc cancer page of fig comparison of os between gastric neoplasm with different proportions of nec components and gastric adenocarcinoma in theneoadjuvant group a overall survival comparison between ghminen and gastric adenocarcinoma b overall survival comparisonbetween ghminen and gastric adenocarcinoma c overall survival comparison between ghminen plus pure nec and gastricadenocarcinoma d overall survival comparison between pure nec and gastric adenocarcinomaneoadjuvant chemotherapy could not benefit the survivalof patients with mixed tumors containing nec components however because only eight patients wereincluded in the neoadjuvant group vans results arequestionable in our study among patients receivingneoadjuvanttherapy no significant difference in osbetween mixed tumor and gastric adenocarcinoma wasobserved even for the pure nec group with the highestnec contentthere was no significant differencesuggesting that neoadjuvant therapy may have a positiveeffect on these neoplasmsalthough this is only a singlecenter retrospectivestudy the sample we reported is considerable for thisrare disease which can provide new ideas for clinicaland basic research in addition we proposed treatingall gastric neoplasms containing nec components asa whole and found that neoadjuvanttherapy mayhave a good effect on these neoplasms in the futurewe will conduct more genomics studies to confirmour ideas this study also has its limitations due tothe lack of recurrence and detailed chemotherapy information we were unable to compare progressionfree survival and analyse the effects of differentchemotherapy regimens as a retrospective study despite our performing psm in advance selection biascannot be completely avoided in addition since theexact proportion of each componentin the mixedtumor could not be obtained we could not determine 0cchen bmc cancer page of whether there is a cutoff value for the diagnosis ofthe mixed tumor with nec componentless than so we could only treat all mixed tumors withnec component as a wholesour study demonstrated that gastric neoplasms withnec components regardless of the proportion of components have poorer overall survival than gastric adenocarcinomaindicating a higher degree of malignancythan gastric adenocarcinoma among the patients receiving neoadjuvant therapy the difference in overallsurvival was not significant which was in sharp contrastwith the results of the surgery group suggesting thatneoadjuvant therapy may have a good effect on theprognosis of these malignancies therefore for this typeof malignancy we should adopt more aggressive andpowerful treatments than those used for gastric adenocarcinoma to improve the prognosis of patients neoadjuvant chemotherapy may be a good way to improve theefficacy offor these tumors at advancedstagestreatmentsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072817additional file table s1 comparison of clinicopathologicalcharacteristics before and after psm of 30ghminen patients insurgical group table s2 comparison of clinicopathologicalcharacteristics before and after psm of ghminen patients in surgicalgroup table s3 comparison of clinicopathological characteristics beforeand after psm of 70ghminen plus pure nec patients in surgicalgroup table s4 comparison of clinicopathological characteristics beforeand after psm of pure nec patients in surgical group table s5 comparison of clinicopathological characteristics before and after psm of 30ghminen patients in neoadjuvant group table s6 comparison ofclinicopathological characteristics before and after psm of ghminen patients in neoadjuvant group table s7 comparison of clinicopathologicalcharacteristics before and after psm of 70ghminen plus pure necpatients in neoadjuvant group table s8 comparison of clinicopathological characteristics before and after psm of pure nec patients in neoadjuvant groupabbreviationsajcc american joint committee on cancer ct computed tomography ghminen gastric highgrade mixed neuroendocrinenonneuroendocrineneoplasm gnec gastric neuroendocrine carcinoma hpf high power fieldminen mixed neuroendocrinenonneuroendocrine neoplasmnec neuroendocrine carcinoma nen neuroendocrine neoplasmnet neuroendocrine tumor mri magnetic resonance imaging os overallsurvival petct positron emission tomography computed tomographypfs progressionfree survival psm propensity score matching who worldhealth anizationacknowledgmentsthanks to dr zhongwu li of the department of pathology peking universitycancer hospital and his colleagues for their assistance in pathologicaldiagnosis and review thanks to all colleagues in the department ofgastrointestinal surgery of peking university cancer hospital and dr jianghong from the statistics department for their assistance in this studyauthors contributionsall authors contributed to the study conception and design jc performeddata collection and wrote the manuscript aw wrote and t revised hemanuscript kj helped with statistical analysis and prepared the illustrationszb edited the manuscript jj conceived the study and reviewed themanuscript all authors read and approved the final manuscriptfundingthis work was supported by the national science foundation for youngscientists of china beijing youth talent plan qml20191101 andscience foundation of peking university cancer hospital thefunders had no role in study design data collection and analysis decision topublish or preparation of the manuscriptavailability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe study was approved by the ethics committee of peking universitycancer hospital and the patients written consent was also obtained writteninformed consent for publication was obtained and stored in pekinguniversity cancer hospitalconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived may accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin matsubayashi h takagaki s otsubo t iiri t kobayashi y yokota t advanced gastric glandularendocrine cell carcinoma with 1year survivalafter gastrectomy gastric cancer park jy ryu mh park ys park hj ryoo by kim mg prognosticsignificance of neuroendocrine components in gastric carcinomas eur jcancer la rosa s inzani f vanoli a klersy c dainese l rindi g histologiccharacterization and improved prognostic evaluation of gastricneuroendocrine neoplasms hum pathol ishida m sekine s fukagawa t ohashi m morita s taniguchi h neuroendocrine carcinoma of the stomach morphologic andimmunohistochemical characteristics and prognosis am j surg patholrayhan n sano t qian zr obari ak hirokawa m histological andimmunohistochemical study of composite neuroendocrineexocrinecarcinomas of the stomach j med investig jiang sx mikami t umezawa a saegusa m kameya t okayasu i gastriclarge cell neuroendocrine carcinomas a distinct clinicopathologic entityam j surg pathol ohike nan la rosa s who classification of tumours of endocrineans 4th ed lyon iarc press amin mb edge sb ajcc cancer s
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"Nonsmall cell lung cancer is the most common cause of cancer death worldwide highlighting the need fornovel therapeutic concepts In particular there is still a lack of treatment strategies for the group of elderly and frail patientswho are frequently not capable of receiving standard therapy regimens Despite comprising the majority of lung cancerpatients this group is underrepresented in clinical trials This applies also to elderly and frail patients suffering fromunresectable stage III NSCLC who are unfit for chemotherapy and therefore cannot receive the standard therapycomprising of radiochemotherapy and the recently approved subsequent durvalumab consolidation therapy These patientsoften receive radiotherapy only which raises the concern of undertreatment The TRADEhypo trial aims at optimizingtreatment of this patient group by combining radiotherapy with concomitant durvalumab administration therebyemploying the immunepromoting effects of radiotherapy and determining safety feasibility and efficacy of this treatmentMethods design In this prospective phase II clinical trial durvalumab therapy will be combined with either conventionallyfractionated CONgroup or hypofractionated HYPOgroup thoracic radiotherapy A safety stopandgo leadin phase willassess safety of hypofractionated radiotherapy with respect to severe pneumonitis in small patient cohorts before ing fullenrollment Tumor tissue blood and stool samples will be collected before and during the study period to investigate theimmunological mechanisms responsible for checkpoint inhibitor efficacy and immunepromoting effects of radiotherapyContinued on next page Correspondence FarastukBozmehrmeduniheidelbergde1Department of Thoracic Oncology Thoraxklinik at University Hospital ofHeidelberg R¶ntgenstrae Heidelberg Germany2Translational Lung Research Center Heidelberg TLRCH Member of theGerman Center for Lung Research DZL Im Neuenheimer Feld Heidelberg GermanyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBozmehr BMC Cancer Page of Continued from previous pageDiscussion Preclinical data suggests that irradiationinduced immunogenicity can be further increased if applied in ahypofractionated setting potentially boosting the expected synergistic effect with immune checkpoint inhibition in restoringthe immune antitumor response If proven safe and efficient a hypofractionated radiation schedule can provide a considerablymore practicable option for the patient Taking into consideration the intend to develop a combination treatment strategy thatcan be made available to patients soon after proving to be efficient and the potentially elevated toxicity of a hypofractionatedradiotherapy approach this trial was designed as a twotrialsinone design An accompanying translational research program isplanned striving to gain insights into the tumorhost biology and to identify suitable biomarkers to predict therapy responseTrial registration Clinicaltrialsgov NCT04351256 Registered April EudraCT Registered October Keywords Nonsmall cell lung cancer NSCLC Radioimmunotherapy Immune checkpoint inhibition AntiPDL1 monoclonalantibody Hypofractionated radiation Geriatric risk profileBackgroundLung cancer is the most common cause of cancer deathworldwide with nonsmall celllung cancer NSCLCrepresenting of cases [ ] Improving therapeutic strategies is thus of imminent importance especially considering elderly and frail patients With a medianage of about years at diagnosis lung cancer clearly is adisease of the elderly yet this group is underrepresentedin clinical trials and these patients are frequently not capable of receiving standard treatment protocols due to ageand tobaccoassociated comorbidities []attracting both immunocompetentIn recent years the advent of immunotherapy has pavedthe way for novel therapeutic concepts including the combination of radiotherapy with immune checkpoint inhibitioneg Programmed cell death ligand PD1 PDL1 Thisapproach is of particular interest as it utilizes synergistic effects While immune checkpoint inhibitors can restore thepatients antitumor immunity through T cell activationradiotherapy may further boostimmunemediated anticancer mechanisms by exposing tumorassociated antigensand byantigenpresenting cells and tumoricidal effector cells [ ] Indeedfor patients with unresectable stage III NSCLC the PACIFICtrial has revealed a profound clinical benefit treatment withthe antiPDL1 monoclonal antibody durvalumab after chemoradiotherapy with remarkably low toxicities [ ] As aresult sequential treatment with durvalumab after chemoradiotherapy has become the new standard treatment for locally advanced unresectable NSCLC However about ofpatients do not receive chemotherapeutic agents presumablydue to significantly higher rates of age and comorbidityrelated adverse events AE under chemoradiotherapy [] Thus elderly and frail patients often receive radiotherapyalone raising the serious concern of undertreatment and theneed for new therapeutic concepts [ ]Considering the immunepromoting effects of radiotherapy a combination with durvalumab therapy mayimprove response rates in these potentially undertreatedpatients Moreoverif applied early concomitant localdatathatsuggestradiotherapy with systemic immunotherapy may particularly increase control of distant micrometastases Preclinicalirradiationinducedimmunogenicity can even be further increased if appliedin a hypofractionated setting with single doses ¥ GrayGy in line with a radiation dosedependent abscopal[] While a hypofractionated radiationeffectschedule is also considerably shorter and more convenient for the patient safety of concurrent immunoradiotherapy is a concern as both therapy modalities maycause severe pneumonitisIn this prospective phase II clinical trial we thereforeaim to determine feasibility and treatment efficacy ofdurvalumab treatment combined with thoracic radiotherapy TRT in previously untreated NSCLC stage IIIpatients unable to receive radiochemotherapy Strivingto develop a combination treatment strategy thatifproven safe and efficient can be quickly made availableto patients a twotrialsinone design was chosen thatcombines durvalumab with either conventionally fractionated CONgroup or hypofractionated thoracicradiotherapy HYPOgroup This study not only aims toincrease the efficacy of radiotherapy by utilizing theimmunesensitizing effects elicited by PDL1 inhibitionbut will also provide biomaterials that will be analyzedwith respect to immunological mechanisms responsiblefor checkpoint inhibitor efficacy and immunepromotingeffects of radiotherapy as well as potential biomarkersMethodsdesignStudy designThe TRADEhypo trialis a prospective randomized label multicenter phase II trial with a safety stopandgo leadin phase Fig During the leadin phasepatients in the HYPOgroup who will receive durvalumab in combination with hypofractionated thoracicradiotherapy will be closely evaluated with regard totoxicity defined as pneumonitis ¥ grade within 0cBozmehr BMC Cancer Page of Fig Study design of the TRADEhypo trial Patients will be enrolled according to eligibility criteria and treated with either a hypofractionated TRTregimen HYPOgroup or conventionally fractionated TRT CONgroup in combination with durvalumab For the HYPOgroup a safety stopandgophase with a design precedes full enrollment Whenever this arm is for recruitment patients will be allocated to this arm until the cohort isclosed whenever HYPOarm is closed for Stop Go decision evaluation based on the toxicity assessment of this regimen weeks after the end of TRTpatients are allocated to the CONarm When the study proceeds to expansion phase patients will be allocated to treatment arms by randomizationusing biased coin algorithm An efficacy interim analysis will be performed after patients have been enrolled in each armweeks after radiotherapy in small cohorts n beforeproceeding with full enrollment into this arm Fig respectrelated biomarkersto treatmentinduced changes and immuneStudy settingThe TRADEhypo trial will recruit patients from participating centers across Germany over a period of months Start of recruitment was planned for April but was delayed to May due to the Covid19pandemic A full list of sites can be obtained at clinicaltrialsgov NCT04351256Study objectivesThe primary objective of this study is to evaluate safetyand tolerability of conventionally fractionated CONgroup and hypofractionated HYPOgroup TRT incombination with durvalumab in patients with unresectable stage III NSCLC unfit for chemotherapy Moreoverefficacies of the two modes of radiotherapy will be evaluated with respect to response rates Further parameterswill be determined in order to assess efficacy safety andquality of life QoL in both treatment arms by recordingincidence and severity of adverse events AEs as well asspecific laboratory abnormalitiesExploratory endpoints include assessment of vulnerability and analyses of tumor tissue blood and stoolsamples that are collected during the clinical trial withCharacteristics of participantsA total of patients will be included into this studyPatients potentially eligible for trialinclusion will beapproached and asked to participate as they present inthe clinic Before a patients participation in the clinicalstudy the investigator must obtain written informedconsentEach participant must be eligible regarding all inclusion and exclusion criteria set for this trial Table Key inclusion criteria include diagnosis of unresectablestage III NSCLC and nonfeasibility of sequential chemoradiotherapy due to increased oxygenindependentvulnerability as reflected by fulfilling at least one of thefollowing criteria i Performance status Eastern Cooperative Oncology Group [ECOG] scale ii ECOG and Charlson comorbidity index CCI ¥ or iii age ¥ Moreover participants must have at least one measurable site of disease as defined by RECIST as wellas adequate bone marrow hepatic and renal functionPatients having received prior immunotherapy other investigational agents or thoracic radiotherapy within thepast years will be excluded from the study Additionally participants must not have an active or recent history of a known or suspected autoimmune disease or 0cBozmehr BMC Cancer Page of Fig Cohort design of the safety stopandgo leadin phase HYPOgroup The safety leadin phase follows a design in order to carefullyevaluate the toxicity of the treatment in the HYPOgroup with respect of the occurrence of a grade pneumonitis event within weeksafter the end of TRT Two events in the first six patients two events in the first or two events in the first patients will result in terminationof the HYPOgroup Stop If no event is observed within the first two safety cohorts ie the first patients the HYPOarm will be ed forfull enrollment with close toxicity assessment with respect to pneumonitis grade and terminated as soon as two events are reported withinthe subsequent six patients Go Full enrollment in the HYPOarm will only take place if the criteria for the nontoxicity scenario are met ie ¤ event in n patients Goany other medical condition conflicting with the studyinterventions and not have used immunosuppressivemedication For a full list of the inclusion and exclusioncriteria see Table TreatmentDurvalumabPatients will be enrolled based on the in exclusion criteria Two treatment groups will be evaluated Onegroup will receive durvalumab immunotherapy combined with conventionally fractionated TRT CONgroup and the other one with hypofractionated TRTHYPOgroup In both groups durvalumab will be administered intravenously at a fixed dose of mg onday and every weeks thereafter for a maximum of months maximum doses last dose at week untilconfirmed disease progressioninacceptable toxicitywithdrawal of consent or end of the study Fig andTable RadiotherapyAll patients are subjected to preparation of individualpositioning devices and CTbased planning Motionmanagement may comprise either 4DCT or midbreathing CT image acquisition Further imaging modalitiessuch as FDGPETCT may be used when deemed necessary Gross tumor volumes GTV will be contouredand expanded by adequate clinical CTV and planningPTV safety margins in order to account for subclinicaldisease and positioning errors No elective nodal irradiation will be performed As for ans at risk bothlungs spinal cord heart and esophagus must be contoured In the HYPOarm no more than of bothlungs minus GTV should receive Gy in the CONarm no more than of both lungs minus GTVshould receive GyIn the HYPOarm fractions of Gy will be administered total dose Gy corresponding to GyBED αβ In the CONarm fractions of Gywill be administered total dose Gy corresponding to GyBED αβ Dose deviations of ± are acceptable when clinically indicated Radiotherapy isscheduled to start within h after the first administration of durvalumab Dose prescription will follow international reports ICRU and Both 3Dconformal and modulated photon radiation techniquessuch as IMRT and VMATRapdArc are acceptable Allparticipating institution are encouraged to perform regular if no daily positioning control using either portalimaging or onboardCT devicesStudy proceduresIn order to minimize the risk exposure of patients whensubjected to the hypofractionated radiation regimen a 0cBozmehr BMC Cancer Page of Table Complete list of inclusion and exclusion criteriaInclusion criteriacid129 Fullyinformed written consent and locally required authorization obtained from the patient legal representative prior to performing any protocolrelated procedures including screening evaluationscid129 Age ¥ yearscid129 Histologically documented diagnosis of unresectable stage III NSCLCcid129 Nonfeasibility of sequential chemoradiotherapy as determined by the sites multidisciplinary tumor board if there is no tumor board then thisdecision will be made by the investigator in consultation with a radiation oncologist if the investigator is not a radiation oncologist or by the investigator in consultation with an oncologist if the investigator is not an oncologistcid129 Fulfills at least one of the following criteria ECOG ECOG and CCI ¥ Age ¥ yearscid129 Must have a life expectancy of at least weekscid129 FEV1 ¥ of predictedcid129 DLCO ¥ of predictedcid129 FVC or VC ¥ of predictedcid129 At least one measurable site of disease as defined by RECIST criteriacid129 Adequate bone marrow renal and hepatic functioncid129 Female patients with reproductive potential must have a negative urine or serum pregnancy test within days prior to start of trialcid129 Evidence of postmenopausal status or negative urinary or serum pregnancy test for female premenopausal patientscid129 The patient is willing and able to comply with the protocol for the duration of the study including hospital visits for treatment and scheduledfollowup visits and examinationsExclusion criteriacid129 Concurrent enrollment in another clinical study or enrollment within days prior to first dose of treatment unless it is an observational noninterventional clinical study or during the followup period of an interventional studycid129 Prior immunotherapy or use of other investigational agentscid129 History or current radiology suggestive of interstitial lung diseasecid129 Oxygendependent medical conditioncid129 Any concurrent chemotherapy investigational product IP biologic or hormonal therapy for cancer treatmentcid129 Prior thoracic radiotherapy within the past years before the first dose of study drugcid129 Major surgery within weeks prior to enrollment into the study patients must have recovered from effects of any major surgery Local nonmajorsurgery for palliative intent is acceptablecid129 Active or prior documented autoimmune or inflammatory disorders with the following exceptions Patients with vitiligo or alopecia patients withhypothyroidism stable on hormone replacement or any chronic skin condition that does not require systemic therapy Patients without activedisease in the last years may be included but only after consultation with the study physiciancid129 Active uncontrolled inflammatory bowel disease Patients in stable remission for more than year may be includedcid129 Uncontrolled intercurrent illness including but not limited to ongoing or active infection symptomatic congestive heart failure uncontrolledhypertension unstable angina pectoris uncontrolled cardiac arrhythmia interstitial lung disease gastrointestinal conditions associated with diarrheaor psychiatric illnesssocial situations that would limit compliance with study requirement substantially increase risk of incurring AEs or compromisethe ability of the patient to give written informed consentcid129 History of another primary malignancy except for a malignancy that has been treated with curative intent and was not active for ¥ years beforethe first dose of IP and of low potential risk for recurrence or adequately treated nonmelanoma skin cancer or lentigo maligna without evidence ofdisease or adequately treated carcinoma in situ without evidence of diseasecid129 History of leptomeningeal carcinomatosiscid129 History of active primary immunodeficiencycid129 History of allogenic an or tissue transplantationcid129 Clinical diagnosis of active tuberculosiscid129 Positive testing for hepatitis B virus surface antigen or hepatitis C virus RNA indicating acute or chronic infection or for human immunodeficiencyviruscid129 Current or prior use of immunosuppressive medication within days before the first dose of durvalumab The following are exceptions to thiscriterion Intranasal inhaled topical steroids or local steroid injections systemic corticosteroids at physiologic doses not to exceed mgday ofprednisone or its equivalent steroids as premedication for hypersensitivity reactionscid129 Receipt of live attenuated vaccine within days prior to the first dose of IPcid129 Female patients who are pregnant or breastfeeding or male or female patients of reproductive potential who are not willing to employ effectivebirth controlcid129 Known allergy or hypersensitivity to any of the IPs or any of the constituents of the productcid129 Any coexisting medical condition that in the investigators judgement will substantially increase the risk associated with the patients participationin the studycid129 Patient who has been incarcerated or involuntarily institutionalized by court order or by the authorities § Abs S Nr AMGcid129 Patients who are unable to consent because they do not understand the nature significance and implications of the clinical trial and thereforecannot form a rational intention in the light of the facts [§ Abs S Nr 3a AMG]safety leadin phase with stopandgo design will precedefull enrollment into the HYPOgroup Fig Toxicitywill be evaluated with a design that is based on thestatistical assumption that ¤ event in n patientsconforms to a nontoxicity scenario with event beingdefined as the occurrence of pneumonitis grade ¥ within weeks after end of TRT Consequently twoevents in the first six patients or two events in the first 0cBozmehr BMC Cancer Page of Table Schedule of assessmentsProcedure Point in timeInformed Consent eligibility criteria demographicsmedical and historyAllocation RandomizationPrior and Concomitant Medication ReviewDurvalumab administrationRadiotherapy CONa or HYPObAEsFull Physical ExaminationDirected Physical ExaminationVital Signs O2 Saturation and WeightPulmonary function tests12lead ECGECOG Performance StatusPregnancy Test CBC with Differential Serum ChemistryPanel Thyroid function testHBV HCVUrinanalysisTumor ImagingFACTL and G8 screening questionnaireTissueScreening Treatment Cycles Q4WScreening C1D1 C1D4 C2D1 C3D1 C4D1XC5D1C13D1EOT PostTreatmentEOT Safety FU FU Q12WXXXXXXXXXXXXXXXXXXXXXXXXXXXXXcXX cXXXXXXXXXXXXXtogether with staging XWhenever clinically indicatedXXXXXXXXXXXXXXXXWhenever clinically indicatedXdXdXeQ8WeXfXXXtogether with staging XXXgXhXXiOptional ReBiopsy at time of progressionXiXXBlood and stoolaCONgroup radiation scheme Patients receive conventional fractions of Gy Gy within weeks of TRT to be started within h after start ofdurvalumab treatmentbHYPOgroup radiation scheme Patients receive hypofractionated thoracic radiotherapy consisting of Gy Gy within weeks of TRT to be startedwithin h after start of durvalumab treatmentcTo be performed on C2D1 and C3D1 if in accordance with local standarddChest Xray to be performed on cycles and if in accordance with local standardeFirst onstudy CT imaging to be performed weeks after first durvalumab administration Further onstudy imaging to be performed Q8W days ± daysfOnly applicable if EOT not according to already detected disease progressiongIn patients with EOT not due to disease progression tumor imaging will be performed until the start of a new anticancer treatment disease progression deathwithdrawal of consent or the end of the studyhQuestionnaires will be collected until disease progression only and may be collected by telephone callsiBiomarker sample to be taken prior to first study drug medication either during screening or C1D1 visitX or two events in the first patients will result in termination of the HYPOgroup Fig During this safety stopandgo phase patients will beallocated to treatment arms asfollows While theHYPOarm is recruiting patients will exclusively enterthis treatment group During safety evaluation of the sixpatients of a HYPOcohort Stop Go decision patientswill be allocated to the CONgroup only If safety criteria in the HYPOcohort are met the HYPOarm willbe re ed to continue toxicity assessment and patients will solely be allocated to this arm Fig Inorder to avoid any bias patients will be allocated centrally by the study management during this phase If thenontoxicity criteria in the safety cohorts are met thestudy will proceed to theandremaining patients will be randomized into the twotreatment arms using an interactive web responseexpansion phasesystem integrated in the electronic case report formeCRF A possibly uneven distribution of patients between the treatment groups at this stage will be compensated by a randomization strategy based on the biasedcoin method [ ] In the randomization phase patients will be stratified according to tumor stage stageIIIA vs stage IIIBIIICIn total patients will be enrolled per group Aftern patients have been enrolled to the HYPO orCONtreatment arm respectively an interim efficacy analysis for the respective arm will be conducted based on theobjective response rate ORR at weeks after first durvalumab administration In case of insufficient efficacy ofone of the arms ie the number of patients who haveachieved a response is eight out of or lower this treatment arm will be terminated Recruitment will be halteduntil results of the interim efficacy analysis are available 0cBozmehr BMC Cancer Page of Tumor response will be assessed according to RECIST by CT and or MRI scans at baseline weeks afterdurvalumab initiation and then every weeks Safetymeasures willinclude physical examinations performance status ECOG clinical laboratory profiles and continuous assessments of AEs An overview of all studyprocedures is presented in Table An Independent Safety Monitoring Board ISMB willensure the continued safety of participants throughoutthe trial Data management and data quality assurancewill be conducted following the Standard OperationalProcedures of the Institute of Clinical Cancer ResearchIKF at Northwest Hospital GmbH Frankfurt GermanyAn eCRF will be carefully maintained for each participant for data collection also reporting serious and nonserious AEs following the common criteria for adverseevents CTCAE version throughout the entire trialAfter the end of the study participants will be proactively followed up regarding treatmentrelated AEsuntil resolved returned to baseline or considered irreversible until lost to followup or withdrawal of studyconsent All subjects will be followed for survival Patients who decline to return to the site for evaluationswill be offered a followup FU by phone every months as an alternative At the end of the study treatment the investigators are responsible for the furthertreatment of the patient and must ensure that he or shereceives appropriate standard of care or other appropriate therapiesSampling for translational researchIf patients participate in the translational research program blood samples will be collected prior to cycles and and at the time of disease progression or end oftreatment EOT along with stool samples Table Samples of untreated tumor lesions serving as baselinespecimens will be collected as paraffinembedded tissueIf rebiopsies are taken at the time of progression samples should also be also submitted for translationalresearchStudy endpointsThe primary endpoint of the study will be toxicity defined by the occurrence of treatmentrelated pneumonitis grade ¥ The ORR evaluated weeks after firstdurvalumab administration according to RECIST isset as the primary efficacy endpoint Secondary endpoints ofthe occurrence oftreatmentrelated AEs and serious AEs SAEsfrequency of prespecified abnormal laboratory parametersprogressionfree survival PFS and duration of clinicalbenefit metastasisfree survival overall survival OSand QoLcomprisethestudyPatient vulnerability and its association with survivaland outcome will be assessed as an exploratory endpoint To this end the G8screening questionnaire asimple and fast screening tool for identifying geriatricrisk profiles with a strong prognostic value for functionaldecline and OS in older populations with cancer will beused [] Furthermore biomaterials will be collectedduring the trial for correlation analyses on selected molecular parameters and clinical data in order to identifymolecular biomarkers predictive for response to therapyThisto obtainhypothesisgenerating data for future research due to theexplorative character of this trialapproach is deemed appropriateStatistical analysisSample size justificationSafety runin phase HYPOgroup With regard to thepneumonitis grade ¥ rate this phase is designed to distinguish between a toxicity scenario pTox and anontoxicity scenario pTox A sample size ofn will yield a probability of to correctly detectthe toxicity scenario while the nontoxicity scenario willcorrectly be detected with a probability of Theseprobabilities are based on the decision rule that if thenumber of patients with pneumonitis grade ¥ in thiscohort is ¥ recruitment to the HYPOgroup will beterminatedanalysisInterim efficacyregarding ORR andexpansion phase An interim efficacy analysis based onthe ORR will be conducted after n patients in eacharm have completed radiotherapy and the 18th patienthas undergone first radiographic assessment at weeksafter first durvalumab dosePreviously an ORR of after radiotherapy alonehas been reported in a Japanese population of elderly patients with unresectable stage III NSCLC [] Based onthis and the observation that Asian ethnicity is associated with a favorable prognosis we assume for theTRADEhypo trial that an ORR higher than in bothtreatment arms can be demonstrated ie the null hypotheses for arm HYPO and CON are defined as H0HYPO Ï HYPO ¤ and H0CON Ï CON ¤ where ÏHYPO and Ï CON denote the actual ORR in arm HYPOand CON respectively [ ] Under the alternativehypothesis it is assumed that both Ï HYPO and Ï CONamount to Using an optimal Simons twostage design with a onesided significance level of α and apower of 1β for each hypothesis test n patients per arm are required while the interim analysis isconducted after n patients per arm have been recruited to the trial After successfully passing the safetyanalysis in the HYPOgroup if among patients in the 0cBozmehr BMC Cancer Page of HYPO or CONarm the number of patients who haveachieved a response is eight or lower the respective armwill be closed Otherwise an additional number of patients will be enrolled into the respective arm Thenull hypothesis of the respective arm can be rejected ifat least of all patients per arm achieve a responseSample size calculation was done using the R packageOneArmPhaseTwoStudy []To account for an estimated dropout rate of fourpatients will additionally be recruited to each treatmentarm Deviations from planned sample sizes will be handled as described by Englert Kieser allowing strictcontrol of the aspired type I error rate in each arm []Methods of statistical analysis The primary populationfor evaluating all efficacy endpoints and subject characteristics is defined as all patients enrolled according toinitial allocationrandomization intentiontotreat population IIT Secondary efficacy analyses will be carriedout on the perprotocol PP population The safetypopulation comprising all patients who received at leastone dose of the study medication will be used for allsafety endpoints and will be analyzed according to theactual treatment receivedResponse rates with confidence intervals CI and pvalues in both study arms will be estimated taking intoaccount the twostage nature of the design [ ] Secondary endpoints will be evaluated descriptively All toxicities will be summarized by relative and absolutefrequency and severity grade based on CTCAE V50 AEand SAE summary tables will provide the number andpercentage of patients with AEs and the ClopperPearson type CIs All analyses will be done using SASversion SAS Institute Cary NC or higherTrial statusAs of July 15th eight study sites are initiated Firstinitiation coincided with the beginning of the Covid19pandemic in Germany Therefore recruitment of patients was withheld On May 8th recruitment wasresumed after consultation with the ISMB The first patient was enrolled on July 13th renal carcinoma and NSCLCDiscussionIn recent years the concept of restoring the patients antitumor immunity by immune checkpoint inhibition hasrevolutionized cancer therapy especially in advancedmelanomaImmunecheckpoint molecules efficiently regulate T cell activation and thus enable tumor cells to evade the immunesystem for example by hijacking the PD1 PDL1 interaction to downregulate effector T cells [ ] To dateseveral human monoclonal antibodies pharmacologicallyblocking these interactions have been implemented incancer therapy such as the antiPD1 antibody pembrolizumab that has been approved in combination withchemotherapy for nonsquamous NSCLC irrespective ofPDL1 expression [ ]Several studies have shown that immune checkpointinhibition and radiotherapy in combination can act synergistically to further boost antineoplastic effects [] Although in a large phase III trial no benefit ofblockade of cytotoxic T lymphocyteassociated antigen CTLA4 after radiotherapy was observed in metastaticprostate cancer [] clinical trials such as NICOLASNCT02434081 and DETERRED NCT02525757investigating concurrent PDL1directed immunotherapyand chemoradiot
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"objectives to describe the benefits and limitations of using individual and combinations of linked english electronic health data to identify incident cancersdesign and setting our descriptive study uses linked english clinical practice research datalink primary care cancer registration hospitalisation and death registration dataparticipants and measures we implemented case definitions to identify first site specific cancers at the most common sites based on the first ever cancer diagnosis recorded in each individual or commonly used combination of data sources between and we calculated positive predictive values and sensitivities of each definition compared with a gold standard algorithm that used information from all linked data sets to identify first cancers we described completeness of grade and stage information in the cancer registration data setresults gold standard cancers were identified positive predictive values of all case definitions were ¥ and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity for case definitions that used cancer registration alone or in combination was ¥ for the four most common cancers and ¥ across all cancer sites except bladder cancer using cancer registration alone for case definitions using linked primary care hospitalisation and death registration data sensitivity was ¥ for the four most common cancers and ¥ for all cancer sites except kidney oral cavity and ovarian cancer when primary care or hospitalisation data were used alone sensitivities were generally lower and diagnosis dates were delayed completeness of staging data in cancer registration data was high from minimum in and in for the four most common cancerss ascertainment of incident cancers was good when using cancer registration data alone or in combination with other data sets and for the majority strengths and limitations of this study º this is the first study to present comprehensive information on the implications of using different individual and combinations of linked electronic health data sources in england to identify cases of the most common incident cancers º using a gold standard algorithm that combined all available data from multiple sources as a comparator we were able to estimate both positive predictive values and sensitivity values for a range of pragmatic case definitions º we described similarities and differences in values between age groups sexes and calendar years the impact of choice of sources on diagnosis dates and mortality rates and completeness of stage and grade in cancer registration data º a key limitation was that our gold standard algorithm is not validated and may be affected by differences in clinical diagnosis and coding of invasive cancers between data sourcesof cancers when using a combination of primary care hospitalisation and death registration dataintroductionthe clinical practice research datalink cprd provides de identified primary care data linked to additional secondary health data sources under a well governed framework1 use of linked data helps researchers to answer more epidemiological questions and increase study quality through improved exposure outcome and covariate classification2 in the field of cancer epidemiology cprd primary care data linked to hospital episode statistics admitted patient care strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access data hes apc office of national statistics ons mortality and national cancer registration and analysis service ncras cancer registration data are used to analyse factors contributing to the risk of cancer and the consequences of cancer and its treatment use of linked data reduces the sample to the common source population and data coverage period for each included data set and has cost and logistical implications which are greatest for ncras data research teams therefore commonly choose not to use all available linked data3 cancer epidemiology studies can also be conducted using ncras and hes apc data provided by national health service nhs digital and public health england phe without linkage to cprd primary care data4 this provides national coverage at the expense of the detailed health data that are available in primary care recordsvalidation studies assessing concordance between cprd gold hes apc and ncras data have estimated high positive predictive values ppvs for cprd gold data and varying proportions of registered cancers that are not captured in cprd gold and hes apc5 the most up to date analysis by arhi et al included the five most common cancers and all papers focussed on concordance between cprd gold only and ncras existing evidence therefore does not provide a complete assessment of the benefits and limitations of using different combinations of data sources within the context of practical study designs national data are available describing completeness of data fields within the cancer registry data in each collection year9 and over time for all cancers combined4 missingness for individual years has been associated with age comorbidities and clinical commissioning groups10 we aim to describe and compare the benefits and limitations of using different combinations of linked cprd primary care data hes apc ons mortality and ncras cancer registration data for conducting cancer epidemiology studies our analyses focus on incident cancer ascertainment as it is a common and important outcome in cancer epidemiology and it is more difficult to distinguish between secondary recurrent and primary cancers at a second site in these data sets we have compared definitions of the most common cancers based on the first ever cancer recorded in individual or combinations of data sets with a gold standard definition comparing information from all four data sets we also describe the availability of stage grade and treatment variables over time in the cancer registration data for the cprd linked cohort this reflects real life study design and will help researchers to decide which combination of data sources to use for future studiesmethodsstudy design and settingwe completed a concordance study using linked2 english cprd gold hes apc ons mortality and ncras data cprd gold data were extracted from the january monthly release and the 13th update to cprds linked data the study period was from january to december with december matching the end of the ncras coverage periodthe cprd gold database includes de identified records from participating general practices in the uk who use vision software1 general practice staff can record cancer diagnoses using read codes or in free text comments boxes though the latter are not collected by cprd diagnoses will typically be entered duringfollowing a consultation or from written information that is returned to the practice from secondary care cprd gold data are linked to hes apc ons mortality and ncras through a trusted third party for english practices that have agreed to participate in the linkage programme2 hes apc data are collected by nhs digital to co ordinate clinical care in england and calculate hospital payments12 admissions for and related to cancer diagnoses are recorded using international classification of diseases version icd10 codes national cancer registration data are collected by ncras which is part of phe in accordance with the cancer outcomes and services data set13 which has been the national standard for reporting of cancer in england since january data include icd10 codes to identify the cancer site and more detailed information such as stage and grade ons mortality data includes dates and causes of deaths registered in england recorded using icd10 codesparticipants exposures and outcomesour underlying study population included male and female patients registered in cprd gold practices who were eligible for linkage to hes apc ncras and ons mortality data and had at least days of follow up between january and december start of follow up was defined as the latest of the current registration date within the practice and the cprd estimated start of continuous data collection for the practice up to standard date end of follow up was determined as the date the patient left the practice ons mortality date of death or practice last collection dateidentification and classification of cancer codeswe used code lists to classify cancer records in each of cprd gold hes apc and ons mortality data as one of the most common sites other specified cancers history of cancer secondary cancers benign tumours administrative cancer codes unspecified and incompletely specified cancer codes https org data incompletely specified cancer codes could be mapped to cancer site eg icd10 code c689 malignant neoplasms of urinary organ unspecified was considered consistent with both bladder and kidney cancer for ncras we accessed coded records for the most common cancers we included cancers recorded in the clinical or referral file for cprd gold cancers recorded in any diagnosis field for hes apc and the underlying or strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure gold standard algorithm to identify incident site specific cancers using all data sources hes hospital episode statistics ncras national cancer registration and analysis service ons office of national statisticsmost immediate cancer cause of death in ons mortality datacancer case definitions based on individual sources and combinations of sourceswe developed alternative cancer case definitions mirroring those commonly used in epidemiology studies based on identifying the first malignant cancer excluding administrative codes and benign tumours recorded in various combinations of data sources ncras alone ncras and hes apc all sources cprd gold hes apc and ons mortality cprd gold alone hes apc alone multiple malignant cancers recorded on the index date in cprd gold or hes apc were reclassified as multiple site cancer and were not considered as individual site cancer records for positive predictive value and sensitivity calculations multiple codes recorded in different sources on the same date were reclassified as the site identified in the ncras data if available and as multiple site cancer if not for each case definition we only examined the first malignant cancer per individual where this occurred within the study period and at least year after the start of follow upgold standard cancer case definitionwe developed a gold standard algorithm that classifies incident records of the most common cancers by comparing the first malignant cancer identified in each individual source figure cancers recorded in ncras alone with no contradictions ie records for first cancers at different sites were considered true cases whereas cancers recorded in hes apc alone or gold alone required internal confirmation within that source in the form of another code for cancer consistent with the same site or with site unspecified within months and no contradictory codes eg for cancers at other sites in this period where cancer records were present in data source we considered a site specific cancer to be a true case a if it was recorded as the first cancer in ncras and the total number of data sources with records for cancer at that site was equal to or greater than the number of data sources with contradictory records ie records for first cancers at different sites or b where the cancer was not present in ncras if there were more data sources in total with records for cancer at that site than data sources with contradictory recordswe used ncras data to identify stage grade and treatment where available in the cancer registry only cohort binary surgery chemotherapy and radiotherapy variables were derived using individual records of treatment from the first year after diagnosisstatistical analysisfor each cancer site and each individual or combined data source we combined our applied study definitions strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access with our gold standard definition to classify each applied study definition as a true positive false positive or false negative recordwe used these categories to calculate sensitivity and positive predictive value overall and stratified by age categories to and calendar year and sex we calculated differences in diagnosis dates for true positives by subtracting the gold standard index date from the index date for each source and combination of sourceswe used kaplan meier methods to describe mortality over time for cancers identified using each definition the ons mortality death date was used for these analyseswe used the ncras only definition to calculate proportions of patients with complete stage and grade and recorded cancer treatment modalities over timepatient public involvementpatients and the public were not involved in conceiving designing or conducting this study and will not be consulted regarding the dissemination of study resultsresultsof research quality patients in the cprd gold january build were eligible for linkage to hes ons mortality and ncras data in set were excluded due to unknown sex of the remainder and had at least year of follow up between january and december and were included in the study population using the gold standard algorithm incident cases of cancer were identified the number of patients identified with each cancer is presented in online supplementary appendix table half n82 of these patients were male aged to aged to and aged or olderfigure presents ppvs for each case definition comparing the first recorded cancer in each combination of data sources with the gold standard algorithm when using ncras data alone to of cancers were confirmed by the algorithm for out of cancer sites the ncras only case definition gave the highest ppv case definitions using data sources not including ncras generally had lower ppvs ranging from to for individual cancer sites for the four most common cancers breast lung colorectal and prostate ppvs were at least for all case definitions minimal differences in ppvs were observed between age groups years and sexes online supplementary appendix figures figure presents sensitivity values for each case definition sensitivity was generally higher for the case definitions that included ncras data ranging from to for individual cancer sites except bladder cancer identified using ncras data alone and ¥ for the four most common cancers breast lung colorectal and prostate sensitivity was also generally high for definitions using a combination of cprd gold hes apc and ons mortality data ranging from to ¥ for the four most common cancers sensitivity was lower for case definitions that used cprd gold alone range to for individual cancer sites or hes apc alone range to sensitivity values for cprd gold alone and hes apc alone increased slightly in younger patients and more recent years no differences were observed between men and women online supplementary appendix figures post hoc analysis suggested that the low sensitivity of cprd gold only definitions for kidney cancer sensitivity n false negatives was driven by missing n1136 or incompletely specified urinary organ cancer codes n1108 in cprd gold rather than contradictory information about the first cancer record n625 these incompletely specified codes are less likely to be used for bladder cancers n85 than kidney cancers n1108 bladder cancers that were not recorded in ncras data n3445 were commonly recorded in both hes apc and cprd gold n2228 or in hes apc only with a subsequent unspecified or bladder cancer record in hes apc within months n995 table describes the number of days median iqr and 5th95th percentile lag between the date of incident cancers from the gold standard definition and the date of cancer arising from each case definition ie the first record within the specific combinations of data sources used case definitions using ncras alone and combinations of ¥ data sources captured cancers close to the gold standard date median lag ¤ days for all cancer sites whereas median lags were generally longer for the case definitions using cprd gold alone and hes apc alonefigure describes mortality over time following incident cancer diagnoses ascertained from each case definition minimal differences in mortality were observed between cancers identified from different case definitions where variability was observed cancers identified using cprd gold only had the lowest mortality rates eg kidney cancer and cancers identified using hes apc only or ncras only had higher mortality rates eg prostate cancer and bladder cancer respectivelyfigure describes completeness of grade and stage for cancers identified using ncras only recording of grade was highly variable between cancers with gradual increases in completeness over time completeness of staging information was low in earlier calendar years but improved substantially from around especially for the four most common cancers minimum in and in post hoc logistic regression models adjusted for year and cancer site indicated that completeness of stage and grade were associated with each other and these variables were least complete in patients aged stage data was more complete for higher grade tumours whereas grade data was more complete for lower stage tumours online supplementary appendix figure online supplementary appendix figure describes recording of treatment modalities identified using ncras strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure positive predictive value of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers defined using the first ever record in each combination of sources confirmed by a gold standard algorithm that considers confirmatory and contradictory data from each source cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure sensitivity of cancer diagnoses for each combination of sources when compared with the main gold standard algorithm percentage of incident cancers identified using the main gold standard algorithm that considers confirmatory and contradictory data from each source that are identified using the first ever record in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system nhl non hodgkin's lymphoma cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service onsoffice of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0celitnecrep hthtrqi inademelitnecreprqi inadem hthtelitnecrep htht inademrqielitnecrep hthtcpaseh dlogdrpc ytil atromsnodna cpaseh dlogdrpc cpasehdnasarcn secruos fonoitanbmoc hcae nii iidrocer reve tsrfi ot etad ssongaddradnats dog nammorf syad n ili emti l ebatopen access recnac lanoitan sarcn atad erac tneitapdetti mda scitsitats edospei latipsoh cpaseh knil atadhcraeser ecitcarp lacniilc drpc metsys suovren lartnec sncl tluafed ybnoitinfieddradnats dog eht sa emas eht si etad ssongad sa nwohs tonnoitinfied secruos ll iia setis recnac nommoc tsom ruofdcii nosrev sesaesdi fonoitacfissacliscitsitats lanoitan rof ecfifo snoi atadnoitartsgerrecnac ecvres ssyanadna liinoitartsgeri lanoitanretni eht imorf sedoc gndnopserroc ot gndrocca deredro era setis recnaci snoitinfiedde ilii ppa dna etad ssongaddradnats dog nam neewteb syadil fo rebmun ot ot ot ot ot cl amoeym epitluml ot ci ameakuel ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot elitnecrep hthtsarcn inademrqi ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot ot c ytivac laroc laegahposeoc hcamots cc latcerooclrecnac amonaeml tnangilamc saercnapc gnulc suretuc etatsorpc seiravoc yendkic tsaerbci xvreccc sncnarbic reddablc amohpmyl s'inkgdo hnonc idoryhtc revlistrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access figure mortality following first ever record of cancer in each combination of sources cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites cns central nervous system cprd clinical practice research datalink hes apc hospital episode statistics admitted patient care data ncras national cancer registration and analysis service nhl non hodgkin's lymphoma ons office of national statisticsstrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen accessfigure completeness of grade and stage for cancers identified using ncras data only cancer sites are ordered according to corresponding codes from the international classification of diseases version icd10 four most common cancer sites grading information is not applicable to braincns sarcoma or haematological cancers and not required by in the national data standard cosd for prostate cancer core staging is not applicable to haematological and gynaecological cancers other types of staging are recommended by cosd cns central nervous system cosd cancer outcomes and services data set ncras national cancer registration and analysis service nhl non hodgkin's lymphomastrongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0copen access only missing records may indicate that the patient did not receive that treatment modality or that the treatment modality was not recordeddiscussionstatement of principal findingswe investigated the use of different sources of electronic health record data to identify incident cancers for all case definitions using individual or combined data sources a minimum of of incident site specific cancers were confirmed using the gold standard algorithm this rose to of the four most common cancers use of cancer registration data alone or in any combination of data sources captured at least of site specific cancers identified by the gold standard algorithm excepting bladder cancer and of cases for the four most common cancers combining cprd gold hes apc and ons mortality data captured at least of site specific cancers excepting kidney oral cavity and ovarian cancers and captured of cases for the four most common cancers sensitivity was much more variable when using primary care or hospital data alone and dropped to when identifying bladder cancers using cancer registration data alone use of primary care or hospital data alone resulted in a small lag in identifying cancers of interest compared with the gold standard dates but other case definitions captured cancers close to the gold standard date finally while we observed minimal changes in ppvs and sensitivities between and completeness of ncras cancer registration stage and grade data increased markedly from onwards for specific cancer types demonstrating that initiatives to improve data can have a profound impact on the quality of the data4 completeness of cancer treatment recording was difficult to assess due to the absence of a missing categorystrengths and weaknesses of the studythe main strength of this study is that we have developed a gold standard algorithm using the entirety of the evidence available from cprd to demonstrate the impact of choice of data sets in identifying incident cancers for real life studies we have also assessed the value of using ncras cancer registration data to measure stage grade and cancer treatment modalitiesa limitation of the study is that our gold standard algorithm is not validated we feel that we were justified in pre weighting ncras data as more reliable that other data sources as ncras is a highly validated data set that matches merges and quality checks data from multiple sources4 we did not consider ncras to be the outright gold standard as it is plausible that ncras does not identify all tumours diagnosed and treated in primary and secondary care for most cancer sites our gold standard algorithm identified a small proportion of cancers that are recorded in hes apc cprd gold or ons mortality data but not in ncras these tumours may have been diagnosed and coded as invasive in primary or secondary care but not by ncras been incorrectly coded in hes apc cprd gold or ons mortality data not have been notified to ncras eg tumours treated in private hospitals or be the result of linkage errors between the data sets the proportion of cancers identified in hes apc but not in ncras is particularly high for bladder cancer this is likely to be the result of difficulties inconsistencies and changes in the pathological definition and coding of cancers over time in ncras which are greatest for bladder cancer4 this explanation is supported by the higher mortality rates that we observed in bladder cancer cases identified in ncras compared with other data sources to identify incident cancers we required months of research quality follow up in cprd gold prior to inclusion in the study previous research has demonstrated that historic data is generally incorporated within the patient record with this time frame15 the identification of first ever cancers will also have been affected by different lengths of follow up data available in linked data sources as ncras data collection started in hes apc in and ons mortality data in and by the inclusion of all diagnostic codes in hes apc assuming that the first ever primary or secondary record identified incident cancer reassuringly ppvs for liver and brain cancer were high for all individual and combinations of data sets suggesting that these were not unduly misclassified as primary incident cancers despite being common sites for metastases requiring internal confirmation within months for cancers recorded in cprd gold alone in our gold standard definition is more likely to discount cancers with poorer prognoses and those recorded in the last months of follow up our data cut only included ncras data for the top cancers earlier cancers at other sites will have been missed in this studyit is also important to note that as the gold standard algorithm uses data recorded after the first record of the cancer site in any source index date it cannot be used to identify outcomes in applied studies and follow up of cohort studies with cancer as an exposure would need to start at least months after diagnosis our first ever cancer record in any source definition would be more appropriate for most studiesstrengths and weaknesses in relation to other studies discussing important differences in resultsthe most up to date study describing concordance between linked cprd gold hes apc and ncras data sets demonstrated that to of the five most common cancers recorded in cprd gold are not confirmed in either hes apc or cancer registration data and to of registered cancers are not recorded in cprd gold8 for cancers recorded in both sources the diagnosis date was a median of to days later in cprd gold than in the cancer registration data using cprd gold alone to identify these strongman a0h et a0al bmj open 202010e037719 101136bmjopen2020037719 0ccancers marginally over represented younger healthier patients and identified to fewer deaths in the first years after diagnosis use of hes apc only identified a higher proportion of patients with the correct diagnosis date than cprd gold but over represented older patients and those diagnosed through the emergency route the majority of registered cancers were picked up using both cprd gold and hes apc ranging from for lung cancer to for breast cancer previous research demonstrated similar results with substantial differences between cancer types5 additionally a study using data from to found that using hes data in addition to ncras data identified an additional and of surgically treated colorectal lung and breast cancer cases respectively16our study is consistent with these results and provides more complete and practical evidence of the strengths and limitations of using individual and combinations of linked data sets to identify and characterise the most common incident cancerswe have also demonstrated the added value of using cancer registration data to measure stage and grade of incident cancers from about onwards levels of data completeness of staging information in the cprd extract in were similar to those reported by the united kingdom and ireland association of cancer registries ukaicr9meaning of the study possible explanations and implications for clinicians and policymakersuse of ncras cancer registration data maximised the proportion of cases confirmed as true positive based on all available linked information and captured the highest proportion of true positive cases highly complete staging and grading information is available from this source from approximately case definitions based on a combination of cprd gold hes apc and ons mortality data also had acceptable validity for the majority of cancer sites including the four most common cancersthese findings should be considered when deciding which data sources to include in research studies and which sources to use to define cancer exposures outcomes and covariatesunanswered questions and future researchfurther research is required to investigate the validity of cancer recorded in cprd gold and hes apc that are not recorded in the ncras data and to understand differences in cancer data recording with cprd gold and cprd aurum cprds recently launched primary care database based on records from practices that use emis software17 further investigation would be required to confidently identify subtypes of cancer either using codes available in each data set eg colon and rectal cancer or additional information available in hes apc or ncras data use of ncrass recently open accesslaunched systemic anti cancer therapy sact18 and national radiotherapy data sets will also improve ascertainment of therapies for futu
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" MTBHsp70 at both flanks. Histopathology Abdominal walls and intestines from mice were fixed for at least 24 h in PBS-buffered 10% formalin. Tissues were routinely embedded in paraffin. 5 ?m thick sections were stained routinely with H&E. For staining tumor-infiltrating T cells mice were perfused with 4% paraformaldehyde (PFA) in PBS and tumor nodules were fixed in 4% PFA/PBS for additional 2 hours washed and infiltrated with 30% sucrose/PBS at 4°C. 6 ?m thick frozen sections were stained with rat anti-mouse CD8 (BD Biosciences 1:100 dilution) or rat anti-mouse Foxp3 (eBioscience 1:12 dilution) followed by polyclonal rabbit anti-rat immunoglobulin/HRP (Dako 1:750 dilution). Signal was developed with diaminobenzidine (DAB Dako). Images were acquired on a Zeiss Axio A1 microscope. All histopathological and immunohistochemical samples were reviewed and the quantitation of the cellular infiltrate was performed in a blinded manner to the observer. Statistical analysis Statistical differences between three or more experimental groups were analyzed using One-Way ANOVA followed by Turkeys multiple comparison tests when mean of each group is compared with that of every other group or followed by Dunnetts multiple comparison tests when mean of each group is compared with that of a control group. Statistical differences between two experimental groups were analyzed using Students t-test. Survival was analyzed with the Log-rank test. Prism 6.0 software (GraphPad Software) was used for all the statistical analysis. Abbreviations DC: Dendritic cell; scFv: Single-chain antibody variable fragment; MSLN: Mesothelin; MTB: Mycobacterium tuberculosis; Hsp: Heat shock protein; i.p.: Intraperitoneal; i.d.: Intradermal; BMDCs: Bone marrow-derived dendritic cells; APCs: Antigen-presenting cells; PBMCs: Peripheral blood mononuclear cells; PBLs: Peripheral blood leukocytes; LPS: Lipopolysaccharide; H&E: Haematoxylin and eosin; PFA: Paraformaldehyde; DAB: Diaminobenzidine; mAb: monoclonal antibody. Competing interests The authors declare that they have no competing interests. Authors contributions JY played a role in the design of the experiments acquisition analysis and interpretation of the data and writing the manuscript. PR JN YY NHA MN GJ-M XT SK HC PU BF TC and PL participated in the performance of experiments. SK and TB were involved in design of the experiments. RB was involved in data analysis. ER was involved in setting up murine ovarian cancer model. SO provided the murine ovarian cancer model. NS provided the plasmid that encodes an scFv fragment specific to MSLN and the recombinant P4 scFv protein. GD NS and SO gave constructive input on experimental design and data analysis. JG played a role in conception and design of the fusion protein. MP and JG were involved in the conceptualization and design of the study analysis and interpretation of datasets and in writing the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1: Figure S1 scFvMTBHsp70 binds to 40L mesothelioma cells. 40L cells were stained with scFvMTBHsp70 or MTBHsp70 followed by mouse anti-MTBHsp70 and Donkey anti-mouse Alexa Fluor 594. Cells were observed using a Nikon Eclipse TiE fluorescence microscope. A Representative pictures from three independent experiments. Scale bar 10 ?m. B Images were analyzed using the NIS-Elements AR Microscope Imaging Software. Mean Fluorescence Intensity was analyzed using ImageJ. P values were determined using One-Way ANOVA followed by Turkeys multiple comparison tests. ****p?<?0.0001. Click here for file Additional file 2: Figure S2 scFvMTBHsp70 or MTBHsp70 plus P4 scFv treatment does not lead to infiltration of inflammatory cells into abdominal or intestinal mesothelial tissues. Samples of abdominal wall and intestine were prepared from C57BL/6 mice that had previously received multiple i.p. injections of scFvMTBHsp70 MTBHsp70 plus P4 scFv or saline as described in the Methods section. Sections of these tissues were stained with H&E and images were acquired on a Zeiss Axio A1 microscope. Representative images from 3 animals per treatment group are shown. No detectable level of mononuclear cell or granulocyte infiltrate within mesothelial tissues was seen in any sampled tissues. Scale bar 20 ?m. Click here for file Additional file 3: Figure S3 scFvMTBHsp70 treatment does not affect numbers of tumor-infiltrating CD8+ or Foxp3+ T cells. (A) Representative images of intratumoral CD8+ and Foxp3+ T cells from saline (n?=?3) scFvMTBHsp70 (n?=?3) or MTBHsp70 plus P4 scFv (n?=?3) -treated mice. Mouse spleen sections were used as positive controls: CD8+ and Foxp3+ T cells are clearly evident in the sections. Scale bar 20 ?m. (B) Numbers of CD8+ and Foxp3+ cells were quantified from 35 randomized fields. Click here for file Additional file 4: Figure S4 Validation of in vivo depletion of CD8+ cells in FVB/NJ mice. Mice were injected i.p. with 200 ?g of anti-CD8 mAb or an isotype-matched irrelevant rat IgG2a as described in Methods. All the mice were bled from the tail vein and the depletion of CD8+ cells was examined by flow cytometry analysis of peripheral blood cells stained with fluorophore-conjugated anti-CD8 on days 7 and 28 after tumor inoculation. (A) Representative results of flow analyses on 10 mice per group and reported as the percentage of CD8+ cells in lymphocytes. (B) CD8+ cells in the mice treated with isotype IgG2a or anti-CD8 mAb were compared. ***p< 0.001. Click here for file Acknowledgments This manuscript is dedicated to the memory of Janet Gelfand a victim of ovarian cancer. The authors gratefully acknowledge the continuing support for this work from the Edmund C. Lynch Jr. Cancer Fund Arthur Luxenberg Esq. Perry Weitz Esq. and the VIC Mesothelioma Research and Resource Program at MGH and the Friends of VIC Fund. PU and NHA were supported by the Prof. Dulcie V. Coleman Studentship at Imperial College London. We thank Oliver Mitchell John Cao Lujia Zhou Rumbidzai Mushavi and Sayinthen Vivekanantham for their technical assistances Dr. Yuhui Huang for his useful comments Michael Waring Dr. Michael Santuosuosso and Dr. Ravi Mylvaganam for their technical advice Dr. Musie Ghebremichael for his advice in statistical analysis and Mahnoor Valibhoy for her assistance with the schematic figure. 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" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearmans correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturers instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearmans correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time à power time à power¼¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci pgml and pgml ci pgml respectively the median level of il p40 before the mwa treatment was pgml ci pgml there was a slight increase to pgml ci pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci pgml and increasedto pgml ci pgml days afterthe mwa treatment and to pgml ci pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci pgml there was a significant increase at h postmwa with a median level of pgml ci pgml the median level ofil1 before the mwa treatment was pgml ci pgml and a significantincrease wasnoted days after the mwa treatment pgml ci pgml the median level of il6before the mwa treatment was pgml ci pgml and significantly increased daysafter the mwa treatment pgml ci pgml the median level ofil8 before themwa treatment was pgml ci pgml and increased significantly to pgml ci pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci pgml and increasedsignificantly days after the mwa treatment pgml ci pgml the median level oftnfα before the mwa treatment was pgml ci pgml and increased significantlyto pgml ci pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of energy time à power time à power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional 10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci ci ci ci ci ci ci ci ci ci h postmwa pgml ci ci ¼ ci ci ci ci ci ci ci ci il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa ¼ 2fold vs premwa days postmwa pgml ci ci ci ci ci ¼ ci ¼ ci ¼ ci ¼ ci ¼ ci days postmwa pgml ci ci ci ci ci ci ci ci ci ci effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 its mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of in situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaenergyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearmans rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the energyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wascancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a cancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled abscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as differentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmeds work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors contributionsjz conceptualization data curation writingoriginal draft and writingreview and editing ql conceptualization and writingreview and editingmm conceptualization and writingreview and editing brconceptualization and writingreview and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writingreview and editing rl conceptualization data curation formalanalysis visualization writingoriginal draft and writingreview and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the six one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim
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Ofï¬cial Case Reports Journal of the Asian Paciï¬c Society of RespirologyRespirology Case ReportsEndobronchial metastases from a primary embryonalcarcinomaChiKang Teng1ChihYen Tu121Division of Pulmonary and Critical Care Medicine Department of Internal Medicine China Medical University Hospital Taichung Taiwan2School of Medicine China Medical University Taichung Taiwan3Division of Pathology China Medical University Hospital Taichung Taiwan WenChien Cheng12 ChiehLung Chen1 TingHan Chen1 YunShan Lin3 AbstractWe report the case of a 24yearold man who presented with chief complaintsof shortness of breath and haemoptysis chest radiography revealed completecollapse of the left lung Bronchoscopy revealed an endobronchial tumourwith complete obstruction of the left main bronchus Cryosurgical excisionwas performed tissue pathology conï¬rmed the diagnosis of metastatic embryonal carcinoma The patient underwent a right orchiectomy followed by ableomycin etoposide cisplatin BEP chemotherapy regimenKeywordsCryosurgery embryonal carcinoma endobronchialmetastases endobronchial tumourCorrespondenceWenChien Cheng Division of Pulmonary and Critical Care Medicine Department of Internal MedicineChina Medical University Hospital No YudeRoad North Dis Taichung City TaiwanEmail wcchengdrgmailcomReceived July Revised July Accepted July Associate Editor James HoRespirology Case Reports e00644 101002rcr2644IntroductionLung metastases from extrapulmonary malignancies areobserved frequently in clinical practice by contrastendobronchial metastases EBMs from extrapulmonarymalignancies are rare and may have distinct histopathological etiologies [] Primary lung cancer is the most common cause of endobronchialtumours Extrapulmonarymalignancies that are frequently associated with metastasesto the central airways include tumours of breast colorectalthyroid origin [] Although mediastinalrenal orlymphadenopathy is frequently observed in associationwith testicular seminoma EBMs from embryonal carcinomas are extremely rare [] In this report we present acase of EBM from a primary embryonal carcinomaCase ReportA 24yearold man with no relevant past medical historypresented to our hospital with a chief complaint of shortness of breath lasting for several days Upon questioningthecoughexperiencedrevealedpatientthatheanendobronchialrevealedleft main bronchushaemoptysis shortness of breath and occasional chest painfor the past several days He reported no fever chills coldsweats weight loss or decreased appetite A chest radiograph at admission revealed complete collapse of the leftlung Fig 1A Computed tomography CT was notable forleft pleural masses an endobronchial tumour obstructingthe left main bronchus and complete collapse of the leftlung and a soft tissue mass lesion in the right scrotumBronchoscopytumourobstructing theFig 1B Theendobronchialtumour was excised with bronchoscopiccryosurgery a followup chest radiograph revealed someimprovement in the status of the left lung Immunohistochemical staining of the tumour tissue revealed that the cellswere thyroid transcription factor1 TTF1negative sallike protein SALL4positive and cluster of differentiation CD30positive These ï¬ndings were consistentwith a ï¬nal diagnosis of metastatic embryonal carcinomaFig We checked the levels of tumour markers in thepatient including those of alphafetoprotein AFP betahuman chorionic gonadotropin BhCG and lactate dehydrogenase LDH Each tumour marker was found to be The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of RespirologyThis is an access under the terms of the Creative Commons AttributionNonCommercialNoDerivs License which permits use and distribution in any mediumprovided the original work is properly cited the use is noncommercial and no modiï¬cations or adaptations are made Vol Iss e00644Page 0cEBM from embryonal carcinomaCK Teng et alFigure Chestradiograph and bronchoscopic view of the endobronchial metastasesEBM A Complete collapse of the left lungon chest radiograph B Bronchoscopic viewof the endobronchial tumour within the leftmain bronchusFigure Tumour pathology of metastatic embryonal carcinoma A Embryonal carcinoma with a complex glandular growth pattern The characteristic large cohesive and highly pleomorphic tumour cells with moderate amounts of amphophilic cytoplasm overlapping nuclei vesicular chromatinand frequent mitoses are shown as indicated by the arrows original magniï¬cation B Immunohistochemical staining with antithyroid transcription factor1 TTF1 highlighting cells in the alveolar space original magniï¬cation C Immunohistochemical staining with antisallikeprotein SALL4 revealed diffuse nuclear staining original magniï¬cation D Immunohistochemical staining with anticluster of differentiation CD30 highlighting diffuse membranous staining original magniï¬cation presentin high levels AFP ngmL BhCG mIUmL and LDH IUL The patientunderwent a right orchiectomy followed by a BEPbleomycin etoposide cisplatin chemotherapy regimenDiscussionWe report here the case of a young man with an EBMfrom a primary embryonal carcinoma who presentedshortness of breath and haemoptysis chest radiography atpresentation revealed complete collapse of the left lungtumoursLikewise manykindsLung metastases from extrapulmonary malignancies arereported relatively frequently in clinical practice howeverEBMs from extrapulmonary malignancies are rare [] Primary lung cancer is the most common cause ofendobronchialofextrapulmonary primary tumours have been associatedwith EBM primarily breast colon and renal carcinomas[] EBMs from testicular seminomas are also extremelyrare The majority of testicular tumours arise from testicular germ cells and are frequently composed of multiple celltypesaretumours most ofie mixedtypethese The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0cCK Teng et alEBM from embryonal carcinomaTable Reports of previous cases of EBMsLocationDiagnostic methodPathologyzt¼rk []MoreiraMeyer []Case Case The oriï¬ce of right upper lobeRight main bronchusLeft main bronchus main carina andright main bronchus Fibreoptic bronchoscopy Mixed GCTFibreoptic bronchoscopy Mixed GCTVideobronchoscopyEmbryonal carcinomazsu []The oriï¬ce of the right upper lobeFibreoptic bronchoscopyTesticular seminomaTuran []Varkey []Our caseand right intermediary lobeRight intermediate bronchusLeft main bronchusLeft main bronchusEBM endobronchial metastases GCT germ cell tumourembryonic carcinomas or seminomas There are only a few published reports of primary testicularembryonic carcinomas resulting in EBMs []The mostofcommon symptomsendobronchialtumours are haemoptysis and coughing with shortness ofbreath and wheezing reported less frequently Howeversome patients are asymptomatic [] In our patient symptoms on presentation included haemoptysis and shortnessof breathResults from chest radiography in patients with EBMcan be quite variable and may include mediastinal lymphadenopathy hilar masses atelectasis and multiple pulmonary nodules chest radiographs may also be normal onpresentation [] Our patient presented with complete collapse of the left lung that was revealed initially by chestradiographyHowever the full diagnosis cannot be made based onlyon symptoms and chest radiographs it can be difï¬cult todistinguish between primary lung cancer and tumours ofextrapulmonary origin based on these ï¬ndings alone Toconï¬rm the diagnosis we obtained a bronchoscopic biopsyspecimen to examine the tumour tissue The ï¬exible bronchoscopy ï¬breoptic bronchoscopy can be performedunder sedation without general anaesthesia as comparedwith rigid bronchoscopy The former is a simple techniquewhich is well tolerated and most commonly performed asan outpatient day procedure [] The patient underwentbronchoscopic cryosurgery under sedation in our bronchoscopy room to excise the tumour the ï¬nal pathology reportconï¬rmed the diagnosis of metastatic embryonal carcinomaWe had evaluated the presence of AFP BhCG andLDH tumour markers Elevated AFP levels can be secretedby germ cell tumours GCTs including embryonal carcinoma yolk sac tumour or teratoma In GCTs detectableRigid bronchoscopyBronchoscopyFibreoptic bronchoscopyand cryosurgerySomatictype GCTEmbryonal carcinomaEmbryonal carcinomaBhCG elevation is observed in both seminomas and nonseminomas The serum level of LDH was directly correlated with tumour burden in nonseminomatous GCTswhich is also useful for the surveillance of patients withadvanced seminoma [] The tumour markers in ourpatient showed elevated levels of AFP BhCG and LDHThis was compatible with the diagnosis of embryonal carcinoma MoreiraMeyer also evaluated the patienttumour markers and found elevated levels of AFP ngmL and BhCG mIUmL The elevatedlevels of both tumour markers contribute to the diagnosisof metastatic embryonal carcinoma [] zsu onlyevaluated the patients BhCG level which was found to beelevated mIUmL and the ï¬nal diagnosis wasmetastatic testicular seminoma []On comparison with previous case reports Table ours was the ï¬rst case in which the tissue was obtainedusing cryosurgery Other reports obtained tissue samplesusing forceps biopsy alone or forceps biopsy combinedwith argon plasma coagulation APC to control bleedingCryobiopsy provided us with enough sample to performmore extensive immunohistochemical staining Cryobiopsyhas more successful diagnostic results than forceps biopsiesdue to larger and highquality artefactfree samplesHaemorrhage was observed to be similar during both procedures [] Further study on this topic will be needed toevaluate which of the diagnostic methods result in superioroutcomesIn conclusion EBMs from primary GCTs notably thoseassociated with total or partial collapse are extremely rareWe have presented this case to emphasize the importanceof distinguishing EBM from primary lung carcinoma andto report the ï¬rst case in which metastatic embryonalcarcinoma was diagnosed by bronchoscopic cryosurgery The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0cEBM from embryonal carcinomaDisclosure StatementAppropriate written informed consent was obtained forpublication ofand accompanyingimagesreportcasethisReferences zt¼rk A Aktas¸ Z and Yılmaz A Endobronchialmetastasis of mixed germ cell tumors two cases TuberkToraks Lee SH Jung JY Kim DH Endobronchialmetastases from extrathoracic malignancy Yonsei Med J Ikemura K Lin DM Martyn CP Endobronchialmetastasis from extrapulmonary neoplasms analysis ofclinicopathologic features and cytological evaluation bybronchial brushing Lung MoreiraMeyer A BautistaHerrera D Hern¡ndezembryonal EndobronchialGonz¡lez MCK Teng et alcarcinomaJ BronchologyInterv Pulmonol zsu S Erol MM Oztuna F Endobronchial metastasis from testicular seminoma Med Princ Pract tumoraltesticular germ cell Turan D Akif zg¼l M Kirkil GEndobronchial metastasis ofEurasian J Pulmonol et Varkey B and Heckman MG Diagnosis of a case ofembryonal carcinoma by bronchial biopsy Chest Paradis TJ Dixon J and Tieu BH The role of bronchoscopy in the diagnosis of airway disease J Thorac Dis Aktas Z Gunay E Hoca NT Endobronchialcryobiopsy or forceps biopsy for lung cancer diagnosisAnn Thorac Med Barlow LJ Badalato GM and McKiernan JM Serumtumor markers in the evaluation of male germ cell tumorsNat Rev Urol The Authors Respirology Case Reports published by John Wiley Sons Australia Ltdon behalf of The Asian Paciï¬c Society of Respirology 0c'
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"Methods A literature search was undertaken until July 2013 to identify the comparative studies evaluating disease-free survival rates and survival rates. The pooled odds ratios (OR) and the 95% confidence intervals (95% CI) were calculated with the fixed or random effect models. Results Six retrospective studies were included in our meta-analysis. These studies included a total of 546 patients: 235 patients were treated with VATS and 311 patients were treated with open thoracotomy. The VATS and the thoracotomy did not demonstrate a significant difference in the 1-3-5-year survival rates and the 1-year disease-free survival rate. There were significant statistical differences between the 3-year disease free survival rate (p?=?0.04) which favored open thoracotomy. Conclusions The VATS approach is a safe and feasible treatment in terms of the survival rate for metastatic lung cancer compared with the thoracotomy. The 3-year disease-free survival rate in the VATS group is inferior to that of open thoracotomy. The VATS approach could not completely replace open thoracotomy. The authors have no support or funding to report. Introduction Metastasectomy is considered a beneficial treatment for a patient with metastatic lung cancer whose primary tumor has been well controlled[1].After surgery 5-year survival rates of 30% to 50% could be achieved depending on the underlying primary cancer[2][4].In practice the surgical approaches to pulmonary metastases are variable. Video-assisted thoracoscopic surgery (VATS) is an emerging technique; many procedures that had previously required a thoracotomy have been performed with the minimally invasive VATS. VATS has been used for the treatment of pulmonary metastases. The routine use of VATS for the treatment of respectable metastatic lung cancer remains controversial. Critics of the VATS approach have argued that it might not be an equivalent oncological operation[5] [6]. A prospective study by Cerfolio[7]found that 22% of the nodules that could be detected by thoracotomy were missing by VATS.Whether the VATS approach can provide a satisfactory outcome is unknown. An evidenced-based investigation of the VATS approach is needed we undertook this meta-analysis to achieve a more objective assessment of the published studies and to provide a more accurate comparison between VATS and thoracotomy for metastatic lung cancer. Methods Search Strategy Electronic searches were of the MEDLINECochrane Controlled Trial Register (CENTRAL) Ovid MEDILINE PubMed and Embase databases were performed until July 2013.The following MeSH search headings were used: metastatic lung cancer pulmonary metastases video-assisted thoracic surgery thoracotomy and comparative study.We searched the reference lists of relevant studies reviews editorials lettersand meeting s. We used the Science Citation Index to cross-reference for further studies that met our criteria. Study Selection The studies included in this meta-analysis were based on our predetermined criteria as follows: (1) clinical trials that include the full text of the paper published in peer-reviewed English journals or reports of presentations at major thoracic surgery meetings; (2) comparison of the efficacy of VATS to that of thoracotomy in patients with metastatic lung cancer; and (3) similarity in the patients' baseline characteristics. Data extraction and quality assessment Two independent reviewers (Siyuan and Wenya) assessed the quality and the risk of bias of the included trials as follows: (1) the studies that did not include a comparative group with surgery as a form of intervention were excluded; (2) the trials focusing on patients undergoing surgery for primary lung cancer were excluded; (3) the studies on robotic video-assisted thoracic surgery were excluded; (4) if there was an overlap between authors centers or patient cohorts evaluated in the published literature only the most recent report was included; (5) studies published more than 20 years ago were excluded because of the significant technological changes that has occurred. The s were evaluated with the Downs and Black quality assessment method[8]. Discrepancies between the two investigators were resolved by discussion and consensus with a senior investigator. The final results were reviewed by two senior investigators (Lin and Jiang).The disease-free survival was defined as the date of the initial metastasectomy until the date of a recurrence. Statistical and sensitivity analyses The meta-analysis was performed using the RevMan 5.1.0. software package. The odds ratio (OR) or the mean difference with 95% confidence intervals (95% CI) was calculated for the dichotomous outcomes and the continuous outcomes respectively. A P value<0.05 was considered a significant difference in the value between the two groups. We used the I2 statistic to investigate the heterogeneity among the studies.The heterogeneity was explored by X2 and I2; I2<25% and I2>50% reflect a small and large inconsistency respectively. P<0.05 was considered significant. If there were a statistical difference in terms of the heterogeneity (P?0.05) a random-effect model was selected to pool the data. Otherwise a fixed-effect model was used. Taking into account the presence of different sample sizes of the included studies a sensitivity analysis was performed to compare the of 1-year survival rate and the 3-year disease free survival rate between VATS and open thoracotomy. Publication bias A funnel plot was used to explore bias. Asymmetry in the funnel plot of trial size against treatment effect was used to assess the risk of bias. Results Description of the studies Six retrospective cohort studies the met our criteria were included in this meta-analysis. A total of 546 patients were included in the six studies;235 patients were allocated to the VATS group whereas 311 were allocated to the open thoracotomy group to evaluate their survival rate.The search algorithm results of the search strategies and selection criteria are shown in Fig 1. The patient characteristics and evaluation index are shown in . .0085329.g001 Identification of studies for inclusion. .0085329.t001 Study Design Country NO(V/O) Gender (M/F) Mean age (years) Assessment score Nakajima2001[28] OC Japan 45/55 V59/41 O34/21 V55±15 O55±14 13 Mutsaerts2002[29] OC Netherlands 8/12 NR NR 19 Nakas2009[30] OC UK 25/27 V16/9 O 19/8 V69 O66 16 Carballo2009[31] OC USA 36/135 V18/18 O82/53 V58.5 O49 15 Gossot2009[32] OC France 31/29 V21/10 O13/16 V43 O40 18 Chao2012[33] OC Taiwan 90/53 V49/41 O35/18 NR 13 V VATS; O Open thoracotomy; NR Not reported; OC observational cohort. Assessment of Recurrence and Survival Six studies documented the 1-year survival rateand there was no significant heterogeneity among the six studies (x2?=?3.79 P?=?0.58I2?=?0%).A fixed effect model was used.The combined result is shown in Fig 2(OR?=?1.15; 95%CI 0.721.84; p?=?0.58). Because of the heterogeneity in sample size the sensitivity analyses were conducted using larger sample sizes. There was no difference between the two surgical methods with an OR of 1.00(95%CI 0.551.79) and with heterogeneity(?2?=?3.23P?=?0.07 I2?=?69%). Five studies reported the 3-year survival rate and heterogeneity was identified through the five studies (x2?=?11.32P?=?0.02I2?=?65%); and a random effect model was adopted (OR?=?1.07; 95%CI 0.502.27; p?=?0.86) (Fig 3). Three studies compared the 5-year survival rate (OR?=?0.96; 95%CI 0.342.71; p?=?0.93) with certain heterogeneity(x2?=?8.86P?=?0.01I2?=?77%) (Fig 4). .0085329.g002 1-year survival rate. Forest plot of the Odds Ratio(OR) of the 1-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g003 Figure 3 3-year survival rate. Forest plot of the Odds Ratio(OR) of the 3-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g004 Figure 4 5-year survival rate. Forest plot of the Odds Ratio(OR) of the 5-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. Four studies compared the 1-year disease free survival rate (OR?=?1.31; 95% CI 0.792.19; p?=?0.30)finding no significant heterogeneity among these studies (x2?=?1.82P?=?0.61I2?=?0%) (Fig 5) and four studies compared the 3-year disease free survival rate (OR?=?0.59; 95% CI0.380.91; p?=?0.02) finding no significant heterogeneity (x2?=?1.82P?=?0.61I2?=?0%) between the patients who underwent VATS and those who underwent open thoracotomy (Fig 6). Because of the heterogeneity in the sample size sensitivity analyses were conducted using larger sample size studies; however there was no difference between the two surgical methods with an OR of 1.71 (95% CI1.022.89) and with heterogeneity (?2?=? 3.07P ?=?0.22 I2?=?35%). There were significant 3-year disease free survival rate benefits with open thoracotomy. We attempted to evaluate the 5-year disease free survival rate.Only two studies reported these ratesand the published data were not sufficient for the combined analysis. .0085329.g005 Figure 5 1-year disease-free survival rate. Forest plot of the Odds Ratio(OR) of the 1-year disease free survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. .0085329.g006 Figure 6 3-year disease-free survival rate. Forest plot of the Odds Ratio(OR) of the 3-year survival rate following VATS versus open thoracotomy for metastatic lung cancer.The estimate of the OR of each individual trial corresponds to the middle of the squares and horizontal line gives the 95% CI.On each linethe numbers of events as a fraction of the total number randomized are shown for both treatment groups.For each subgroupthe sum of the statistics along with the summary OR is represented by the middle of the solid diamonds.A test of heterogeneity between the trials within a subgroup is given below the summary statistics. Publication bias Publication bias might exist when nonsignificant findings remain unpublishedthus artificially inflating the apparent magnitude of an effect.The funnel plots of the study are shown in Figure 7.The funnel plots of the 1-year survival rate following VATS and thoracotomy for the treatment of metastatic lung cancer showed asymmetry which suggested that there was some publication bias. .0085329.g007 Figure 7 Funnel plot of the outcome of 1-year survival rate. Discussion Many tumors can metastasize to the lungand colorectal and breast tumors are the most common primary tumors[9].Pulmonary resection has been shown to be beneficial for patients with resectable and isolated pulmonary metastases[10]. Traditional open thoracotomy and VATS are two principally different surgical methods for pulmonary metastasectomy.The selection of an approach depends more on the theoretical knowledge and personal experience of the surgeon than on the evidence. Over the past two decades several studies have demonstrated the benefits of VATS that included less postoperative pain shorter hospital stays a smaller degree of immunosuppression and enhanced recovery and the ability to tolerate adjuvant therapy[11][13]. Whether the long-term advantages are comparable to those of open thoracotomy is not well documented. The major deficiency of the VATS approach is that nodules might be undetected by VATS that might be detected by manual palpation during thoracotomy; such missing nodules are not imaged on a preoperative CT scan. The VATS approach has long been controversial because VATS does not consistently detect all the metastases and it is recognized that complete resection remains a major determining factor of survival [14].The detection rate of HRCT for pulmonary metastases is 7884%[15][17].Kayton[18]found that 35% of the pathologically verified metastases were missed by CT. In the International Registry of Lung Metastases study of 5206 patients the 5-year survival rate was 36% for complete resection compared with 13% incomplete resectoin[19]. It is not certain whether the nodule imaged on a CT scan and resected by VATS is the correct one [14]. Those who disagree with the use of VATS hypothesize that VATS-related recurrence is commonly observed including port-site recurrence and resection stump recurrence[20]. Johnstone reported 23 cases of port-site chest wall recurrence related to VATS[21]. They hypothesized that the thoracoscopic approach should only be used in patients with a solitary lesion and when resection is requried for diagnostic purposes. The surgeons who favor the VATS approach advocate that VATS minimizes pain and trauma to the patients and that the VATS group might have an improved tolerance of chemotherapy which would likely ensure delivery of planned post-resection adjuvant therapy without a reduction in dosage or delay. The standard surgical procedure for pulmonary metastases is wedge resection that usually does not require manipulation of the pulmonary hilum which is appropriate for the VATS approach.They hypothesiezd that a lesion overlooked by CT but detected by palpation might not result in a survival gain[22] [23] and may be partially compensated for by carefully follow-up.Flores[24] hypothesized that the VATS group might demonstrate a great number of metachronous tumors over time;however the metachronous lesions in each group was similar. Our work suggests that thoracoscopic resection of metastatic lung cancer is a safe and curative procedure with 13 and 5-year survival rates comparable to those of thoracotomy. Patients with metastatic lung cancer are likely to relapse in the lung and after lung metastasectomy by VATS patients might benefit from a second metastasectomy. We hypothesize that earlier chemotherapy and radiation are essential to maximizing survival. Our study might be subject to pretreatment selection bias because most of the patients selected for open thoracotomy had multiple lesions and high risk and were not suitable for treatment with VATS.The missing lesions perhaps skewed the data more toward VATS as an equivalent procedure. We were also interested in the recurrence of cancerand the disease-free survival rates were evaluated. This study demonstrates a similar 1-year disease-free survival rate;however the 3-year disease-free survival rate is inferior for three reasons. First unrelated cancer deaths were included in our analysis of the 13 and 5-year overall survival which might account for VATS having a comparable overall survival rate but an inferior disease-free survival rate. Secondthe patients in the VATS group might have lesions that are missed and there are more likely to relapse in the lung leading to the inferior 3-year disease-free survival rate.Third some of our included studies were in the early period of VATS development when the technology was immature and some of the complications can now be prevented with more experience. Schaeff[25] reported 23 cases of port-site recurrence associated with VATS that occurred before 1998.The number of cases studied was small and the observation period was limitied. Spiral computed tomography has a far higher detection rate today than it did 20 years ago;so small lesions can be accurately localized before surgery[26] which ensures the success of VATS. With advances in imaging technology palpaiton during open thoracotomy is becoming less important.The latest VATS technology has a high-definition resolution and the flexible-tip thoracoscope enables complete inspection of the pleural cavity.These advancements ensure that VATS is an ideal method for patients with a solitary and relatively small peripheral lesions.Tamas[27] hypothesizes that palpation is necessary in a therapeutic metastasectomy as opposed to a diagnostic procedure.Whether patients with multiple lesions should be treated with open thoracotomy or VATS is controversial. This study is the first meta-analysis of the oncological outcome of thoracoscopic surgery for the treatment of metastatic lung cancer. In our work we observed that VATS might be a promising treatment for metastatic lung cancer. No randomized trials existing to guide doctors in the field of metastatic lung cancer currently. A prospective randomized study of the different surgical strategies is needed. Limitation No randomized controlled trials existing to comparing VATS with thoracotomy have been conducted. Heterogeneity was observed between the sample size and the years covered. Most studies are limited to small observational studies and single-institution case series. For these reasonsthere are only a total of 546 patients were included in the two groups for a study period spans more than a decade. Two of the studies comprise almost 65% of the patients and one study has only 20 patients; there are potential sources of bias in our work.Additional randomized controlled trials in the studies we accessed would have increased the strength of our results.There is a bias for the English language. Conclusion In our meta-analysis we found that for patients with metastatic lung cancer comparing VATS with thoracotomy showed almost equivalent survival rates. The VATS can not replace open thoracotomy completely. Further study is neededand a large multicenter randomized trial comparing VATS and thoracotomy would be ideal. Supporting Information Checklist S1 PRISMA Checklist. (DOC) Click here for additional data file. References 1 RuschVW (2010) Pulmonary metastasectomy: a moving target. J Thorac Oncol5: S13013120502246 2 CassonAG PutnamJB NatarajanG JohnstonDA MountainC et al (1992) Five-year survival after pulmonary metastasectomy for adult soft tissue sarcoma. Cancer69: 6626681730117 3 van HalterenHK van GeelAN HartAA ZoetmulderFA (1995) Pulmonary resection for metastases of colorectal origin. Chest107: 152615317781341 4 KandiolerD KromerE TuchlerH EndA MullerMR et al (1998) Long-term results after repeated surgical removal of pulmonary metastases. Ann Thorac Surg65: 9099129564899 5 McCormackPM BainsMS BeggCB BurtME DowneyRJ et al (1996) Role of video-assisted thoracic surgery in the treatment of pulmonary metastases: results of a prospective trial. Ann Thorac Surg62: 213216 discussion 216217.8678645 6 SaishoS NakataM SawadaS YamashitaM SaekiH et al (2009) Evaluation of video-assisted thoracoscopic surgery for pulmonary metastases: 11-years of experience. Surg Endosc23: 556118437482 7 CerfolioRJ BryantAS McCartyTP MinnichDJ (2011) A prospective study to determine the incidence of non-imaged malignant pulmonary nodules in patients who undergo metastasectomy by thoracotomy with lung palpation. Ann Thorac Surg91: 16961700 discussion 17001691.21619965 8 DownsSH BlackN (1998) The feasibility of creating a checklist for the assessment of the methodological quality both of randomised and non-randomised studies of health care interventions. J Epidemiol Community Health52: 3773849764259 9 KondoH OkumuraT OhdeY NakagawaK (2005) Surgical treatment for metastatic malignancies. Pulmonary metastasis: indications and outcomes. Int J Clin Oncol10: 818515864692 10 PorterGA CantorSB WalshGL RuschVW LeungDH et al (2004) Cost-effectiveness of pulmonary resection and systemic chemotherapy in the management of metastatic soft tissue sarcoma: a combined analysis from the University of Texas M. D. Anderson and Memorial Sloan-Kettering Cancer Centers. J Thorac Cardiovasc Surg127: 1366137215115994 11 PetersenRP PhamD BurfeindWR HanishSI TolozaEM et al (2007) Thoracoscopic lobectomy facilitates the delivery of chemotherapy after resection for lung cancer. Ann Thorac Surg83: 12451249 discussion 1250.17383320 12 PaulS AltorkiNK ShengS LeePC HarpoleDH et al (2010) Thoracoscopic lobectomy is associated with lower morbidity than open lobectomy: a propensity-matched analysis from the STS database. J Thorac Cardiovasc Surg139: 36637820106398 13 WhitsonBA GrothSS DuvalSJ SwansonSJ MaddausMA (2008) Surgery for early-stage non-small cell lung cancer: a systematic review of the video-assisted thoracoscopic surgery versus thoracotomy approaches to lobectomy. Ann Thorac Surg86: 20082016 discussion 20162008.19022040 14 EckardtJ LichtPB (2012) Thoracoscopic versus open pulmonary metastasectomy: a prospective sequentially controlled study. Chest142: 1598160222677347 15 AmbrogiV PaciM PompeoE MineoTC (2000) Transxiphoid video-assisted pulmonary metastasectomy: relevance of helical computed tomography occult lesions. Ann Thorac Surg70: 1847185211156082 16 MargaritoraS PorziellaV D'AndrilliA CesarioA GalettaD et al (2002) Pulmonary metastases: can accurate radiological evaluation avoid thoracotomic approach? Eur J Cardiothorac Surg21: 1111111412048094 17 ParsonsAM DetterbeckFC ParkerLA (2004) Accuracy of helical CT in the detection of pulmonary metastases: is intraoperative palpation still necessary? Ann Thorac Surg78: 19101916 discussion 19161918.15561000 18 KaytonML HuvosAG CasherJ AbramsonSJ RosenNS et al (2006) Computed tomographic scan of the chest underestimates the number of metastatic lesions in osteosarcoma. J Pediatr Surg41: 200206 discussion 200206.16410133 19 Long-term results of lung metastasectomy: prognostic analyses based on 5206 cases. The International Registry of Lung Metastases. J Thorac Cardiovasc Surg113: 37499011700 20 MutsaertsEL ZoetmulderFA RutgersEJ (2001) Port site metastasis as a complication of thoracoscopic metastatectomy. Eur J Surg Oncol27: 32732811373113 21 JohnstonePA RohdeDC SwartzSE FetterJE WexnerSD (1996) Port site recurrences after laparoscopic and thoracoscopic procedures in malignancy. J Clin Oncol14: 195019568656265 22 TreasureT (2007) Pulmonary metastasectomy: a common practice based on weak evidence. Ann R Coll Surg Engl89: 74474817999813 23 RothJA PassHI WesleyMN WhiteD PutnamJB et al (1986) Comparison of median sternotomy and thoracotomy for resection of pulmonary metastases in patients with adult soft-tissue sarcomas. Ann Thorac Surg42: 1341383741009 24 FloresRM IhekweazuUN RizkN DycocoJ BainsMS et al (2011) Patterns of recurrence and incidence of second primary tumors after lobectomy by means of video-assisted thoracoscopic surgery (VATS) versus thoracotomy for lung cancer. J Thorac Cardiovasc Surg141: 596421055770 25 SchaeffB PaolucciV ThomopoulosJ (1998) Port site recurrences after laparoscopic surgery. A review. Dig Surg15: 1241349845574 26 ChenYR YeowKM LeeJY SuIH ChuSY et al (2007) CT-guided hook wire localization of subpleural lung lesions for video-assisted thoracoscopic surgery (VATS). J Formos Med Assoc106: 91191818063512 27 MolnarTF GebitekinC TurnaA (2010) What are the considerations in the surgical approach in pulmonary metastasectomy? J Thorac Oncol5: S14014420502249 28 NakajimaJ TakamotoS TanakaM TakeuchiE MurakawaT et al (2001) Thoracoscopic surgery and conventional open thoracotomy in metastatic lung cancer. Surg Endosc15: 84985311443456 29 MutsaertsEL ZoetmulderFA MeijerS BaasP HartAA et al (2002) Long term survival of thoracoscopic metastasectomy vs metastasectomy by thoracotomy in patients with a solitary pulmonary lesion. Eur J Surg Oncol28: 86486812477479 30 NakasA KlimatsidasMN EntwisleJ Martin-UcarAE WallerDA (2009) Video-assisted versus open pulmonary metastasectomy: the surgeon's finger or the radiologist's eye? Eur J Cardiothorac Surg36: 46947419464921 31 CarballoM MaishMS JaroszewskiDE HolmesCE (2009) Video-assisted thoracic surgery (VATS) as a safe alternative for the resection of pulmonary metastases: a retrospective cohort study. J Cardiothorac Surg4: 1319239710 32 GossotD RaduC GirardP Le CesneA BonvalotS et al (2009) Resection of pulmonary metastases from sarcoma: can some patients benefit from a less invasive approach? Ann Thorac Surg87:"
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"PAX6 expression was obviously weakened in A549 PAX6 KD and H1299 PAX6 KD cells. To elucidate whether PAX6 expression has any effect on the growth of lung cancer cells RNAi was used to generate pax6 knock-down (PAX6 KD) cell lines. We selected two target cell lines: H1299 which showed high levels of PAX6 expression and A549 which showed low levels of expression. In the present study pGCSIL-pax6 shRNA-GFP was infected into H1299 and A549 cells. Cells were also infected with pGCSIL-NS shRNA-GFP (PAX6 NS) as a negative control (NC). To determine the function of PAX6 H1299 H1299NC A549 or A549NC cells were used as controls in all assays. The PAX6 mRNA level in H1299 PAX6 KD and A549 PAX6 KD cells was determined by real-time PCR to confirm whether PAX6 expression was specifically inhibited through RNAi in A549 and H1299 cells. As shown in B PAX6 expression in A549 PAX6 KD cells was inhibited by 8090% compared to cells infected with lentivirus-mediated NS-shRNA. We found similar results in H1299 PAX6 KD cells. PAX6 mRNA expression in these cells was also inhibited by 9095% as compared to NC cells (**P<0.01; C). PAX6 protein expression in these cells was detected by Western blotting. As shown in D PAX6 protein in H1299 PAX6 KD and A549 PAX6 KD cells was not readily detected whereas a clear PAX6 protein band was evident in the control cells. Inhibition of PAX6 expression leads to a decline in cell proliferation PAX6 is a critical transcription factor that plays an important role in regulating proliferation and differentiation during human embryonic development [3]. A cell proliferation assay was performed to determine whether PAX6 plays a role in cellular growth. A549 PAX6 KD H1299 PAX6 KD and control cells were seeded in 96-well plates and cell proliferation activity was measured using a Cell Proliferation Assay kit. A549 and H1299 cell growth was obviously suppressed when PAX6 expression was inhibited by RNAi (A and B). As shown in A and B the decrease in cell growth caused by the inhibition of PAX6 expression in H1299 was much stronger than that in A549 cells. These different results may be attributable to the different PAX6 expression levels between H1299 and A549 cells displayed in A and D. .0085738.g002 The lentivirus-mediated pax6-shRNA knockdown of PAX6 expression could suppress lung cancer cell growth. A -B A549 PAX6 KD H1299 PAX6 KD cells and control cells were seeded in 96-well plates and an MTS assay was performed. The absorbance at 490 nm (y -axis) was measured at 24-h intervals up to 120 h. C -D Colony formation efficiency in A549 PAX6 KD H1299 PAX6 KD cells and control cells. The y -axis represents the normalized colony formation rate relative to A549 (C) or H1299 (D) cells. E -F A soft -agar assay was performed to investigate the effects of PAX6 on tumorigenesis in vitro. The y -axis represents the normalized soft -agar colony formation rate relative to A549 (E) or H1299 (F) cells. The data are expressed as the means ± SEM from three separate experiments. Times of the experiments are listed above the graph.*P <0.05 **P <0.01. Reduced colony formation and soft-agar colony formation in PAX6 KD cells Colony formation represents a loss of contact inhibition or the ability to maintain cell growth and movement despite contact with surrounding cells. To clarify whether PAX6 could confer a loss of contact inhibition cells infected with pGCSIL-pax6 shRNA-GFP as well as their control cells were seeded into 6-well plates and cultured for 7 days. After 2% gentian violet staining colonies containing more than 50 cells were counted under a light microscope. As displayed in C and D the inhibition of PAX6 expression in A549 and H1299 cells led to an obvious decrease in the number of foci generated compared to the control cells (**P<0.01). To further study the function of PAX6 soft-agar colony formation was analyzed to determine whether PAX6 contributed to anchorage-independent colony formation in lung cancer cells. The rate of soft-agar colony formation declined in A549 PAX6 KD and H1299 PAX6 KD cells compared to NC cells (**P<0.01 *P<0.05; E and F). PAX6 expression increased cell growth by promoting faster progression into S phase of the cell cycle To detect the effect of PAX6 on cell cycle progression the cell cycle progression of A549 PAX6 KD H1299 PAX6 KD A549 PAX6 NC H1299 PAX6 NC A549 and H1299 cells was analyzed by flow cytometry. As displayed in A the percentage of cells entering S phase was decreased in the A549 PAX6 KD cell line along with an increase in the population of G0-G1 phase cells. A similar result was observed in H1299 PAX6 KD H1299 PAX6 NC and H1299 cells (B). In these experiments PAX6 expression led to cell growth by inducing cell cycle progression. .0085738.g003 PAX6 expression promoted cell cycle progression. A B Cell cycle analysis. Cells were stained with propidium iodide (PI) and analyzed for cell cycle phase distribution. The histogram was the statistical data from three independent experimental replicates. *P <0.05 **P <0.01. In this study the expression level of cyclin D1 a relevant cyclin regulating G1-S progression [15] [16] was detected in A549 PAX6 KD and H1299 PAX6 KD cells. As indicated in A cyclin D1 expression was decreased in A549 PAX6 KD cell lines compared to control cells."
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"Vitamin D is a fatsoluble vitamin vitamin D is essential to sustain health and it protects againstosteoporosis It is crucial to the human bodys physiology in terms of muscular movement and neurological signaltransmission and to the immune system in defense against invading pathogensCase presentation This was a case of a 26yearold Sudanese woman who presented with a 2year history ofanosmia recurrent nasal polyps back pain and chronic fatigue She was diagnosed as having a case of vitamin Ddeficiency and responded well to treatmentConclusion There is an association between vitamin D deficiency and recurrent allergic nasal conditionsKeywords Vitamin D deficiency Allergy Nasal polyps Backache Chronic fatigabilityBackgroundVitamin D is a fatsoluble vitamin it is naturally presentin some foods and as dietary supplements It is also produced endogenously through exposure to ultraviolet raysfrom sunlight Vitamin D obtained from sun exposurefood and supplements is biologically inert and mustundergo two hydroxylations in the body for activationTheand produces hydroxyvitamin D 25OHD also known as calcidiolThe second occurs in the kidney and forms the physiologically active 125dihydroxy vitamin D 125OH2D alsoknown as calcitriol []first occursin theliverVitamin D is found in cells throughout the body vitamin D is essential to sustain health and it protectsagainst osteoporosis It is crucial to the human bodysphysiology in terms of muscular movement and neurological signal transmission and to the immune system indefense against invading pathogens []Although there are different methods and criteria fordefining vitamin D levels the criteria Holick proposed Correspondence drmuhanadkamalhotmailcom1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum SudanFull list of author information is available at the end of the have been widely accepted In this proposal vitamin Ddeficiency is defined as blood level of less than ngmlinsufficiency of vitamin D is defined as blood levels ranging between and ngml and sufficiency if greaterthan or equal to ngml [] About one billion peopleglobally have vitamin D deficiency and of the population has vitamin D insufficiency The majority ofaffected people with vitamin D deficiency are the elderlyobese patients nursing home residents and hospitalizedpatients Vitamin D deficiency arises from multiplecauses including inadequate dietary intake and inadequate exposure to sunlight Certain malabsorption syndromes such as celiac disease short bowel syndromegastric bypass some medications and cystic fibrosis mayalso lead to vitamin D deficiency []Vitamin D deficiency is now more prevalent than everand should be screened in highrisk populations Manyconflicting studies now show an association betweenvitamin D deficiency and cancer cardiovascular diseasediabetes autoimmune diseases and neuropsychiatricdisorders [ ] The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of two functional endoscopic sinusCase presentationThis was a case of a 26yearold Sudanese woman married who has a 3yearold boy This woman presentedto our ear nose and throat ENT department complaining of anosmia for the past years She had a history ofsurgeriesFESSs for nasal polyps the first one was years agoand the second one was years prior to presentationShe complained of being highly sensitive to different irritants including dust weather change perfumes andpetsShe also stated that she attended more than three different physicians due to generalized fatigue and gettingtired easily after simple daily activity in addition to sleeping for more than hours a dayShe attended an orthopedic clinic for unspecified lower back pain that was notrelated to any type of trauma or physical activity a lumbosacral magnetic resonance imaging MRI was done andrevealed no abnormal findingsShe mentioned that she isknown to be anxious most of the time and aggressive toward simple reactions from her family members She hadno psychiatric history and was not using any medicationsShe was not known to be diabetic or hypertensive orto have any chronic illnesses she was not on any regularmedication She is a housewife of high socioeconomicstatus she is well educated graduated from dentalschool with a bachelors degree but currently notemployed She has never consumed tobacco or alcoholshe practiced regular cardio exercisesOn examinationshe looked healthy well not pale or jaundiced Herpulse rate was 74minute and her blood pressure was Her body mass index BMI was All systems examinations were normal except for bilateral nasalpolyps Complete blood count CBC renal function testREF electrolyte liver function test LFT thyroid function test TFT urine analysis general urine test antinuclear antibody ANA and rheumatoid factor RFwere all normal An imaging profile included lumbosacral MRI a computed tomography CT scan of her sinuses and electrocardiogram ECG which were normalexcept for bilateral nasal polyps and severe sinusitis thatlooked allergic to fungi in natureShe underwent FESSsurgery to remove the polyps and clean out her sinusesup to weeks after surgery she used nasal steroidsmometasone furoate two times a day but hersymptoms regarding anosmia were not improved MRIof her brain and a CT scan of her sinuses were done andboth revealed normal features A vitamin D deficiencywas suggested and the laboratory results revealed a lowvitamin D level of ngml Treatment with vitamin Dsupplement was prescribed at international unitsIU weekly for weeks and then IU maintenancedose daily she was advised to take food rich in vitaminD and get exposed to sunlight for minutes three timesa week after the loading dose of supplement She was atregular followup for months at rates of weekly for thefirst month every weeks for the second month andmonthly for the rest of the followup period At eachvisit she was assessed with clinical history and examination It was noticed that the symptoms of tirednesssleeping anosmia and back pain were dramatically improving during that period At the months followupher blood level of vitamin D was normal she describedher condition as free from all symptoms and shereturned back to normal physical activityDiscussion and sThis was a nonclassical case of vitamin D deficiency ofa 26yearold woman who presented with chronic anosmia and recurrent nasal polyps She was diagnosed ashaving a case of vitamin D deficiency and respondedwell to vitamin D replacement therapy This case correlated an association between decreased levels of vitaminD and recurrent nasal polyps that led in time to chronicanosmia as a result of chronic high sensitivity reactionstriggered by our patients autoimmune system The literature links chronic rhinosinusitis with nasal polypsCRSwNP with asthma and allergic rhinitis but the cellular and molecular mechanisms that contribute to theclinical symptoms are not fully understood Sinonasalepithelial cell barrier defectsincreased exposure topathogenic and colonized bacteria and dysregulation ofthe host immune system are all thought to play prominent roles in disease pathogenesis []Despite all the previous surgical and medical interventions over the past years our patients condition did notimprove and she still complained of anosmia A study revealed that this patient was experiencing excessive allergicreactions that led to recurrent nasal polyps It is wellknown that classical clinical effects of vitamin D deficiencyare bones and musculoskeletalrelated disorders severallines of evidence demonstrate the effects of vitamin D onproinflammatory cytokines regulatory T cells and immune responses with a conflicting interpretation of theeffects of vitamin D on allergic diseases []The working diagnosis was suggested in relation tosome musculoskeletal symptoms and chronic fatigue especially when the imaging profile for her lower back andall routine investigations were normal It has been suggested that clinicians should routinely test for hypovitaminosis D in patients with musculoskeletal symptomssuch as bone pain myalgias and generalized weaknesswhich might be misdiagnosed asfibromyalgia andchronic fatigue [] The most common causes of anosmia were assessed as well and they were negative theseincluded sinonasal diseases post infectious disorder andposttraumatic disorder and congenital defects and disorders caused by neurodegenerative disease [] 0cIbrahim and Elnimeiri Journal of Medical Case Reports Page of Authors contributionsMI analyzed and interpreted the findings of the case report and was themajor contributor in writing the manuscript ME reviewed the report andadded valuable comments All authors read and approved the finalmanuscriptFundingNoneAvailability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from Albasar Institutional Review BoardConsent for publicationWritten informed consent was obtained from the patient for publication ofthis case report and any accompanying images A copy of the writtenconsent is available for review by the EditorinChief of this journalCompeting interestsThe authors declare that they have no competing interestsAuthor details1Community Medicine and Epidemiology Faculty of Medicine Ibn SinaUniversity Khartoum Sudan 2Preventive Medicine and EpidemiologyAlneelain University Khartoum SudanReceived March Accepted July Thus blood level for vitamin D was requested and theresults were of low D levelIn the past history of the previous nasal polyps surgeries our patient noted that there was no anosmia and hermain complaints were classic complaints of sinusitis including sneezing nasal blockage and headache Soonafter surgery her symptoms improved except for theallergyrelated symptoms despite usage of inhaled steroids spray She stated that at the last time the presentation was different since it was only anosmia indicatingthat there was significant inflammation that affected thesmell receptors around the olfactory epithelium Afterthe last nasal polyps and sinuses drainage surgery thesymptoms related to allergic reactions including chronicsneezing did not improve for up to weeks and she wasstill suffering from hyposmia although that was a fairpostoperative period for recoveryThe symptoms of anosmia and sneezing and othersystematic symptoms gradually started to improve aftervitamin D supplements indicating that the main reasonbehind her symptoms was vitamin D deficiency She wasfollowed up for up to months after establishment ofvitamin D supplements and at the last followup she hada normal sense of smell and she was free from backpain fatigue and allergyrelated symptomsThis was a nonclassical presentation as our patientwas young and she did not have alkaline phosphatasecalcium and phosphorus abnormalities [] that are expected in cases of vitamin D deficiencyThis case revealed an association between decreasedlevels of vitamin D and recurrent nasal polyps that ledto anosmia as a result of hypersensitive reactions produced by the bodys systemsAlthough vitamin D deficiency is prevalent measurement of serum 25OHD level is expensive and universalscreening is not supported However vitamin D testingmay benefit those at risk for severe deficiencyIt is highly recommended to consider vitamin D deficiency among all patients with unspecified symptoms orin cases of nondiagnosed disorder regardless of the presenting complaintIn there is an association between vitaminD deficiency and recurrent allergic nasal conditionsAbbreviationsCRSwNP Chronic rhinosinusitis with nasal polyps CT Computedtomography ECG Electrocardiogram ENT Ear nose and throatFESS Functional endoscopic sinus surgery BMI Body mass indexCBC Complete blood count RFT Renal function test LFT Liver function testTFT Thyroid function test ANA Antinuclear antibody RF Rheumatoid factorIU International unitReferencesNational Institutes of Health Vitamin D fact sheet for health professionals httpsodsodnihgovfactsheetsVitaminDHealthProfessionalen2Accessed Apr National Institutes for Health NIH Vitamin D fact sheet for consumers httpsodsodnihgovfactsheetsVitaminDConsumer Accessed Dec Kuriacose R Olive KE Vitamin D insufficiencydeficiency managementSouth Med J httpsdoi101097SMJSizar O Khare S Givler A Vitamin D deficiency Treasure Island Stat PearlsPublishing httpswwwncbinlmnihgovbooksNBK532266 PMID Accessed Dec Wlliam B Fatme A Meis M Targeted 25hydroxyvitamin D concentrationmeasurements and vitamin D3 supplementation can have important patientand public health benefits Eur J Clin Nutr httpsdoi101038s4143002005640Hanmin W Weiwen C Dongqing L Xiaoe Y Xiaode Z Nancy O et alVitamin D and chronic diseases Aging Dis httpsdoi1014336AD20161021 Whitney W Ropert P Robert C Chronic rhinosinusitis with nasal polyps JAllergy Clin Immunol Pract httpsdoi101016jjaipThacher TD Clarke BL Vitamin D insufficiency Mayo Clin Proc httpsdoi104065mcp20100567Kennel KA Drake MT Hurley DL Vitamin D deficiency in adults when totest and how to treat Mayo Clin Proc httpsdoi104065mcp20100138Sanne B Elbrich M Duncan B Antje W Veronika S Joel D et al Anosmia aclinical review Chem Senses httpsdoi101093chemsebjx025Shikino K Ikusaka M Yamashita T Vitamin Ddeficient osteomalacia due toexcessive selfrestrictions for atopic dermatitis BMJ Case Rep httpsdoi101136bcr2014204558AcknowledgementsNot applicablePublishers NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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" patients who have undergone radical cystectomy for urinary bladder cancer are not sufficientlyphysically active and therefore may suffer complications leading to readmissions a physical rehabilitationprogramme early postoperatively might prevent or at least alleviate these potential complications and improvephysical function the main aim of the canmore trial is to evaluate the impact of a standardised and individuallyadapted exercise intervention in primary health care to improve physical function primary outcome and habitualphysical activity healthrelated quality of life fatigue psychological wellbeing and readmissions due tocomplications in patients undergoing roboticassisted radical cystectomy for urinary bladder cancermethods in total patients will be included and assigned to either intervention or control arm of the study allpatients will receive preoperative information on the importance of early mobilisation and during the hospital staythey will follow a standard protocol for enhanced mobilisation the intervention group will be given a referral to aphysiotherapist in primary health care close to their home within the third week after discharge the interventiongroup will begin weeks of biweekly exercise the exercise programme includes aerobic and strengtheningexercises the control group will receive oral and written information about a homebased exercise programmephysical function will serve as the primary outcome and will be measured using the sixminute walk test secondaryoutcomes are gait speed handgrip strength leg strength habitual physical activity healthrelated quality of lifefatigue psychological wellbeing and readmissions due to complications the measurements will be conducted atdischarge ie baseline postintervention and year after surgery to evaluate the effects of the intervention mixedor linear regression models according to the intention to treat procedure will be usedcontinued on next page correspondence andreaporserudkise1department of neurobiology care sciences and society division ofphysiotherapy karolinska institutet stockholm sweden2allied health professionals function medical unit occupational therapy andphysiotherapy karolinska university hospital stockholm swedenfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cporserud bmc cancer page of continued from previous pagediscussion this proposed randomised controlled trial has the potential to provide new knowledge withinrehabilitation after radical cystectomy for urinary bladder cancer the programme should be easy to apply to otherpatient groups undergoing abdominal surgery for cancer and has the potential to change the health care chain forthese patientstrial registration clinicaltrialsgov clinical trial registration number nct03998579 first posted june keywords abdominal surgery behaviour bladder neoplasm complications exercise physical activity primaryhealth care process evaluation the most common treatment for solid cancer tumoursis surgery often in combination with chemotherapy orradiotherapy or both minimising postoperative complications is important in health care for the individual patient and in reducing health care costs for society earlymobilisation at the ward and physical activity at homeafter discharge are important activities to reduce complications common complications after abdominal surgery are postoperative pulmonary complications andvenous thrombosis [ ] which generally are thought tobe partially avoidable with early mobilisationafter radical cystectomy for urinary bladder cancerthere is a high risk for postoperative complications thecomplications could be directly related to the patientshigh age to a high degree of comorbidity or both the major risk factor for developing urinary bladdercancer is smoking most of the patients are men andthe median age of undergoing a radical cystectomy is years [ ] as much as of patients are at severe nutritional risk before a radical cystectomy afterroboticassisted radical cystectomy rarc for urinarybladder cancer ofthe patients need to bereadmitted to hospital after discharge because of complications [ ]there is strong evidence that aerobic physical activityhas a positive impact on health survival and quality oflife qol patients diagnosed with cancer shouldfollow the general recommendations on physical activityand exercise for health [ ] yet most patients areinsufficiently active consequently with an increasing number of cancer survivors the importance of supporting high physical function and qol increases research has shown that exercise has a positive effect onhealthrelated qol hrqol in patients who have completed active cancer treatment moreoverin patients living with or beyond a diagnosis of cancerbehaviouraleg goalsetting andgraded tasks are important components in the exerciseinterventions with high adherence and positive physicaloutcomes support methodsin a recent study we evaluated the activity board®phystec sweden as a method to enhance mobilisationand recovery after abdominal surgery for cancer theactivity board is a tool based on techniques to supportbehaviour change [ ] the evaluation showed thatthe activity board resulted in a higher level of mobilisation in the group with the activity board compared withthe group who received standard treatment although evidence for exercise after abdominal surgery isscarce a few studies have evaluated exercise programmes for patients postoperatively atthe hospitalward with promising results [ ]despite the lack of exercise interventions after surgeryit has been shown that functional performance after aradical cystectomy for urinary bladder cancer correlatesto overall survival a large proportion of patientswith urinary bladder cancer do not achieve the recommendations on physical activity and exercise it isalso common that patients who undergo radical cystectomy have not performed physical exercise for a longtime before surgery finally after surgery patientsreport a low level of physical exercise recently tworeviews have been published on physical and psychological interventions to improve healthrelated outcomesin this patient group [ ] both reviews include thesame two postoperative exercise studies [ ] onelarger rct showed that early physical exercise and enhanced mobilisation after radical cystectomy positivelyaffected some domains of hrqol in addition in apilot study we tested a model for physical rehabilitationafter radical cystectomy the model consisted of weeks of individually tailored exercise after dischargefrom the hospital the exercise programme conductedat the hospital showed both short and longterm effectson physical function and hrqolconsequently the few studies within the field raiseseveral research questions for future exercise interventions in patients with urinary bladder cancer undergoingradical cystectomy current recommendations proposethe following areas supervised exercise after dischargethe optimal type of exercise fidelity and adherence ofthe intervention if shortterm outcomes are sustainedclinical relevance longterm outcomes and readmissionsto hospital [ ] we also need to understand thekinds of support that are optimal use behaviour change 0cporserud bmc cancer page of screened for eligibility in medical records by the responsible researcher rr and given written information by a registered nurse at a preoperative meetingafter days the rr will phone the patient provideoral information and then ask about participation informed consent will be signed before surgery the rrkeeps a protocol for enrolment the patient flowchartis depicted in fig eligibility criteriainclusion criteria will be patients who are planned for ararc for urinary bladder cancer the patients shouldbe able to talk and understand swedish without an interpreter be mobile with or without a walking aid and livein the stockholm regionstrategies and implement the intervention as a part ofthe patients clinical pathway through the healthcare system [ ]in summary patients who have been treated forurinary bladder cancer are not sufficiently physicallyactive and suffer from readmissions to hospital due tocomplications therefore there is a need for developing a physical rehabilitation programme to supportpatients who have a radical cystectomy in the earlypostoperative period in this paper we present a studyprotocol for the canmore trial a physical rehabilitation programme after rarc for urinary bladdercancerimpact ofamethodsdesignmain objectiveis to evaluatethe main aim of the canmore trialthestandardised and individuallyadapted exercise intervention in primary health carephcfunction primary outcome and habitual physical activity hrqol fatiguepsychological wellbeing and readmissions due tocomplications in patients undergoing rarc for urinary bladder cancerto improve physicalhypothesiswe hypothesise that the canmore programme is morebeneficial than homebased exercises in increasing physical function primary outcometrial designthe canmore trialis a randomised controlled trialrct with a singleblinded design the interventiongroup will receive a 12week h twice a week standardised and individually adapted exercise intervention inphc and behavioural support for daily physical activitythe control group will receive a homebased exerciseprogramme as well as recommendations on daily physical activity based on general guidelines we will followthe spirit standard protocol items recommendationsfor interventional trials statement and guidelinesfor reporting the study protocol the clinical trial registration number for this trial is nct03998579study settingthe study will be conducted in two settings a universityhospital and a phc context in region stockholmrecruitment and screeningparticipants will be recruited through theme canceratthe karolinska university hospital solna andscreened for eligibility recruitment will be performedconsecutively based on power analysis patientswill be included potential participants will initially befig patient flowchart 0cporserud bmc cancer page of exclusion criteriapatients with planned palliative surgery or cognitive impairment identified by screening of medical records willnot be includedrandomisation assignment of interventionpatients who fulfil the criteria for inclusion will afterhaving given their oral and written consent be randomised in the alea system operated by the clinical trials office cto atthe centre for clinical cancerstudies theme cancer karolinska university hospitalsolna swedenrandomisation will be conducted in blocks of patients stratified by sex and age ¥ years a confirmation email will be sent to the entering investigatorand an enrolment log will be filed at the centre the patient will receive the next consecutive code number inthe trial and treatment arm according to the randomisation schemeislogic model for the canmore programmeit is recommended that programme design should bebased on a theory to improve evidence synthesis the theoretical framework underpinning the canmoreprogrammethe movement continuum theorymct and the evidencebased calore taxonomy forbehavioural change techniques the mct posits that anindividual has three stages of movement capability amaximum a current and a preferred the canmoreprogramme identifies the patients current physical function capability and intervenes regarding the patientsneed for function several models and theories are supporting a behaviour change which results in increasedphysical activity however research has shown thatit is most often not necesssary for a complete theorybut instead the different components in the theory thatsupport the behaviour to consider the patients need forsupport the calore taxonomy for behavioural changetechniques has been added the taxonomy is recommended to be used to improve the specification of interventions behavioural techniques proven effective tosupport behaviour change are goalsetting graded tasksselfmonitoring feedback and reward all of these areused in the canmore programmea conceptual framework visualising the inputs theoryintervention components and its intermediate and possible longterm outcomes is depicted in a logic modelfig interventionall patients receive preoperative information on the importance of early mobilisation and postoperative individual physiotherapy during the hospital stay the activityboard is used for enhanced mobilisation before discharge from the hospital the patients receive standardised information about avoiding the lifting of heavyobjects and the importance of physical activity the patients are then randomised to either the intervention orthe control groupintervention grouppatients in the intervention group get a referral to aphysiotherapist in phc close to where they live the patients can choose from phc settings spread throughout the stockholm region within the third week afterdischarge the patients begin weeks of biweekly exercise the patients pay for their primary care visits physiotherapists in the targeted primary care units receive afig logic model of the intervention 0cporserud bmc cancer page of leaflet and a short education before starting comprisinginformation about rarc restrictions potential adverseevents the trial process and the exercise programmethe physical exercise is individually targeted but basedon international recommendations for persons with cancer disease the exercise programme includes aerobicexercise aiming at moderate intensity minsessionand strengthening exercises comprising endurance training with à repetitions see additional file theprogramme is gradually increased based on the patientscapability the programme also includes exercises forabdominal muscles including pelvic floor exercises tominimise the risk of developing stoma hernia however to avoid heavy strain on the surgery wounds restrictions regarding abdominal muscles are followed weeks postoperatively the exercise programme hasbeen approved by the responsible medical surgeons inaddition to the structured exercise sessions the patientsare advised to take daily walks in their neighborhoodthe number of recommended steps per day is set together with the physiotherapist based on the patientscapability to support the patient individual goalsettingfeedback and selfmonitoring of daily steps are usedsimilarly to those of the activity board at the end ofthe exercise periodthe physiotherapist recommendscontinued physical activity according to their clinicalroutinesanasapplicationactivitydiarypedometeroraphoneoutcomesthe measurements will be conducted using validated instruments at discharge ie baseline postintervention months and year after surgery table with thepurpose to receive information on the patients healthand physical function before surgery eg to adjust forin the analysis measurements will also be conductedbefore surgery all measurements will be conducted byexperienced physiotherapist blinded to the interventiona protocol for the measurements has been developedand the physiotherapist will receive specialised trainingby the research staffprimary outcomephysical function the primary outcome will be measured using the validated sixminute walk test 6mwt[ ] the test reproduces activity of daily living at asubmaximal level which is particularly applicable to elderly patients the patients are asked to walk as faras possible for min the number of meters m oxygensaturation and heart rate measured with a pulse oximeter will be recorded at the end of the test according tostandard procedures the primary outcome variablewill be walking distance in meterscontrol groupthe control group will receive oral and written information of a gradually increased homebased exerciseprogramme that includes daily walks and sittostandexercises they will also receive information on supportive techniques to improve physical activity suchsecondary outcomesgait speed will be assessed with the 10m walk test the test is used to determine walking speed in metersper second ms over a short distance the test is performed as three 10m walks without assistance one testwalk one in preferred walking speed and one in thepreop testingxbaselinexposttestingx1year followupxxxxxxxxxxxxxxxxxxxxxxxxxxxxxtable outcome measures and test occasionsvariablephysical functionmeasure6min walk testgait speedleg strength10m walk testchair stand testhandgrip strengthjamar hand dynamometerhabitual physical activityprevious physical activity levelahealthrelated quality of lifefatiguepsychological wellbeingpainactivpal3 microstanford brief activity surveyeortc qlqc30eortc qlqblm30piper fatigue scalehadsnrslength of hospital stay dayscomplicationsmedical recordsmedical recordsxxxxxxxxreadmissionsaprevious physical activity level is only used for adjustment purposes in the analysismedical records 0cporserud bmc cancer page of fastest speed possible time is measured for the intermediate m to allow for acceleration and decelerationthe outcome variable is msgrip strength will be assessed with the validated jamarhydraulic hand dynamometer the patients will sitin a chair and hold the dynamometer the test is performed three times for each hand and a mean value foreach hand is calculated the outcome variable is the gripstrength reading in kgleg strength will be assessed with the 30s sec chairstand test the patient is asked to rise from a chairas many times as possible for s the outcome variableis the number of sittostand transitionshabitual physical activity will be measured usingthe activpal3 micro activity monitor pal technologies ltd glasgow uk [ ] the activpal is asmall device which when attached to the thigh provides information based on position and accelerationof the body the information is then transferred tobody posture the transition between postures stepping and stepping speed the activity monitor is attached to the anterior midline ofthe thigh withdressing and does not provide feedback to the patientthe monitor will be worn for seven consecutive daysafter discharge from hospital and after the intervention period outcome variables will be time spentsittinglying standing stepping numbers of stepcounts and sittostand transitionsselfreported previous physical activity level will bemeasured using the 2item stanford brief activitysurvey sbas the sbas assesses the usualamount and intensity of physical activity during thepast year that a person performs the first item describes five patterns of work activity ranging frommostly sedentary to hard physical labour the seconditem describes five patterns ofleisuretime physicalactivity ranging from sedentary to regular vigorousintensity aerobic activities for respondents who areretired and have no job or regular work they wouldselect the response not applicable for both itemsthe outcome variable was categorical and rated on a5point scalehrqol will be assessed using the eortc qlqc30 with addition of the eortc qlqblm30 questionnaire the eortc qlqblm30 is explicitlydeveloped for patients with muscleinvasive urinarybladder cancerfrom theworst to the best for functional health statusand from the best to the worst for symptoms outcome variables will range from to fordifferent domains scoring rangesfatigue will be assessed using the piper fatigue scale the questionnaire consists of items and scoringranges from noneto severe the score ispresented in four domains plus a total fatigue scoreoutcome variables will range from to psychological wellbeing will be assessed using the hospital anxiety and depression scale hads thescale consists of questions with each scored on ascale from to where represents more symptomsthe questions are equally divided into the domains anxiety or depression each domain can result in a maximum score of outcome variables will range from to pain will be assessed using the numeric rating scalenrs the nrs is an eleven point scale and scoring ranges from no pain to worst pain the nrsis verbally delivereddata on length of stay at the hospital and frequency ofreadmission to hospital due to complications will be extracted from patient medical records readmissions willbe extracted as and days after surgery and complications will be registered using the claviendindo classification [ ]ethicsthe project is approved by the regional board of ethicsin stockholm dnr and the swedishethical review authority dnr the new model for rehabilitation is compared withsimilar care the patients are given in todays care delivery yet the control group will receive less attentionthan the intervention group we find it unethical to askthe patients to come to phc pay their visit and only receive for example stretching exercises in addition itwill be challenging to motivate the physiotherapists inphc to offer such treatmentsample sizefunction evaluatedthe primary outcome is physicalwith the validated 6mwt based on data from our pilotstudy we calculate an increase in m in theintervention group m in the control group and astandard deviation of m to obtain a statistical powerof with a type error set at patients areneeded in each group however as the test is highlycorrelated with sex and age we will stratify the analysis and increase the sample sizefor the secondary outcome readmissions due to complications we will estimate readmissions in theintervention group compared with the proportion of patients being readmitted today which is in the control group to obtain a statistical power of with atype error of patients are needed in eachgroup taken all this into account and guard againstdropout patients in each group will be includedin the study 0cporserud bmc cancer page of data management and study databasedata will be entered using an electronic system pheedit which is based on the sas system provided and operated by the cto at the centre for clinical cancerstudies theme cancer karolinska university hospitalsolna sweden a data management plan is delivered bycto documenting the database and all procedures fordata management the investigator verifies that all dataentries in the case report forms crfs are accurate andcorrect if certain assessments according to the protocolare not performed for any reason or if certain information is not available not applicable or unknown this willbe indicated in the crf by the investigator the investigator is required to sign off all reported datasource datain this study physicaltests movement sensor datapatientreported outcome measures and medical recordswill be regarded as source datacodingin the study database patients will be identified onlythrough the unique randomisation number all data willbe stored with coded identification and no access to thepatients id patient identification will not be revealed intext files logbooks with identification numbers and therespective codes will be stored in a locked environmentat the local centre that is not accessible to personnel notinvolved in conducting the trialstatistical analysisdescriptive statistics will be performed to ensure comparability between data at baseline to evaluate the effectof the intervention mixed models or linear regressionmodels spss inc chicago il usa according to theintention to treat procedure and with an alpha level of will be used significance of main or interaction effects will be explored using the bonferroni posthoc multiple comparison test in the case of skewed distributionlogarithmic transformations or corresponding nonparametric statistics will be used to assess the effect of theinterventionimplementation processbecause the intervention design is intricate we will inaddition to testing effects of the canmore programmeon patientlevel outcome measures also observe andgather information on factors that might have influencedthe implementation of the programme the evaluation of the implementation process will be based on themedical research council guide for process evaluationof complex interventions the knowledge gainedcan also be used to offer recommendations on whichstrategies to use when implementing the canmoreprogramme in other clinical settings and on a largescalethe initial strategies for the process evaluation will include meetings with surgeons the head of the surgicalward and phc clinics discussions and involvementwith physiotherapists and nurses at the surgical wardand physiotherapists at phc clinics and education ofthe canmore programme and outcome measures tophysiotherapists who will be involved in the intervention a leaflet for the patients an extended educationalleaflet and a short education for the physiotherapistshave been developedto study what is delivered measures of fidelity doseadaptation and reach will be assessed fidelity relative tothe canmore programme will be evaluated as the extentto which the programme was delivered as expecteddose will be assessed as the quantity of the interventionthe canmore programme and the education of physiotherapists in phc implemented adaptation such aschanges done to fit different phc settings will be reported in a questionnaire reach will be assessed regarding how many eligible patients signed an informedconsent form and how many in the intervention groupfulfilled the canmore programme in addition adverseevents will be registerediethe extentcontext includes external factors that may act as abarrier or facilitator to both implementation itself andthe patient level effect assessing barriers and facilitators to programme implementation will also involveevaluating programme feasibilitytowhich patients and health care staff regard the canmore as satisfactory in terms of content and complexitydifficulty we plan to conduct an interviewstudy on patients experiences of the intervention inaddition we plan to collect information on possiblebarriers that might have influenced the implementation ofthe programme and facilitators that mighthave supported it at the various clinical sites bothqualitative structured observationfocus groups andindividual semistructured interviews and quantitativequestionnaires and enrolment files methods will beused to assess how the intervention was delivered aswell as experiences of the different stakeholders patients physiotherapists and other health care staff aswell as managersby using several sources for data collection triangulation can be achieved which supports the trustworthinessof the study the consolidated framework for implementation research cfir will be used in the currentstudy to guide the investigation of context ie potentialbarriers and facilitators of the implementation processconstructs that we believe specifically impact the implementation outcomes in the present study and guidelinesforand observation protocolsinterview questions 0cporserud bmc cancer page of published by cifr will be followed httpcfirguidetoolshtmldiscussionto our knowledge this is the first study to examine theeffects of individually targeted exercise in phc compared with traditional advice on home exercise trainingafter rarc moreover the study will include a processevaluation of factors thought to influence the implementation of the programmealthough there is evidence that exercise is beneficialto improve physical function physical activity hrqolreduce fatigue and perhaps reduce complications it isessential to design feasible and easy to implement interventions in a health care setting our intervention is individually targeted and designed based on currentglobal guidelines for physical activity and exercise strategies for behavioural support and adapted to fitwithin the phc structure the length of the interventionis based on exercise principles and what is feasibleto conduct within phc yet this study does not tell uswhether the length of the intervention is the optimallength for best health benefits nor if a booster sessionafter the intervention period is needed the dose of exercise has been previously tested in a pilot study andshowed a positive effect on physical function was safeand had no adverse events after the pilot study werevised the exercise programme to be individually basedbut still include aerobic and musclestrengthening exerto ourcises the addition of behaviourprogrammeselfmonitoring and feedback has been shown to be associated with increased physical activity behaviour setting graded taskseg goalsupportat discharge from the hospital the standard care forpatients includes information on the importance ofphysical activity in this study the control group receivea light intervention ie recommended daily walks andlegstrengthening exercises instead of the standard carethe exercise recommendation is low dose and not specific but can still affectthe results by producing asmaller difference between the groups because there isstrong evidence for the effect of physical activity we findit unethical not to give the control group any advice onphysical activity at discharge many patients are feeblebecause of surgery and the postoperative period at hospital which can also result in fear of movement thesepatients require supervised physical exercise as in theintervention group in this studythe process evaluation using measures offidelitydose adaption adherence and reach as well as patientperspectives and experiences of the programme can helpexplain the results in additionit can speed up theprocess of translating findings from research settings toclinically representative settings the programme fitswell within the healthcare system and the exercises aregeneric to those recommended for cancer survivors andthe tools for motivational support are generic for thewhole population if the programme is proven effectiveit should be generalisable to other patient groupsthatis notthere are some limitations first due to the difficultiesof doubleblinding we have a singleblind design inwhich a physiotherapistinvolved in theprogramme is conducting the measurements secondwe foresee a long recruitment period that can lead to achange in health care staffphysiotherapists in phc toensure quality we plan to have continued contact withthe clinics and a new educational structure for trainingif neededcystectomyin summary this proposed rct has the potential toprovide new knowledge within rehabilitation after radicalcancer theprogramme should be readily applied to other patientgroups undergoing abdominal surgery for cancer andhas the potential to change the health care chain forthese patientsfor urinary bladdersupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071405additional file exercise programmeabbreviationscanmore cancer mobilisation rehabilitation cfir consolidated frameworkfor implementation research crf case report form cto clinical trials officehads hospital anxiety depression scale hrqol healthrelated quality of lifemct movement continuum theory ms meters per second phc primaryhealth care qol quality of life rarc roboticassisted radical cystectomyrct randomised controlled trial rr responsible researcher sbas stanfordbrief activity survey sec seconds spirit standard protocol itemsrecommendations for interventional trials 6mwt sixminute walk testacknowledgementsnot applicableauthors contributionsmh and ap conceived the idea for this study and designed it along with theother authors all authors ap pk er ma lh mnb and mh were involvedin drafting and revising the manuscript all authors will be involved in datacollection analysis or manuscript preparation as the study proceeds allauthors read and approved the final manuscri
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" clinical trials have been conducted to clarify the beneficial effects of vd3 1α25dihydroxy vitamind3 also known as calcitriol treatment in prostate cancer however the molecular mechanisms underlying theseeffects are not fully understood recent studies on igfbp3 have indicated its intracellular functions in cell growthand apoptosis the aim of this study was to confirm the benefits of lowdose vd3 treatment and clarify themolecular mechanisms underlying these beneficial effects in prostate cancer cellsmethods the molecular effects of simultaneous treatment of lncap cells and their genetically modified cell lineswith low concentration of docetaxel and vd3 were biologically and biochemically analyzed to further determinethe effects of vd3 treatment on igfbp3 induction system cells were temporarily treated with vd3 in combinationwith a transcriptional inhibitor or protein synthesis inhibitor bcl2 protein and its mrna behavior were also observed inigfbp3 expressionmodified lncap cells to determine the involvement of igfbp3 in the suppression of bcl2 by vd3treatmentresults changes in igfbp3 expression levels in lncap cells indicated that it mediated the inhibition of cell growthinduced by vd3 treatment igfbp3 was also found to be a mediator of the enhanced cytotoxicity of prostate cancercells to vd3 in combination with the anticancer drug we further identified the distinct property of the igfbp3induction system wherein temporal vd3 stimulationinduced prolonged igfbp3 expression and vd3 treatmentinduced increase in igfbp3 expression were optimized based on the protein concentration rather than the mrnaconcentration meanwhile bcl2 expression was downregulated by vd3 treatment in an igfbp3independent manner these findings indicate the molecular mechanisms of igfbp3 induction stimulated by vd3 and igfbp3independent bcl2 suppression by vd3 treatment in prostate cancer cells the results could prompt a reevaluation ofvd3 usage in therapy for patients with prostate cancerkeywords vitamin d nonlinear igfbp3 induction bcl2 suppression prostate cancer treatment correspondence satorusasagawatokushukaijp1molecular biology laboratory research institute nozaki tokushukai hospitaltanigawa daito osaka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cigarashi bmc cancer page of vitamin d has a central role in calcium and skeletalhomeostasis [ ] its pleiotropic role both in physiological and pathological phenomena such as cell growthimmune function and tumorigenesis has also been examined [] which revealed that exposure of cancercells to vitamin d significantly reduces the cell growthrate in multiple cancer types [] indeed recent epidemiologicalinvestigations have reported that highervitamin d concentration could prevent multiple types oftumorigenesis consistent with such finding for example an increase in colon cancer incidence with lowervitamin d dietary habits has been reported [ ]however suppressive effect on prostate cancer is stillunder discussion [ ]in turntraditional methodscurrently prostate cancer is one of the most commonthecancers in men worldwide clinical use ofprostatespecific antigen psatest dramatically improved the screening sensitivity of prostate cancer compared to that ofthenumber of patients with earlystage prostate cancer hasrapidly increased since the mid1990s [ ] unlikeother cancer types most cases of prostate cancer haveslow progression or have nonprogressive indolentsymptom and are localized in the prostate thus they areunlikely to cause poor physical condition or death therefore patients with prostate cancer need a less burdensome treatment in order to avoid potential harmfrom excessive treatmentalthough the impact of vitamin d as a single agent onprostate cancer has been investigated its significance remains under discussion [ ] meanwhile the synergistic or additive effects of vitamin d and its derivativeswith anticancer drugs on prostate cancer have beenclinically studied and encouraging results have been reported [ ] however the results of larger trials thatevaluated the synergistic effect of vitamin d in combination with docetaxel one of the firstline anticancerdrugs in prostate cancer chemotherapy showed limitedor nonsignificant benefit of vitamin d efficacy in castration or androgen deprivation therapyresistant prostatecancer [ ] furthermore overconsumption of 1α25dihydroxy vitamin d3 vd3 also known as calcitriolthe biologically active form of vitamin d3 from food orprolonged treatment with vd3 derivatives could triggerhypercalcemia resulting in physiological side effects therefore to date vd3 has not been proactivelyused in the treatment of patients with prostate cancerthe biological function of vitamin d is mainly mediatedby vitamin d receptor vdr which acts as a transcriptional factor vitamin d receptor elements vdreon the promoter region of target genes are recognizedand transcriptionally activated by vitamin dcoupledvdr consistent with the diverse physiological functionof vd3 vdre was identified not only in the gene related to calcium and skeletal homeostasis but also in thegene related to fundamental cellular functions includingcell growth igfbp3 is one of the families of sixhigh affinity igfbps and was originally found in plasmaas a stabilizer and transporter of igfs in the bloodstream interestingly vdre was found on the prothe igfbp3 gene and recent studies havemoter ofrevealed that igfbp3 functions inside the cell as wellregulating cell growth and apoptosis [ ]methodsthis study aimed to investigate igfbp3 induction byvitamin d treatment and determine its role in prostatecancer treatment with vitamin d in combination withanticancer drugs in order to provide molecular biologicalevidence of benefit of vitamin d and to suggest effectivevitamin d usage in prostate cancer treatmentchemicals and reagentsdihydrotestosterone dht and calcitriol vd3 purchased from tokyo chemical industry tokyo japanwere resolved in ethanol as a stock solution pei maxmolecular weight was purchased from polysciences pa usa the other chemicals and reagentswere purchased from wako pure chemicalosakajapan and sigmaaldrich st louis mo usaincluding testosteronecharcoal stripping of fetal bovine serum fbsfbs was purchased from gibco waltham ma usato deplete hormonesin fbsdextrancoated charcoal powder was added to theserum and the mixture was incubated with rotation at degree overnight thereafter the mixture was centrifugedto pellet charcoal and the supernatant was filtered througha 022μm polyvinylidene difluoride membrane thecharcoalstripped serum was used for all experimentstotalthe concentrations oftestosterone and totalvitamin d in the serum were determined using a totaltestosterone test kit abbott japan chiba japan and atotal vitamin d test kit roche basel switzerland according to manufacturers instructions the concentrations of total testosterone in the pre and posttreatmentserum were nm and less than nm limit of detection respectively the concentrations of total vitamin d in the pre and posttreatment serum were and nm respectively thus the basal concentrationsin the culture medium supplemented with fbs wereless than nm total testosterone and approximately nm total vitamin dcell culturethe lncap cell line was obtained from american typeculture collection and cultured in dulbeccos modified 0cigarashi bmc cancer page of eagle medium dmem sigmaaldrich supplementedwith 10charcoalstripping fbs the 293ft celllinewas purchased from invitrogen waltham ma usaand cultured in dmem supplemented with fbs mm of lglutamine sodium pyruvate and nonessentialamino acids the cells were cultured at a temperature of °c in co2humidified condition the mycoplasma contamination was routinely checked and confirmed as negativecell growth assaydmem supplemented with charcoalstripping fbs was used for the cell growth assay the cells were seededat à cells per well in a 6well plate the next daythe medium was replaced with ml of fresh mediumand nm dht andor nm vd3 were added the cellculture was continued throughout the indicated periodthe cultured cells were trypsinized and the number ofcells was assessed using an automated cell countercountess iitm fl invitrogen each assay was repeatedat least three times and similar results were obtainedwestern blottingthe antibodies used for western blotting were mouseantiigfbp3 santa cruz biotechnology mouseantiβactin santa cruz biotechnology mouseantibcl2 santacruz biotechnology and horseradish peroxidaseconjugated secondary antibodies jackson immunoresearch laboratories westgrove pa usa the collected cells were resuspendedin ripa buffer supplemented with protease and phosphatase inhibitors roche basel switzerland and lysedusing bioruptortm ii sonicator cosmo bio tokyojapan cell lysates were resolved by nupagegels invitrogen and transferred onto polyvinylidenefluoride membrane millipore burlington ma usathe signals were developed using enhanced chemiluminescence reagent perkinelmer waltham ma usaand luminograph i atto tokyo japan was used forimage capture quantification of band signal wasanalyzed using cs analyzer software atto at leasttwo biological replicates of each experiment were performed with similar resultsrealtime reverse transcriptionpolymerase chain reactionthe total rna from the cultured cells was extractedusing trizol reagent invitrogen according to the manufacturers instruction the rna was reverse transcribedby the primescript rt reagent kit takara bio shigajapan using oligodt quantitative reverse transcriptionpolymerase chain reaction rtpcr reaction wasperformed using tb green premix ex taq ii takaraand genespecific primers supplementary table bycfx96 touch realtimepcr system bioradlaboratories hercules ca usa gene expression datawere normalized against glyceraldehyde 3phosphate dehydrogenase or hprt1 as internal control at leastthree biological replicates of each experiment were performed and similar results were obtainedflow cytometric analysis of cell cycle and apoptosisfor cell cycle analysis and apoptotic cell detection flowcytometric analysis was performed using the guavaeasycyte plus flow cytometry system millipore andguava cell cycle reagent and annexin v fitc apoptosiskit millipore according to manufacturers instructionas previously described at least three biologicalreplicates of each experiment were performed and similar results were obtainedlentiviral construction and transductionbackbone vectors plko1 puro plasmid andplenti cmv puro dest w118 plasmid were provided by drs bob weinberg eric campeau andpaul kaufman respectively via addgene ref pentr1a plasmid and lentiviral packaging mix plp1plp2 and plpvsvg were purchased from invitrogenfulllength igfbp3 was cloned from lncap cell complementary dna cdna using kod fx neo toyoboosaka japan with a specific primer set supplementarytable and inserted into the pentr1a vector thenthe sequence was confirmed the igfbp3 cdna was introduced into the plenti cmv puro dest vector by recombinaseenzymeinvitrogen to generate the lentiviral expression vectorspecific short hairpin rnas shrnas were designedusing invitrogen or biosettia websites the selectedtarget sequence oligos supplementary table wereannealed and inserted into the plko1 puro vector according to addgenes instruction the lentiviral expressionvector or shrna vector was cotransfected with the lentiviral packaging mix into the 293ft cells using pei maxinstead of lipofectamine according to invitrogensinstruction twentyfour hours posttransfectionthemedium was replaced with the culture medium forlncap cells one day later the lentiviruscontaining supernatants were collected and filtrated through a 045mmpolyvinylidene fluoride filter milliporereaction using lr clonaseiilentivirus infectionone day before infection à lncap cells wereplated into a 10cm dish then the culture medium wasreplaced with the lentiviruscontaining supernatant andculture was continued twentyfour hours post infectionreplaced with fresh culturemedium two days later the medium was replaced withfresh culture medium that contained μgml of puromycin and culture was continued until the noninfectedthe medium was 0cigarashi bmc cancer page of control cells were completely killed the puromycinselected cells were then subjected to each assaystatistical analysisresults are presented as mean ± standard deviation unless otherwise specified pairs of groups were comparedusing a twotailed unpaired students ttest onewayanalysis of variance was used for multiplegroup comparisons rather than specifying three a pvalue was considered statistically significant all statistical analyses were performed using excel software microsoftredmond wa usa and statcel3 addin for exceloms publishing tokyo japanresultsvd3 reduces cell growth rateconsistent with a previous report that treatmentwith vd3 inhibits growth of prostate cancer cells ourresults showed that vd3 treatment reduced the cellgrowth rate in a dosedependent manner and nm fig 1a left as shown below igfbp3 induction activity of vd3 was reached to plateau at nm concentration fig 4a on the other hand testosterone hasbeen reported to stimulate the growth of prostate cancer and the results of this study confirmed that dhttreatment from very low concentration nm stimulated cell growth rate and its activity was reached toplateau at less than nm concentration fig 1a centerthe purpose of our study was to investigate the role ofvd3igfbp3 induction system in cell growth inhibitionand to propose the potency oflowdose vd3 usagewhich could evade sideeffect of vd3 treatment such ashypercalcemia in therapy for patients with prostate cancer thus nm of dht and nm of vd3 concentrations were chosen as minimum but stably workingconcentration for following experiment previouslyithas been demonstrated that simultaneous treatmentwith vd3 and dht enhanced the reduction of cellgrowth rate compared to treatment with vd3 alone anda similar result was reproduced with lowdose dht nm and vd3 nm in this study fig 1a right tofurther characterize growth inhibitory effect with lowdose of dht nm and vd3 nm cell cycle andapoptotic analyses were performed with flow cytometrythe cell cycle analysis revealed that there was no significant change in the cell cycle phase distribution amongfig cellular response of lncap cells treated with vd3 and dht a effect of combined treatment of vd3 and dht on cell growth in lncapcells left vd3 treatment reduced cell growth in a dosedependent manner center dht treatment increased cell growth at lowerconcentrations right simultaneous treatment of vd3 and dht enhanced the reduction of cell growth compared to treatment with vd3 aloneb change in cell cycle phase by vd3 or dht treatment neither vd3 nor dht treatment significantly changed the cell cycle phase c induction ofapoptosis by vd3 or dht treatment neither lowdose vd3 nor dht treatment influenced apoptosis at shortterm d induction of igfbp3expression by vd3 or dht treatment vd3 treatmentinduced igfbp3 expression and cotreatment with dht enhanced the expression level ofigfbp3the ratio indicates the density of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium condition the uncropped fulllength blot images are presented in supplementary fig 5a 0cigarashi bmc cancer page of control no treatment dht nm vd3 nm anddhtvd3 treatment conditions fig 1b suggestingthat lowdose of vd3 or dhtvd3 treatment did notarrest the cell cycle at a specific phase previous reportshave shown that longterm vd3 treatment has apoptosisinducible activity and dht has apoptosis inhibitingactivity in a dosedependent manner in lncapcells however it was unclear or controversial whetherlowerconcentration of vd3 and dht at shorttermcould influence apoptosis respectively in lncap cellsthus the apoptosis assay was performed with nm ofdht and nm of vd3 for shortterm and found thatneither lowerdose dht vd3 nor dhtvd3 treatmentfor shortterm influenced apoptosis fig 1c these results suggested that the decrease in the cell number induced by lowdose vd3 or dhtvd3 treatment wasmainly due to a decrease in the cell growth rate to further address what was occurring at the molecular levelduring lowdose dht andor vd3 treatment the genesknown to regulate the cell cycle and inducible by dhtandor vd3 treatment were chosen and messengerrna mrna induction was quantitatively measuredfig 1d upper supplementary fig 1a the quantitativertpcr results showed that igfbp3 mrna inductionwas positively correlated to cell growth suppression inresponse to lowdose vd3 or dhtvd3 treatment andthe expression strength was dramatically sensitive tovd3 or dhtvd3 treatment consistent with thatigfbp3 protein was markedly induced by vd3 treatment and it was enhanced by simultaneous treatment ofvd3 with dht a similar response was observed in theexpression of ar the receptor of dht which wasknown to a one oftarget of vdr supplementaryfig 1bmultiple recent studies have revealed that igfbp3functions in cellular response including cell growth andapoptosisin an insulinlike growth factor igfindependent manner considering these findings we believethat igfbp3 can be a key molecule for vd3 treatmentin prostate cancer cellsigfbp3 was a dominant factor in cell growth suppressionto confirm ifigfbp3 dominantly suppresses cellgrowth in lncap cells we applied the gainoffunctionand lossoffunction approach using a lentivirus systemfirst we generated igfbp3overexpressing lncapcells and found that the expression of igfbp3 mrnawas about higher compared to that by lowdosedhtvd3 treatment fig 2a as an infection controlegfpoverexpressing lncap cells were also generatedand it was confirmed that lentivirus infection per se didnot induce igfbp3 expression using these cell linesthe effect of igfbp3 on cell growth was observed fig2b results showed that the cell number of egfpoverexpressed cells treated with nm of dht and nm of vd3 for days was decreased to comparedwith that of untreated cells and the igfbp3overexpressing cells showed comparable cell growth decreasewithout dhtvd3 treatment next we generatedshrna for igfbp3 shigfbp3expressing lncap cellsthe knockdown of igfbp3 mrna and protein inducedby lowdose dhtvd3 treatment was confirmed inshigfbp3expressing lncap cells fig 2c using thiscell line the effect of lowdose dht and vd3 treatmenton cell growth was observed as we expected the suppressive efficacy of lowdose vd3 on cell growth wasweakening and simultaneous treatment with nm ofdht and nm of vd3 increased cell growth fig 2dtaken together these data indicated igfbp3dominantfactor of cell growth suppression induced by lowdosevd3 treatment in lncap cellsacceleration of anticancer drug effect by vd3as previously reported and we demonstrated abovevd3 alone is not cytotoxic at physiological and pharmacological concentrations meanwhile simultaneous treatment with vd3 has been reported to improve theefficacy of anticancer drugs including docetaxel howeverits molecular mechanisms were remained not fully uncovered here we supposed that igfbp3 might be amediator of vd3induced sensitization to anticancerdrugs in prostate cancer cells to confirm this hypothesis lncap cells were treated with lowdose of dhtvd3 in combination with several concentrations of docetaxel to determine the appropriate concentration ofdocetaxel for evaluating the vd3 effect we first screenedthe concentration of docetaxel based on cytotoxic activity and found that a docetaxel concentration nmkilled bulk of the cells treated fig 3a here ic50 ofdocetaxel was nm in our assay and it was consistent with previous reports nm thus a docetaxel concentration nm was chosen to observe theeffect of combinatoriallowdose dhtvd3 treatmentfor the following assay to evaluate the synergistic effectof dhtvd3 on cytotoxicity by docetaxel lncap cellswere treated with or nm of docetaxel withor without dhtvd3 and results showed that lowdosedhtvd3 with docetaxel reduced the living cell numberat the concentration range of nm but the effectwas masked when nm docetaxel was applied fig 3bsimilarlylowdose dhtvd3 with cisplatin reducedthe living cell number at the concentration range of nm supplementary fig to see if these enhancedcytotoxicity effects were dependent on igfbp3 igfbpoverexpressed or shigfbp3expressed lncap cellswere analyzed in the same manner indeed in igfbp3overexpressed cells the living cell number was reducedby docetaxel without dhtvd3 addition fig 3c 0cigarashi bmc cancer page of fig igfbp3 mediates the effect of vd3 on cell growth in lncap cells a overexpression of igfbp3 in lncap cells lncap cells were infectedwith lentivirus containing the igfbp3 gene and its overexpression was confirmed by quantitative reverse transcriptionpolymerase chainreaction b suppression of cell growth by igfbp3 igfbp3overexpressed cells were cultured as they were and control cells were cultured withdhtvd3 for days and then the cell number was measured c knockdown of igfbp3 in lncap cells lncap cells were infected with lentiviruscontaining shrna for igfbp3 and igfbp3 knockdown was confirmed by quantitative reverse transcriptionpolymerase chain reaction andwestern blotting the ratio indicates the density of igfbp3 band normalized by corresponding βactin band d the igfbp3 knockdown cells weretreated with dht andor vd3 for days and then the cell number was measured the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5bcorrespondingly in the shigfbp3expressed cells reduction of living cell number by dhtvd3 addition wascanceled rather the cell living number was increasedliving cell number despitefig 3d the increase ofdhtvd3 addition in docetaxeltreated shigfbp3expressed cells was assumed by cancelation of vd3 effect and emerging of dht effect on cell growth basedthese findings lowdose dhtvd3induced enhancedcytotoxicity by docetaxel on lncap cells was dependenton igfbp3 expressioncharacterization of the igfbp3 induction mechanismas demonstrated above igfbp3 had a pivotal role inlowdose dhtvd3induced enhanced cytotoxicity byantitumor drugs to further dissect the igfbp3 induction mechanism and to provide the molecular evidenceof vd3 treatment for clinical research mechanisms ofigfbp3 induction by dht and vd3 were analyzed aspreviously reported in prostate cancer cells vd3 treatment induces cyp24a1 as well an enzyme that catalyzes vd3 to its inactive form as a negative feedbackfactor the induced cyp24a1 limits the efficacy of vd3meanwhile activated ar induced by dht treatmentsuppresses cyp24a1 transcription thus cancelling thenegativefeedback loop to inactivate vd3 consistentwith that cyp24a1 induction by vd3 treatment and itssuppression by simultaneous treatment with dht wereconfirmed even when we applied lowdose dht and 0cigarashi bmc cancer page of fig vd3 enhanced cytotoxicity of docetaxel a dosedependent cytotoxicity of docetaxel lncap cells were treated with docetaxel for daysand then the living cell number was measured ic50 was nm b simultaneous treatment of vd3 with dosedependent docetaxelsimultaneous treatment of vd3 with docetaxel reduced the living cell number at nm range c igfbp3 overexpression was conducive todhtvd3 treatment on docetaxel treatment igfbp3overexpressed or control cells were treated with dhtvd3 and nm of docetaxel andcultured for days and then the cell number was measured d igfbp3 knockdown canceled the effect of vd3 on docetaxel treatment in igfbp knockdown cells dhtvd3 treatment increased the cell number compared to that of the notreatment control and the cell number wasalmost equal to that of the dht treated sample the concentration of docetaxel used was nm the all experiments were performed in serumcontaining medium conditionvd3 which were enough to induce and suppresscyp24a1 expression supplementary fig meaningthat h lowdose dhtvd3 treatment could cancel thecyp24a1driven negativefeedback loop to further dissect the mechanism of igfbp3 expression in lncapcells the cells were treated with vd3 alone or dhtfixed in nm and vd3 in a dosedependent manner and nm when treated with vd3alone the induced igfbp3 reached a plateau at to nm in contrast when treated with vd3 together withdht the amount of induced igfbp3 was increased according to the increment of vd3 concentration fig 4athese results indicated that lowdose dht could improve igfbp3 induction activity of vd3 throughcyp24a1 suppressionclinically highdose vd3 or its derivatives for treatment can cause hypercalcemia thus its continual usageshould be carefully monitored to avoid sideeffect ofvd3 here we wondered if continual vd3 treatmentfig 4btop herewould be required for maintaining igfbp3 induction inprostate cancer cells to address this lncap cells weretreated with vd3 alone or lowdose dhtvd3 for or days followed by washout which was done by replacing the culture medium and continuing the culturefor days in totalintracellularigfbp3 protein was observed by western blottinginterestingly 1day treatment of vd3 or dhtvd3 induced stable igfbp3 expression fig 4b bottom although treatment of vd3 alone showed mild igfbp3induction compared to that by dhtvd3 note thatigfbp3 showed similar strength of expression between and days of vd3 or dhtvd3 treatment this resultclearly indicated that temporal vd3 treatment couldinduce prolonged stable igfbp3 expressionthis nonlinear response suggested the presence of aunique molecular property underlying the igfbp3 expression mechanism generally nonlinear cellular response such as sustained protein expression by temporal 0cigarashi bmc cancer page of fig characterization of igfbp3 expression induced by vd3 and dht treatments a effect of dosedependent vd3 treatment with or withoutdht on igfbp3 induction lncap cells were treated with vd3 alone at the indicated concentration or with vd3 and nm dht top westernblotting image of igfbp3 and bottom quantified graph of igfbp3 induction open circles vd3 alone filled circles dht vd3 b effect oftemporal treatment of dhtvd3 on igfbp3 induction top schematic time course of temporal treatment of vd3 and bottom western blottingimage of igfbp3 the ratio indicates the density of igfbp3 band normalized by corresponding βactin band c effect of mrna transcription andprotein synthesis on the stability of igfbp3 expression induced by dhtvd3 lncap cells were treated with vd3dht for day followed byactinomycin d actd μm or cycloheximide chx μm for another day after vd3dht was washed out or left as is top quantification graphof igfbp3 mrna induction middle western blotting image of igfbp3 and the combination of treatments bottom the ratio indicates thedensity of igfbp3 band normalized by corresponding βactin band the all experiments were performed in serumcontaining medium conditionthe uncropped fulllength blot images are presented in supplementary fig 5ctriggered by positivefeedback loopstimulation wasmechanismin which protein synthesis and transcriptional regulation was included as hysterical responsedriver thus to further dissect if protein synthesis ortranscriptional regulation or both are involved in nonlinear vd3igfbp3 induction actinomycin d and cycloheximide which are transcriptionalinhibitor andprotein synthesis inhibitor respectively were added withvd3 or dhtvd3 and behavior of igfbp3 proteinand its mrna was observed experimentally actinomycin d or cycloheximide was added day after treatmentwith vd3 alone or dhtvd3 and the culture was continued for another day when interfered with μm ofactinomycin d there was no change in igfbp3 mrnaor protein expression fig 4c by contrast when interfered with μm of cycloheximide the igfbp3 proteinwas immediately reduced howeverthe mrna wasunexpectedly increased several times higher than thosewithout cycloheximide interference fig 4c the unexpected mrna increase became stronger when dhtvd3 washout was performed ahead of the cycloheximideinterference these cellular responses on igfbp3 induction interfered by actinomycin d and cycloheximidesuggested that the cells had a protein abundancebasedpositivefeedback loop to maintain the total amount ofigfbp3 via transcriptional controligfbp3independent bcl2 suppression by vd3as shown above although lowdose dht andor vd3treatment did not induce apoptosis fig 1d vd3 treatment rendered lncap cells sensitive to the antitumordrugs fig 3b and supplementary fig suggesting thatany apoptosisrelated factor might be influenced to addressinvestigated the behavior ofidea wethis 0cigarashi bmc cancer page of apoptosisrelated molecules in response to lowdosevd3dht treatment consistent with previous report although the concentration of vd3 was higher thanthat we used here bcl2 protein an antiapoptotic molecule was downregulated by lowdose vd3 treatmentfig 5a compared to that by vd3 alone it seemed thatbcl2 downregulation by dhtvd3 was not seemed tobe enhanced unlike to igfbp3 expression suggestingthat bcl2 downregulation by vd3 was igfbp3 induction independent manner to see whether bcl2 downregulation was igfbp3dependent or not the behaviorof the expression of bcl2 protein and mrna was observed in shigfbp3expressedcells interestingly despitethe igfbp3 disappearance bcl2 downregulation wasobserved according to vd3 or dhtvd3 treatmentfig 5b bottom and it was not significantly different atinigfbp3expressionenhancedprotein and mrna expression level compared to that inshctrlexpressing cells moreover in order to confirmthatconditionshcyp24a1 expression cells were established in whichvd3 effect on igfbp3 induction was expected tostrengthen the knockdown of cyp24a1 under the vd3treatment condition was confirmed by qrtpcr supplementary fig indeed the amount of igfbp3 protein wasincreased in shcyp24a1expressing cellscompared to that in shctrl cells fig 5c bottom despiteigfbp3 expression downregulation bcl2 protein by lowdose vd3 or dhtvd3treatment was not observed compared to that in shctrlcells fig 5c bottom also the expression of bcl2mrna was not significantly changed fig 5ctopthese results suggested that the downregulation of bclenhancement offig igfbp3independent reduction of bcl2 protein expression induced by vd3 a western blotting image of bcl2 reduction by vd3treatment the ratio indicates the density of bcl2 band normalized by corresponding βactin band b effect of igfbp3 suppression on bcl2reduction by vd3 treatment the cells were infected with lentivirus encoding of shrna for igfbp3 and then treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band c effect of igfbp3 overexpression on bcl2 reduction byvd3 treatment the cells were infected with lentivirus encoding of the cyp24a1 gene then the cells were treated with vd3 and dht topquantification graph of bcl2 mrna expression and bottom western blotting image of bcl2 and igfbp3 induced by vd3 and dht treatmentthe ratio indicates the density of bcl2 band normalized by corresponding βactin band the all experiments were performed in serumcontainingmedium condition the uncropped fulllength blot images are presented in supplementary fig 5d e 0cigarashi bmc cancer page of protein by lowdose vd3 treatment was independentof igfbp3 induction besides the mrna of bcl
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environmental exposure to arsenite as3 has a strong association with the development ofhuman urothelial cancer uc and is the 5th most common cancer in men and the 12th mostcommon cancer in women muscle invasive urothelial cancer miuc are grouped into basalor luminal molecular subtypes based on their gene expression profile the basal subtype ismore aggressive and can be associated with squamous differentiation characterized byhigh expression of keratins krt1 and and epidermal growth factor receptoregfr within the tumors the luminal subtype is less aggressive and is predominatelycharacterized by elevated gene expression of peroxisome proliferatoractivated receptamma pparÎ and forkhead box protein a1 foxa1 we have previously shown thatas3transformed urothelial cells ast exhibit a basal subtype of uc expressing genesassociated with squamous differentiation we hypothesized that the molecular subtype ofthe ast cells could be altered by inducing the expression of pparÎ andor inhibiting theproliferation of the cells nontransformed and ast cells were treated with troglitazonetg pparg agonist μm pd153035 pd an egfr inhibitor μm or a combination oftg and pd for days the results obtained demonstrate that treatment of the ast cellswith tg upregulated the expression of pparÎ and foxa1 whereas treatment with pddecreased the expression of some of the basal keratins however a combined treatment oftg and pd resulted in a consistent decrease of several proteins associated with the basalsubtype of bladder cancers krt1 krt14 krt16 p63 and tfap2a our data suggeststhat activation of pparÎ while inhibiting cell proliferation facilitates the regulation of genesinvolved in maintaining the luminal subtype of uc in vivo animal studies are needed toaddress the efficacy of using pparÎ agonists andor proliferation inhibitors to reduce tumradestage of miuca1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation mehus aa bergum n knutson pshrestha s zhou xd garrett sh activation of pparÎ and inhibition of cellproliferation reduces key proteins associated withthe basal subtype of bladder cancer in as3transformed urotsa cells one e0237976 101371 pone0237976editor karl x chai university of central floridaunited statesreceived may accepted july published august copyright mehus this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the paper and its supporting informationfilesfunding seema somji und school of medicineand health sciences pilot grant das allundergraduate research and core facilities ndinbre idea program p20 gm103442 from thenational institute of general medical sciences nih one 101371 pone0237976 august one 0ccompeting interests the authors have declaredthat no competing interests existactivation of luminal pathway in basal bladder cancerintroductionbladder cancer bc is the ninth most common cancer diagnosed worldwide and in theamerican cancer society estimated that about new cases of bc would be identified inthe us and about deaths would occur from bladder cancer among bcs urothelialcell carcinomas uc are the most common being the second most diagnosed cancer of thegenitourinary tract behind prostate cancer [ ] it is the 5th most common cancer in menand the 12th most common cancer in women urothelial cancers are classified as muscle invasive miuc or nonmuscleinvasivenmiuc nonmuscleinvasive urothelial cancers have a lower tendency to progress whereasmiucs have a high rate of metastasis and a year survival rate of approximately bothmiuc and nmiuc have been subtyped into various groups with the basal and luminal subtype being the most prominent the luminal subtype of human uc includes the majority ofthe early stage noninvasive ucs and a significant number of miucs this subtype isenriched for papillary histology is less aggressive and has a more favorable patient outcome [ ] basal classified tumors have a poorer overall survival compared to luminal tumors they often exhibit squamous differentiation are aggressive and found exclusively inmiuc that metastasize and spread to distal ans about of miucs arise independent of the papillary pathway have poor outcomes and an overall year survival rate of environmental exposure to arsenite as3 has a strong association with the developmentof human uc the increased risk of uc correlates to the same endemic areas of the worldwhere populations have been identified with arsenicinduced skin cancer [] we havedeveloped a cell culture model of arsenicinduced urothelial cancer by exposing the immortalized nontumorigenic urothelial cell line urotsa to arsenite these transformed cell lines produce tumors in athymic mice that express genes for keratin krt and asignature pattern highly similar to the basal subtype of human miuc [ ] the tumorshave a histology similar to urothelialtransitional cell carcinomas with focal areas of squamousdifferentiation [ ] which is associated with poor prognosis [ ]the molecular mechanism driving a tumor towards a basalsquamous subtype is currentlyunknown in a recent study yamashita show that transcription factor activatingprotein alpha tfap2a is expressed at high levels in basalsquamous bladder cancerenriched in areas of squamous differentiation and is associated with increase lymph nodemetastasis and distant recurrence of the disease the study also shows that increased expression of tfap2a can facilitate the expression of other transcription factors such as tumor protein p63 tp63p63 also known as p63 which is known to be associated with the basalsubtype of uc palmbos demonstrated that p63 binds to the transcriptional regulatory regions of the gene ataxiatelangiectasia group d complementing gene atdc alsoknown as trim29 and krt14 thus increasing their expression the study further showedthat both krt14 and trim29 promote the invasion of the basal subtype of uc in vitro and invivo the luminal subtype of uc is associated with the expression of the transcriptional factorsforkhead box protein a1 foxa1 gata binding protein gata3 and peroxisome proliferatoractivated receptor gamma pparÎ [ ] the activation of pparÎ with an agonistcan represses the expression of tfap2a and inhibit squamous differentiation in vitro the exact role of pparÎ signaling in carcinogenesis is somewhat unclear however theexpression of pparÎ in bladder cancers is a favorable prognostic marker both in vivoand in vitro studies indicate that pparÎ ligands such as troglitizone can promote differentiation inhibit cellular proliferation induce autophagy and enhance apoptosis in bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer[] likewise suppressing cellular proliferation with epidermal growth factor receptoregfr inhibitors has been used preclinically to reduce basallike muscle invasive bladdertumor growth although the egfr inhibitors did not have the same efficacy in nonbasalliketumors the goal of this study was to determine if the activation of pparÎ and inhibition of cellproliferation in the urotsa parent and the as3transformed urotsa isolates would repressthe expression of genes involved in maintaining the basalsquamous type of bladder cancerand induce genes that were associated with the luminaldifferentiated state of bladder cancermaterials and methodsanimalsathymic nude ncrnunu mice purchased from envigo were used in these studies themice were housed four to a cage at Ëc under a 12hour lightdark cycle food and water wasavailable ad libitum mouse heterotransplants of the urotsa transformed cell lines as3 andas4 and the rt4 cell line were produced by subcutaneous injection at a dose of x cellsin the dorsal thoracic midline of athymic nude ncrnunu mice this study adhered to allrecommendation dictated in the guide for the care and use of laboratory animals of thenih tumor sizes were assessed weekly and the animals were sacrificed when the size of thetumor was approximately cm or when dictated by clinical conditions euthanizationwas done by co2 asphyxiation and conformed to the american veterinary medical association guideline on euthanasia death was confirmed by ascertaining cardiac and respiratoryarrest following which the ans and tumor were harvested care of taken to ensure thatthere was no distress to the animals during the procedure the protocol was approved by theuniversity of north dakota animal care committee iacuc16122ccell culturethe urotsa parent cells and two of the as3transformed isolates as3 and as4 were cultured in in dulbecos modified eagles medium dmem supplemented with vv fetalbovine serum as described previously the cells were subcultured at a ratio usingtrypsinedta and the cultures were fed fresh growth medium every three days the urotsaparent cell line has been authenticated using short tandem repeat str analysis the as3transformed isolates used in the current study have been previously characterized for its ability to form colonies in soft agar form spheroids when grown in ultralow attachment flasksand form tumors when injected subcutaneously in immunecompromised mice [ ]the as3 can also form tumors upon intraperitoneal injection for drug treatmentsurotsa parent and the as3transformed isolates as3 and as4 were grown to confluence inserum containing medium following which the cells were incubated with a serum freemedium consisting of a mixture of dmem and hamss f12 growth medium for h thecells were then exposed to either dimethyl sulfoxide dmso the drug vehicle troglitizonetg μm a pparÎ agonist pd153035 pd μm an epidermal growth factor receptoregfr inhibitor or a combination of tg and pd tgpd for and hours the concentrations of the drugs were chosen based on preliminary studiesvisualization of dapistained cellstoxic effects of tg and pd on the urotsa cells was determined by visualization of 406diamidino2phenylindole dapistained nuclei as described previously by this laboratory atthe indicated time points the cell monolayers were washed twice with phosphate buffered one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancersaline pbs following which the cells were fixed for min with ethanol and rehydratedwith 1ml pbs the rehydrated cells were stained with 10μl dapi 10μgml in distilled waterrna isolation and realtime pcr analysistotal rna was isolated using tri reagent molecular research center as described previously the expression of various genes was assessed with realtime reverse transcriptionpolymerase chain reaction rtpcr using primers that were purchased commercially frombiorad laboratories the genes along with the catalog number of the primers are listed insupplemental material s1 table total rna μg was transcribed to cdna using theiscript cdna synthesis kit biorad laboratories the amplification of the cdna was performed using the itaq universal sybr green supermix biorad laboratories with μlcdna and μm primers in a total volume of μl in a cfx96 realtime detection systembiorad laboratories amplification was monitored by sybr green fluorescence cyclingparameters consisted of a s hotstart followed by cycles of denaturation at Ëc for sannealing at Ëc for s and extension at Ëc for s which gave optimal amplificationefficiency the resulting levels were normalized to βactin expression assessed by the sameassay the threshold cycles cts for βactin and the resulting delta cts for the target genes arereported in s2 tablewestern blot analysiswestern blot analysis was performed as described previously the cell pellets were dissolved in 1x radioimmunoassay precipitation assay ripa lysis buffer supplemented withpmsf protease inhibitor cocktail and sodium orthovandate santa cruz biotechnology thecell suspension was sonicated and the lysate was centrifuged to remove cellular debris proteinlysates were quantified using the pierce bca protein assay kit thermoscientific pierceprior to loading samples were reduced and denatured the protein extracts were separated ontgx anykd sdspolyacrylamide gels purchased from biorad laboratories and transferred toa μm hybondp polyvinylidene difluoride membrane using semidry transfer the blotswere blocked in trisbuffered saline tbs containing tween20 tbst and wvbovine serum albumin bsa for min at room temperature the membranes were probedovernight at Ëc with the primary antibody diluted in wv bovine serum albumin allantibodies were purchased from commercial suppliers and were validated against known positive and negative expressing cell lines by western analysis prior to use in experimental protocols the source of the antibody along with their catalog numbers are reported in s3 tableafter washing times for minutes each wash in tbst the membranes were incubated withthe antimouse or antirabbit secondary antibody for min at room temperaturethe blots were visualized using the clarity¢ western ecl blotting substrate bioradlaboratoriesimmunohistochemical stainingserial sections were cut at μm and immersed in preheated target retrieval solutiondako in a steamer for min the sections were allowed to cool to room temperature andimmersed into tbst for min the primary antibodies used in this study along with theirdilutions and catalogue numbers are listed in s3 table the primary antibodies were localizedusing dako peroxidase conjugated envision plus for rabbit or mouse primary antibodies atroom temperature for min liquid diaminobenzidine dako was used for visualizationcounter staining was performed for sec at room temperature using readytousehematoxylin dako slides were rinsed in distilled water dehydrated in graded ethanol one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancercleared in xylene and coverslipped two pathologists judged the presence and degree ofimmunereactivity in the specimensstatistical analysisall experiments were performed in triplicate and the results are expressed as the mean ± semstatistical analyses were performed using graphpad prism1 software version using oneway anova with tukeys or dunnetts posthoc testing for gene expression statistics wererun on the delta cycle threshold δct values that were generated from normalization to βactin levels unless otherwise stated the level of significance was p005resultseffect of troglitizone and pd153035 on the viability and morphology ofurotsa parent and ast cellsthe urotsa parent and ast cells as3 and as4 were treated with either dmso controltroglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr as seenin fig 1a1d there was no change in the morphology of the urotsa parent cells with varioustreatments there was a change in the morphology of the as3 and as4 cells when treatedwith tg fig 1f and 1j and tgpd fig 1h and 1l the cells looked more differentiatedformed mounds and resembled the intermediate cells of the bladder there was a decrease inthe number of urotsa parent cells treated with pd and tgpd when compared to the cellstreated with tg alone or with dmso fig 1m there was also a decrease in the number ofas4 cells when treated with tg and tgpd when compared to the dmso treated cells fig1o there was no significant decrease in the number of as3 cells in any of the treatmentgroups fig 1n an examination demonstrated the lack of dead cells in the treated groups andthe decrease in cell number compared to the dmso group could be due to lack of proliferationandor increased differentiation of cellseffect of pparÎ activation and egfr inhibition on the expression ofluminal genesthe transcription factors pparÎ foxa1 and gata3 play a role in the establishment of theluminal subtype of bladder cancer [ ] studies done by varley have shown thatagonistdependent activation of pparÎ with simultaneous inhibition of egfr phosphorylation in normal human urothelial cells increases the effectiveness of the pparÎ agonist in thepresent study we investigated the effect of the pparÎ agonist tg and an egfr inhibitor pdon the expression of luminal transcriptional factors in the urotsa parent cells and the astcells expression levels for the target genes in this study are reported for a hr hr and hr timecourse for the parent as3 and as4 cells s1 s2 and s3 figs respectively for simplicity the hr gene and protein levels are reported in the main body of the manuscripttreatment with tg increased the expression of pparÎ in the urotsa parent fig 2a i iv andv cell line a similar effect was seen in as3 fig 2b i iv and v and as4 fig 2c i iv and vcell lines treatment with pd did not induce the expression of pparÎ in the urotsa parentfig 2a iv and v or as3 fig 2b iv and v cells but there was a small increase in pparÎ protein in the as4 cells fig 2c iv and v treatment with both tg and pd tgpd increasedthe expression levels of pparÎ mrna in the urotsa parent cells but there was no increase inthe protein levels there was no increase in mrna expression in the as4 cells but there was aslight increase in the protein levels fig 2c i iv and v one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig morphology and viability of urotsa parent and ast cells the urotsa parent ad and ast cells as3 eh and as4 il were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr the measurements were performed in triplicatesand the values reported are mean percentage of control ± sem ordinary oneway anova was performed followed by tukeys posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g001foxa1 gene and protein expression in the urotsa parent cells treated with tg wasreduced fig 2a ii iv and vi however the expression was increased in the as3 and as4cells fig 2b ii iv and vi and 2c ii iv and vi at the protein level treatment with pd decreasedfoxa1 protein in the parent cells but the levels were elevated in the as3 cells tgpdreduced the expression of foxa1 in the urotsa parent cells but it increased the expression offoxa1in the as4 cells at the protein leveltreatment of the urotsa parent cells with tg pd or tgpd did not increase the mrnalevels of gata3 but there was an increase in the protein levels after treatment with tg andtgpd when compared to the dmso treated group fig 2a iii iv and vii in as3 and as4cells there was an additive reduction of gata3 protein by using the combined tgpd treatment fig 2b iv and vii and 2c iv and vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression of luminal markers in urotsa parent and ast cells the urotsa parent aivii as3 bivii and as4 civii weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ciiii western blot analysis was used to measure protein levels a b civ and theintegrated optical density iod of each protein band was calculated a b cvvii gene expression was normalized to βactin and gene andprotein are plotted as foldchange relative to the dmso control amplification was below detectable levels for pparÎ in dmso as3 so adelta cycle threshold δct value of was assigned which is higher than the highest delta ct detected for pparÎ in that cell linetriplicate measurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova wasperformed followed by tukeys posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g002effect of tg and pd on the phosphorylation and expression levels of egfrthe effect of tg and the egfr inhibitor pd was determined on the expression and phosphorylation of egfr in the urotsa parent and ast cells only the combination of tgpdreduced the expression of egfr mrna in the urotsa parent cells however all three treatments decreased the protein levels fig 3a i ii and iii there was no basal phosphorylation ofegfr in the urotsa parent cells and none of the treatments had any effect on the phosphorylation levels fig 3a ii the expression of egfr in the ast cells varied with a decrease inas3 cells and an increase in as4 cells fig 3b i ii and iv and fig 3c i ii and iv respectivelyboth the transformed cell lines had basal phosphorylation of the egfr pegfr and treatment with pd decreased the pegfr levels in as3 fig 3b ii and iii and as4 fig 3c ii andiii which indicates that the pd treatment was effectiveeffect of the pparÎ agonist and inhibition egfr phosphorylation on theexpression of keratinsstudies performed by varley have shown that inhibition of the egfr with simultaneous activation of pparÎ signaling switches normal human urothelial cells from a squamous metaplastic phenotype to a transitional differentiated state with the repression ofkrt14 and the upregulation of krt13 and krt20 therefore we wanted to determineif the urotsa parent and the ast cells would revert more from a basal phenotype to amore transitionalintermediate phenotype when treated with tg and pd the mrnaexpression data is shown in fig and the protein expression data is shown in fig for theurotsa parent cells there was a decrease in expression of krt1 krt5 and krt14 with alltreatments fig 4a i ii and vi and fig 5a i ii and iv the protein for krt1 was undetectedin the urotsa parent cells the expression of krt6 and krt16 increased with tg butdecreased with pd and tgpd fig 4a iii iv v and vii and fig 5a iii and v the krt6antibody does not distinguish between the krt6a krt6b and krt6c isoforms and recognizes protein made by these three genes thus it is not known which isoform is beingexpressed at the protein level there was a decrease in expression of krt13 in the urotsaparent cells fig 4a viii and fig 5a i and vi in the as3 cells the expression levels of thebasal krts with various treatments was similar to the urotsa parent cells fig 4b ivii andfig 5b ivi with the exception of krt1 protein which was expressed in the as3 cells andits expression decreased with tg and tgpd treatment in the as4 cells the expression ofthe krts was similar to as3 with the exception of krt16 fig 4c iviii treatment withtg decreased the expression of krt16 the protein levels for the all the krts was similarto the mrna level with the exception of krt5 krt13 and krt16 krt5 showed anincrease in expression with pd and tgpd treatment and krt16 which showed anincrease in expression with pd fig 5c ivii in the as3 and as4 cells krt13 geneexpression was reduced however the protein levels were elevated from all three treatmentsfig 4b viii fig 4c viii and fig 5b i vii fig 5c i vii one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig expression and phosphorylation of epidermal growth factor receptor the urotsa parent aiiii as3 biiv and as4 ciiv weretreated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real time rtpcranalysis was performed to verify gene expression a b ci western blot analysis was used to measure protein levels a b cii and the integratedoptical density iod of each protein band was calculated aiii b and ciii and iv phosphorylatedegfr was not detected in the urotsa parentcell line gene expression was normalized to βactin and gene and protein are plotted as foldchange relative to the dmso control triplicatemeasurements of gene and protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed bytukeys posthoc test bars with differing letters indicate significant differences p 101371 pone0237976g003effect of pparÎ activation and egfr inhibition on expression oftranscriptional factors p63 and tfap2a and the oncogene trim29associated with squamous differentiationrecently tfap2a has been implicated in the development of squamous differentiation inbasal cancers and activation of pparÎ is shown to represses the expression of tfap2a we therefore investigated the effects of pparÎ activation and egfr inhibition on the expression of tfap2a in urotsa parent and ast cells our results demonstrate that tg reducedtfap2a protein within the parent and as3 cells while the mrna and protein was elevatedin the as4 cells from tg exposure treatment with pd as well as tgpd decreased the one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig gene expression of keratins the urotsa parent aiviii as3 biviii and as4 civiii were treated witheither dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr real timertpcr analysis was performed to verify gene expression a b civiii gene expression was normalized to βactin andare plotted as foldchange relative to the dmso control triplicate measurements of gene levels were performed and arereported as mean ± sem ordinary oneway anova was performed followed by tukeys posthoc test bars withdiffering letters indicate significant differences p 101371 pone0237976g004expression of tfap2a in the urotsa parent fig 6a i iv and v as3 fig 6b i iv and v andas4 cells fig 6c i iv and vtranscription factor p63 is another protein associated with human bladder cancersenriched in basalsquamous markers [ ] there was a decrease in the expression of p63 inthe urotsa parent cells with all treatments fig 6a ii iv and vi in the as3 cells the expression of p63 was low and treatment with tg increased its expression however treatment withpd and tgpd decreased its expression fig 6b ii iv and vi the expression of p63 in as4cells increased at the mrna level with tg and tgpd treatments however the protein levelswere decreased when compared to the dmso control fig 6c ii iv and vithe expression of trim29 a gene associated with the basal gene expression program was also determined in the urotsa parent and the as3transformed cells for the urotsaparent and as3 cells there was a decrease in the expression of trim29 in cells treated withpd and tgpd fig 6a and 6b iii iv and vii for as4 pd and tgpd treatments increasedtrim29 protein fig 6c iv and viiexpression of trim29 tfap2a and p63 within tumors formed fromurotsa as3 as4 and rt4 cellsurotsa as3 and as4 are considered to be of the basal molecular subtype while rt4 cellsare considered to be of the luminal molecular subtype of bladder cancer cells therefore wewanted to confirm the in vivo expression of trim29 tfap2a and p63 within the nondifferentiated basalsquamous areas of tumors originating from urotsa as3 and as4 cells incomparison to the expression in tumors originating from the luminal rt4 cells all three ofthese proteins were enriched within the nondifferentiated areas of tumors developed from theurotsa as3 and as4 cells fig 7a 7b 7d 7e 7g and 7h signs a lower expression wasobserved in the welldifferentiated areas of the urotsa as3 and as4 tumors fig 7a 7b7d 7e 7g and 7h� asterisks there was low to no staining in the rt4 cells for trim29tfap2a and p63 fig 7c 7f and 7idiscussionthe classification of uc into various subtypes has implications in the overall patient management of the disease with the basal subtype having a worse outcome when compared to theluminal subtype the molecular mechanisms involved in the development of these subtypesare not yet established however the role of some transcriptional factors is established withpparÎ playing an important role in the activation of the luminal specific genes [ ] andtfap2a playing a role in the development of the basal subtype of uc in addition studieswith normal human urothelial cells show that activation of pparÎ with an agonist along withinhibition of cellular proliferation via the egfr pathway can switch cells from a squamousmetaplastic phenotype to a more transitional differentiated phenotype combination therapiesusing egfr inhibitors and pparÎ agonists show promising results against some urothelialtumors in vivo as well as against other cancers based on these studies we sought todetermine if the urotsa parent and the as3transformed urotsa cell lines that are one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancer one 101371 pone0237976 august one 0cactivation of luminal pathway in basal bladder cancerfig protein expression of keratins the urotsa parent aivi as3 bivii and as4 civii were treated with either dmso control troglitizone tg μm pd153035 pd μm or tg and pd tgpd for hr western blot analysis was used to measure protein levels ai bi ci and the integratedoptical density iod of each protein band was calculated aiivi b and ciivii protein levels are plotted as foldchange relative to the dmso controltriplicate measurements of protein data were performed and are reported as mean ± sem ordinary oneway anova was performed followed by tukeysposthoc test bars with differing letters indicate significant differences p 101371 pone0237976g005molecularly characterized as a basal subtype of bc could convert to a luminal transitionalcell type when treated with a pparÎ agonist andor an egfr inhibitor as this could affect theoverall outcome of the diseasethe urotsa parent cells when grown in serum expresses many genes that are associatedwith the basal subtype in this study treatment of these cells with the pparÎ agonist tginduced the expression of pparÎ as well as gata3 but not foxa1 in mortal human urothelial cells treatment with the pparÎ agonist shows an increase in the expression of the transcription factor foxa1 and this is contrary to what is seen in our study these differencescould be due to the cell type since we are using immortalized cells the as3transformedurotsa cells showed an induction in the expression of pparÎ and foxa1 when treated withtg however the expression of gata3 in these transformed lines was not consistent in bccell lines studies by others have shown that have shown that gata3 and foxa1 cooperatewith pparÎ activation to drive the differentiation of a basal bladder cancer subtype to a moredifferentiated luminal subtype other studies have also linked the expression of foxa1 tothe development of the luminal subtype of urothelial cancer [] in our studies treatmentwith tg did result in the differentiation of the as3transformed cells based upon morphological changes and induced the expression of pparÎ as well as foxa1 both of which are factorsknown to drive luminal differentiation of bladder cancer cell linessignaling via the pparÎ pathway is essential for the growth arrest and terminal differentiation of adipocytes and other normal epithelial [] and cancer cells [] whereassignaling via the egfr receptor plays a role in cell proliferation the keratins play an essentialrole in the differentiation of epithelial cells and different keratin profiles are expressed at different stages of differentiation the normal bladder has three different stages of differentiationand these stages are marked by the expression of krt14 krt5 and krt20 krt14 isexpressed in a subset of basal cells that are thought to play a role in regeneration as well astumorigenesis whereas other basal cells and intermediate cells in the normal bladderexpress krt5 the expression of krt20 is restricted to the most differentiated cells theumbrella cells these differentiation stages of the bladder are shared by bladder cancersand malignant transformation can occur in any of the different cell types of the bladder theexpression of krt14 is seen in the least of the differentiated tumors and its expression correlates to poor prognosis krt14 whereas the expression of krt20 is restricted to differentiated bladder cancers and its expression is associated with good prognosis a study doneby varley showed that normal human urothelial cells in culture express krt14 andlack the expression of krt13 and krt20 activation of pparÎ in these cells induced theexpression of krt13 and decreased the expression of krt14 this effect was significantlyenhanced when
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" bridge to surgery bts using a selfexpandable metallic stent sems for the treatment of obstructivecolorectal cancer improves the patients quality of life this study aimed to examine prognostic factors ofobstructive colorectal cancermethods we analyzed stage iiiii resectable colon cancer cases cur a retrospectively registered between january and december overall patients with cur a obstructive colorectal cancer were evaluated ofthem underwent emergency surgery es group and of them after bts with sems placement bts group wecompared surgical results and prognoses between the two groupsresults a total of patients underwent endoscopic sems placement which technical success of andmorbidity rate of primary anastomosis rates were in es and in bts p postoperativecomplication in es and in bts p pathological findings of lymphatic invasion in es and in bts p venous invasion were in es and in bts p and recurrence of in esand in bts the 3year overall survival was significantly different between two groups es 868bts bts is worse than es logrank test p venous invasion independently predicted worsened recurrencefreeand overall survivals the vascular invasiveness was correlated with tumor progression after sems placement and thesurvival rate was lower in bts sems potentially worsens prognostic outcomes in stage iiiii obstructive colorectalcancerkeywords bowel obstruction colorectal cancer selfexpandable metallic stent colorectal cancer crc remains the leading cause ofcancerrelated deaths worldwide because several patientsare initially diagnosed during advanced stages correspondence ohtakmedkindaiacjp1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japanfull list of author information is available at the end of the approximately of patients with crc were diagnosed with acute colonic obstruction [] severe malignancy with bowel obstruction needs urgent surgicalintervention which includes primary lesion resectionand stoma creation leading to increased morbidity andmortality and a potential failure to achieve completeoncological resection [ ]an endoscopic procedure with selfexpandable metallic stent sems is an acceptable bridge to surgery bts the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc surgery page of treatment for acute colonic obstruction [] preoperative sems placement provides an opportunity to perform medical resuscitation comorbidity optimizationbowel preparation tumor staging and observation ofproximal lesions the procedure prevents highriskemergency surgeries and increase oncological resectionand primary anastomosis rates [ ] after the inclusion of colonic sems placement as bts in the coverageof the national health insurance in japan several physicians joined the colonic stent safety procedure researchgroup and developed skills to provide safe treatmentthe largest multicenter prospective study demonstratedthe feasibility and safety of sems placement as bts inpatients with malignant colorectal obstruction the oncological safety and minimalinvasiveness ofthis procedure have confirmed that sems placement asa bridge to elective surgery is not recommended as astandard treatment for symptomatic leftsided malignantcolonic obstruction [ ] several studies reportedthat prognostic factors of malignant colonic obstructionin sems placement had oncological disadvantages compared with those in emergency surgery es [ ] incontrast several trials showed that sems placement as abridge to elective surgery did not improve the survivalrates [] how sems placement worsens prognosticoutcomes remains unclear [ ]this study aimed to evaluate the induction of curativesurgery in patients with malignant colorectal obstructionafter a sems placement and its longterm results andprognostic factors postoperatively compared to patientswithout sems placement we demonstrated prognosticfactors and overall survival os and recurrencefreesurvival rfs rates for curative surgery after a semsplacementmethodspatientsmedical records of patients who underwent primarycolorectal resection at higashiosaka city medical centerbetween january and december werereviewed all participants provided written informedconsent oralintake and symptoms before and aftersems placement were assessed in table using thecolorectal obstruction scoring system cross from to we recruited patients with all class oftable the colorectal obstruction scoring system crosspatients symptom and their condition of an oral intakesolid meal low residue and full diet without symptomcrosscross as stent insertion candidate from we excluded patients with cross and based on updatedstent insertion guideline malignant colorectal obstruction was diagnosed through clinical examinationcross radiography and computed tomography surgery was performed using three approaches es comprised laparotomylymph node dissection as possibleand primary anastomosis on the same day between and bts after sems placement comprised standbylaparoscopy d3 lymph node dissection and primaryanastomosis since january overall patientswith stage iiiii cur a obstructive colorectal cancerwere evaluated of them underwent emergency surgery as es group and of them after bts with semsplacement as bts group we compared surgical resultsand prognoses between the two groupssems devices and the procedurepatients were endoscopically treated with placement ofan uncovered wallflex enteral colonic stent boston scientific corporation natick ma usa or nitis enteralcolonic uncovered stent taewoong inc gimpo southkorea placements were performed as presented in thepreintroduction publicity announcement placement details were mentioned on the website as a brief guideline obstruction structures were determined using aguide wire and a contrast tube was inserted into theproximal colorectal lumen obstructions were measuredusing contrast agents and then the endoscopist determined the number size and type of stent pathologicalbiopsies were recommended after sems locations andintraluminal or extraluminal marking using an endoscopic clip were recommended via visual recognition ofthe endoscopist dilatation of the colonic obstructionbefore sems placement was generally not allowedhistological findingsparaffinembedded specimens were obtained from a cohort of patients diagnosed by the union for international cancer control stage iiiiisurvival definitionsos was defined as the duration from surgery to anydeath or last followup diagnosis of recurrence was calculated based on recist according to the chemotherapy criteria rfs was defined as the durationfrom surgery to any recurrence includes local recurrenceor distant metastasissolid meal low residue and full diet with symptomliquid or enteral nutrient intakeno oral intakerequiring continuous decompressionstatistical analysisstudents ttest and wilcoxon test for continuous variables and the Ï2 and fishers exact tests for categoricalvariables were conducted survival curves were generated using the kaplanmeier method and compared 0cohta bmc surgery page of table baseline characteristics and outcomes of endoscopicsems placementtable comparison of baseline characteristics in patientsundergoing emergency surgery and bridge to surgerybts n es n pmalefemalemedian range genderagelocationmalefemalemedian rangececumascendingtransversedescendingsigmoidrectumlength of obstructionmedian range cmtechnical successprocedurestentingmorbiditymortalityclinical successthrough the scopethrough the wirewall flex cmnitis cmoverall cda iiicda vcrossbts n a claviendindo classificationusing a logrank test univariate and multivariate survival analyses were performed using the cox proportional hazards regression model all statistical analysesused jmp version sas institute cary nc or statistical scripting language r httpwwwrprojectpvalues of ¤ twosided were considered statistically significant this prognostic study complied withthe reporting recommendationsfor tumor markerprognostic studies resultsa total of patients underwent endoscopic semsplacement which was technically safe for malignantcolorectal obstruction with the technical success rate of the clinical success rate was and the patientssymptoms and oral intake dramatically improved afterthe sems placement shown in table a total of patients were reviewed and patients underwentes and bts respectively as shown in table baselineclinical characteristics were balanced between the twogroups moreover cases of patients underwent es on the same day as in open surgery the median waiting period for surgery was days for bts thegenderagelocationtype of operationcecumascendingtransversedescendingsigmoidrectumstandbyemergency duration tooperationmedian range dayssurgical procedurelaparotomy laparoscopy timemedian range minblood lossmedian range ml before afterstoma creation morbidity30day complication cda iii anastomosticleakagehospital stayaclaviendindo classificationmedian range days primary anastomosis ratios were in es and in bts p postoperative complication rateswere in es and in bts p postoperative hospital stay was shorter in bts days comp patients withpared to esobstructive crc showed significantinpostoperative complication rate and hospital stay withsems placement operative procedures were dramatically changed and the primary anastomosis rate improved after the sems placementimprovement daysthe pathological tissue type accounted for of differentiated types shown in table tumordepth was similar between the two groups lymphatic vesselinvasion ratios were in es and in bts p and venous invasion ratioswere in es and in bts p recurrence rates were cases in es and cases in bts nodenegative patients stage iimorewhereas nodepositive patients stage iii more frequently had liver metastasis in the kaplanmeier survival analysis in fig 1a the 3year rfs waslung metastasisfrequentlyhad 0cohta bmc surgery page of table comparison of pathological characteristics of emergency surgery and bridge to surgerypt factortotal lymph nodespn factorhistologicallymphatic invasionvenous invasionsurgical clearancet4b t4a t3median rangen0 n1 n2 n3tub1 tub2 othersly v cur a b csignificantly different between the two groups es bts which was significantly low inbts than that in es logrank test p the3year os rate was also significantly different between thetwo groups es bts p shown in fig 1b the relationship between lymph node metastasis and sems placementwas also evaluated the pathological nodenegativestage ii 3year rfs rate was not different betweenthe two groups es bts as shown infig 2athe pathological nodepositivestage iii 3year rfs rate was different between thetwo groups es bts as shown in fig 2bthe stage ii 3year os rate was not different between thetwo groups es bts as shown in fig 2cwhereas stage iii 3year os rate was different between thetwo groups es bts as shown in fig 2dthese results suggestthat vascular invasiveness andpathological nodepositive status were correlated withtumor progression after sems placementthein contrastthuses n bts n p survival rate was affected by poor prognosis in the btsgroupresults of adjusted multiple cox proportional hazard regression for rfs and os in all stages and stage iii diseaseare presented in table after adjusting for possible confounders venous invasion and bts independently predicted poor rfs in all stages and venous invasionindependently predicted poor rfs in stage iii disease venous invasion and bts were also significantly associatedwith os in stage iii diseasediscussionacute colonic obstruction requires emergent surgicalintervention a mandatory conventional treatment skillemergent surgicalis associated with highmorbidity mortality and stoma creation rates affectingthe quality of life of patients malignant colorectal obstruction is not only an intestinal obstruction but alsoan advanced stage crc their prognosis was poorerthan that in patients with nonocclusive disease becausetreatmentfig kaplanmeier survival curves in patients undergoing emergency surgery vs bridge to surgery a recurrencefree survival b overall survival 0cohta bmc surgery page of fig kaplanmeier survival curves in patients undergoing emergency surgery vs bridge to surgery a rfs nodenegative patients b rfs nodepositive patients c os nodenegative patients d os nodepositive patientsof highly invasiveness and distant metastasis [ ]chen revealed that the prognosis in patients withperforation associated with obstruction was poor early intervention in the clinical setting before the colonic perforation has been established endoscopic placement of colonic stents improves the high decompressioneffect and reduces clinical symptoms high postoperative complication rates were correlatedwith poor prognosis in patients with cancer in severalans [] reducing complication rates can improve the prognosis our results showed high clinicalsuccess rate after sems placement and high primarysurgicalinterventions howeveranastomosis rate stentrelated complications requiredemergentthe stentplacement is safe and feasible in this study moreoverthe laparoscopic rate was high and postoperative complication rate was clinical results including shortterm outcomesin bts after sems were verifiedthrough a metaanalysis [ ]the prognosis was poor in patients with stent perforation and increased local recurrence rate after the colonic stent placementthe longtermprognosis in patients with colorectal obstruction afterbts was not different compared with that in patients however 0cohta bmc surgery page of table multivariate analysis of recurrencefree survival at all stages and stage iiipvaluevariablesrecurrence free suvivalall stages hazard ratio ± sd cistage iii hazard ratio ± sd cipvaluees vs semspt factor t3t4pn factor n0n1n2verous invasion v01v2lymphatic invasion ly01ly2hr ± hr ± hr ± hr ± hr ± overall suvivales vs semspt factor t3t4pn factor n0n1n2verous invasion v01v2lymphatic invasion ly01ly2hr ± hr ± hr ± hr ± hr ± without obstruction [] according to the europeansociety of gastrointestinal endoscopy clinical guidelinethat considers the risk of perforation due to colorectalstents only limited uses are allowed therefore colorectalstent placement is not a standard treatment []the prognostic outcomes of bts in this study were significantly worse than those of es particularly in lymphnodepositive patientslymphatic and venous invasionseemed to be a significant prognostic factor althoughreduced postoperative complication rate would improve the prognosis our results were contradictoryafter the stent replacement these results suggestedthat stent placement leads to poor prognosis a concern that colonic stents may be associated with adverse effects of mechanical expansion also exists mechanical expansion may be associated with thegrowth of solid tumors particularly lymphatic andvenous invasion [ ]we found that recurrence and os were associatedwith high vascular invasion after a colonic stent placement venous invasion was an independent factor for recurrence and prognosis the ck20 mrna level anepithelial marker is significantly increased in peripheralblood serum suggesting stent deployment into the vasculature alliteratively ki67 level associated withcellular proliferation and p27 gene assisting cell cycleprogression were measured using specimens obtainedbefore and after sems insertion next the ki67 leveldecreased in the specimen after an sems placementcompared with that before and cell proliferation wassuppressed the prognostic nutritionalindex andserum albumin levels were significantly decreased afterstenting suggesting its disadvantage as bts theduration from stent placement to surgery was daysoncological and nutritional factors might change in theblood and contribute to poor prognosis during the waiting period mechanical expansion of the replacement hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± hr ± should be minimized to prevent perforation and molecular cytological factors to improve the materials expansion and establishment of new mechanism are necessaryin colorectal obstruction [ ]thisisafirstretrospectivethese findings should be considered in light of severallimitationsnonrandomized small sample sized study from a single institution thereby the heterogeneity of the surgical strategy may have affected the prognostic factors secondalthough validated endoscopic procedures were validated stent devices used in this study had differentlengths types and thickness and obtained from differentvendors lastly we performed stent placement in the patients with cross and who are not indicated forstent insertion until to investigate the oncological longterm prognosis ofcolonic sems placement as a bridge to elective surgerylarge sample size and prospective randomized controlledstudies are warranted to develop a treatment strategy forcrc with obstructionvascular invasiveness was correlated with tumor progression after a sems placement and os and rfs rateswere lower in bts sems placement potentially worsensprognostic outcomes in stage iiiii malignant colorectalobstructionabbreviationsbts bridge to surgery crc colorectal cancer cross colorectal obstructionscoring system es emergency surgery esge european society ofgastrointestinal endoscopy jsccr japanese society for cancer of the colonand rectum recist response evaluation criteria in solid tumors version os overall survival rfs recurrencefree survival sems selfexpandablemetallic stent wses world society of emergency surgeryacknowledgementsthe authors thank for contribution as endoscopic technical adviser kenkonishi md phd from department of surgery hyogo prefecturenishinomiya hospital 0cohta bmc surgery page of authors contributionsall authors have read and approved the manuscript ko protocolproject development data collection and management andmanuscript writingediting mi protocolproject developmentmanagement and manuscript writingediting mu protocolprojectdevelopment and data collection and management jm se jm and ikdata collection and management yt and sn data collection ki andtn data analysis and manuscript writingediting mt and ty dataanalysis and managementfundingauthors have no grant support and no financial relationship for this studyavailability of data and materialsthe datasets used and analyzed during this study are available from thecorresponding author upon reasonable requestethics approval and consent to participateall procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional researchcommittee and with the helsinki declaration and its later amendmentsor with comparable ethical standards all participants or their guardians haveprovided their written informed consent and that the study protocol wasapproved by higashiosaka city medical center ethical committee on humanresearch assignment number consent for publicationno applicablecompeting intereststhe authors declare that they have no conflict of interest to discloseauthor details1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japan 3thoracic surgeryhigashiosaka city medical center osaka japan 4digestive surgery kawasakimedical school okayama japan 5gastroenterology higashiosaka citymedical center osaka japanreceived june accepted august referencesjemal a bray f center mm ferlay j ward e forman d global cancerstatistics ca cancer j clin pubmed pmid epub eng winner m mooney sj hershman dl feingold dl allendorf jd wright jd incidence and predictors of bowel obstruction in elderly patients withstage iv colon cancer a populationbased cohort study jama surg pubmed pmid pubmed central pmcid pmc45 epub engjullumstro e wibe a lydersen s edna th colon cancer incidencepresentation treatment and outcomes over years color dis pubmed pmid epub engcheynel n cortet m lepage c benoit l faivre j bouvier am trends infrequency and management of 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on selfexpandable metallic stents as a bridgeto surgery for malignant colorectal obstruction in japan efficacy and safetyin patients surg endosc pubmed pmid epub engsaida y sumiyama y nagao j uramatsu m longterm prognosis ofpreoperative bridge to surgery expandable metallic stent insertion forobstructive colorectal cancer comparison with emergency operation discolon rectum suppls44 pubmed pmid epub engtomita m saito s makimoto s yoshida s isayama h yamada t selfexpandable metallic stenting as a bridge to surgery for malignant colorectalobstruction pooled analysis of patients from two prospectivemulticenter series surg endosc pubmed pmid pubmed central pmcid pmc6342866 epub eng chen hs sheenchen sm obstruction and perforation in colorectaladenocarcinoma an analysis of prognosis and current trends surgery pubmed pmid epub eng huang x lv b zhang s meng l preoperative colonic stents versusemergency surgery for acute leftsided malignant colonic obstruction ametaanalysis j gastrointest surg pubmed pmid epub engkim hj huh jw kang ws kim ch lim sw joo ye oncologic safetyof stent as bridge to surgery compared to emergency radical surgery forleftsided colorectal cancer obstruction surg endosc pubmed pmid epub engshigeta k baba h yamafuji k kaneda h katsura h kubochi k outcomesfor patients with obstructing colorectal cancers treated with onestagesurgery using transanal drainage tubes j gastrointest surg pubmed pmid epub engvan hooft je bemelman wa oldenburg b marinelli aw lutke holzik mfgrubben mj colonic stenting versus emergency surgery for acute leftsided malignant colonic obstruction a multicentre randomised trial lancetoncol pubmed pmid epub engvan hooft je fockens p marinelli aw timmer r van berkel am bossuyt pm early closure of a multicenter randomized clinical trial of endoscopicstenting versus surgery for stage iv leftsided colorectal cancer endoscopy pubmed pmid epub engsloothaak da van den berg mw dijkgraaf mg fockens p tanis pj vanhooft je oncological outcome of malignant colonic obstruction inthe dutch stentin trial br j surg pubmed pmid epub eng gorissen kj tuynman jb fryer e wang l uberoi r jones om localrecurrence after stenting for obstructing leftsided colonic cancer br j surg pubmed pmid epub eng ormando vm palma r fugazza a repici a colonic stents for malignantbowel obstruction current status and future prospects expert rev meddevices pubmed pmid epub englauro a binetti m vaccari s cervellera m tonini v obstructing leftsidedcolonic cancer is endoscopic stenting a bridge to surgery or a bridge tonowhere digestive diseases and sciences pubmed pmid epub engschwartz lh litière s de vries e ford r gwyther s mandrekar s recist 11update and clarification from the recist committee europeanjournal of cancer oxford england p pubmed pmid pubmed central pmcid pmc5737828 epub eng mcshane lm altman dg sauerbrei w taube se gion m clark gm reportingrecommendations for tumour marker prognostic studies remark eur jcancer pubmed pmid epub eng hashiguchi y muro k saito y ito y ajioka y hamaguchi t japanesesociety for cancer of the colon and rectum jsccr guidelines for thetreatment of colorectal cancer int j clin oncol pubmed pmid epub eng cortet m grimault a cheynel n lepage c bouvier am faivre j patterns ofrecurrence of obstructing colon cancers after surgery for cure a population 0cohta bmc surgery page of based study color dis pubmed pmid epub eng nojiri t maeda h takeuchi y funakoshi y kimura t maekura r predictive value of btype natriuretic peptide for postoperative atrialfibrillation following pulmonary resection for lung cancer eur jcardiothorac surg pubmed pmid epub engpubmed pmid pubmed central pmcid pmc4128744 epub engkaragiannis gs poutahidis t erdman se kirsch r riddell rh diamandis epcancerassociated fibroblasts drive the progression of metastasis throughboth paracrine and mechanical pressure on cancer tissue mol cancer res pubmed pmid pubmed central pmcidpmc4399759 epub eng nojiri t inoue m yamamoto k maeda h takeuchi y funakoshi y b maruthachalam k lash ge shenton bk han af tumour celldissemination following endoscopic stent insertion br j surg pubmed pmid epub eng matsuda a miyashita m matsumoto s sakurazawa n kawano y yamahatsuk colonic stentinduced mechanical compression may suppresscancer cell proliferation in malignant large bowel obstruction surg endosc pubmed pmid epub eng haraguchi n ikeda m miyake m yamada t sakakibara y mita e colonic stenting as a bridge to surgery for obstructive colorectal canceradvantages and disadvantages surg today pubmedpmid epub eng zhu z li b liao w lv n chen y shu x novel predictive nomogram foridentifying difficult guidewire insertion in patients with malignant colorectalobstruction and sphincterotomeassisted guidewire insertion for improvingthe success rate of selfexpandable metal stent insertion front oncol pubmed pmid pubmed central pmcid pmc7237730epub eng miyasako y kuwai t ishaq s tao k konishi h miura r newlydeveloped selfexpandable nitis md colonic metal stent for malignantcolonic obstruction world j gastrointest surg pubmedpmid pubmed central pmcid pmc7215972 epub engpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationstype natriuretic peptide as a predictor of postoperative cardiopulmonarycomplications in elderly patients undergoing pulmonary resection for lungcancer ann thorac surg pubmed pmid epub eng cowie mr struthers ad wood da coats aj thompson sg poolewilsonpa value of natriuretic peptides in assessment of patients withpossible new heart failure in primary care lancet pubmed pmid epub eng cuthbertson bh card g croal bl mcneilly j hillis gs the utility of btypenatriuretic peptide in predicting postoperative cardiac events and mortalityin patients undergoing major emergency noncardiac surgery anaesthesia pubmed pmid epub engsabbagh c browet f diouf m cosse c brehant o bartoli e isstenting as a bridge to surgery an oncologically safe strategy for themanagement of acute leftsided malignant colonic obstruction acomparative study with a propensity score analysis ann surg pubmed pmid epub engtung kl cheung hy ng lw chung cc li mk endolaparoscopic approachversus conventional open surgery in the treatment of obstructing leftsidedcolon cancer longterm followup of a randomized trial asian j endoscsurg pubmed pmid epub eng gianotti l tamini n nespoli l rota m bolzonaro e frego r aprospective evaluation of shortterm and longterm results from colonicstenting for palliation or as a bridge to elective operation versus immediatesurgery for largebowel obstruction surg endosc pubmed pmid epub eng yang sy park yy han yd cho ms hur h min bs oncologicoutcomes of selfexpandable metallic stent as a bridge to surgery andsafety and feasibility of minimally invasive surgery for acute malignantcolonic obstruction ann surg oncol pubmed pmid epub engvan hooft je van halsema ee vanbiervliet g beetstan rg dewitt jmdonnellan f selfexpandable metal stents for obstructing colonic andextracolonic cancer european society of gastrointestinal endoscopy esgeclinical guideline endoscopy pubmed pmid epub eng ansaloni l andersson re bazzoli f catena f cennamo v di saverio s guidelenines in the management of obstructing cancer of the leftcolon consensus conference of the world society of emergency surgerywses and peritoneum and surgery pns society world j emerg surg pubmed pmid pubmed central pmcid pmc3022691epub eng arezzo a balague c targarona e bhi f giraudo g ghezzo l colonic stenting as a bridge to surgery versus emergency surgery formalignant colonic obstruction results of a multicentre randomisedcontrolled trial esco trial surg endosc pubmedpmid epub eng amelung fj burghgraef ta tanis pj van hooft je ter b f siersema pd critical appraisal of oncological safety of stent as bridge to surgery inleftsided obstructing colon cancer a systematic review and metaanalysiscrit rev oncol hematol pubmed pmid epub eng domingo s puertolas s graciavilla l puertolas ja mechanical comparativeanalysis of stents for colorectal obstruction minim invasive ther alliedtechnol pubmed pmid epub engsuzuki n saunders bp thomasgibson s akle c marshall m halligan scolorectal stenting for malignant and benign disease outcomes incolorectal stenting dis colon rectum pubmed pmid epub eng voutouri c mpekris f papageis p odysseos ad stylianopoulos t roleof constitutive behavior and tumorhost mechanical interactions in thestate of stress and growth of solid tumors plos one 201498e104717 0c"
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gastroenteropancreatic neuroendocrine neoplasms gep nens as well as neuroendocrine tumors gep nets are heterogeneous tumors that originate from peptidergic neurons and neuroendocrine cells previously described as carcinoid tumors in most nets are indolent tumors compared with other epithelial malignancies however they are reported to have the potential to metastasize even in well differentiated tumors and are resistant to therapies1 data from the surveillance epidemiology and end results seer database indicate that the incidence of nets has increased significantly approximately times reaching casesyear of which gep nets account for approximately to of all nets and the correspondence qiang feng department of colorectal surgery national cancer centernational clinical research center for cancercancer hospital chinese academy of medical sciences and peking union medical college no panjiayuan south road chaoyang district beijing peoples republic of china email fengqiang2008vipsinacomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars256723 cancer management and research wu this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphp 0cwu dovepressincidence and prevalence of colorectal nets are inferior only to those of colorectal adenocarcinoma146 in addition the tumor sites varied markedly by race with the incidence of rectal nets among asian populations increasing from per in to per in which was among white populations147 similarly the incidence of rectal neuroendocrine tumors rnets grew fastest among all nets by approximately times compared with the incidence in accounting for of all nets which makes it the second most common net in china8significantly higher than that for localized colorectal nets endoscopic resection including endoscopic mucosal resection emr and endoscopic submucosal dissection esd and surgery including transanal excisions as well as surgical resections are both effective methods for metastatic tumors somatostatin analogs ssas radiation radiofrequency ablation rfa chemotherapy targeted therapy and peptide receptor radionuclide therapy prrt are all alternatives however the 5year survival rate of nets with lymph node metastasis lnm or distant metastasis is still disappointing with fiveyear overall survival rates of approximately and respectively6911the prognostic factors of colorectal nets have been explored by numerous studies tumor stage location size grade lymphovascular invasion and the status of resection margins are major factors that have been reported to be associated with lnm and poor prognosis12 however these studies were highly heterogeneous which affected the accuracy of the metaanalysis and the effectiveness of these scurrently controversies remain in the treatment of colorectal nets experts from the chinese society of clinical oncology csco agreed that colonic nets greater than cm and less than cm could be completely resected endoscopically when the t stage was less than t2 but the national comprehensive cancer network nccn recommends that these tumors be treated by surgery in accordance with the guidelines for colon adenocarcinoma13the aim of this study was to evaluate the outcomes of colorectal nets explore the risk factors for lymph node metastasis in colorectal nets and identify the prognostic factors for survival outcomesmethodsclinical data collectionbetween and a total of consecutive patients received treatments for colorectal nets in our center we constructed a database of retrospectively collected data from patients medical records including clinical characteristics pathological reports recurrence and survival during the followup periodfor radical resection with lymph node dissection lymph node metastasis was detected by pathological evaluation for local excision such as endoscopic mucosal resection emr endoscopic submucosal dissection esd or transanal excision tae lnm was evaluated through computed tomography ct or magnetic resonance imaging mri before the treatment and during the followup periods the diagnosis of a metastatic lymph node was based on the following criteria size criteria short axis diameter of lymph nodes was greater than mm for round lymph nodes and greater than mm for ovoid lymph nodes morphological abnormalities irregular contour and margin unclear border heterogeneous internal echoes or signal intensity1617 the tumor diameter refers to the longest diameter of the tumor according to pathology reports for patients with distant metastases tumor diameter was determined by endoscopic findings before treatmentpathological diagnosisthe tumor stage was classified according to the american joint committee on cancer ajcc cancer staging manual 7th edition and 8th edition1819 and the tumor grade was classified according to the classification20 for patients before we revised their pathology results and found that they were all neuroendocrine carcinomas necs therefore we classified them as having g3 grade tumors the mitosis count n23 was expressed as the number of mitotic cells in ten highpower fields hpfs from hematoxylin and eosin hestained slides examined with microscopy according to enetswho guidelines g1 grade mitotic image hpfs g2 grade mitotic image hpfs and g3 grade mitotic images2010 hpfs the ki index was calculated as the percentage of cells labeled by immunohistochemistry according to enetswho guidelines g1 grade ki67 positive index g2 grade ki67 positive index to and g3 grade ki67 positive index20 the expression levels of chromogranin a cga n101 and synaptophysin syn n109 were scored according to the percentage of positive cells and the intensity of cell staining the positive cell percentage score was based on the following system points no positive cells point positive cells accounting for to points positive cells accounting for to points submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu positive cells accounting for to and points positive cells accounting for to the positive cell staining intensity score was based on the following system points negative point weakly positive points moderate positive and points strong positive the two scores were multiplied together points for negative to points for weak positive to points for moderate positive and to points for strong positive inclusion criteriapatients who were treated in our center for localized and metastatic colorectal nets from to exclusion criteria patients who had colorectal nets combined with other malignancies patients for whom the pathological diagnosis was mixed adenoneuroendocrine carcinoma patients for whom there was insufficient clinical inappropriate pathology reports information or from outside hospitalsrisk factors for lymph node metastasis and prognostic factors related to survival were investigated in all patientsthe primary endpoint was progressionfree survival pfs which was defined as the interval between initial treatment and the first documentation of disease progression or deathstatistical analysisstatistical analysis was performed using spss for mac version spss chicago il usa continuous data are described as means±sds in this study the risk factors for lnm were assessed using pearsons Ï2 test in univariable analysis and logistic regression analyses in multivariable analysis the 5year overall survival os and progression free survival pfs were analyzed with the kaplanmeier method variables were compared with the log rank test and the multivariable analysis for survival outcomes was conducted using the cox proportional hazards model statistical significance was accepted for p values female ratio was the frequencies of grade g1 g2 and g3 nets were and respectively of the patients patients were resected locally by emr by esd and by transanal excision in addition nets were surgically resected including radical resections multivisceral resections and palliative resections due to distant metastasis the remaining patients were treated by systemic treatment due to distant metastasis the most commonly used chemotherapeutic regimen in our center was the ep regimen etoposide and cisplatin as the firstline chemotherapy and the secondline chemotherapy was variable and included the xelox regimen oxaliplatin and capecitabine the folfox regimen oxaliplatin calcium folate and 5fu and everolimus temozolomide and tegafurgimeraciloteracil and combinations between them the tumor diameter was less than cm in patients and the distance from the anal verge was less than cm in patients lnm was found in cases and distant metastasis occurred in patients two patients had radiologically determined lnm after tae in the followup period and one of them went on to undergo radical surgery the other patient was also found to have liver metastasis therefore he was treated with chemotherapy the clinical and histopathological characteristics are summarized in table risk factors for lnmthe risk factors for lnm through univariate analysis were tumor location in the colon p0001 tumor diameter ¥ cm p0001 t stage p0001 tumor grade p0001 and the positive degree of syn p0012 and cga p0049 table in multivariable analyses tumor diameter ¥ cm or ci p0040 and tumor grade g3 or ci p0001 were independent risk factors for lnm in colorectal ntes tumor location in the colon or ci p0083 and tumor grade g2 or ci p0066 might be independent risk factors for lnm even though the p value was greater than table resultsclinical and histopathological characteristics figure a total of patients were included in our study figure the age of the patients was ± years and the male risk factors for survival outcomesthe median followup period was months range months a total of patients died in this cohort in patients with distant metastasis before treatment patients died during chemotherapy cancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepressfigure flowchart of the selection of patientspatients died after multivisceral resections and patients died after palliative resections due to tumor progression in patients without distant metastasis before treatment patients died due to the recurrence of distant metastasis at the liver peritoneum lung pleura and brain and patient died of severe lung infections the 5year progressionfree survival pfs and overall survival os rates of all patients were and respectively the prognostic factors for the 5year pfs and os rate in all patients were age neoadjuvant chemotherapy tumor diameter tumor location tumor grade lnm cga level and treatment method table in the multivariable analysis age ¥ hr ci p00020001 and lnm yes hr ci p00180025 were independent risk factors for 5year pfs and os the cga level [moderate positive hr ci p0010 and strong positive hr ci p0007] were independent risk factors for 5year pfs tumor diameter ¥ cm hr ci p0063 and tumor grade g3 hr ci p0090 were independent risk factors for 5year pfs even though the p value was greater than table comparison of two age groupsin univariable analyses patients were in the 65year group and patients were in the ¥65year group the proportion of tumors with a diameter ¥ cm was significantly higher in the ¥65year group than in the 65year group vs p0016 there were also significantly more patients with lnm in the ¥65year group vs p0041 for t stage the proportion of earlystage t1 patients in the ¥65year group was significantly less than that in the 65year group although p was vs p0086 for treatments there were significantly more patients who were treated with systematic chemotherapy in the ¥65year group vs p0040different operative methods for t1n0m0 colorectal netsthere was no significant difference in tumor grade tumor location surgical margin relapse or 5year os except for tumor diameter p0012 the diameters of tumors resected by emr esd and transanal excision were ± cm ± cm and ± cm respectively table submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table clinical and histopathological characteristics of colorectal nets n135table continued variablesage¥sexmalefemalebmi¥smoking historydrinking historyfamily cancer historyyesnoneoadjuvant chemotherapyyesnoneoadjuvant radiotherapyyesnodistance from the anal verge cm¥tumor locationrectumcolonappendixtreatmentemresdtransanal excisionradical resectionmultivisceral resectionpalliative resectionsystemic treatmenttumor diameter cm¥t stage after chemotherapyxn variableslnmnegativepositivedistant metastasisnegativepositivetumor gradeg1g2g3cga n101negativesyn n109negativen kaplanmeier survival curveskaplanmeier survival curves for os and pfs according to tumor grade diameter location and cga level are shown in figures discussionnets have a relatively good prognosis with a median os time reported to be years months and a 5year os rate of however the survival outcomes varied significantly at different stages of nets the 5year os rate of stage i and ii tumors was reported to be as high as and but dropped to and for stage iii and iv tumors respectively22 according to epidemiological data from the seer database and gkr joint cancer registry the 5year os rates of lymph node metastases stage iiib and distant metastases stage iv are and respectively1610 the 5year os rates of all patients and patients without distant metastasis were and respectively and the 5year pfs rates were and respectively lymph node metastasis is the most important factor that determines the prognosis of nets and the prediction of lymph node continuedcancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepresstable univariable analysis for risk factors for lymph node metastasis n table multivariable analysis for risk factors for lymph node metastasis n135lnm n Ï2 valuep valuen hr cip valueage¥tumor diameter¥ tumor locationrectumcolonappendix tumor gradeg1g2g3t staget0t1t2t3t4cganegativesynnegative notes by logistic regression analyses p values abbreviation hr hazard ratiomost nets are at the g1 phase and g2 or g3 phases account for only to of all nets and have been reported to be risk factors for lnm by numerous studies1223 a multicenter clinical study in china showed that pathological type g3 nec is an independent risk factor affecting the prognosis of patients with rectal nets p similarly sohn et al3 found that the lnm rate of g1 phase rectal nets was only but it increased remarkably to at the g2 phase in our study our results showed that histological tumor grades g2 p and g3 p0001 were independent risk factors for lymph node metastasis lymph node metastasis occurred in of patients with g3 tumors and with g2 tumors but only with g1 tumors the 5year variablessexmalefemaleage¥bmi¥smoking historyyesnodrinking historyyesnotumor locationrectumcolonappendixtumor diameter¥t staget0t1t2t3t4 tumor gradeg1g2g3cga n101negativesyn n109negative notes by pearsons Ï test p values metastasis is necessary for clinicians to choose a suitable treatment the aim of our research was to explore the predictive factors for lymph node metastasis of colorectal nets and assess the current therapeutic algorithmsubmit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table prognostic factors for survival outcomestable continued variables5year pfs 5year os Ï2 valuep valueg3lnmyesnocga negativesyn negativetreatment variablessexmalefemalebmi¥age¥smoking historyyesnodrinking historyyesnoneoadjuvant radiotherapyyesnoneoadjuvant chemotherapyyesnotumor locationrectumcolontumor diameter cm¥t stagextumor gradeg1g25year pfs 5year os Ï2 value p value continuedemresdtransanal excisionradical or palliativesurgerysystemic treatmentnotes kaplanmeier method and log rank test p values os rate decreased sharply from to when the tumor grade increased from g3 to g1 however tumor grade was not significant in survival outcomes possibly because tumor grade affects survival outcomes indirectly by directly affecting lymph node statustumor size has been reported to be a strong predictive factor for lymph node metastasis previous studies have shown that a tumor less than mm is usually limited to the submucosa with a low metastasis risk of less than and the 5year survival rate can reach approximately to according to the enets guidelines surgical treatment is recommended if rnets are greater than cm g1g3 or are g3 phase with or without metastasis endoscopic resection is feasible when the tumors are less than cm g12 phase and t1 stage15 the treatment of rnets in western countries and in china is similar but there have been controversies regarding the treatment of 2cmsized cnets chinese experts agree that endoscopic resection can be considered for cnets less than cm however there is cancer management and research submit your manuscript wwwdovepresscom dovepress 0cwu dovepresstable multivariable analysis for survival outcomes5year pfsos hr cip valueage ¥tumor diameter¥tumor locationrectumcolonappendixlnmnoyescganegativesynnegativetumor gradeg1g2g3neoadjuvant chemotherapynoyestreatmentemresdtaesurgerysystemic treatmentnotes by the cox proportional hazards model p values abbreviation hr hazard ratio0019987800003633e1700003066e29200001826e4500343994900007412e29010015970800001686e30no explicit mention of treatment in the enets guidelines and experts from the nccn recommended surgical resection instead of endoscopic resection1315 in our study survival curves were significantly better p0001 among patients with tumors less than cm figure a tumor diameter greater than cm was an independent risk factor for lnm p0040 table and we believe this was due to the small sample size however patients with tumors less than cm had lnm and patients with tumors less than cm also developed lnm the lnm in small nets might be due to the tumor cells extending to the submucosal layer which has abundant lymphatic vessels for them to spread through previous studies have reported that small nets also have malignant potential26 therefore even if tumor size was submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress wu table comparison of different treatment methods for t1n0m0 colorectal netsemresdtransanal excisiong gradeg1g2g3tumor diameter±±±tumor locationrectumcolonappendixsurgical marginpositivenegativerelapse cases5year os rate notes by anova p values Ï2f valuep valuea strong predictive factor for lnm lnm should not be predicted only by tumor size and further examinations such as eus or ct can help us to evaluate the status of lymph nodes more specificallychromogranin a cga and synaptophysin syn are two neuroendocrine differentiation ned immunohistochemistry markers frequently used in nets in univariable analysis there was a significant difference for colorectal nets with different expression levels of cga in terms of both risk factors for lnm and survival outcomes both p005 in multivariable analysis moderate positive p0010 and strong positive cga p0007 were independent risk factors for 5year pfs which has been proven in a wide variety of retrospective analyses27 in addition prospective clinical trials radiant1 and have been performed to assess the prognostic value of cga in advanced nets and the results showed shorter os for patients with elevated cga31 however the increase in the expression level of cga was not proportional to the increase in the lnm rate or 5year os rate and cga was negative in patients with lnm which may affect the accuracy of the prognostic value of cga lindholm et al35 also found that a relevant portion of nets do not show elevated cga levels the major problem is that several confounding factors can including gastrointestinal and affect cga levels figure a pfs curves according to tumor grade b os curves according to tumor gradecancer management and research submit your manuscript wwwdovepresscom dovepress ab050100150200020406080100progressionfree survivalg1g2g3 0cwu dovepressfigure a pfs curves according to tumor diameter b os curves according to tumor diameterfigure a pfs curves according to tumor location b os curves according to tumor locationfigure a pfs curves according to age b os curves according to agesubmit your manuscript wwwdovepresscom dovepress cancer management and research ababab 0cdovepress wu figure a pfs curves according to cga level b os curves according to cga levelcardiovascular disorders or proton pump inhibitor ppi consumption and a variety of other nontumor reasons36 regarding syn there was only a significant difference in the univariable analysis for lnm previous studies have shown that patients with a low level of synaptophysin had a better os rate than those with a high level however the small sample size limited the accuracy of the results37 based on the findings of this research and previous studies38 it can be suggested that ned might affect the survival outcomes of colorectal net patients and markers especially cga might be suitable for clinicians to predict the prognosis of patientsthe location of the tumor is also an important factor affecting the prognosis and treatment of nets tumors in the colon are more common in necs and generally have a worse prognosis with higher metastatic potential than tumors in the rectum the outcomes of neuroendocrine tumors from the right hemicolon of the midgut and from the left hemicolon from the hindgut are not the same the welldifferentiated biological behavior of the left hemicolon is closer to that of the rectum a recent chinese multicenter study found that more than of colonic nets of midgut origin are necs or mixed adenoneuroendocrine carcinomas manecs39 according to statistics from the seer database the 5year survival rate of patients with rnets is which is significantly higher than that of colonic nets in our study the lymph node metastasis rate in the colon was which was significantly higher than that in the rectum the 5year os rate and pfs rate of individuals with lnm in the appendix and in the rectum were significantly better than those of individuals with lnm in the colon p0005 and p0003 respectively which was consistent with previous studies1 based on the findings of this research and previous studies it can be suggested that colonic nets should be completely resectedin our research the survival outcomes of patients years and older in our study were worse than those of patients younger than years p0001 we also found that tumors from elderly patients ¥ years were larger and more advanced than those from younger patients years both p0001 the reason for the poor prognosis in elderly patients may be that elderly patients have lower tolerance to surgical trauma and side effects of chemotherapy because of their weakened an physiological functions which leads to multiple complications4142 therefore when we encounter elderly patients minimally invasive therapies such as laparoscopic surgery could help reduce surgical trauma and chinese herbs can relieve and reduce the adverse events of chemotherapy43for nets in the colon the recommended treatment varies among different guidelines but surgical resection is generally recommended because of the greater likelihood of malignant behavior than with rectal nets endoscopic resection may be considered if the tumor diameter is less than cm and does not reach the muscularis propria for nets in the rectum eus is required before surgery surgical resection is recommended when the tumor diameter is more than cm g3 grade t3 to t4 stage or when there is peripheral lymph node metastasis when the tumor diameter is less than cm g1 or grade and t1 stage endoscopic resection is feasible in other cases the treatment method is determined according to the depth of tumor invasion assessed by eus21cancer management and research submit your manuscript wwwdovepresscom dovepress 050100150200020406080100months progressionfree survivalnegativeweak positivemoderate positivestrong positivep0023050100150200020406080100months overall survivalnegativeweak positivemoderate positivestrong positivep0038ab 0cwu dovepressthis study has some limitations including its retrospective design and the relatively small number of patients included although lnm should be evaluated after radical resection with lymph node dissection we analyzed the risk factors for lnm by ct or mri in those who underwent local excision before the treatment and during the follow up periods and we believe the results are reliable because this study lasted more than years we could investigate the longterm survival outcomes and prognostic factors after different treatments even with the small number of patients finally further studies should be performed to validate our main sthe clinical and pathological characteristics of rectal and colon neuroendocrine tumors are different
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Lasting and SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaMarissa R Keever1 Pan Zhang2 Courtni R Bolt1 Adrienne M Antonson1Haley E Rymut1 Megan P Caputo1 Alexandra K Houser1 Alvaro G Hernandez3Bruce R Southey1 Laurie A Rund1 Rodney W Johnson14 andSandra L RodriguezZas12456 Department of Animal Sciences University of Illinois at UrbanaChampaign Urbana IL United States Illinois InformaticsInstitute University of Illinois at UrbanaChampaign Urbana IL United States Highthroughput Sequencingand Genotyping Unit Roy J Carver Biotechnology Center University of Illinois at UrbanaChampaign Urbana ILUnited States Neuroscience Program University of Illinois at UrbanaChampaign Urbana IL United States Departmentof Statistics University of Illinois at UrbanaChampaign Urbana IL United States Carl R Woese Institute for GenomicBiology University of Illinois at UrbanaChampaign Urbana IL United StatesThe prolonged and sexdependent impact of maternal immune activation MIA duringgestation on the molecular pathways of the amygdala a brain region that uencessocial emotional and other behaviors is only partially understood To address thisgap we investigated the effects of viralelicited MIA during gestation on the amygdalatranscriptome of pigs a species of high molecular and developmental homology tohumans Gene expression levels were measured using RNASeq on the amygdalafor 3weekold female and male offspring from MIA and control groups Amongthe genes that exhibited significant MIA effect a prevalence of differentiallyexpressed genes annotated to the neuroactive ligandreceptor pathway glutamatergicfunctions neuropeptide systems and cilium morphogenesis were uncovered Genesin these categories included corticotropinreleasing hormone receptor glutamatemetabotropic receptor glycoprotein hormones alpha polypeptide parathyroidhormone receptor vasointestinal peptide receptor neurotensin proenkephalinand gastrinreleasing peptide These categories and genes have been associatedwith the MIArelated human neurodevelopmental disorders including schizophreniaand autism spectrum disorders Gene network reconstruction highlighted differentialvulnerability to MIA effects between sexes Our results advance the understandingnecessary for the development of multifactorial therapies targeting immune modulationand neurochemical dysfunction that can ameliorate the effects of MIA on offspringbehavior later in lifeKeywords immune activation pigs RNAseq neuropeptides glutamatergic pathway GABAergic pathwayINTRODUCTIONThe maternal immune response triggered by pathogens and other environmental stressors duringgestation can also elicit an indirect response by the fetal immune cells Kroismayr Odorizzi and Feeney Prins Viral infection during gestation for exampleactivates a cytokinerelated signaling cascade and molecules from this process can cross theEdited byNo¨lia Fern ndezCastilloCentre for Biomedical NetworkResearch CIBER SpainReviewed bySilvia PellegriniUniversity of Pisa ItalyTewarit SarachanaChulalongkorn University ThailandCorrespondenceSandra L RodriguezZasrodrgzzsillinoiseduSpecialty sectionThis was submitted toNeurogenomicsa section of the journalFrontiers in NeuroscienceReceived May Accepted July Published August CitationKeever MR Zhang P Bolt CRAntonson AM Rymut HE Caputo MPHouser AK Hernandez AGSouthey BR Rund LA Johnson RWand RodriguezZas SL Lastingand SexDependent Impactof Maternal Immune Activation onMolecular Pathways of the AmygdalaFront Neurosci 103389fnins202000774Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaplacenta and reach the fetal brain The resulting maternalimmune activation MIA can impactfetal developmentalprocesses and exert longterm postnatal eï¬ects in the oï¬springRutherford The relationship between MIAand neurodevelopmental disordersincluding schizophreniaspectrum disorders SSD and autism spectrum disorders ASDand neurodegenerative disorders such as Alzheimers diseaseAD in oï¬spring has been established Knuesel Canetta Mattei These diseases sharesome behavior symptoms comorbidities such as eating disordersand genetic and environmental ie MIA agents Canitanoand Pallagrosi The previous neurological disorders havebeen associated with abnormal structure and dysregulationof the amygdala Schumann FernandezIrigoyen and share genes and molecular mechanismsincluding histocompatibility complex MHC genes Andersand Kinney glutamatergic and GABAergicassociatedgenes Bourgeron Marin Li andmitochondrial activity processes Pieczenik and Neustadt Sragovich socialinteractionThe fetal amygdala is susceptible to ammatory signals andthe plasticity of this brain structure to MIA can lead to alterationsof the developmental trajectory These disruptions may havelonglasting and maladaptive consequences for the oï¬springdue to the significant role that the amygdala plays in manyneurological pathways Located in the forebrain the amygdalauencescognition neuroendocrinebehavior learning memory emotion and autonomic systemsThe amygdala also modulates the response of these processesto stressors including pathogenic infections and those resultingfrom management practices such as weaning Tian The amygdala experiences high uptake of gonadalhormones and is anatomically connected to other sexuallydimorphic nuclei Therefore this brain region is involved inregulation of several dimorphic functions such as aggressionsexual behavior gonadotropin secretion and integration ofolfactory information Hines Evidence supports thediï¬erential activation of the amygdala to stimuli between malesand females Killgore and YurgelunTodd includingdiï¬erences in the sexual responses and emotional memoryHamann and diï¬erential vulnerability to insult Baird Due to the interconnected and multiregulatorynature of this brain structureinsults to the amygdala canimpact the individuals social locomotor and feeding behaviorPetrovich and Gallagher growth and reproductivephysiology health status and immunological response tosecondary stressorsRecent studies lend support to the link between MIA andaltered amygdala function Carlezon In miceMIA elicited by polyinosinicpolycytidylic acid [PolyIC]increased the synaptic strength of glutamatergic projectionsfrom the prefrontal cortex to the amygdala Li In ï¬eld tests mice exposed to MIA spentless timein the center and traveled a higher distanceindicativeof a higher anxiety behavior incidence than the controlcounterparts These ï¬ndings suggest that the change in thebalance between excitation glutamatergic and inhibitiontherefore aï¬ecting brain circuitsspike output offeedforward GABAergic modiï¬ed theamygdala neuronsthatcould regulate behavior in SSD and ASD A candidate genestudy of the eï¬ects of social stress during gestation reportedthatthe expression of a corticotropinreleasing hormonereceptor in the amygdala of 10weekold pigs was higherin females than in males Rutherford Thisstudy concluded that prenatal stress substantially increasedanxietyrelated behaviorstheimpact of maternal stressors during gestation on speciï¬camygdala molecular proï¬les and associated neurological orbehavioral disorders in the oï¬spring later in life highlightthe complexity of the molecular mechanisms underlying thepathophysiology of MIAin female pigs Studies ofetalvirusadvantages ofrodents when consideringstudying a pig modelto pigsResearch on the lasting eï¬ects of MIA in pigs complementsAntonson et althe insights oï¬ered by rodent modelsstem Theratherfrom the greater homology of humansan physiologythan toin particular brain growth andsize development anddevelopment processesMurphy A pigmodel that has oï¬ered insights into MIA employs porcinereproductivePRRSVtothein the brain andmicroglia ie macrophagelike cellsisin neonatal pigsAntonson associated with behavioralelicit MIA Thisand respiratorysyndromechallengeactivatesimmunechangesThe study of MIA elicited by PRRSV allows for thecharacterization of the impact of a live viral pathogen thatselfreplicates in the host evoking extended activation ofimmune pathways PRRSV challenge during gestation is a wellcharacterized replicable and eï¬ective method for inducingMIA in pigs Antonson In additionPRRSV outbreaks impose a major economic burden to thelivestock industry PRRSV is an enveloped singlestranded RNAvirus thatinfects alveolar macrophages causing interstitialpneumonia and increased serum levels ofthe cytokinesinterleukin betainterleukin and tumor necrosis factoralpha Antonson The persistent repercussionsof MIA on the molecular pathways ofthe pig amygdalaare yet to be investigated Moreover the potentially distinctvulnerability to the prolonged eï¬ects of MIA between sexesremains unknownThe overarching goal of the present study is to advancethe understanding of the impact of MIA on the molecularmechanisms ofthe amygdala Three supporting objectivesare explored a characterization of prolonged transcriptomechanges elicited by viral MIA in pigs a species that hashigh neurodevelopmental homology with humansandfood production valueidentiï¬cation of molecularpathwaysthat present diï¬erential vulnerability to MIAbetween sexes and c understanding the eï¬ect of MIA onmolecular interactions assisted by gene network inferenceThe ï¬ndings from these complementary analyses supportthe use of multipleto amelioratethe potential detrimental eï¬ect of MIA on the oï¬springphysiology and behaviortherapeutictargetsbFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaMATERIALS AND METHODSAnimal ExperimentsAll experimental procedures used published protocols Antonson The animal studies were approved by theIllinois Institutional Animal Care and Use Committee IACUCat the University of Illinois and are in compliance with the USDAAnimal Welfare Act and the NIH Public Health Service Policy onthe Humane Care and Use of AnimalsCamborough gilts born and raised at the University of Illinoisat UrbanaChampaign herd were inseminated at days ofage using PIC boar sperm Antonson All gilts were PRRSV negative and were moved at gestationday GD into diseasecontainment chambers maintainedat ¦C and a h lightdark cycle with lights on at AM The gilts were fed daily kg of a gestational diet andhad ad libitum water access One week after acclimation fourgilts were intranasally inoculated with live PRRSV strain P129BV School of Veterinary Medicine at Purdue University WestLafayette IN United States using mL of median tissueculture infectious dose TCID50 diluted in sterile Dulbeccosmodiï¬ed Eagle medium DMEM mL total volume Thefour gilts in the Control group were intranasally inoculatedwith an equal volume of sterile DMEM PRRSV inoculationcorresponded to the last third of gestation in pigs and humansduring initiation of rapid fetal brain growth Antonson PRRSV and Control groups were housed in separatecontainment chambersThe rectal temperatures and diet consumption of the giltswere recorded daily until farrowing Antonson The PRRSVinoculated gilts were oï¬ered the maximumfed daily and feed refusal was measured The Control giltswere fed the same amount consumed by the PRRSVinoculatedgilts on the previous day The daily body temperature andfeed intake levels were compared using a mixedeï¬ects modelanalyzed with PROC MIXED SAS Institute Inc Cary NCUnited States The model included the eï¬ects of gilt treatmentand replicate while accommodating for heterogeneity of variancebetween MIA groupsFarrowing was induced with an intramuscular injection of mg of Lutalyse dinoprost tromethamine Pï¬zer New YorkNY United States on GD in consideration that the averagegestation length is approximately days Antonson Gilts farrowed in individual farrowing crates ofstandard dimensions m After farrowing thegilts were fed twice a day up to kg of a nutritionallycomplete diet for the lactating period and water remainedavailable ad libitum Pigs received intramuscular injections ofiron dextran mgpig Butler Schein Animal Health DublinOH United States and Excede for Swine mgpig ZoetisParsippany NJ United States to control for respiratory diseasesThe pigs remained with their mothers until PD The bodyweight of pigs was measured daily and analyzed using the mixedeï¬ects model in SAS PROC MIXED SAS Institute Inc CaryNC United States The model included the eï¬ect of MIA andthe random eï¬ect of gilt accommodating for heteroscedasticitybetween pig treatment and sex groups The impact of MIA wasstudied at PD because this is a common age to wean pigsThe study of transcriptome proï¬les from older pigs could beconfounded with changes in diet and environment associatedwith weaning while proï¬les from younger pigs would hinder theassessment of the prolonged eï¬ects of MIARNA Extraction and SequencingA balanced experimental design was studiedincluding pigs evenly distributed between maternal PPRSV activatedMPA group of pigs and Control gilts CON group of pigseach group encompassing males and females denoted Maand Fe respectively At PD pigs were removed fromthe farrowing crate and anesthetized intramuscularly using atelazolketaminexylazine drug cocktail mg of tiletamine mg of zolazepam reconstituted with mL ketamine gL and mL xylazine gL Fort Dodge AnimalHealth Fort Dodge IA United States at a dose of mLkgbody weight following protocols Antonson Following anesthetization pigs were euthanized using anintracardiac injection of sodium pentobarbital mgkg bodyweight Fata Plus Vortech Pharmaceuticals Dearborn MIUnited States Pig brains were extracted the amygdalae wererecognized using the stereotaxic atlas of the pig brain Felix dissected out ï¬ash frozen on dry ice and stored at ¦Cfollowing published protocols Antonson RNA wasisolated using EZNA isolation kit following the manufacturersinstructions Omega Biotek Norcross GA United States TheRNA integrity numbers of the samples were above indicatinglow RNA degradation The RNASeq libraries were preparedwith TruSeq Stranded mRNAseq Sample Prep kit Illumina IncSan Diego CA United States The libraries were quantitatedby qPCR and sequenced on one lane on a NovaSeq for cycles from each end of the fragments using NovaSeqS4 reagent kit FASTQ ï¬les were generated and demultiplexedwith the bcl2fastq v220 conversion software Pairedend reads nt long were obtained and the FASTQ ï¬les are availablein the National Center for Biotechnology Information GeneExpression Omnibus GEO database experiment accessionnumber GSE149695RNA Sequence Mapping and DifferentialExpression AnalysisThe average Phred quality score of the reads assessed usingFastQC Andrews was across all read positions andtherefore no reads were trimmed The pairedend reads fromthe individual samples were aligned to the Sus scrofa genomeversion Sscrofa Pruitt using kallisto v0430Bray with default settings The normalized trimmedmean of Mvalues gene expression values were described usinga generalized linear model encompassing the eï¬ects of the MIAgroup MPA or CON levels sex Fe or Ma levels and MIAbysex interaction and analyzed using edgeR version in the R v environment Robinson Genessupported by transcripts per million TPM by each MIAsex combination were analyzed to ensure adequate representationacross comparisonsFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaOrthogonal pairwise contrasts between MIA and sex groupswere evaluated in addition to testing for the eï¬ects of MIAbysex interaction and main eï¬ects of MIA and sex Thefour groups compared in the contrasts identiï¬ed by treatmentfollowed by the sex levels are MPA_Fe MPA_Ma CON_Fe andCON_Ma The Pvalues were adjusted for multiple testing usingthe BenjaminiHochberg false discovery rate FDR approachBenjamini and Hochberg categoriesamongtheMFand KEGG pathways The GeneFunctional Enrichment and NetworkInferenceapproaches were used to identifyTwo complementaryoverrepresented functionalgenesexhibiting diï¬erential expression across MIA and sex groupsCaetanoAnoll©s GonzalezPena et al2016ab Functional categories investigated included GeneBPs GO molecularOntology GO biological processesfunctionsSetEnrichment Analysisapproach implemented inthesoftware package GSEAP Subramanian was used to identify category overrepresentationwith gene over and underexpressed while considering allgenes analyzed The normalized enrichmentscore NESofin the Molecular Signature DatabaseMSigDB was calculated using the maximum deviation ofthe cumulative sum based on the signed and standardizedfold change The statistical signiï¬cance ofthe enrichmentwas assessed using the FDRadjusted Pvalue computed from permutationscategoriesGSEAtheThe overrepresentation of functional categories was alsoevaluated among genes that exhibited a significant MIAbysex interaction or main eï¬ect using the Database forAnnotation Visualization and Integrated Discovery DAVID Huang The enrichment of Direct GOcategories in the DAVID database was assessed The Susscrofa genome was used as the background for enrichmenttesting and enrichmentis reported using the ExpressionAnalysis Systematic Explorer EASE score that was computedusing a onetailed jackknifed Fisher hypergeometric exacttest Functional categories were clustered based on geneannotation and the statisticalissummarized as the geometric mean of the log10 EASE scoresofthe categories Delï¬no Serao Delï¬no and RodriguezZas signiï¬cance of clustersWeighted Gene Coexpression NetworkAnalysis and Gene Network VisualizationAn approach complementary to the identiï¬cation of diï¬erentiallyexpressed genes was used to uncover coexpression networksusing Weighted Gene Coexpression Network AnalysisWGCNA version Langfelder and Horvath The input data were voomtransformed read count valuesgenerated using the limma package version Ritchie in R version Genes were ï¬ltered to removethose with low expression levels or no variation across samplesper developer recommendations The number of genes usedfor network analysis was genes Considering potentialfor interaction patterns a sexdependent softthresholdingpower was used to call for network topology analysis Thelowest power values that support a scalefree topology powerused were for the CON_MaMPA_Ma contrast and for the MPA_FeMPA_Ma contrast The Pearson correlationcoeï¬cient ofthe normalized expression values was usedto identify modules of connected genes The minimummodule size was set to with the deepSplit set to and themergeCutHeight set to Module proï¬les were identiï¬edusing the correlation between the eigengene of each moduleand pig group Enrichment of functional categories among thegenes in each module proï¬le was explored with DAVID using theSus scrofa genome as background and testing included an FDRmultiple test adjustmentFurther understanding of the impact of the MIAbysexinteraction was gained through the reconstruction of genenetworks using the BisoGenet package Martin inthe Cytoscape platform Shannon Information fromgene and protein interactions annotated in databases includingBIOGRID HPRD DIP BIND INTACT and MINT was usedto visualize relationships between genes Salwinski Alfarano Mishra Stark Kerrien Licata Networks highlightingdiï¬erences in gene levels associated with MIA within sex iethe contrasts MPA_MaCON_Ma and MPA_FeCON_Fe werecompared The network framework includes genes that exhibiteda significant MIAbysex interaction eï¬ect FDRadjusted P and are annotated to enriched functional categoriesThe framework genes were identiï¬ed by full nodes with sizereï¬ecting the diï¬erential expression level between the MPAand CON groups The network edges depict known molecularrelationships curated in the BisoGenet databases The frameworkgenes were connected through correlated genes listed in theBisoGenet database of molecular interactions that did not reachsignificant MIAbysex interaction eï¬ect The comparison ofthese networks oï¬ered insights into the simultaneous eï¬ect ofMIA across interacting genes and enabled the detection of sharedand distinct coregulation patterns between MPA and CONpigs across sexesRESULTSMaternal Immune Activation andSequencing MetricsThe diï¬erences between MPA and CON giltsin rectaltemperatures and daily diet consumption indicated the activationof the maternal immune system in response to PRRSV Thediï¬erence in body temperature between CON and MPA giltson GD was ¦C standard error ¦C P Thediï¬erence in feed refusal between CON and MPA gilts on GD was g standard error g P A significantincrease in rectal temperatures and decrease in feed intakeP was observed within h of inoculation and returnedto baseline levels within days for body temperature and within days for feed intake At days of age CON pigs were kgFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the Amygdalaheavier than MPA pigs standard error P whileno significant sex or interaction eï¬ects were detectedThe sequencing of the RNA samples produced billionsequenced reads and million pairedend reads per sampleThe number of reads was consistent across MIA and sexgroups coeï¬cient of variation and the eï¬ects ofMIA sex and MIAbysex interaction were tested on genes that surpassed the minimum number of reads per MIAsex combinationTranscriptome Changes Associated WithMaternal Immune Activation That AreSexDependentOverall genes exhibited a significant FDRadjusted P MIAbysex interaction eï¬ect and among these genes hada significant eï¬ect at FDRadjusted P The proï¬le ofthese genes indicated that the eï¬ect of MIA diï¬ered betweenfemales and males Fortysix genes that presented a MIAbysex interaction eï¬ect are listed in Table together with theirexpression pattern and Pvalue The majority of the genes inTable including neurotensin NTS displayed a reversal inthe expression level between CON and MPA groups across sexesie opposite Log2[fold change] sign across sexes An extendedlist including genes that exhibited a MIAbysex interactioneï¬ect at FDRadjusted P is provided in SupplementaryFile S1 Table AAnother frequent pattern among the genes that displayed aMIAbysex interaction eï¬ect was characterized by a consistentexpression proï¬le between CON and MPA across sexes albeitthe magnitude diï¬ered between sexes Table For exampleglycoprotein hormones alpha polypeptide CGA was overexpressed in CON relative to MPA but the diï¬erential washigher in males than in females Other genes presentingthis pattern included guanylatebinding protein GBP1transthyretin TTR aldehyde dehydrogenase family memberA2 ALDH1A2 hemoglobin subunit beta HBB and basichelixloophelix family member e22 BHLHE22GRPNotable is the significant MIAbysex interaction eï¬ecton genes associated with neuropeptides and hormones andgenes that participate in glutamatergic processes Genes underexpressed in MPA relative to CON males while presentingthe opposite pattern in females Table included NTS theneuropeptide gene proenkephalin PENK the neuropeptidegene gastrinreleasing peptidethe neuropeptiderelated gene vasoactive intestinal peptide receptor VIPR2corticotropin releasing hormone receptor CRHR2 neuronderived neurotrophic factor NDNF reelin RELN glutamatemetabotropic receptor GRM4 solute carrier family member SLC17A6 calcium voltagegated channel auxiliarysubunit alpha delta CACNA2D3 EFhand domain familymember D1 EFHD1 glutathione peroxidase GPX3parathyroid hormone receptor PTH1R thyroid hormoneresponsive THRSP and CGA The CGA gene codes for thealpha subunit protein of the hormones chorionic gonadotropinCG luteinizing hormone LH folliclestimulating hormoneFSH and thyroidstimulating hormone TSHFunctional and Network Analysis ofGenes That Exhibit SexDependentAssociations With Maternal ImmuneActivationThe genes expressing significant MIAbysex interaction eï¬ectswere analyzed for functional enrichment Table presentsthe clusters of most enriched and informative categoriesfrom the DAVID analysis and the complete list of categoriesis in Supplementary File S1 Table B The categories inTable encompass genes presenting the mostfrequentinteraction proï¬le characterized by underexpression in CONfemales relative to males but overexpression in MPA femalesrelative to males These genes include KEGG Autoimmunethyroid diseaseCluster and BP brain developmentGO0007420 Cluster Enrichment results from GSEA complemented the ï¬ndingsfrom DAVID Highly enriched informative categories amonggenes that have a MIAbysex interaction eï¬ect are presentedin Table and the extended list of categories is presented inSupplementary File S1 Table C The categories in Table in Table including ion homeostasissupport pathwaysTable and regulation of voltagegated calcium channelactivity processesenrichmentinteraction pathwayofthe neuroactiveand the hormoneactivity processesinclude genes such as CGA and VIPR2 that were identiï¬edin Table ligand receptorand neuropeptideTable Notablythethe diï¬erentialNetwork visualization furthered the understanding ofthe impact of MIA on the relationships among genes thatexhibited a significant MIAbysex interaction eï¬ect Thenetworks in Figures depictthe relationships betweengenes in the enriched neuroactive ligand receptor pathwaythat highlightexpression between CONand MPA in males and females ie CON_MaMPA_Maand CON_FeMPA_Ferespectively Red andrectangular nodesblueframework genes andthe known associations between genesedgesrepresentbased on curated databases of molecularinteractionsRed and blue nodes denote over or underexpressionof the gene in CON relative to MPA and the size is anthe diï¬erential expressioninverse logarithmic function ofPvalue Thediï¬erentialexpression pattern and connectivity among genes highlightsthe discrepancyelicited by MIAbetween the sexesin network modulescontrastsrepresentsimultaneousstudytheofTranscriptome Changes Associated WithMaternal Immune ActivationOverall genes exhibited diï¬erential FDRadjusted P expression between MPA and CON pigs irrespective of sexTable lists notable highly diï¬erentially expressed genesis in Supplementary File S1 Tableand the complete listD The majority of these genes were overexpressed in MPArelative to CON pigs Among the genes overexpressed in MPAcompared to CON pigs were islet amyloid polypeptide IAPPFrontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaTABLE Genes exhibiting significant FDRadjusted Pvalue maternal immune activationbysex interaction effectGene symbolPvalueaCON FeCON Ma MPA FeMPA MaCON FeMPA FeCON MaMPA MaCON FeMPA MaCON MaMPA FeRGS16CGAPOMCGPX3RELNVIPR2ANKRD34CGBP1GRM4CCDC136SLC17A6BTBD11TTRCACNA2D3CRHR2NDNFCXCL12USP43CCDC17KCNIP4CAMK2N2ALDH1A2GRPPENKSYT12PTH1RHBBESYT1EFHD1BHLHE22ZFP37SLC2A2THRSPNR4A3LOC396781C1QTNF1RAB27ANTSGVIN1SSTR1CCDC9BCCDC33CCDC162PPTHSYNPO2LCHGB5E115E115E115E115E115E115E115E115E1153E0911E0844E0850E0848E0728E0628E0662E0664E0671E0672E0696E0614E0515E0516E0529E0537E0567E0585E0596E0510E0412E0415E0431E0433E0444E0445E0456E0479E0479E0486E0488E0414E0314E0314E0315E0318E03aLog2[fold change] between two maternal immune activationsex groups MPA PRRSVinduced maternal immune activation CON control Fe females Ma malesankyrin repeat domain ANKRD24interferoninducedtransmembrane protein IFITM1 and IFITM3 cathepsinC CTSC mitogenactivated protein kinase kinase MAP2K7heparan sulfateglucosamine 3sulfotransferase HS3ST5secreted phosphoprotein SPP1 immunoglobulin heavy chainIGHG and transforming acidic coiledcoilcontaining protein TACC1 Among the genes underexpressed in MPA relative toCON pigs are insulinlike growth factor IGF2 cellular retinoicacidbinding protein CRABP2 and aldehyde dehydrogenase family member A1 ALDH1A1Frontiers in Neuroscience wwwfrontiersinAugust Volume 0cKeever et alGestational Activation of the AmygdalaFunctional Analysis of Genes AssociatedWith Maternal Immune ActivationTable presents the top significant clusters of informativeenriched categoriesfrom the DAVID analysis of genesTABLE Most enriched DAVID clusters and supporting functional categoriesenrichment score ES among the genes presenting significant maternalimmune activationbysex interaction effectaCategory Category identiï¬er and namePvaluebFDRPvalueCluster KEGGBPKEGGKEGGKEGGCluster BPBPBPBPBPCluster BPCluster BPBPES ssc05320Autoimmune thyroid diseaseGO000250¼Antigen processing andpresentation of peptide orpolysaccharide antigen via MHC class IIssc04514Celladhesion moleculesCAMsssc05323Rheumatoid arthritisssc05164Inï¬uenza AES GO0051050¼Positive regulation oftransportGO0051049¼Regulation of transportGO0050801¼Ion homeostasisGO0048878¼Chemical homeostasisGO0030001¼Metal ion transportES GO0048871¼Multicellular anismalhomeostasisES GO0061564¼Axon developmentGO0007420¼Brain development290E06190E03450E04340E01230E03320E02480E03200E02560E02170E01440E03350E01450E03130E02280E02450E02320E01360E01480E01570E01200E04370E01640E05130E03170E01310E01aBP biological process KEGG KEGG pathway bFalse discovery rate adjustedPvalueTABLE Enriched informative categories NES using GSEA among thegenes based on the overall maternal immune activationbysex interactionaCategory Category identiï¬er and namebNES PvalueKEGGKEGGMFBPBPBPKEGGBP 10E10 10E10 10E10 10E10ssc04080Neuroactive ligand receptorinteractionssc04912GnRH signaling pathwayGO0005179¼Hormone activityGO0006970¼Response to osmoticstressGO0019221¼Cytokine mediatedsignaling pathwayGO1901385¼Regulation of voltagegated calcium channel activityssc04020Calcium signaling pathway 12E01GO0085029¼Extracellular matrix 18E01assembly 91E02 12E01cFDRPvalue83E0299E0222E0135E0154E0154E0154E0155E01aMF molecularfunction KEGG KEGG pathway BP biological processbNormalized enrichment score negative values indicate genes underexpressionin CON females relative to males but overexpression in MPA females relative tomales cFalse discovery rate adjusted Pvaluediï¬erentially expressed between MPA and CON groupsacross sexes the extended list of categories is presented inSupplementary File S1 Table E Some categories identiï¬edby the DAVID analysis are consistent with the categoriesdetected at more significant levels among the genes presentingan MIAbysex interaction eï¬ect Table and include theBP angiogenesisand KEGG autoimmunethyroid disease and EpsteinBarr virus infection pathwaysTable Also enriched Supplementary File S1 Table Ewere the BP homeostatic GO0042592 MF ion bindingGO0043167 and BP anatomical structure formation inmorphogenesis GO0048646GO0001525The GSEA enrichment results within the gene expressionpatterns of CON relative to MPA groups complemented theï¬ndings from DAVID The most informative enriched categoriesare presented in Table and the extended list of categories ispresented in Supplementary File S1 Table F Enriched clustersof genes overexpressed in CON relative to MPA detected byGSEA were the BP enrichment of microtubule bundle formationGO0001578 and cilium morphogenesis GO0060271Transcriptome Differences BetweenSexes Independent of Maternal ImmuneActivationOverall genes were diï¬erentially expressed between malesand females FDRadjusted P These genes exhibiteda consistent diï¬erential expression between sexes irrespectiveof the MIA group The complete list of genes diï¬erentiallyexpressed between sexes at FDRadjusted P is available inSupplementary File S1 Table G and the majority were overexpressed in males relative to females Among the previousgenes excluding those that presented MIAbysex interactioneï¬ect a selection of informative genes is listed in Table Genesoverexpressed in males relative to females included eukaryotictranslation initiation factor 1A Ylinked EIF1AYleptinreceptor LEPR luteinizing hormone beta polypeptide LHBLIM homeobox LHX9 luteinizing hormone beta polypeptideLHB and immunoglobulin family member IGSF1Informative categories among the DAVID clusters ofenriched categoriesthe genes diï¬erentially expressedbetween sexes are listed in Table a complete list i
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" the ampactivated protein kinase ampk is an evolutionarily conserved regulator of cellular energyhomeostasis as a nexus for transducing metabolic signals ampk cooperates with other energysensing pathwaysto modulate cellular responses to metabolic stressors with metabolic reprogramming being a hallmark of cancerthe utility of agents targeting ampk has received continued scrutiny and results have demonstrated conflictingeffects of ampk activation in tumorigenesis harnessing multiomics datasets from human tumors we seek toevaluate the seemingly pleiotropic tissuespecific dependencies of ampk signaling dysregulationmethods we interrogated copy number variation and differential transcript expression of ampk pathway genesacross diverse cancers involving over patients cox proportional hazards regression and receiver operatingcharacteristic analyses were used to evaluate the prognostic significance of ampk dysregulation on patientoutcomesresults a total of and seven ampk pathway genes were identified as having loss or gainoffunction featuresthese genes exhibited tissuetype dependencies where survival outcomes in glioma patients were most influencedby ampk inactivation cox regression and logrank tests revealed that the 24ampkgene set could successfullystratify patients into high and lowrisk groups in glioma sarcoma breast and stomach cancers the 24ampkgeneset could not only discriminate tumor from nontumor samples as confirmed by multidimensional scaling analysesbut is also independent of tumor node and metastasis staging ampk inactivation is accompanied by the activationof multiple oncogenic pathways associated with cell adhesion calcium signaling and extracellular matrixanization anomalous ampk signaling converged on similar groups of transcriptional targets where a commonset of transcription factors were identified to regulate these targets we also demonstrated crosstalk between procatabolic ampk signaling and two proanabolic pathways mammalian target of rapamycin and peroxisomeproliferatoractivated receptors where they act synergistically to influence tumor progression significantlycontinued on next page correspondence alvinalaiuclacukinstitute of health informatics university college london euston roadlondon nw1 2da uk the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchang and lai bmc cancer page of continued from previous page genetic and transcriptional aberrations in ampk signaling have tissuedependent pro or antitumorimpacts pancancer investigations on molecular changes of this pathway could uncover novel therapeutic targetsand support risk stratification of patients in prospective trialskeywords ampk glioma lossoffunction tumor metabolism pancancer the ampactivated protein kinase ampk is an evolutionary conserved key player responsible for energy sensing and homeostasis orthologous copies of ampkprevail universally as heterotrimeric complexes wherethe human genome encodes two genes for the α catalyticsubunit two regulatory subunit genes and three Πsubunit genes historically ampk was discovered as a crucial regulator of lipid metabolism since then ampkis implicated in a wide variety of fundamental metabolicprocesses as well as in metabolic diseases such as cancerand diabetes the first link between ampk and cancer was identified through the tumorsuppressive function of lkb1 which is upstream of the mtor pathway theroles of ampk werepharmacologically demonstrated by the application ofmetabolic inhibitors such as the antidiabetic metforminand the mimetic of amp aicar [] numerousstudies have since compellingly established the promiscuous nature of these pharmacological agents wherebythe inhibition of cancer cell proliferation occurs throughnonspecific ampkindependent avenues [ ]tumorsuppressivestressorssuchasin contrastof metabolicto the tumorsuppressive results frompharmacological studies genetic experiments on cancercells have credibly demonstrated that ampk activationis crucial for tumor progression and survival [] amyriadoxygendeprivation nutrient starvation and oxidative stress exists within the tumor microenvironment metabolic reprogramming during carcinogenesis would thus triggerampk activation to enable cells to survive under conditions of stress typically found in the tumor microenvironment hence conferring an overall tumorpromotingeffect ampk is also shown to support cancer growthand migration through crosstalk with other prooncogenicpathways for instance overexpression of oncogenes mycand src or the loss of the tumor suppressor folliculincould lead to ampk activation []genetic and pharmacological studies have paved theway for our understanding of the function of ampk incancer however anti and proneoplastic features ofampk remain controversial potentially due to the oversimplification of ampkmodulated processes in in vitroand nonhuman invivo models the genetic and clinicallandscape of ampk signaling has not been systematically investigated thus our study aims to address anunmet need to rigorously investigate the role of ampkin diverse cellular context using multiomics data fromactual tumors where we examined somatic copy numberalterations transcriptional and clinical profiles of tumorsfrom cancer types our analyses of clinical samples atscale would complement evidence from pharmacologicaland genetic studies to better elucidate the multifacetedand cellspecific nature of ampk signaling on tumorprogressionmethodsampk pathway genes and cancer cohortsninetytwo ampk pathway genes were retrieved fromthe kyoto encyclopedia of genes and genomes keggdatabase additional file clinical genomic and transcriptomic datasets of cancers involving patients were downloaded from the cancer genome atlastcga copy number variation differential expressionmultidimensional scaling and survival analysesdetailed methods of the above analyses were previouslypublished and thus will not be repeated here as per the guidelines [] to summarize discrete amplification and deletion indicators for copy number variation analyses were obtained from gistic geneleveltables gistic values of and were annotatedas shallow amplification and shallow deletion heterozygous events respectively gistic values of and were annotated as deep amplification and deep homozygous deletion events respectively multidimensionalscaling analyses and permutational multivariate analysisof variance permanova were performed using the rvegan package survival analyses were performed usingcox proportional hazards regression and the logranktest sensitivity and specificity of the 24ampkgene setwere assessed using receiver operating characteristicanalyses differential expression analyses were performed on patients stratified into high 4th quartileand low 1st quartile expressing groups using the genesetto determine the transcriptional effects ofanomalous ampk signalingpathway and transcription factor analysesgenes that were differentially expressed degs betweenthe 4th and 1st quartile patient groups were mapped to 0cchang and lai bmc cancer page of kegg gene ontology and reactome databases using gprofiler to ascertain biological processes and signaling pathways that were enriched the enrichr tool was used to map degs to the chea and encodetranscription factor tf databases to identify tfs thatwere significantly enriched as regulators of the degsirs2pik3r1sirt1 tbc1d1calculating the 24ampkgene score peroxisomeproliferatoractivated receptors ppar score andmammalian target of rapamycin mtor scoreampk scores were calculated from the mean expressionthe following genes slc2a4 foxo3 ppp2cbofpik3cd cab39l ccna1 fbp1 fbp2 foxo1 hmgcrppargc1appp2r2c mlycd pfkfb3 ppp2r2b prkaa2 leprcab39 irs1 and pfkfb1 ppar scores for each patientwere calculated by taking the mean expression of pparsignature genes plin5 pparg acadm gk cpt2scp2 acaa1 apoa1 ppara acox2 angptl4fabp3 plin2 aqp7 acsl1 fabp5 acadl andpck2 mtorpi3kakt scores for each patientwere calculated using the following equation mtorpi3kakt score akt mtor gsk3 s6k s6 pten all figures were generated using r version andadobe illustrator version cs6resultspancancer genomic and transcriptional alterations ofampk pathway genesfocusing on the genomic and transcriptomic landscapeof genes associated with ampk signaling retrievedfrom kegg across cancer types involving patients additional file we interrogated somatic copynumber alterations scna and mrna expression seeadditional file for a flowchart illustrating the study design to determine the effects of genomic alterations inampk pathway genes we classified genes as havinghighlevel amplifications gains lowlevel amplificationsdeep homozygous deletions and shallow heterozygousdeletions to evaluate pancancer patterns of scnaswe considered genes that were gained or lost in at least of samples within a cancer type and in at least onethird of cancer types ie at least seven cancer types atotal of genes were recurrently amplified while genes were recurrently lost fig additional file ampk is the central regulator of cellular energy levelswhich controls a number of downstream targets an example being the nuclear receptor hnf4a remarkablyhnf4a was found to be the most amplified gene identified as being recurrently amplified in of samplesin all cancers fig additionalfile this isfollowed by cftr cancer types and four other genesthat were amplified in cancer types adipor2 lepfile fig additionalprkag2 and rhebincontrast ppp2r2a was the most deleted gene found in of samples across cancers followed by the deletion of slc2a4 in cancers and five additional genesfoxo3 ppp2cb ppp2r2d ppp2r5c and ppp2r5ein cancer types fig additional file among allcancer types the highest number of amplified ampkpathway genes was observed in esophageal carcinomaesca genes followed by bladder cancer blca genes and lung cancer genes in both lung squamouscell carcinoma [lusc] and adenocarcinoma [luad]fig glioma tumors gbmlggin contrast hadonly five genes that were recurrently amplified fig in terms of somatic deletions lusc and esca both had genes deleted while no recurrent deletions were observed in papillary renal cell carcinoma kirp fig lossoffunctionevents differentialwe reasoned that scnas associated with transcriptional alterations could be considered as putative gainorexpressionanalyses between tumor and nontumor samples in eachcancer revealed that and genes were significantlyupregulated and downregulated in at least seven cancertypes respectively additional file of these differentially expressed genes seven and genes were alsorecurrently amplified and deleted respectively venn diagram in fig both gene sets were mutually exclusiveie the genes either had gainorfunction or lossoffunction features but not bothmolecular underpinnings of patient survival involvingputative lossoffunction ampk pathway componentswe next investigated the impact of transcriptional dysregulation ofthe putative gain and lossoffunctiongenes identified previously on patient survival outcomesacross all cancer types employing cox proportional hazards regression we observed that all genes sevengainoffunction and lossoffunction genes wereprognostic in at least one cancer type fig 2a thehighest number of prognostic genes was observed inglioma gbmlgg tumors genes while none ofthe genes were significantly associated with overallsurvival outcomes in esca and cholangiocarcinomachol fig 2a intriguingly although esca had thehighest number of scnas fig none of the genesharbored prognostic information suggesting that alterations in ampk signaling components have minimalroles in driving tumor progression and patient outcomes fbp1 was significantly associated with overallsurvival outcomes in cancers while ppp2r2c andppp2r2b in cancers fig 2a fbp2 is the least prognostic gene in only one cancer type cervical squamouscell carcinoma and endocervical adenocarcinoma cescfig 2a 0cchang and lai bmc cancer page of fig the landscape of somatic copy number alterations of ampk pathway genes heatmaps depict a fraction of samples within each cancertype that harbor somatic deletions and b somatic amplifications fortynine genes are recurrently deleted in at least of tumors within eachcancer and in at least seven cancer types fortysix genes are recurrently amplified in at least of tumors within each cancer and in at leastseven cancer types stacked bar charts on the yaxes illustrate the fraction of samples that possess copy number variation of a gene underconsideration grouped by shallow and deep deletions or amplifications stacked bar charts on the xaxes illustrate the fraction of samples withineach cancer type that contain shallow and deep deletions or amplifications the bar charts on the right of each heatmap depict the number ofcancer types with at least of samples affected by gene deletions and amplifications the venn diagrams demonstrate the identification of putative loss and seven gainoffunction genes from gene sets that are somatically altered and differentially expressed cancer cohorts analyzedwith corresponding tcga abbreviations are listed in parentheses bladder urothelial carcinoma blca breast invasive carcinoma brca cervicalsquamous cell carcinoma and endocervical adenocarcinoma cesc cholangiocarcinoma chol colon adenocarcinoma coad esophagealcarcinoma esca glioblastoma multiforme gbm glioma gbmlgg head and neck squamous cell carcinoma hnsc kidney chromophobekich pankidney cohort kipan kidney renal clear cell carcinoma kirc kidney renal papillary cell carcinoma kirp liver hepatocellularcarcinoma lihc lung adenocarcinoma luad lung squamous cell carcinoma lusc pancreatic adenocarcinoma paad sarcoma sarcstomach adenocarcinoma stad stomach and esophageal carcinoma stes and uterine corpus endometrial carcinoma ucec number ofsamples for each cancer type are indicated in parentheses blca brca cesc chol coad esca gbm gbmlgg hnsc kich kipan kirc kirp lihc luad lusc paad sarc stad stes and ucec given the prevalence of lossoffunction phenotypes indetermining clinical outcomes fig 2a we proceeded toexamine the combined impact of all lossoffunctiongenes on patient survival and oncogenic dysregulationto determine the extent of ampk pathway variationacross the cancers we calculated pathway scores foreach of the tumor samples by taking the meantranscript expression values of the genes slc2a4foxo3 ppp2cb pik3cd cab39l ccna1 fbp1fbp2 foxo1 hmgcr irs2 pik3r1 sirt1 tbc1d1ppargc1a ppp2r2c mlycd pfkfb3 ppp2r2bprkaa2 lepr cab39 irs1 and pfkfb1 we observedinteresting patterns when cancers were ranked from lowto high based on their median pathway scores fig 2bgbmlgg had the highest median pathway score whileblca and cesc were found at the lower end of thespectrum fig 2b as expected kaplanmeier analysisrevealed a significant difference in overall survival between glioma patients p stratified by low andhigh 24gene pathway scores fig 2c interestingly thecontribution of ampk signaling in cancer prognostication is cancertype dependent as in gliomalogranktests revealed that patients with high 24gene scores hadsignificantly improved survival outcomes in breast cancer p and sarcoma p fig 2c incontrast high expression of the genes was associated 0cchang and lai bmc cancer page of fig prognostic significance of ampk loss and gainoffunction genes a heatmap illustrates significant hazard ratio values from coxproportional hazards regression analyses on the lossoffunction and seven gainoffunction genes across all cancers b the distributions of ampkgene scores in each cancer are illustrated in the boxplot cancers are sorted from low to high median scores refer to fig legend forcancer abbreviations c kaplanmeier analyses and logrank tests revealed the prognostic significance of the 24ampkgene set in four cancertypes patients are stratified into q1 1st quartile and q4 4th quartile groups based on their 24gene scores for logrank tests dmultidimensional scaling analyses of the 24gene set depicted in 2dimensional space significance differences in the distribution between tumorand nontumor samples are confirmed by permanovawith increased mortality rates in stomach adenocarcinoma p fig 2c these results were in agreement when independently validated using the coxregression approach breast hazard ratio [hr] p glioma hr p sarcomahr p and stomach hr p cancers additionalfile since the 24genescore could be used to stratify patients into high andlowrisk groups we predict that when considered together gene expression values could discriminate tumorfrom nontumor samples although analysis could notbe performed on sarcoma this dataset only had twonontumor samples multidimensional scaling analysesand permanova tests of breast p gliomap and stomach p cancers revealed significantseparation between tumor and nontumor 0cchang and lai bmc cancer page of samples in twodimensional space fig 2d overall thissuggests that the 24gene set could be harnessed as adiagnostic biomarker for early cancer detectionconsistent with our previous observation that highpathway scores were associated with good prognosis insarcoma fig 2cto determine the independence of the 24gene setfrom other clinicopathologicalfeatures we employedmultivariate cox regression and observed that the gene set is independent of tumor node and metastasistnm staging where available in breast hr p and stomach cancers hr p additional file similarly kaplanmeier analyses andlogrank tests confirmed that the 24gene set allowedfurther risk stratification of patients with tumors of thesame tnm stage breast p and stomach p fig 3a furthermore we observed that within ahistological subtype of sarcomaleiomyosarcoma patients with elevated ampk signaling had significantlybetter3asurvival outcomesp figexploredthepredictivewe nextperformancesensitivity versus specificity of the 24gene set in allfour cancer types using receiver operating characteristicanalysis the area under the roc curve auc is an indication of how well the gene set could predict patientsurvival which ranges from to we found that thecombined model uniting both 24gene set and tnm staging outperformed the 24gene set when considered onits own in breast cancer patients auc vs fig 3b for stomach cancer the 24gene set onlycontributed to a marginally higher auc when used incombination with tnm staging when compared to the24gene set alone auc vs fig 3baucs of the 24gene set in glioma and sarcoma werefig the 24ampkgene set is independent of tumor stage and histological subtype a kaplanmeier analyses of patients grouped by tumornode and metastasis tnm stage breast and stomach cancers or by the histological subtype of leiomyosarcoma and the 24gene score forleiomyosarcoma the logrank test reveals a significant difference in survival rates between 1st and 4th quartile patients b receiveroperatingcharacteristic roc analyses on the 5year predictive performance of the 24gene set roc curves generated by the 24gene set are compared tocurves generated from both 24gene set and tnm staging where available or histological subtype auc area under the curve 0cchang and lai bmc cancer page of and respectively fig 3b within the leiomyosarcoma histological subtype auc was even higherat fig 3boncogenic transcriptional alterations associated withampk pathway inactivationampk pathway inactivation was associated with alteredsurvival outcomes in patients figs and we predictthat this could be due to broad transcriptional dysregulation arising from abnormal ampk signaling to investigate this phenomenon we performed differentialexpression analyses between patients stratified by the24gene set into high 4th quartile and low 1st quartileexpression groups and found that an outstanding number of common genes that were significantly differentially expressed in all four cancer types fig 4a thehighest number of differentially expressed genes degswas observed in stomach cancer genes followedby sarcoma genes glioma genes and breastcancer genes fig 4a additionalfile thedegs were mapped to kegg gene ontology andreactome databases to determine whether they were associated with any functionally enriched pathways intriguingly all four cancer types share similar patterns offunctional enrichments fig 4b and c for instance biological processes associated with cell communicationsignal transduction cell differentiation cell signalingcell adhesion and cell morphogenesis were enriched inall four cancers fig 4c in terms of specific signalingpathways calcium signaling camp signaling and processes associated with extracellular matrix anizationwere among the most enriched fig 4cto further identify potential transcriptional regulatorsof the degs we mapped the degs to encode andchea transcription factor tf binding databases remarkably we identified common tfs shared across allfour cancers that were implicated as direct binding partners of the degs fig 4c five tfs suz12 smad4rest ezh2 and nfe2l2 were found to be enriched inall four cancers suggesting that transcriptional dysregulation of tumors with aberrant ampk signaling involveddirect physical associations of these tfs with targetdegs fig 4c curiously foxm1 and e2f4 wereenriched only in glioma tumors which deserves furtherexploration in the next section overall our analysesdemonstrated that impaired ampk signaling resulted incommon patterns of oncogenesis which affect the severity of cancer and consequently mortality ratesinpatientsdownstream targets of ezh2 nfe2l2 rest smad4 andsuz12 were associated with survival outcomespathways modulating energy homeostatic may transducesignals to influence other cognate signaling modulesezh2 nfe2l2 rest smad4 and suz12 were all implicated as common transcriptional regulators of degsin glioma sarcoma breast and stomach cancers suggesting that altered ampk signaling converged on similargroups of transcriptional targets of all the target degsof the aforementioned tfs and geneswere found to be common targets of ezh2 nfe2l2rest smad4 and suz12 respectively in all four cancers fig 5a concatenating all five gene sets yielded unique genesie genes that were binding targets ofmore than one tf were considered only once to gainfurther insights into how ampk inactivation influencestumor progression we performed cox regression analyses to determine the association between each of the genes and survival outcomes the highest number ofprognostic genes was observed in glioma genes good prognoses and five adverse prognoses fig 5b incontrast out of genes were associated with adverseprognosis in stomach cancer fig 5b these observations were consistent with the 24ampkgene set beingpositive and negative prognostic factors in glioma andstomach cancer respectively fig which mirrored thebehavior of degs identified as a result of aberrantampk signaling fig 4c of the genes only andten were significantly associated with survival outcomesin sarcoma and breast cancer respectively fig 5b collectively our results suggest that the ampk pathwayand its interaction with other signaling modules are keydeterminants of patient outcomes in multiple cancertypesprognostic significance of joint ampk pathway activityand transcriptional levels of five oncogenic tfs inpatients with gliomahaving discovered the importance of the 24ampk geneset we sought to explore the crosstalk between ampksignaling and tf activity in glioma as previously mentioned glioma had the highest 24ampkgene scorefig 2b with a vast majority of the genes conferringprognostic information fig 2a moreover of the transcriptional targets of the five common tfs identifiedin patients with altered ampk signaling were significantly associated with survival outcomes in glioma fig5b additionally tfs foxm1 and e2f4 were identifiedto be enriched only in glioma tumors fig 4c thus wepredict that a joint model uniting ampk and tf expression profiles would allow further delineation of patientsinto additional risk groups and if so allowing combinedtargeting of ampk and candidate tfs for therapeuticaction as done previously we calculated ampk scoresfor each patient based on the mean expression of the genes interestingly we found that ampk scores weresignificantly negatively correlated with tf expressionlevels in glioma e2f4 rho p ezh2 0cchang and lai bmc cancer page of fig see legend on next page 0cchang and lai bmc cancer page of see figure on previous pagefig ampk inactivation drives oncogenic transcriptional alterations in diverse biological processes and signaling modules a venn diagramillustrates the number of differentially expressed genes degs between 1st and 4th quartile patients as stratified using the 24ampkgene set infour cancer types a total of degs were common in all four cancers b dot plots depict the number of significantly enriched pathways andbiological processes upon the mapping of degs to kegg gene ontology and reactome databases each dot represents an enriched event contologies that exhibit similar patterns of enrichment across four cancers are shown degs are also mapped to encode and chea transcriptionfactor tf databases to determine enriched tf binding associated with degsrho p foxm1 rho p smad4 rho p and suz12rho p fig 6a we subsequentlycategorized patients into four groups using the mediancutoff of the ampk scores and tf expression values lowlow highhigh low ampk score and high tfexpression and high ampk score and low tf expression logrank tests revealed that patients stratified intothe four groups had survival rates that were significantlydifferent e2f4 p ezh2 p foxm1p smad4 p and suz12 p fig 6b for e2f4 ezh2 foxm1 and suz12patients with low ampk scores and high tf expressionperformed the worst e2f4 hr p ezh2 hr p foxm1 hr p and suz12 hr p fig6c for smad4 patients within the lowlow categoryhad the highest mortality rates hr p fig 6ccrosstalk between ampk and other anabolicrelatedpathways ppar and mtorampks antianabolic and procatabolic activities maywork in concert with other metabolic pathways toinvestigate the synergistic effects of ampk and two proanabolic pathways peroxisome proliferatoractivated receptors ppar and mammalian target of rapamycinmtor signaling in tumor progression we calculatedppar and mtor pathway scores detailed in themethods section for each glioma tumor low ampkscores were associated with poor outcomes in gliomafig to evaluate ampk and ppar or mtor ascombined models patients were separated into fourgroups using the median cutoff as mentioned previously interestingly when ampk and ppar scores werecollectively used for patient stratification we found thatpatients with low ampk and high ppar scores had thehighest death rates hr p confirmingthat ppar hyperactivation is associated with poor outcomes in glioma tumors with low ampk activity fig in contrast when considering mtor activitypatients with low ampk and low mtor scores performed the worst hr p fig theresults overall suggest that the ampk pathway could actsynergistically with ppar and mtor signaling to influence cancer progression significantlydiscussionwhile the role of ampk in energysensing is wellunderstood its full potential in metabolic diseases suchas cancer remains an open topic of debate despite extensive efforts spent on elucidating the role of ampksignaling [ ] there remains no consensus onwhether ampk promotes or suppresses tumor progression exploiting a rich reservoir of pancancer datasetsafforded to us by tcga we performed a thoroughexamination of genomic and transcriptomic profiles of ampk pathway genes in diverse cancer types ourcurrent understanding of ampk signaling is fueled bygenetic studies in cell lines and animal models although useful in determining causal relationships resultsfrom in vitro cell lines and animal models may have limited translational relevance as they do not accurately reflect human pathology animal models may offeradditional mechanistic insights but limitations in ethicsand costs remain moreover the complexity of humancancers is not accurately modeled in animals less than of results from animal models are translated to clinical trials despite analyses on tumor genetic datasets providing mostly correlative outcomes they remainvaluable in understanding diseasespecific molecularpathology when interrogated at scale on large patientgroups [] and when results are considered in relation to those obtained from celllines and animalmodelsemploying pancancer population data our studyidentified conserved and unique patterns of ampk signaling across diverse cancer types analyses at two molecular levels genetic and transcriptional yielded amore comprehensive depiction of ampk signalingwhere we identified genes that were both somatically altered and differentially expressed these putative lossor gainoffunction genes are more likely to impacttumor progression as they are altered at both macromolecular levels as reported in other studies we confirmedthat ampk signaling could either be oncogenic ortumor suppressive depending on the cellular context intuitively since ampk is antianabolic its function maynot be fitting for tumor growth and proliferation this isconsistent with reports demonstrating ampks tumorsuppressive activity [ ] a study on lymphoma demonstratesthewarburg effect and hypoxia signaling in mice that ampk downregulation induces 0cchang and lai bmc cancer page of fig see legend on next page 0cchang and lai bmc cancer page of see figure on previous pagefig prognostic significance of degs targeted by enriched tfs a venn diagrams illustrate the extent of overlap between degs targeted byezh2 nfe2l2 rest smad4 and suz12 across four cancers b forest plots depict degs that are significantly associated with overall survivaloutcomes hazard ratios are denoted as purple squares while pink bars represent the confidence intervals significant wald test p values areindicated in blueits loss ofampk is proposed to act as a metabolic gatekeeper tolimit cancer cell division hencefunctionwould contribute to tumor aggression because of theloss in metabolic checkpoints [ ] ampk regulatesthe tumorsuppressive function of the serinethreoninekinase lkb1 ablation of lkb1 results in enhanced riskof developing gastrointestinal lung and skin squamouscell cancers [ ] moreover ampk is shown to inhibit pi3kaktmtor signaling which is activated inmany cancers [ ] also metabolic inhibitors such asmetformin which indirectly activates ampk could suppress tumor growth via autophagy induction and mtorinhibition [ ] metformin is shown to inhibit theproliferation of estrogen receptor α erα negative andpositive breast cancer cell lines through ampk stimulation however when tested in mice models metformin contributes to enhanced tumor progression andincreased angiogenesis providing us with a glimpse ofpotential proneoplastic effects of ampk activation in our study we observed that high levels of ampkpathway activity were associated with better outcomes inglioma breast cancer and sarcoma fig corroboratingprevious results on the tumorsuppressive function ofthe opposite is true in stomachampk converselyfig prognostic relevance of candidate tfs and the 24ampkgene set in glioma a scatter plots illustrate significant negative correlationsbetween ampk scores and tf expression levels in glioma patients are separated and colorcoded into four categories based on median ampkand tf scores density plots appended to the y and xaxes demonstrate the distribution of ampk and tf scores b logrank tests are performedon the four patient grou
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clinical manifestations of sarscov2 infection include more frequently fever and cough failure can occur in persons with additional comorbidities liver dysfunction is one of the most striking affections among patients suggesting that sarscov2 may represent a new king of liver aggressor however the molecular process underlying this phenomenon is 0cstill unclear in this work we overview the most recent findings between the molecular biology of the virus pathogenic mechanisms and its relationship to liver disease observed in patients abbreviations aado2 ace2 aih alt ast covid19 ggt gi gtex alveolararterial oxygen gradient angiotensinconverting enzyme autoimmune hepatitis alanine transaminase aspartate aminotransferase coronavirus infectious disease gamma glutamyl transpeptidase gastrointestinal genotypetissue expression metabolicassociated fatty liver disease nonstructural proteins open reading frame preproofcomplex disease in many severely ill patients in other infected subjects an infection is keywords sarscov2 liver liver impairment covid19 ace2 mafld nsp orf introduction sarscov2 is the etiological agent of the disease known as covid19 which causes a disease characterized by pneumonia cough fever occasional diarrhea headache cardiac injury and in some patients liver alterations covid19 has been found to be an extremely reported to be so severe that it can lead to a disproportionate and mortal reaction of the immune system known as a cytokine storm all of these factors make covid19 highly unpredictable it is what specialists call a multisystem disease 0caround the world cases of liver dysfunction denoted by elevated hepatic enzymes in serum such as ast aspartate aminotransferase and alt alanine transaminase have been documented among patients infected with sarscov2 there is still no certainty whether the covid19related liver damagedysfunction is due mainly to the viral replication per se drugs toxicity or other coexisting comorbidities whether sexrelated difference could help to explain why infected men are more healthy or harmful relationship between the liver and its viral aggressor in this paper we describe a brief overview of the implications for researchers in the field of it is important to understand how liver function can be altered by direct infection with this predisposed to develop covid19associated liver dysfunction than infected women to analyze if there is any genetic predisposition related to impaired liver function during the respiratory virus which mechanisms of viral pathogenesis are involved to evaluate disease and of course the crosstalk between viral and cellular proteins that mediate this preproofliver disease of the most recent findings between the molecular biology of the virus this emerging viral illness is typically characterized by fever dry cough myalgia headache sore throat diarrhea and may be aggravated with shortness of breath and respiratory failure the angiotensinconverting enzyme ace2 the functional receptor of the spike glycoprotein of sarscov2 is widely distributed in the anism historically hamming and colleagues reported ace2 expression in the surface of lung alveolar epithelial cells enterocytes of the small intestine arterial and venous endothelial pathogenic mechanisms and its relationship to liver disease observed in patients clinical characteristics and liver injury in patients with covid19 0ccells and arterial smooth muscle cells posterior transcriptomic and proteomic analyses confirmed their findings and added high ace2 expression in adipose tissue bone marrow duodenum endometrium heart kidney small intestine smooth muscle testis and thyroid ace2 is also expressed in liver but in lesser extent one of the most worrisome severe cases of covid19 regarding the gastrointestinal gi tract and liver over covid19associated liver injury is defined as any liver damage that occurs during disease progression andor covid19 treatment in patients with or without a history of previous complications is the unusual formation of blood clots in many patients with covid19 even those who were receiving anticoagulants researchers at mount sinai in new york published studies suggesting that clots in the lungs play an important role in the most of covid19 patients develop gi symptoms such as anorexia diarrhea nausea and vomiting and a significant proportion present with altered liver function tests preproofdecreased albumin levels are associated with severe infection and poor prognosis still ast elevation is the most common abnormality in patients presenting with covid19 observed more frequently in men and is mainly documented in more severe cases liver disease in general the incidence of increased liver biochemical markers in hospitalized patients with covid19 mainly ast and alt and slightly elevated bilirubin varies between to of cases the increase in liver enzymes is there are no reports of acute or subacute liver failure in patients with covid19 the largest cohort study that included cases of covid19 from china showed that had preexisting chronic liver disease lei and colleagues reported that impaired liver function was related to mortality in covid19 patients elevated ast was more frequent and significant than the increase of alt in severe 0chospitalized patients moreover elevated ast was shown to be associated with highest mortality risk in the study reported by yijin wang they found that of covid patients had elevated ast activity the median levels of alt were ul vs ul respectively ast were ul vs ul respectively in abnormal and normal aminotransferase groups liver enzymes abnormality were associated with disease severity protein levels in addition they found by ultrastructural examination of coronavirus ps in hepatocytes in covid19 cases sarscov2 infected hepatocytes displayed as well as a series of laboratory tests including higher alveolararterial oxygen gradient aado2 higher gamma glutamyl transpeptidase ggt lower albumin and lower total conspicuous mitochondrial swelling endoplasmic reticulum dilatation and glycogen granule decreased histological findings showed apoptosis and binuclear hepatocytes preproofshowed a disease course similar to that reported in non immunosuppressed population coronavirus the interaction of its proteins with cellular proteins and consequently the immunosuppressive therapy for autoimmune hepatitis aih developing covid19 taken together both ultrastructural and histological evidence indicated a typical lesion of viral infection all these findings by different reports demonstrates that sarscov2 infection in liver is a crucial cause of hepatic impairment in covid19 patients however alteration of cellular metabolism that give rise to systematic alterations and metabolic report from alessio gerussi demonstrated that patients under today the cellular and molecular mechanisms that are altered by infection with this changes are still unknown 0cmolecular biology of sarscov2 coronaviruses are enveloped viruses that contain a positively polarized unsegmented rna genome belonging to the coronaviridae family and the order of nidovirales they are distributed in humans and other mammals the size of the sarscov2 virions is approximately to nm in diameter [] sarscov2 has a genome that consists polymerase rdrp which is nsp12 and is responsible of the replication and transcription of the virus which are encoded by the various genetic loci on the genome at the center of the virion lies a nucleocapsid composed of the genomic rna and the nucleocapsid protein the virus glycoprotein s consists of two subunits s1 which is at the amino terminus and that encodes for structural proteins and a larger region that encodes two open reading frames orf 1a and 1b which together encode for the nonstructural virus proteins from nucleocapsid protein which is within the phospholipid bilayer and nonstructural proteins nsp1 to nsp16 the virions have a structural sspike protein outer spiky glycoprotein mmembrane protein a type iii transmembrane glycoprotein nof nucleotides encoding amino acids and it is composed of a region preproofvirus as well as protein m which is a type iii transmembrane glycoprotein and participates in the cellular membrane rearrangements for the replication and transcription complexes among the encoded proteins is an rnadependent rna provides the receptor binding site and s2 which is at the carboxyl terminus responsible for membrane fusion the envelope protein e has a role in the assembly and release of the nonstructural proteins have several functions during de viral cycle for example nsp 0cthe virus enters the cell by endocytosis through the interaction between envelope glycoprotein s with the cell receptor ace2 and with the participation of the type ii transmembrane serine protease tmprss2 once it enters the cell the n protein with viral genome are released within the cytoplasm then cellular proteases degrade the capsid and the virus genome is left free next the polyprotein containing the viral proteins that are how does the virus select which cells to infect viruses can infect only certain species of hosts and only permissive cells within that host permissive cells make all the necessary proteins and viral factors to allow virus to replicate once a virus gets inside a cell it hijacks the cellular processes to produce virally encoded proteins that will replicate the viruss genetic material viral replication may cause exocytosis will translate into viral proteins this entire process will occur in the cell cytoplasm the processed to form the replication complex is translated and then the complementary strand of negative sense pregenomic rna is synthesized to be used as a template to replicate the structural proteins that will be synthesized in the endoplasmic reticulum membrane to assembly the viral p and finish the cycle through the release of the viruses by positive sense viral genome furthermore the replication and transcription complex will lead to a series of smaller positive sense subgenomic rnas these are the ones that preproofboth sarscov and the new sarscov2 are very similar in structure and pathogenicity but the major structural protein s protein is slightly different between them compared to other beta coronaviruses the presence of a furinlike cleavage site in sarscov2 enables the s protein priming and facilitates an increase on the efficiency of the spread of sarscov2 as is reported wide world 0cbiochemical changes producing cell damage called cytopathic effects like other coronaviruses sarscov2 requires cellular receptors to initiate its internalization to the cells that carry these factors li sarscov2 uses the angiotensinconverting enzyme ace2 as a host cell receptor sarscov2 spike s protein binds ace2 with significantly high affinity in addition the main host protease that suggested to promote the pathogenesis of this coronavirus program httpsportalgdccancergov they compared ace2 expression levels across human tissues between males and females and between younger and older persons in these individual tissues furthermore other reports have analyzed the correlations between ace2 in order to provide insights into the mechanism of sarscov2 infection li analyzed the expression of ace2 in various normal human tissues using the datasets from the genotypetissue expression gtex project and the cancer genome atlas tcga transmembrane serine protease other host proteases such as furin have also been mediates sprotein activation on primary target cells and initial viral entry is the type ii preproofreported by li ace2 expression levels showed no significant difference between have no significant association with sex age or race is the liver a direct target for sarscov2 males and females between younger and older persons or between asian and nonasian races this finding suggests that the infection risk of sarscov2 and sarscov may expression levels and immune signature enrichment levels in individual tissues as as we expected because the systemic manifestations of covid19 it has been reported that sarscov2 has an anotropism beyond the respiratory tract including the kidneys 0cliver heart and brain and possibly that anotropism influences the course of covid19 disease and aggravates preexisting conditions the ace2 protein is found at high levels in the gi tract as the colon biliary system and liver on the other hand it is well documented a sarscov2 rna shedding in the gi tract supporting its tropism for architecture express ace tmprss1 receptors the presence of these two host factors in the liver suggests that a direct viral cytopathic effect occurs also in sars infection the presence of viral rna in liver tissue was documented but not as extensively as the new coronavirus data published by gordon suggest that mitochondrial proteins interact directly with the virus which helps to understand the potential mechanism by which elevated ast the gi tract and liver cells and these may be sites of active viral replication and either direct or indirect tissue injury indeed a large part of the cells distributed in the liver preproofeffect the exacerbated inflammatory response in covid19 may play a central role in profiles are detected in these patients furthermore in addition to this intracellular more recently identified the clinical and laboratory characteristics of covid19 patients with abnormal liver transaminases and they reported that sarscov2 is able to which high levels of il6 have been reported which are involved in both infect liver cells and cause liver impairment by direct cytopathic effect inflammatory and repair responses in liver disease mechanisms of liver pathogenicity 0cif sarscov2 replication has direct adverse effects on liver function it is still unknown findings in liver biopsy of patients killed by covid19 showed moderate micro vesicular steatosis and mild portal and necroinflammatory activity this seems to indicate that a direct injury occurred while the infection that could have been directly caused by sarscov2 another possibility is that a druginduced liver injury occurred to date there are the following possible mechanisms figure infection the massive release of cytokines by the immune system in response to the viral infection can result in a cytokine storm and symptoms of sepsis that are the cause of death in of fatal covid19 cases in these cases uncontrolled inflammation induces multian damage leading including liver failure biomarkers of inflammation such as creactive protein pcr serum ferritin ldh ddimer il6 il2 are have been found to be significantly elevated in immune damage from exacerbated inflammation in response to sarscov2 preproofpathogenesis of sars cov related liver disease more studies should be liver is a potential target for direct infection with this virus to understand the performed for evidence of viral mechanisms of replication in different cell anelles as cytoplasm endoplasmic reticulum golgi apparatus and lipidrafts cov2 enters cells through the ace2 molecule which is abundantly expressed in the liver and in particular in bile epithelial cells based on this expression the direct cytopathic effect due to active viral replication in various liver cells sarscritically ill patients with covid19 into hepatocytes and liver histology characterization it is also important to know in cells their capacity to efficiently produce both infectious and defective non 0cinfective whole virions there are not yet enough data to know the viral dynamics in the different tissues and the associated pathogenesis anoxia respiratory failure is one of the main characteristics of covid19 anoxic hypoxic hepatitis is common in patients with severe symptoms reactivation of preexisting liver disease patients with preexisting chronic liver medications such as tocilizumab and baricitinib used to combat the adverse immune cholestatic liver disease various studies such as lopinavir ritonavir remdesivir chloroquine tocilizumab uminefovir traditional chinese medicine so it is important to consider that they could be potentially hepatotoxic in some patients druginduced liver damage dili initial clinical guidelines recommended antiviral agents for covid19 so a variety of drugs have been administered in disease may be more susceptible to liver damage from sarscov2 biological preproof genetic factors genetics may well be one of the determining factors in some reaction may also cause hbv reactivation and induce eventual impairment of liver function in those patients with hbv on the other hand it is still unknown whether sarscov2 infection exacerbates cholestasis in people with underlying patients who become seriously ill with covid19 but until now we cannot be sure it is possible for example that the genetic variation that makes an individual more susceptible to high blood pressure or diabetes also makes him more vulnerable to the virus it will be important to find out what role genetic factors predisposing to liver steatosis have and their sensitivity to severe symptoms of covid19 ji and 0ccolleagues showed that subjects with metabolicassociated fatty liver disease mafld have a higher risk of covid19 severity disease and abnormal liver blood tests than patients without mafld in contrast louise biquard demonstrated that mafld is not associated with changes in liver expression blood test abnormalities reported by ji and colleagues is thus likely not explained by concluding remarks the scenario is not yet complete which does not allow us to establish or understand the natural history of the disease and the participation or commitment of the liver in this disease certainly the application of new technological platforms such as singlecell increased hepatic sarscov2 uptake still several contradictory reports will help of genes implicated in sarscov2 infection the observed persistence of liver to find the real role of genetic factors in the evolution of this disease preprooftranscriptomic assays will allow us to quickly know the commitment of each cell type in affected ans and the meaning of viral dynamics in the various affected systems including the liver however as we have already learned from the old hepatotropic viruses history still is ongoing and we have much to learn and understand about the virologic characteristics of this emerging rna virus that allow us to develop specific antivirals such as the case of hcv and the vaccine to decrease the impact of this acute infection declarations of interest none ethical approval not required 0c references chen n zhou m dong x qu j gong f han y epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan china httpsdoi101002path1570 wang d eraslan b wieland t hallström b hopf t zolg dp a deep proteome and transcriptome abundance atlas of healthy human tissues mol syst hamming i timens w bulthuis mlc lely at navis gj van goor h tissue distribution of ace2 protein the 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h clinical and chai x hu l zhang y han w lu z ke a specific ace2 expression in invest httpsdoi101172jci137244 death cases with covid19 medrxiv httpsdoi1011012020022620028191 immunological features of severe and moderate coronavirus disease j clin preproofcholangiocytes may cause liver damage after 2019ncov infection biorxiv patients medrxiv httpsdoi1011012020040120047381 httpsdoi10110120200203931766 herold t jurinovic v arnreich c hellmuth jc von bergweltbaildon m klein m level of il6 predicts respiratory failure in hospitalized symptomatic covid grein j ohmagari n shin d diaz g asperges e castagna a compassionate use of remdesivir for patients with severe covid19 n engl j med httpsdoi101056nejmoa2007016 u s food and drug administration fact sheet for health care providers emergency 0cuse authorization eua of hydroxychloroquine sulfate supplied from the strategic national stockpile for treatment of covid19 in certain hospitalized patients varona pérez j rodriguez chinesta jm riesgo de reactivación de la hepatitis b asociado al tratamiento con corticoides frente a sarscov2 covid19 rev clÃnica española httpsdoi101016jrce202004012 ji d qin e xu j zhang d cheng g wang y nonalcoholic fatty liver httpsdoi101016jjhep202003044 sarscov2 in metabolicassociated fatty liver disease j hepatol diseases in patients with covid19 a retrospective study j hepatol preproof biquard l valla d rautou pe no evidence for an increased liver uptake of httpsdoi101016jjhep202004035 0cfigure legends preprooffig1 proposed mechanisms of liver pathogenicity of sarscov2 in infected cells sars cov2 infection2 cytokinestorm3 drugeffects4 hypoxia5 previousliverdamagebiochemicallabmarkerswhite bloodcellsgenomereleasereplicationtranslationvirionassemblyviral proteinsmaturevirus release\uf0e9aado2mitochondrialproteinshypoxicisquemicliverinjuryliver damagelopinavirritonavirremdesivirchloroquinetocilizumaboxidativeimbalancesteatosisace2s proteincytopathiceffect\uf0e9gmcsf\uf0e9il6\uf0e9il1β\uf0e9il2\uf0e9il8\uf0e9ccl2\uf0e9ccl3\uf0e9ccl5\uf0e9cxcl \uf0e9alt\uf0e9ast\uf0eaalbumin\uf0e9pcr\uf0e9ldh\uf0e9ddimer\uf0e9ferritin\uf0e9bilirubin 0c'
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" chemotherapy is one of the most commonly used treatments for patients with advanced colon cancer yet the toxicity of chemotherapy agents such as fluorouracil 5fu limits the effectiveness of chemotherapy ginsenoside rg3 rg3 is an active ingredient isolated from ginseng rg3 has been shown to display anticancer effects on a variety of malignancies yet whether rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer remains unknown the present study was designed to ascertain whether rg3 is able to enhance the anticolon cancer effect of 5fu the results revealed that combined treatment of rg3 and fu significantly enhanced the inhibition of the proliferation colony formation invasion and migration of human colon cancer cells sw620 and lovo in vitro we also found that combined treatment of rg3 and fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway and arrested the cell cycle of the colon cancer cells in g0g1 by promoting the expression of cyclin d1 cdk2 and cdk4 in addition the pi3kakt signaling pathway in colon cancer cells was suppressed by rg3 and 5fu in vivo rg3 synergized the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude mice similarly combined treatment of rg3 and 5fu altered the expression of colon cancer protein in vivo and in vitro collectively the present study demonstrated that ginsenoside rg3 enhances the anticancer effect of 5fu in colon cancer cells via the pi3kakt pathwaycorrespondence to dr xiangbo chen endoscopy center the quanzhou first hospital affiliated to fujian medical university east street licheng quanzhou fujian pr chinaemail coloboyeahnetkey words ginsenoside rg3 colon cancer 5fluorouracil pi3kaktintroductioncolon cancer is a common malignant tumor of the digestive tract located in the colon which mainly occurs at the junction of the rectum and the sigmoid colon statistics show that the highest incidence of colon cancer is in the age group of years the ratio of male to female is and the incidence of colon cancer ranks third among all cases of gastrointestinal tumors the 5year survival rate of patients with colon cancer is approximately yet the 5year survival rate of patients with advanced stage disease is as low as since the early symptoms of patients with colon cancer are not obvious only about of patients can be diagnosed at the early stage of the disease chemotherapy is one of the most important treatments for patients with advanced colon cancer of which fluorouracil fu is the most widely used 5fu inhibits the proliferation invasion and migration of tumor cells by interfering with the nucleic acid metabolism of tumor cells but it is also toxic to normal cells causing serious adverse reactions even endangering the life safety of patients severely limiting its clinical application previous research has shown that 5fu combined with other agents may reduce the required dosage of 5fu consequently reducing the adverse reactions caused by 5fu without affecting or even improving the efficacy of chemotherapy compared with chemical drugs and biopharmaceuticals multicomponent multitarget and less adverse reactions are unique advantages of traditional chinese medicine for the treatment of diseases in patients with colon cancer chinese medicine can improve patient immunity reduce the side effects of radiotherapy and chemotherapy or enhance drug sensitivity inhibiting the expression of oncogenes helps to inhibit the migration of cancer cells and has a good effect on the treatment of colon cancer ginsenoside rg3 rg3 an active ingredient isolated from ginseng is a tetracyclic triterpenoid saponin that inhibits neovascularization induces tumor cell apoptosis and selectively inhibits tumor cell metastasis and enhances immune function previous studies have shown that rg3 exhibits an inhibitory effect on proliferation invasion and migration of human tumor cells such as lung cancer gastric carcinoma and prostate cancer in colon cancer rg3 was found to activate the ampk signaling pathway to accelerate apoptosis in colon 0chong effects of ginsenoside rg3 on colon cancercancer cell line ht29 in vitro and also to block colon cancer progression by targeting inhibition of cancer stem cells and tumor angiogenesis in vivo although numerous studies have shown that rg3 increases the efficacy and decreases the toxicity of chemotherapeutic drugs and suppresses the chemotherapeutic resistance in cancer its combination with chemotherapeutic agent fu to achieve extra benefits in anticolon cancer treatment warrants detailed investigationin the present study the effects of a combined treatment of rg3 and 5fu on the biological properties of sw620 and lovo cells were investigated in vivo and in vitro we found that a combined treatment of rg3 and 5fu not only enhanced the inhibition of colon cancer cell proliferation migration and invasion but also promoted apoptosis of colon cancer cells and arrested the cells in the g0g1 phase in addition it was also found that rg3 could synergize the capacity of 5fu to inhibit the growth of human colon cancer xenografts in nude mouse and the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt pathway in colon cancer cellsmaterials and methodscell lines and agents sw620 ccl atcc american type culture collection manassas va usa and lovo ccl229 atcc cell lines were cultured with dmem medium cat no thermo fisher scientific inc supplemented with fetal bovine serum fbs cat no thermo fisher scientific inc and penicillinstreptomycin cat no thermo fisher scientific inc the cell lines used in the present study were cultured at Ëc with co2rg3 cat no sigmaaldrich merck kgaa and 5fu cat no sigmaaldrich merck kgaa were dissolved in dmso for the cell experiments the diluted culture solution of rg3 or 5fu was dissolved in dmso to achieve the experimental concentration and was administered to the cells for h for animal experiments pbs diluted rg3 or 5fu was dissolved in dmso to the experimental concentration the experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinamtt assay a total of 2x103 cellswell were inoculated in a 96well culture plate containing the indicated medium dmem plus fbs we evaluated the viability of the sw620 and lovo cells by mtt assay in short after h of culture mtt µl mgml which was dissolved in dmso was added to the cells and incubated the cell supernatant was removed and then µl dmso was added after min the optical density od570 was determined using a plate reader elx808 biotek instrumentscell colony formation assay a total of 2x103 cellsml were seeded in 6well plates with ml mediumwell and medium was exchanged once every days cells were routinely cultured for about weeks when visible clones appeared in the well the culturing was stopped the supernatant culture medium was drawn washed with pbs times and fixed with formaldehyde for min the supernatant was drawn stained with crystal violet for min and slowly rinsed with sterile water plates were placed in a sterile purification table and images were captured after drying the relative proliferation was measured by measuring the absorbance at nm using a plate reader elx808 biotek instrumentswestern blot analysis ripa lysate buffer cat no p0013c beyotime institute of biotechnology shanghai china was used to extract total cellular protein and the bca kit cat no p0009 beyotime institute of biotechnology was used to determine the protein concentration then cell lysates of sw20 and lovo cells were separated by sdspage with µg total protein and transferred to a pvdf membrane the following primary antibodies were selected as follows antincadherin antibody ab18203 dilution antiecadherin antibody ab1416 dilution antimmp ab38898 dilution antiactivecaspase antibody ab2324 dilution antiactivecaspase3 antibody ab2302 dilution antiapaf1 antibody ab2324 dilution antipi3kp85 antibody ab191606 dilution antipi3k110 antibody ab32569 dilution antipanakt phospho t308 antibody ab38449 dilution antipanakt antibody ab8805 dilution antipdk1 antibody ab52893 dilution and antigapdh ab9484 dilution the secondary antibody was selected as follows goat antirabbit ab150077 dilution or goat antirat ab150117 dilution the blocking protocol was with skim milk for h at room temperature the primary antibody was incubated overnight at Ëc and the secondary antibody was incubated for h at room temperature the beyoecl plus kit cat no p0018s beyotime was used for the chromogenic protein bands with beckman coulter immunoassay system unicel dxi beckman coulter and imagej v2147 national institutes of health was used for the densitometric analysis of protein bands all antibodies were purchased from abcam unless otherwise statedtranswell invasion experiment the cell density was adjusted to 05x106 cellsml and then the cells were added to a 24well transwell upper chamber corning corning ny usa medium containing fbs gibco thermo fisher scientific inc was added into the lower transwell chamber and the transwell was incubated at Ëc for h the transwell was taken out and the medium was removed it was washed twice with pbs methanol was added and dried after being fixed for min after the membrane was dried it was stained with crystal violet for min and the relative migration was determined by measuring the absorbance at nm using a plate reader elx808 biotek instruments inccell scratch test a total of 5x105 cells were placed in a 6well plate mlwell a scratch was made as far as possible perpendicular to the back of a horizontal line by using tips against a ruler tips should be vertical and cannot be tilted the cells were washed with pbs for three times and the scratched cells were removed and serumfree dmem was added cells were cultured at Ëc in a co2 incubator for h and images were captured in and h using an ckx41 olympus inverted microscope magnification x100 olympus corp 0concology reports figure effect of the combined treatment of rg3 and 5fu on proliferation of colon cancer cells in vitro sw620 and lovo cells were treated with different doses of a rg3 mmoll or b 5fu µmoll and then the mtt assay was used to detect cell viability c and d after treatment with rg3 mmoll or 5fu µmoll or their combination the colony formation of the colon cancer cells was photographed wt group was used as a baseline for cell viability and cell colony formation three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the wt group fu fluorouracil rg3 ginsenoside rg3 flow cytometric analysis cells that had been treated in different manners were collected and precooled ethanol prechilled pbs and waterfree configuration was added at Ëc overnight then the cells were washed with pbs and stained with propidium iodine pi for cell cycle macsquant® analyzer flow cytometer miltenyi biotec was used to detect the cell cycle and the annexin v fitcpi kit invitrogen thermo fisher scientific inc was used for flow cytometry to detect apoptosisanimal experiment human colon cancer cells 5x10602 ml in the logarithmic phase were selected a total of female nude mice weeks of age g shanghai lingchang biological technology co ltd that were adaptive for feeding [room temperature of Ëc half day light and night dark cycle air humidity of ] for one week were selected mice were anesthetized [ sodium pentobarbital mgkg intraperitoneal ip] and then the lateral skin of the nude mice was selected as a cell inoculation site to inoculate 5x10602 ml human colon cancer cells at the logarithmic phase of growth when the tumor tissue grew to a volume of approximately mm3 then the mouse were randomly assigned to the solvent group equal amount of pbs dmso rg3 group mgkg gavage administration once every two days 5fu group mgkg ip injection once every two days and rg35fu group combined rg3 and 5fu group administration after weeks of treatment the mice were sacrificed using cervical dislocation and breathing and heartbeat for min were observed to determine death and tumor tissues were extracted and weighed with an analytical balance bsa124s beijing sartorius instruments ltd beijing china all animal experiments were approved by the ethics committee of quanzhou first hospital affiliated to fujian medical universitystatistical analysis all data are expressed as mean ± standard deviation and spss ibm corp was used to analyze the data student's ttest was used to compare differences between two groups and multiple groups were compared with oneway anova followed by duncan test as a post hoc test p005 was assigned to indicate that a difference was statistically significantresultscombined treatment of rg3 and fu enhances inhibition of cell proliferation after treatment with different doses of rg3 or 5fu mtt assay was used to measure the cell viability the results revealed that the cell viability of sw620 and lovo cells was significantly and gradually decreased with an increasing dose of rg3 thus we chose mmoll rg3 for subsequent experiments fig 1a as shown in fig 1b the proliferative activity of the colon cancer cells in the combined treatment group of rg3 and fu was significantly lower than 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on migration and invasion of colon cancer cells in vitro treatment of colon cancer cells with combined treatment of rg3 mmoll and 5fu µmoll inhibited the migration a and invasion b abilities of the sw620 and lovo cells scale bar µm c western blot analysis was used to detect the expression of emtrelated protein ncadherin ecadherin and mmp9 the solvent group was used as a baseline for the migration and invasion of cells three independent repetitions for each experiment were performed p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 emt epithelialmesenchymal transition mmp matrix metalloproteinase that of the 5fu treatment alone group in addition the cell viability of sw620 and lovo cells gradually decreased with the increasing dose of 5fu however after treatment with the combination of mmoll rg3 and µmoll fu for h the cell viability of sw620 cells was only which was not conducive to subsequent protein detection experiments thus mmoll rg3 and µmoll 5fu were chosen for subsequent experimentationcell clone formation assays were also used to detect in vitro proliferation of colon cancer cells as shown in fig 1c and d the number of colonies formed by the colon cancer cells treated with rg3 and fu was significantly lower than that of rg3 or fu alone these findings indicated that combined treatment of rg3 and 5fu enhanced the inhibition of colon cancer cell proliferation in vitrocombined treatment of rg3 and fu enhances the inhibition of cell migration and invasion the ability of tumor cells to invade and migrate is the key to tumor progression in the present study we compared the effects of different treatment conditions on the invasion and migration of colon cancer cells it was demonstrated that the invasion and migration ability of the colon cancer cells treated with rg3 combined with fu was significantly lower than that of rg3 or 5fu alone fig 2a and b epithelialmesenchymal transition emt is the source of tumor cell ability to acquire higher invasion and migration capacity thus we determined the levels of three emtrelated proteins ncadherin ecadherin and mmp9 and found that the expression of ncadherin and mmp9 protein in the rg35fu group was significantly lower than that of rg3 or fu alone group but 0concology reports figure effect of the combined treatment of rg3 and 5fu on the apoptosis of colon cancer cells in vitro a the percentage of apoptotic sw620 and lovo cells in the different groups b apoptosisrelated proteins [cleaved clcaspase clcaspase and apaf1] were assessed by western blot analysis in sw620 and lovo cells three independent repetitions for each experiment were carried out p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 apaf1 apoptotic protease activating factor the expression of ecadherin protein was significantly higher fig 2ccombined treatment of rg3 and fu promotes apoptosis of colon cancer cells fist we found that the apoptosis of the colon cancer cells treated with rg3 combined with 5fu was significantly higher than that of rg3 or fu alone fig 3a the levels of apoptosisrelated proteins in the sw620 and lovo cells were assessed by western blot analysis as shown in fig 3b expression levels of apaf1 cleaved clcaspase and clcaspase protein in colon cancer cells sw620 and lovo treated with rg3 were significantly increased and the expression of these apoptosisrelated protein in colon cancer cells following fu treatment was significantly higher than that treated with rg3 more importantly expression levels of these apoptosisrelated proteins in colon cancer cells treated with the combination of rg2 and 5fu were significantly higher than levels treated with rg3 or 5fu alonewe analyzed the cell cycle distribution of the colon cancer cells after treatment with the different agents as shown in fig 4a the percentages of colon cancer cells in the g0g1 phase treated with the rg3 and 5fu combination were significantly higher than the percentages following rg3 or 5fu alone similarly we also detected cell cycleassociated protein by western blot analysis as shown in fig 4b the expression levels of cyclin d1 cdk2 and cdk4 protein in colon cancer cells which were treated with the rg3 and 5fu combination were significantly lower than levels following treatment with rg3 or 5fu alonecombined treatment of rg3 and fu suppresses pi3kakt signaling in colon cancer cells the pi3kakt signaling 0chong effects of ginsenoside rg3 on colon cancerfigure effect of the combined treatment of rg3 and 5fu on cell cycle progression of colon cancer cells in vitro a flow cytometry was used to analysis the cell cycle in colon cancer cells after treatment with rg3 mmoll or 5fu µmoll or the combination b cell cycleassociated protein cyclin d1 cdk2 and cdk4 were assessed by western blot analysis three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 cdk cyclindependent kinase pathway is a signaling pathway involved in cancer cell proliferation invasion and migration and its abnormal activation can confer high proliferation invasion and migration ability of cancer cells in the present study we found that the expression levels of pp85 p110 ppdk1 and pakt protein in the colon cancer cells which was treated with rg3 and 5fu combination were significantly lower than levels in the cells treated with rg3 or 5fu alone fig these results indicated that the combined treatment of rg3 and 5fu enhanced the inhibition of the pi3kakt signaling pathway in colon cancer cells in vitrocombined treatment of rg3 and fu suppresses tumor growth in nude mice based on the results of in vitro studies we further investigated the effects of the rg3 and 5fu combination on colon cancer cell proliferation and protein expression in nude mice sw620 cells were injected into the armpits of nude mice after weeks of treatment the mice were sacrificed and the weight and volume of tumor tissues were measured it was found that the weight and volume of tumor tissues in the rg35fu group were significantly lower than these parameters in the groups treated with rg3 or 5fu alone fig 6a and bmoreover western blot analysis was used to detect the expression of emtrelated proteins cell cyclerelated proteins and key proteins in the pi3kakt signaling pathway it was found that although the effects of the rg3 and 5fu combination were not as obvious as the in vitro results compared with rg3 of 5fu alone the overall trend in protein expression was consistent fig 6ce these results demonstrated that rg3 0concology reports figure effect of the combined treatment of rg3 and 5fu on pi3kakt signaling in colon cancer cells in vitro a and b western blot analysis was used to detect the expression of key proteins in the pi3kakt signaling pathway after treatment with rg3 mmoll or 5fu µmoll or the combination three independent repetitions were performed for each experiment p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 synergizes the effect of 5fu to inhibit the growth of human colon cancer xenografts in nude micediscussionthe anticancer effect of 5fluorouracil 5fu is exerted mainly by interfering with tumor cell dna replication and it is a commonly used antitumor agent for the treatment of advanced colon cancer however since 5fu displays nonspecific cytotoxicity it also causes damage to normal cells causing irreversible renal dysfunction and severe gastrointestinal reactions these adverse effects limit its clinical application and further improvements in the efficacy of chemotherapy are needed therefore it is urgent to discover a drug that can enhance the chemotherapeutic effects of 5fu and reduce the 5fu toxicity when used in combination with 5fuginsenoside rg3 rg3 is one of the main active ingredients extracted from ginseng research has shown that ginsenoside rg3 has certain inhibitory effects on lung cancer breast and prostate cancer the antitumor mechanism of rg3 was that rg3 reduced the neovascularization probability of tumor recurrence proliferation and metastasis in tumors by inhibiting kdrvegf protein expression and blocking hif1αcox2vegf pathway in the present study we found that the combined treatment of rg3 and 5fu promoted the inhibition of colon cancer cell proliferation in vivo and in vitro tumor growth development and metastasis are closely related to cell proliferation the previous study found that rg3 inhibits the proliferation of tumor cells such as rg3induced egfrmapk pathway deactivation was found to inhibit melanoma cell proliferation by decreasing fut4ley expression rg3 was found to inhibit the proliferation of multiple myeloma cells by inducing the secretion of igf1 promoting tumor cell apoptosis is also a method of inhibiting tumor cell proliferation in the present study we found that the combined treatment of rg3 and fu significantly enhanced the apoptosis of colon cancer cells by activating the apaf1caspase 9caspase pathway in the mitochondrial pathway of apoptosis apoptosisrelated signals release cytochrome c by stimulating the mitochondrial outer membrane cytochrome c enters the cytoplasm which activates caspase9 by binding with apaf1 activation of caspase9 further activates caspase3 while the activated caspase3 can activate caspase leading to apoptosis in addition we also found that the rg3 and 5fu combination enhanced the number of g0g1 phase colon cancer cells and decreased expression of cyclin d1 cdk2 and cdk4 the cell cycle refers to the whole process that the cell undergoes from the completion of one division to the end of the next division and 0chong effects of ginsenoside rg3 on colon cancerfigure effects of the combined treatment of rg3 and 5fu on tumor growth and protein expression of colon cancer cells in vivo after weeks of treatment the mice were sacrificed tumor tissues were excised and the weight a and volume b of tumor tissues were measured ce total protein was extracted from the colon cancer tumor tissues and the expression of proteins was detected by western blot analysis five nude mice in each group and at least tumor tissues were used to evaluate protein expression p005 p001 and p0001 compared with the rg35fu group fu fluorouracil rg3 ginsenoside rg3 the regulation of the cell cycle is mainly achieved by the retention of the g1 phase when a cell is in the g1 phase there is an important node regulating the cell cycle the r point when the cell cycle is before the r point the cell needs the external growth factor to achieve the normal operation of the cell cycle after the cell cycle crosses the r point the cell cycle becomes a process that is controlled autonomously by the cell and no longer depends on the presence of external cytokines cyclin d1 is a g1sspecific cyclin and its main function is to promote the cell cycle from g1 to s by binding and activating the cyclindependent kinase cdk24 a unique cyclindependent kinase of g1 so as to promote cell proliferation invasion and migration of tumor cells are the most important features of malignant tumors and the important causes of death in patients with malignant tumors ncadherin ecadherin and mmp9 are three proteins that play important roles in cell epithelialmesenchymal transition emt whereas emt provides cells the ability to transfer and invade promoting tumor cell emt can inhibit the expression of intercellular junction protein resulting in decreased intercellular connectivity which is beneficial to the invasion and migration of tumor cells to surrounding healthy tissues previous studies have found that rg3 not only inhibits metastasis and invasion of lung cancer cells by inhibiting emt induced by transforming factor 1 but also inhibited the metastasis of prostate pc3m cells by downregulating the expression of aqp1 by downregulating mmp rg3 affected the metastasis and invasion ability of melanoma cells the present study demonstrated that the combined treatment of rg3 and fu significantly suppressed the invasion and migration ability of human colon cancer cell in vitro by altering emtrelated proteinfurthermore we also found that rg3 and 5fu combination inhibited the conduction of the pi3kakt signaling pathway in vivo and in vitro many studies have shown that the occurrence and development of tumors are the result of multifactor multigene and multipathway processes and the cell signal transduction pathway is crucial in the process of tumor development invasion and metastasis the phosphatidylinositol 3kinaseserinethreonine kinase b pi3kakt signaling pathway plays an important role in the regulation of solid tumors [eg liver cancer breast cancer colon cancer gastric cancer neuroblastoma ] and blood tumors [eg leukemia ] pi3k acts as a bridge molecule for the relationship between extracellular signals and cellular responses under the influence of a series of upstream or bypass signaling molecules it acts on the downstream of the effects of a variety of molecules thus promotes cell migration 0concology reports inhibits cell apoptosis accelerates the process of the cell cycle and promotes cell proliferation many previous studies have shown that traditional chinese medicine or traditional chinese medicine monomers can play an antitumor role by inhibiting the pi3kakt signaling pathway in conclusion rg3 enhances 5fu inhibiting proliferation invasion and migration of colorectal cancer cells and helps 5fu block g1 phase induced apoptosis in more colorectal cells all in all our study found that rg3 enhanced the anticancer effect of 5fu on colon cancer cell via pi3kakt pathwayacknowledgementsnot applicablefundingno funding was receivedavailability of data and materialsthe datasets used during the present study are available from the corresponding author upon reasonable requestauthors' contributionsxc made substantial contributions to the conception and design of the study and critically revised it for important intellectual content sh contributed to the acquisition of the data wc zh yw xm yh and zl analyzed and interpreted the data all authors read and approved the final manuscriptethics approval and consent to participateall animal and cell experiments were approved by the ethics committee of the quanzhou first hospital affiliated to fujian medical university quanzhou fujian chinapatient consent for publicationnot applicablecompeting intereststhe authors state that they have no competing interestsreferences siegel rl ward em and jemal a trends in colorectal cancer incidence rates in the united states by tumor location and stage cancer epidemiol biomarkers prev siegel rl miller kd and jemal a cancer statistics ca cancer j clin chen w zheng r baade pd zhang s zeng h bray f jemal a yu xq and he j cancer statistics in china ca cancer j clin chen w zheng r zhang s zeng h zuo t xia c yang z and he j cancer incidence and mortality in china in an analysis based on urbanization level chin j cancer res li j hou n faried a tsutsumi s and kuwano h inhibition of autophagy augments fluorouracil chemotherapy in human colon cancer in vitro and in vivo model eur j cancer sanoff hk carpenter wr freburger j li l chen k zullig ll goldberg rm schymura mj and schrag d comparison of adverse events during fluorouracil versus fluorouraciloxaliplatin adjuvant chemotherapy for stage iii colon cancer a populationbased analysis cancer cheah ky howarth gs bindon ka kennedy ja and bastian sep low molecular weight procyanidins from grape seeds enhance the impact of fluorouracil chemotherapy on caco human colon cancer cells plos one e98921 gao y xiao x zhang c yu w guo w zhang z li z feng x hao j zhang k melatonin synergizes the chemotherapeutic effect of fluorouracil in colon cancer by suppressing pi3kakt and nfbinos signaling pathways j pineal res doi 101111jpi12380 wang sf wu my cai cz li m and lu jh autophagy modulators from traditional chinese medicine mechanisms and therapeutic potentials for cancer and neurodegenerative diseases j ethnopharmacol ernst e traditional chinese medicine for cancer br j cancer sun hy lee jh han ys yoon ym yun cw kim jh song ys and lee sh pivotal roles of ginsenoside rg3 in tumor apoptosis through regulation of reactive oxygen species anticancer res tang yc zhang y zhou j zhi q wu my gong fr shen m liu l tao m shen b ginsenoside rg3 targets cancer stem cells and tumor angiogenesis to inhibit colorectal cancer progression in vivo int j oncol wang j tian l khan mn zhang l chen q zhao y yan q fu l and liu j ginsenoside rg3 sensitizes hypoxic lung cancer cells to cisplatin via blocking of nfκb mediated epithelialmesenchymal transition and sternness cancer lett joo e ha yw and kim ys abstract lb23 molecular mechanisms of ginsenoside rg3 related to apoptosis in human lung and pancreatic adenocarcinomas cancer res lb23 kim bj nah sy jeon jh so i and kim sj transient receptor potential melastatin channels are involved in ginsenoside rg3induced apoptosis in gastric cancer cells basic clin pharmacol kim sm lee sy cho js son sm choi ss yun yp yoo hs yoon dy oh kw han sb and hong jt combination of ginsenoside rg3 with docetaxel enhances the susceptibility of prostate cancer cells via inhibition of nfkappa b eur j pharmacol yuan hd quan hy zhang y kim sh and chung sh 20sginsenoside rg3induced apoptosis in ht29 colon cancer cells is associated with ampk signaling pathway mol med rep liu tg huang y cui dd huang xb mao sh ji ll song hb and yi c inhibitory effect of ginsenoside rg3 combined with gemcitabine on angiogenesis and growth of lung cancer in mice bmc cancer sun my ye y xiao l duan xy zhang ym and zhang h anticancer effects of ginsenoside rg3 review int j mol med longley db harkin dp and johnston pg fluorouracil mechanisms of action and clinical strategies nat rev cancer hokmabady l raissi h and khanmohammadi a interactions of the fluorouracil anticancer drug with dna pyrimidine bases a detailed computational approach struct chem rateesh s luis sa luis cr hughes b and nicolae m myocardial infarction secondary to fluorouracil not an absolute contraindication to rechallenge int j cardiol e331e333 shan x aziz f tian ll wang xq yan q and liu jw ginsenoside rg3induced egfrmapk pathway deactivation inhibits melanoma cell proliferation by decreasing fut4ley expression int j oncol luo y zhang p zeng hq lou sf and wang dx ginsenoside rg3 induces apoptosis in human multiple myeloma cells via the activation of bcl2associated x protein mol med rep cardone mh roy n stennicke hr salvesen gs franke tf stanbridge e frisch s and reed jc regulation of cell death protease caspase by phosphorylation science 0chong effects of ginsenoside rg3 on colon can
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" national economies are increasingly facing the challenge of having to finance the prevention andtreatment of human diseases and of having to compensate for the resulting loss of economic production physicalinactivity is demonstrably closely related to the risk of developing certain disease group physical inactivity results indirect and indirect burdens that the present study intends to quantify in hungary for the period between and methods based on the data of the hungarian public finances this study determines the direct and indirect costsincurred by hungary due to illnesses and through the par method it quantifies the financial burden of physicalinactivity incurred by the hungarian treasuryresults the total financial burden of illnesses in hungary showed a decreasing tendency from to eventhough the year saw an increase in costs compared to similarly while total public expenditure on illnessesassociated with physical inactivity increased by when compared to the total amount attributable to medicalconditions stemming from physical inactivity still showed a decrease of billion huf in the overall period the biggesteconomic burden is posed by cardiovascular diseases hypertension and type diabetess the increase in the economic burden associated with physical inactivity can be attributed to thecombined effect of two factors changes in total expenditure on specific disease groups which showed an increase inthe period under review and changes in the physical activity levels of the hungarian population which showed animprovement over the period under review initiatives in hungary aimed at encouraging an active lifestyle fromchildhood onwards should be continued since beyond the initial impact that has already been felt to some extent inrecent years these initiatives will come to their full fruition in the coming decadeskeywords physical inactivity economic burden parmethod direct costs indirect burden population attributable risk the fundamental change towards a more sedentary lifestyle has a serious impact on peoples health physical inactivity is one of the most important global issues of thetwentyfirst centuryleading to an increased risk ofchronic diseases such as type diabetes cardiovascular correspondence davidpaaretkptehu1university of pecs faculty of health sciences pecs hungaryfull list of author information is available at the end of the disease certain types of cancer rectal colon breastobesity and osteoporosis these diseases may even become the cause of death the world health anizationwho has also identified these medical conditions as themost burdensome noncommunicable diseases of todaysdeveloped world regular moderate physical activity reduces the risk of the most common of these diseases andcontributes to an increased sense of wellbeing [ ] acinactivity ranks as thecording to the who physical the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0c¡cs bmc public health 20suppl page of fourth most significant mortality factor in the world with million deaths a year worldwide [ ]another study suggests that there is a lower likelihoodof health problems among people engaging in regularphysical activity than among those leading a sedentarylifestyle furthermore there is convincing evidence thatregular physical activity increases life expectancy and reduces the likelihood of developing coronary and cardiovascular problems of suffering a stroke or developingcolon cancer inactive and sedentary lifestyles directly affect metabolism bone mineral composition and magnify the healtheffects of cardiovascular disease furthermore thereis epidemiological evidence to suggest that a sedentarylifestyle increases the risk of cancer obesity metabolicand psychosocial problems according to oecd data the average life expectancyof hungarians at birth in was years which is years below the oecd average actually one of the lowest on the list for men this value is years forwomen years both showing an increasing trend in recent years the hungarian government has made anumber of efforts to bring about significant changes in theinactive lifestyle of the hungarian population these include measures to increase the number of physical education lessons and to improve the conditions in pe lessonsat school also the development and construction of sportsfacilitiesincreased funding for sports associations andeven the use of corporate tax incentives for sporting purposes while improving the conditions alone does notresult in a change in the attitudes of the population towards sport it is certainly a prerequisite procedures that quantify the burden on the hungarianeconomy resulting from physicalinactivity are one ofthe ways of measuring the effectiveness of state intervention [ ] this study aims to contribute to this bodyof research and proposes to analyse a longer timespectrummethodsto analyze the economic burden of physical inactivity weneed to start with the burden of diseases on the nationaleconomy as physical inactivity plays a vital role in the onset of several diseases and leads to various causes of deathat a national level diseases have direct and indirect costsdirect costs of diseases include treatments medications sickpay allowances and associated ancilliary coststhat are directly related to the illness the direct costs inhungary are mainly financed by the national health insurance fund nhif since it is called the national health insurance fund administration nhifa but we must not disregard the cost of sick leave and private costs outside of nhifnhifa financing the latterof which are directly borne by members of societyamong the indirect burdens we include items that constitute a loss to the economy or to society as a result ofthe loss of work caused by a disease there was a significant change in this area during the research periodwhile in and there was a longterm loss ofproduction only in jobs on the skillsshortage list or invery special cases by the number of job vacanciesin hungary reached thousand while by july thisnumber rose to thousand people which is of theworkforce our calculations were based on the following assumptions in and in a labor marketcharacterised by an oversupply of labor a frictional unemployment of months groupbased performance expectations and the market of goods and services beingoversupplied people on average worked days a yearand loss was calculated based on gdp per capita thestudy that inspired our calculations had a similarcalculation but we replaced its assumptions with theabovementioned assumptions and we broadened andtightened formulas and corrected data that had becomefact since then however when calculating the results we had to change the assumptions about the labormarket from the previouslyoutlined conditions as by hungarian labor market had become characterizedwith overdemand therefore we had to increase frictiontime as well monthsanother economic burden is presenteesim which isthe term used for the phenomenon when a sick individual goes to work which results in poorer performanceand thus loss of productionour main goalin this research was to quantify theeconomic burden of diseases for the years and and more specifically the costs to thenational economy directly attributable to physicalinactivity in the market years and during our research we treated as relevant secondarydata eurobarometer and and nhifnhifa data from and []in the course of our resreach we examined the typesof medical conditions related to physical inactivity andtheir possible complications the factual data for whichwas obtained from nhif and nhifawith the help of par method population attributable risk the most commonly used method in international research we were able to obtain quantitativemeasurements that were used uniformly in the analysisof data for all three yearspar ¼ pexp 02 rr¾ ¾ pexp 02 rr°°¾ 02 wherepexp prevalence refers to the section of the populationwhere a given risk is present 0c¡cs bmc public health 20suppl page of rr relative risk describes the risk associated with asedentary lifestylewhen using the index it is necessary to break downthe population into physically active and inactive sections and then by determining the relative risk rate wecan estimate the number and cost of illnesses stemmingfrom a physically inactive lifestyle the physical activity indicators ofthe hungarianpopulation showed fluctuations during the period underreview the situation was the worst in when wesaw of the population as physically inactive in ouropinion the health protection effect does not manifestitself in the case of those who never excercise or only doso times a month by this figure dropped to and at the same time the ratio of those engaging inexercise at least times per week increased threefold inthe following years a more negative trend was observed as the rate of inactive people rose to although this is still significantly better than the basefigure for fig with the help of metaanalysis we calculated the relative risk ratio rr an indicator which is prevalent ininternational literature in order to estimate the futureexpenditure stemming from physical inactivity for all affected disease groups such as cardiovascular diseasestroke hypertension colon cancertype diabetesosteoporosis depression gastrointestinal complicationsobesity high triglyceride diseases and deliberate selfharm []the rr is the proportion of the applicaple diseasesamong people with inactive lifestyles divided by the proportion of the applicable diseases among people with active lifestyles on the basis of the rr values it is possibleto quantify the par indicator by disease group for eachyear table in order to allow the data to be compared over timethe data on the burden of illnesses stemming from physicalinactivity for and was recalculated to prices while the total amount of burden imposedby illnesses was recalculated to prices using thefig the ratio of physical activity and inactivity in hungary in sourcespecial eurobarometer special eurobarometer specialeurobarometer 0c¡cs bmc public health 20suppl page of table the cumulative relative risk rate and par values for theexamined disease types in disease typesheart and coronary diseasespar par rrpar strokehypertensioncolon cancertype diabetesosteoporosisdepressiongastrointestinal complicationsobesityhigh triglyceridesdeliberate selfharmsource katzmarzyk aldoori ewing andersen schuch domestic producer and consumer price index of thehungarian central statistical office hcso the economic burden ofresultsat pricesillnessesamounted to more than billion forints huf in of which the direct burden was billion forintsdirect costs accounted for of the burden of illnessesand the billion huf sickness benefit represented justover of total direct costs indirect burden representeda significantly lower percentage amounting to over billion huf the economic burden imposed by sicknessin was of hungarys gdpby the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden of illnesses that year less than of which amounting to billion huf was forsickness allowance expenditues indirect burdens decreased to billion huf the burden of sicknessamounted to of the gdp in by the economic burden of diseases fell to billion huf at prices direct costs accounted for of the total burden that year and of it weresick allowances amounting to billion forints indirectburdens fell to billion huf the burden of sicknessdecreased to of the gdp in by the economic burden of illnesses increasedcompared to but it was still below the initial figure huf billion and it decreased in comparison with the gdp the share of direct costs dropped significantly to within which the sickness benefitrepresented to the value of billion forints atthe same time indirect burdens increased significantlyto billion huf all in all the burden of sickness decreased to of the gdp in between and the economic burden of diseases fell by billion huf which is a total decrease of corresponding to an average annual decrease of and in the meantime the countrys gdp increasedsignificantly altogether at current prices obviously the decrease is due to a number of reasons butthe effect of the increase in physical activity is an important factor among them table in the years examined in the case of disease groupslinked to physical inactivity the burden of illnesses onthe state budget excluding sickness allowance amounted to billion huf and billion hufrespectively of which the lowest value was in however only a part of these can be directly linked tophysical inactivity as many other risk factors play a rolein the development of these diseases as regards therelative weight of each disease group cardiovascular disease is the biggest burden followed by hypertension atthe same time type diabetes was only ranked the fifthfor costs in the first year but by it became thethird largest item only slightly behind high blood pressure expenditure on stroke obesity and deliberate selfharm were almost negligible compared to other diseasegroups table based on the results it can be stated that in theexpenditures in the state budgetfor the diseasegroups examined drastically decreased by approximately billion huf compared to the initial starting positionof billion huf but by the expenditures hadsurpassed the base total from by more than billion huf compared to only type two diabetesand osteoporosis showed an increase and respectively compared to although the latter is dueto the relatively low total expenditure for all other disease groups the level of expenditure declined in absoluteterms resulting in a significant decrease of billionhuf in total expenditurehowever in the case of the picture is more varied if we examine the relative position of certain diseasegroups compared to type diabetes showed themost significant increase to the tune of more than billion huf the other diseases lag behind in terms ofexpenditure cardiovascular diseases and colon cancerare next with an increase of billion forints inaddition there is an increase in the costs associated withosteoporosis stagnation or decrease was observed forthe other disease groups but this could not compensatefor the increase in the costs of the aforementioned diseases the most significant drop in expenditure is observed in hypertension billion huf and hightriglyceride diseases billion huf table focusing on the direct burden of physical inactivitywe can conclude that of the total expenditure ofthe disease groups is directly attributable to physical 0c¡cs bmc public health 20suppl page of table economic burdens of diseases in hungary in huf million in real terms in direct costs statefinancedeconomic burdens of diseasesin hungary medicationmedical aidsgeneral practitioner servicesdental servicesoutpatient carect mrimedical centers exluding vd clinicsdialysishome careinpatient carehighcost medical procedurespatient transportspa treatmentsgovernmental health careexpendituresick leavedisability rehabilitation treatmentcharged tonhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifanhifnhifnhifanhifnhifain totalprivate costsprivate health care expenditureindirect costsexpenditure associated withsick leavehealth insurance managementand other costsfriction due to sickness leading toloss of productionof which reduced pay due to sickpay and sick leaveof which tax loss for the statefriction due to disability leadingto loss of productionindividualemployernhifaemployer individualstateindividualstatesocietypresenteeism costsin totalemployerinactivity the major part is the cost of cardiovasculardiseases and hypertension and these were closelyfollowed by type diabetes by due to the factthat the total expenditure for stroke obesity and deliberate selfharm was also insignificant compared to otherdisease groups their expenditure related to physical inactivity is insignificant in the case of deliberate selfharm the costs cannot be measured even in the order ofone hundred thousand forints table compared to the decrease for the year ofthe direct costs stemming from physical inactivity is larger in proportion than the decrease of total expenditurethis is true of each disease group and for those twogroups type diabetes and osteoporosis where therewas an actual increase in costs the increase was less forrespectivelyphysical inactivityrelated expenses than for overall expenses the largest drop in monetary terms can be observed in the case of hypertension and cardiovasculardiseases over billion huf but there was a decreaseof and billion hufin hightriglyceriderelated diseases and colon cancer howeverpercentagewise nhif achieved the highest cost reduction for high triglycerides and colon cancer closely followed by a decrease in stroke expenditure and deliberate selfharm although inthe last two categories the low sum total spent alsomakes this decrease appear larger percentagewise thanwould be the case with larger totalscompared to the expenditure related to physicalinactivity in shows a decrease of billion huf 0ctotal amountinactivitytotal amountinactivitytotal amountinactivitycardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotal¡cs bmc public health 20suppl page of table total cost incured by nhifa nhif of those disease groups that are associated with physical inactivity and costs directlyattributtable to physical inactivity itself in terms of prices million hufdisease typesat the level of the individual disease groups the amountsvary the most significant decline in absolute terms is inthe high blood pressure and high triglyceriderelated illness groups however the burden of type diabetes increased significantly and there was an increase in coloncancer and osteoporosis disease groups the direction andextent of the changes are mostly comparable to the totalexpenditure amounts at the overall level of the diseasegroups although the changes in the physical inactivity ratenaturally lead to differences in the specific values this isso much so that the total expenditure amounts increasedat the level of all disease groups by but overall theexpenditure related to physical inactivity shows a decreaseof table discussionwe can clearly conclude similarly to other international researches [ ] that physical activityand forms of recreational exercise have a protectiveeffect eg a preventive effect against certain types ofchronic diseases cardiovascularlocomotor disordersdiabetes and certain types of tumors the decreasein physical inactivity has a positive effect on productivity as fewer people avail themselves of sick leave atable changes in total expenditure as reported by nhifa nhif and changes in expenditure directly stemming from physicalinactivity compared to the base level expenditure in in real terms adjusted to prices million hufdisease typescardiovascular diseasesstrokehypertensioncolon cancertype diabetesosteoporosisdepressiondigestive disordersobesityhigh triglyceridesdeliberate selfharmtotaltotal amount inactivity total amount inactivity 0c¡cs bmc public health 20suppl page of study of economic development over the past centuryhas concluded that the advancement of the populations health status is responsible for about ofeconomic growth []in our comparative study we used four samplingpoints between and to demonstrate the burden of diseases at the level of the national economy forthe various loadcarriers in the period under review theeconomic burden decreased significantly overall from of the gdp to the weight of indirect burden increased however as in the currently demanddominated labormarket it is more difficult to replacelost workforce in the period of analysis the number ofemployees in hungary increased with which increased the amount of sick leave and number of sicknessdays but their gdp contribution was significantly higheralthough associated costs and burdens increased innominal terms they decreased in relation to the gdpa large part of diseases burdens are borne by the stateand society followed by households andemployers the proportions are similar to ding in european countries included hungary although we estimate that the burdens on employers arehigher and the burdens on households are lower inhungarysince the physical activity rate of the hungarianpopulation has been fluctuating but overall there is animproving tendency which is also apparent in the savings potential of the examined expenditures categoriescompared to the gdp the amount of spending dependsheavily apart from the physical inactivity rate on thenumber of employees as well as those people who arenot employed can not have sick leavefor exampleoverall government spending depends on the budget ofthe country which is connected to the overall economicsituationcardiovascular diseases accounts for most of the costof physical inactivity in hungary which coincides withmattli in switzerland however of directinactivityto depression inswitzerland while nhifas depression costs account foronly in hungaryattributablecostsarein hungary a number of measures have recently beentaken in order to integrate physical activity and sportinto peoples daily lives such measures include theintroduction of everyday physical education in schoolsor the extensive development of sports infrastructure however the effects of these measures will have amore pronounced effect in the long run several studieshave shown that high physical activity in childhood isnot yet measurable in terms of economic returns lessfrequent use of health care and a lower cost associatedwith using them as some effects such as the high costof sports injuries high rates of childhood illness haveresearch data confirm the facta negative bearing on the rate of return on investmenthowever a longterm change of attitude and opennessto physical activity at later stages in life are where thesemeasures bear a profit so any effort to support childinterhood sports is rewarding [ ] in additionnationalthatthoseparents who are themselves engaged in sport or currently do so are more likely to prefer sporting activitiesamong their children it is important to draw attentionto the fact even minimal physical activity has a healthimproving effect at any stage of life that is whysport and health policies at all times should promoterecreational activities for all ages not just young peoplewe would like to expand the scope of our current research if we could also examine how the patient numbers varied each year by disease group unfortunatelythe data was not available this would be of particularinterest for the year as the expenditure on illnessesshowed a significant increase in real terms compared to which may be due to the fact that more patientsreceived care and treatment but may also be due an increase of the normative provision per person by the government possibly to provide better quality careif we posit based on the eurobarometer data that thephysical activity rate improved compared to wecould also assume that fewer people were treated for theanalysed medical conditions in which would basically have a downward effect on total expenditure at thesame time however the picture is somewhat shaded bythe fact that even if the attitude of the population towards regular physical activity has changed in the lastfew years it is not certain that the number of illnesseswould decrease significantly in such a short period asthe negative effects of a sedentary lifestyle led for decades would not be easily offset by a few years ofexcerciseladen lifestyle this is especially true forolder age groups that is to say a reduction in the number of patients is not realised yet in patient care at thesame time the use of rapidlydeveloping medical technologies is also increasing the financial burden on thebudget as the higher costs of new technologies makemedical care per patient more expensive on the otherhandit should not be fotten that healing can bemade more effective and can lead to higher returns onhuman capitalthe study examined the development and compositionof direct and indirect burdens of disease in hungary andthe costs of physical inactivity to the state budget theseburdens fell in the examined periode which was associated with an increase of gdphowever there was an increase in the economic burinactivity which can beden associated with physical 0c¡cs bmc public health 20suppl page of attributed to the combined effect of two factors changesin total expenditure on specific disease groups whichshowed an increase in the period under review andchanges in the physical activity levels of the hungarianpopulation which showed an improvement over theperiod under review initiatives in hungary aimed at encouraging an active lifestyle from childhood onwardsshould be continued since beyond the initial impactthat has already been felt to some extent in recent years these initiatives will come to their full fruition in thecoming decadesabbreviationsct computed tomography gdp gross domestic product hcso hungariancentral statistical office huf hungarian forint mri magnetic resonanceimaging nhif national health insurance fund nhifa national healthinsurance fund administration oecd anisation for economic cooperation and development par population attributable risk rr relativerisk vd veneral disease who world health anizationacknowledgementsthe authors acknowledge to the nhifas colleagues for their help incollecting the dataset especially to mr zsolt kiss director general to drmihaly palosi head of department to petra fadgyasfreyler head ofdepartment and to valentina beitl analistthe authors would like to express their special thanks to prof attila fabianformer vice state secretary for his cooperative help during the datacollectionabout this supplementthis has been published as part of bmc public health volume supplement level and determinants of physical activity in the v4countries part the full contents of the supplement are available online athttpsbmcpublichealthbiomedcentralcomssupplementsvolume20supplement1authors contributionspa was the leader of the complete research coordinated the different coauthors work systematized the dataset summarised the literature related tothe relative risk ratios of illnesses calculated the par indices and contributedto the s dp has made calculations of par indices the direct costs ofphysical inactivity in the nhifa budget and contributed to the sfrom its results ms has made calculations of the total burdens direct andindirect of illnesses in hungary and contributed to the s from itsresults mh and psz summarised the related literature to the section ak and tsz have revised the results and contributed to the sall authors read and approved the final manuscriptfundingthe research was carried out and the publication costs funded by thesupport of hrdop36216201700003 cooperative research network ineconomy of sport recreation and health the authors declare that thefunding body does not have any role in the design of the study andcollection analysis and interpretation of data and in writing the manuscriptavailability of data and materialsthe data of the state financed direct costs that support the findings of thisstudy are available from national health insurance fund administration butrestrictions apply to the availability of these data which were used underlicense for the current study and so are not publicly available data arehowever available from the authors upon reasonable request and withpermission of national health insurance fundthe datasets of the private ind indirect costs used and analysed during thecurrent study are available from the corresponding author on reasonablerequestethics approval and consent to participatethe ethical approval was granted for the study by ethics committee ofuniversity of p©cs nr participants were informed about theresearch aim and methods before signing the informed consent form theinvestigation conforms to the principles outlined in the declaration ofhelsinkiconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1university of pecs faculty of health sciences pecs hungary 2university ofphysical education budapest hungary 3corvinus university of budapestcorvinus business school budapest hungaryreceived march accepted march published august referencessebestyen a boncz i molnar a korosi l kovi r kriszbacher i olah apentek m sandor j relationship between surgical intervention type and days mortality of elderly femoral neck fracture in the prsence of differentcomorbidities value health 2009123a66kruk j health and economic costs of physical inactivity asian pac j cancerprev reiner m niermann c jekauc d woll a longterm health benefits ofphysical activitya systematic review of longitudinal studies bmc publichealth pratt m norris j lobelo f roux l wang g the cost of physical inactivitymoving into the 21st century br j sports med who global recommendations on physical activity for health switzerlandgeneva who blair sn cheng y holder js is physical activity or physical fitness moreimportant in defining health benefits med sci sports exerc s379tremblay ms colley rc saunders tj healy gn owen n physiological andhealth implications of a sedentary lifestyle appl physiol nutr metab rishiraj n inactivity a bad habit costing our productive lifestyle int j physmed rehabil oecd health status in edited by development ofecoa ¡cs p h©cz r pa¡r d stocker m a fitts©g m©rt©ke a fizikai inaktivit¡snemzetgazdas¡gi terhei magyarorsz¡gon k¶zgazdas¡gi szemle gabnai z m¼ller a b¡cs z b¡ba ©b the economic burden of physicalinactivity at national level [a fizikai inaktivit¡s nemzetgazdas¡gi terhei]eg©szs©gfejleszt©s health dev acs p stocker m fuge k paar d olah a kovacs a economic and publichealth benefits the result of increased regular physical activity eur j integrmed hcso in hcs o editor edn stadat time series of annual data labour market distribution of job vacancies koll¡nyi z imecs o az eg©szs©gbefektet©s budapest demosmagyarorsz¡g special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan special eurobarometer [httpeceuropaeucommfrontofficepublicopinionindexcfmresultdocdownloaddocumentky82432]accessed jan powell ke population attributable risk of physical inactivity phys actcardiovasc health katzmarzyk pt gledhill n shephard rj the economic burden of physicalinactivity in canada cmaj 0c¡cs bmc public health 20suppl page of aldoori wh giovannucci el rimm eb wing al willett wc use ofacetaminophen and nonsteroidal antiinflammatory drugs a prospectivestudy and the risk of symptomatic diverticular disease in men arch fammed andersen lb schnohr p schroll m hein ho allcause mortality associatedwith physical activity during leisure time work sports and c
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" to improve the postoperative prognosis of patients with lung cancer predicting the recurrence highrisk patients is needed for the efficient application of adjuvant chemotherapy however predicting lung cancerrecurrence after a radical surgery is difficult even with conventional histopathological prognostic factors thereby anovel predictor should be identified as lipid metabolism alterations are known to contribute to cancer progressionwe hypothesized that lung adenocarcinomas with high recurrence risk contain candidate lipid predictors this studyaimed to identify candidate lipid predictors for the recurrence of lung adenocarcinoma after a radical surgerymethods frozen tissue samples of primary lung adenocarcinoma obtained from patients who underwent a radicalsurgery were retrospectively reviewed recurrent and nonrecurrent cases were assigned to recurrent n andnonrecurrent n groups respectively extracted lipids from frozen tissue samples were subjected to liquidchromatographytandem mass spectrometry analysis the average total lipid levels of the nonrecurrent andrecurrent groups were compared candidate predictors were screened by comparing the folding change and pvalue ofttest in each lipid species between the recurrent and nonrecurrent groupsresults the average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp a total of lipid species were increased folding change ¥ p and lipid species were decreasedfolding change p in the recurrent group among these candidates increased sphingomyelin smd351 inthe recurrent group was the most prominent candidate predictor showing high performance of recurrence predictionauc sensitivity specificity accuracy we propose smd351 as a novel candidate predictor for lung adenocarcinoma recurrence our finding cancontribute to precise recurrence prediction and qualified postoperative therapeutic strategy for lung adenocarcinomastrial registration this retrospective study was registered at the umin clinical trial registry umin000039202 on 21stjanuary keywords lung adenocarcinoma prognostic factor recurrence prediction lipid mass spectrometry correspondence kahyohamamedacjp1department of cellular and molecular anatomy hamamatsu universityschool of medicine handayama higashi ward hamamatsu shizuoka japan5international mass imaging center hamamatsu university school ofmedicine handayama higashi ward hamamatsu shizuoka japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctakanashi bmc cancer page of lung cancer is one of the leading causes of cancerrelatedmortality worldwide radical resection is the standardtreatment for stage iii nonsmall cell lung cancer nsclc however the postoperative survival rate remainsunsatisfactory despite complete resection among patientswith nsclc who received complete resection experience local or distant disease recurrence thereforeadjuvant chemotherapy should be administered to improve survival after a radical surgery adjuvant chemotherapy has been shown to reduce therisk of death due to lung cancer recurrence [] nonetheless not all patients who underwent radical surgerybenefit from adjuvant chemotherapy because some ofthem are already successfully healed without adjuvantchemotherapy therefore patients highly at risk for recurrence who are likely to benefit from adjuvant chemotherapy should be identified for the efficient applicationof adjuvant chemotherapyadenocarcinoma is the most common histologicaltype of nsclc accounting approximately of allnsclc cases in lung adenocarcinomas severalprognostic factors obtained by histopathological evaluations of surgical specimens have been reported to datesuch as lymph node metastasis pleural invasion lymphatic vessel invasion [ ] blood vessel invasion[ ] adenocarcinoma subtype of micropapillary pattern and spread through air space stas [ ]however predicting lung cancer recurrence after radicalsurgery is still difficult because data on the direct relationship between conventional prognostic factors and recurrence are limited furthermore subjective judgmentsof conventional prognostic factors are considered to hinder accurate recurrence prediction and its retrospectivevalidation accordingly novel recurrence predictors withhigh objectivity are strongly expectedexampleprevious studies demonstrated that lipid metabolismalterations in cancer contribute to cancer cell proliferation and invasion [ ] and some lipids have beensuggested as prognostic factors in several cancer typesforthe number of phosphatidylcholinepc321 in recurrent cases of primary triplenegativebreast cancer tnbc is higher than in that of nonrecurrent cases and thereby pc is suggested as acandidate predictor for tnbc recurrence oleicacid attenuation is correlated with shorter progressionfree period in clear cell renal carcinoma with regard to lung cancer although nsclc is reportedly characterized by drastic changes in phospholipid profiles ascompared to the normal lung tissue and contains different lipid profiles according to the histologic subtypes no lipidomic approach to investigate the prognosticfactor for nsclc has been used based on these previous studies we hypothesized that lung adenocarcinomaswith high recurrence risk have different lipidomes fromthat of lung adenocarcinomas with low recurrence riskand specific lipids that can be considered as candidatesas novel predictive factors for recurrencein this study lipid species that can be considered as potential predictors for lung adenocarcinoma recurrence aftera radical surgery were identified by comparing lipidomes ofprimary lung adenocarcinomas between recurrent andnonrecurrent cases using liquid chromatographytandemmass spectrometry lcmsmsmethodspatients and tissue samplesretrospective frozen tissue samples of primary lungadenocarcinoma obtained from patients who underwentradical surgery from january to december athamamatsu university hospital were examined radicalsurgery was defined as complete resection performedwith lobectomy or pneumonectomy accompanied by systematic lymph node dissection at stage i or ii and ascomplete resection achieved by segmentectomy orwedge resection with or without lymph node samplingat stage i tissue samples of primary tumors were collected immediately after the resection and stored at °c after a rapid freezing in liquid nitrogen histopathological diagnosis was performed by experienced pathologists according to the world health anizationcriteria pathological staging was identified based on the8th edition ofthe tnm classification for lung andpleuraltumors patients were followedup withcomputed tomography ct of the body trunk and biochemicalantigencea every months during the first years thenevery months until more than years after the surgerywhen cea was elevated ¥ ngml without any ctfindings of recurrence head magnetic resonance imaging and systemic positron emission tomography wereperformed for the detection of brain metastasis or bonemetastasiscarcinoembryonicexamination ofin patient selection clinical records of these tissuesamples were retrospectively reviewed patients withpathological stage i or ii indicated for radical surgeryand with major histological subtypes of invasive adenocarcinoma lepidic papillary acinar or solid predominant were analyzed patients who received inductionchemotherapy or radiotherapy and those with other subtypes of adenocarcinoma were excludedthencases withoutand with recurrence wereassigned to nonrecurrent and recurrent groups respectively recurrence was defined as radiological imagingbased findings of distant or locoregional recurrencewithin years whereas no recurrence was defined as nofindings of distant or locoregional recurrence in ¥ yearsafter the radical surgery in the nonrecurrent group 0ctakanashi bmc cancer page of cases with followup period of years were excludedin the recurrent group cases with recurrence in theform of pleural dissemination were excluded assumingthe possible attribution with insufficient surgical marginfinally cases for recurrent and for nonrecurrentgroups were subjected for analysishistological evaluationparaffinembedded tissue blocks were sectioned at μmthick sections stained by hematoxylineosin he wereexamined for adenocarcinoma subtypesizelymph node metastasis and stas d2 stain wasused to evaluate lymphatic vessel invasion and elasticavan gienson stain to evaluate blood vessel invasion allhistologicalsections were reviewed by experiencedpathologiststumorchemicalsmethanol chloroform glacial acetate and ultrapurewater were purchased from wako pure chemical industries osaka japan the 12dilauroylsnglycero3pcavanti polar lipids alabaster al pc 120_120 wasused to calibrate standard lipid levelslipid extraction from the cancer tissueeach weight of the frozen tissue samples was measuredusing sartorius analytical lab balance cpa224s sartorius ag göttingen germany additional file supplemental table after the weight measurement modifiedblighdyer methods were performed for lipid extractiontissue samples were transferred into glass tubes and ml of methanol ml of chloroform and mlof m glacial acetate were subsequently addedthen mmol of pc 120_120 per mg of sampletissue was added and subsequently followed by 10minextraction at room temperature after the extraction ml of chloroform was added and vortexed sequentially ml of m glacial acetate was added andvortexed extracted samples were subjected to centrifugation at rpm for min extracted anic layerswere transferred into new glass tubes and were evaporated until completely dried using mivac duo lv genevacextracted lipid wasdissolved with μl of methanol and μl of the dissolved lipids were diluted again with methanol proportional to the weight of the original tissue samples so thatthe concentration of pc 120_120internal controlwill be as similar as possible among casesipswich england thelipid analysis by liquid chromatographytandem massspectrometry lcmsmsextracted lipids from collected frozen tissue sampleswere analyzed using q exactive¢ hybrid quadrupoleorbitrap¢ massanequipped withspectrometerelectrospray ionization source and connected to an ultimate system thermo scientific μl of the extracted lipid samples were injected and separated onacculaim c18 column mm à mm μmthermo scientific components of mobile phase awere as follows wateracetonitrilemethanol vvv mm ammonium formate and formic acidthe components of mobile phase b were as followsacetonitrileisopropanol vv mm ammonium formate and formic acid for elution the flow ratewas set at μlmin a set of linear gradient startingat solvent b was used and linearly increased to b in min maintained at b until minthen decreased linearly to b from min to min and finished with b for the last min theoverall run time was min ms instrument conditionswere as follows sheath gas flow rate auxiliary flowrate sweep gas flow rate capillary temperature °c slens rf level probe heater temperature °c and spray voltage of kv in positive mode and kv in negative mode fullms mode conditions forquantification were as follows ms scan range resolution agc target à and maximum injection time was ms for identification top datadependent ms2 method with a resolution of was used the agc target was à and themaximum injection time was ms stepped normalizedcollision energies of and for the positivemode and and for the negative mode wereapplied spectral data were acquired in the mz range of mz using an xcalibur v30 software thermoscientifictolerance ppmlipid identification and quantificationlipidsearch¢ software version mitsui knowledgeindustry tokyo japan was used to identify and quantify lipid species parameter settings for identificationwere followings database hcd retention time min search type product_qex precursor tolerance ppm and productidentificationquality filters of a b and c were used quantificationwas performed at mz tolerance of ± with retentiontime range from min to min alignment of theidentified lipid species among cases was performedwith retention time tolerance of molecules that areannotated as redundant lipid names with different calculated mz and retention times were regarded as independentisomersinadditional file duplicationannotatedasdata processingtrend analysis between the nonrecurrent and recurrentgroups was performed by comparing the average totallipid level between the two groups and principal 0ctakanashi bmc cancer page of component analysis pca intensities of lipids recordedin the xcalibur v30 software and monoisotopic peakarea values of lipid species identified by lipidsearch¢software were normalized by dividing with the areavalues of internal control pc 120_120 the total lipidlevel of each case was defined as an accumulation ofnormalized intensities of lipids normalized area valueswere subjected for pcafor respective lipid species pvalues were calculatedusing the student ttest to compare area values betweenthe two groups to screen candidate lipids for recurrence prediction lipidomes were compared between thenonrecurrent and recurrent groups by describing volcano plots with log10 pvalue for vertical axis andlog2 folding change for horizontal axis the foldingchange for a lipid was defined as an average area valueof the recurrent group divided by that of the nonrecurrent group significance was determined at pvaluesof folding change of ¥ or statistical analysisdemographic information and associations with clinicalcharacteristics were evaluated using the fisher exact testcategorical variables or the mannwhitney utest forcontinuous variables the student ttest was used tocompare the average totallipid amounts of the nonrecurrent and recurrent groups and to describe volcanoplots recurrentfree survival rfs was determined asthe time from operation until the first disease recurrenceor death survival curve was described using thekaplanmeier method the optimal cutoff values todiscriminate the two groups were determined using thereceiver operating characteristic roc curve analysisthe area under the roc curves aucs were calculatedto validate the discrimination abilities of candidate lipidsspearmans rank correlation analysis was used to validatethe correlation among candidate lipid predictors allstatistical analyses except for the ttest were performedusing r the r foundation for statistical computingvienna austria version the student ttest wasperformed with ttest of excel¢ microsoft redmond usa pvalues of were considered assignificantresultsclinicopathological characteristics of patient cohortclinicopathological characteristics of patients are shownin table in this study cohort tissue samples from nonrecurrent and recurrent cases were analyzedamong the characteristics of these two groups differences in pathological stage p lymph node metastasis p and blood vessel invasion p were statistically significant the and 2year rfs rateof the recurrent group was and with median rfstime of range monthsrespectivelyfile supplemental fig the medianadditionalfollowup time ofthe nonrecurrent and recurrentgroups was range and range months respectivelytrend analysis between the nonrecurrent and recurrentgroupsthe frozen tissue samples were subjected to lcmsms and the total lipid level of cases was calculated byaccumulating normalized intensities of lipids notablythe average total lipid level of the recurrent group was times higher than that of the nonrecurrent groupp fig a total of lipid species wereidentified and quantified by analyzing the mass spectraldata using a lipidsearch¢ software the full list of identified lipid species is presented as additional file which were also subjected to pca the pca plot didnot show clear separation between the recurrent andnonrecurrent groups however the recurrent group exhibited partial separations between the first three principal components additional file supplemental fig these results suggested differences of lipidome betweenthe recurrent and nonrecurrent groups which urged usto screen lipids to distinguish the two groupsscreening of candidate lipids for recurrence predictionto screen lipids with different levels between the twogroups volcano plots of the identified lipids were described first and lipidomes between the nonrecurrentand recurrent groups were compared fig the volcano plotidentified lipid species with relativeamounts significantly different between the two groupsfolding change ¥ or pvalues thenumber of lipids that increased and decreased in the recurrent group was and respectively these increased or decreased lipid species consisted of varioushead groups additional file increased lipid speciesshown in red decreased lipid species shown in greenthen based on prominent distributions of the volcanoplot we narrowed the candidate lipids increased inthe recurrent group to the following molecules fig biotinylbluephosphoethanolamineceramidecerd420 sphingomyelin smd351 cerd180_240pc monoether phosphatidylcholine mepc346echolesterol ester che241 mepc 408e and che20 as for the lipids that decreased in the recurrent groupthe following four molecules were annotated fig bluearrows pointing to green plots monohexosylceramidehex1cert421 otriglyceride tg150_140_140pc 182_182 and lysophosphatidylcholine lpc120biotinylpe303arrowsplotspointingtoredthe relative amounts of these lipid species were evaluated with their distributions by comparing the two 0ctakanashi bmc cancer page of table clinicopathological characteristics of the nonrecurrent and recurrent groupscharacteristicsmedian age rangenonrecurrent n gender malefemalesmoking history pathological stage iiimedian tumor size mm rangeadenocarcinoma subtypelepidicpapillaryacinarsolidlymph node metastasis pleural invasion lymphatic vessel invasion blood vessel invasion micropapillary component spread through air space driver gene mutationegfr alk surgical procedurelobectomywedge resectionadjuvant chemotherapyindication stage ia3iibreceivedrecurrent stylelocoregionaldistant recurrent n pvalueabbreviations alk anaplastic lymphoma kinase egfr epithelial growth factor receptroups fig 3a and b in all tested lipids distributionsbetween the two groups were well separated enough toestablish the cutoff values whereas only few markedoutliers were foundwe next calculated the cutoff values and auc of these lipids to evaluate their discrimination ability for diseaserecurrence and the following final candidates with topthree auc were selected smd351 cerd420 and tg 150_140_140 table respective lipidspecies can be found in additional file with the followingidentical numbers smd351 cerd420 andtg 150_140_140 these three final lipid candidates were annotated as the following ions [smd351 h] [cerd420 hcoo] and [tg 150_140_140 nh4] in the lipidsearch¢ software additional file msms for [smd351 h] [cerd420 hcoo]and [tg 150_140_140 nh4] demonstrated production peaks corresponding to phosphocholine severalfragments compatible with fragmentation of cerd42 with concomitant oxidation reaction two fragmentsproduced by neutralfatty acid fa140 orfa respectivelyadditional file supplemental fig consequentlythe annotations ofthe final candidates by lipidsearch¢ software were consistent with the results ofmsmsfrom tg 150_140_140loss ofamong these three candidate predictors smd351was found to be positively correlated with cerd420spearmans rank correlation coefficient[rs] p tg 150_140_140 was inversely correlated with smd351 rs p and tg150_140_140 was weakly inversely correlated withcerd420 rs p additional file supplemental fig 0ctakanashi bmc cancer page of conventionalpathologicalvalidation of recurrence prediction ability among thefinal lipid candidatestable shows the sensitivity specificity and accuracyof the final candidate lipid predictors compared withfactorstheprognosticlymph node metastasis and blood vesselinvasionwhich were identified as significant recurrent factorsin this cohort sensitivity of all three candidate lipidpredictors is superior to that of lymph node metastasis patients with lymph node metastasis all of themwere hilar or lobar lymph node metastasis corresponded to those in stage ii among the recurrentgroup in this study cohort half of the study population had stage i whereas the other half had stage iias lymph node metastasis can be detected amongstage ii cases the sensitivity of lymph node metastasiswas consequently lower than those of three candidatelipid predictors which detected both stage i and stageii hencethese three predictors were superior tolymph node metastasis for patient screening whencomparing the candidate lipid predictors and bloodvesselshowed predictionto those of blood vesselabilities higher or equalinvasion only smd351fig comparison of total lipid levels between the recurrent andnonrecurrent groups the average total lipid level of the recurrentgroup was times higher than that of the nonrecurrentgroup p fig volcano plots of identified lipid species each plot represents a lipid species to be identified the relative amount of lipid speciesred plots were increased fc ¥ right side of in the horizontal axis pvalue in vertical axis and that of lipid species greenplots were decreased fc left side of in the horizontal axis pvalue in vertical axis in the recurrent group nineincreased lipids showing prominent distributions and all decreased lipid species were annotated for candidate predictors blue arrowsabbreviations cer ceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepcmonoether phosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine sm sphingomyelin tg triglyceride 0ctakanashi bmc cancer page of fig comparisons of relative amount distributions between the nonrecurrent and recurrent groups are shown for increased a and decreasedb lipid species in the recurrent group boxplots show the upper percentile upper quartile median lower quartile and lower percentilemaximum and minimum values are shown in dots pvalues for significance and fcs are presented for each lipid species abbreviations cerceramide che cholesterol ester fc folding change hex1cer monohexosylceramide lpc lysophosphatidylcholine mepc monoetherphosphatidylcholine pc phosphatidylcholine pe phosphoethanolamine tg triglycerideinvasion in allvalidation points therefore wepropose smd351 as the most hopeful candidate forrecurrence predictiondiscussionin this study candidate lipid predictors for lung adenocarcinoma recurrence after a radical surgery were retrospectively screened and smd351 was found as themost prominent predictor showing that the predictionability was superior to that of conventional pathologicalprognostic factors in this small cohortthe average total lipid level was significantly high inthe recurrent group in this study furthermore thenumber ofincreased lipid species was considerablyhigher than that of decreased lipid species in the recurrent group these results were consistent with that of 0ctakanashi bmc cancer page of table auc rank of candidate lipid predictors determined byroc curverankcutoff valuespeciessmd351cerd420tg150_140_140cerd180_240pc182_182che241pc412biotinylpe303lpc120hex1cert421 omepc408eche201mepc346eauc ci lipids with top three auc were selected as final candidate predictorsboldfaced notationsabbreviations auc area under the roc curve ci confidential interval rocreceiver operating characteristicprevious studies that showed an accelerated lipid synthesis in cancer cells contributing to tumor phenotypes such as cellular membrane building stimulationof signaling pathways for growth and proliferation orsurvival under hypoxic conditions by supporting glycolysis [ ] increased total lipid level in the recurrent group may be biologically plausible because theaggressiveness may be supported by accelerated lipidsynthesisthe number of smd351 and cerd420 two of finalcandidate predictors were increased in the recurrentgroup sm and cer are major bioactive components oflipid rafts on the cellular membrane sm is synthesized from cer by sm synthase sms which transfersthe phosphocholine head group from phosphatidylcholine to cer and results in concomitantly producingtable comparison of sensitivity specificity and accuracyamong the three final candidate predictors and conventionalhistopathological prognostic factorspredictors for recurrencecandidate lipid predictorsspecificitysensitivityaccuracysmd351cerd420tg150_140_140pathological prognostic factorslymph node metastasisblood vessel invasionsmd351 showed the most excellent prediction abilityabbreviations cer ceramide sm sphingomyelin tg triglyceridediacylglycerol dag sm reconversion to cer is catalyzed by sphingomyelinase smase increased smabundance and sms activity have been reported to playa critical role in cell proliferation and survival in severalcancer types [] with regard to lung cancer metabolic changes in sphingolipids are suggested to correlatewith chemoresistance phenotype and the total smlevel in cancer tissues is reportedly lower than that ofthe normallung tissue in patients with nsclc this is speculated in the report that decreased sm abundance in lung cancer tissues may be attributable to highconsumption of serine precursor by highly proliferatingcancer cells cer accumulation in the lungs has beensuggested to participate in both cell apoptosis andtumorigenesis under cigarette smokeinduced oxidativestress taking together these knowledge and significant positive correlation between smd351 h andcerd420 in this study increased synthesis flow of certoward sm in the recurrent group was suggested actually significant increase on the total sm p leveland increased tendency on total cer p anddag p levels in the recurrent group were observed in this study cohort additional file supplemental fig this result supports the suggestion ofstrong synthesis flow of cer toward sm the sm andcer levels were not compared between the tumor tissuesand normal lung tissues in this study because normallung tissue samples were lacking nonetheless increasedsmd351 and cerd420 in the recurrent group in thisstudy is consistent with previous studies [ ]based on the following explanation among lung adenocarcinomas with high sm and cer consumption casesthat can maintain increased sm and cer synthesis havehighly aggressive phenotypes resulting in recurrencedecreased tg 150_140_140 in the recurrent groupwas also included in the final candidate predictors although tg abundance in the lung cancer tissue has not yetbeen explored to date tg level in colon cancer is reportedto be lower as the disease progresses suggesting that energysupply for colon cancer with higher degree of malignancymay depend on tg hydrolysis inconsistent with theprevious study the total tg level in this study revealedno significant difference between the nonrecurrent and recurrent groups p possible explanation for decreased tg 150_140_140 in the recurrent group is thataggressive recurrent lung adenocarcinoma that may preferably consume specific tg species for energy supplythe difficulty of predicting lung cancer recurrenceusing histopathological prognostic factors may be partlyattributed to subjective judgement in addition althoughthe degree of histopathological prognostic factors widelyvaries their judgements have been performed qualitatively [ ] thereby these methods may hinder accurateretrospectiverecurrence prediction and its 0ctakanashi bmc cancer page of between representativerecurrentimages of papillarytype adenocarcinomavalidation conversely excellent prediction ability ofsmd351 that is superior to histopathological factorswas considered for its high objectivity and quantitativevalues actuallyit was difficult to predict recurrentprognosis objectively from the conventional histopathologicalthemost popular tissue subtype with no significant differenceand nonrecurrent cases whereas the mass spectrum intensitiesof [smd351 h] were markedly higher in the recurrent case to help recurrence prediction additional file supplemental fig furthermore as high smd351level was detected in all recurrent cases including stagei and stage ii cases with high specificity and accuracysmd351 was considered to be widely applicable for recurrence prediction in postoperative patients whounderwent radical surgeryseveral limitations in this study should be acknowledged first this retrospective study is performed on asmall sample size due to difficulty of obtaining frozensurgical specimens with clinical information that meetour inclusion criteria thereby verifying the reproducibility of using other validation cohorts was difficult thusidentified lipid predictors did not exceed above the candidate levels and further large cohort studies should beconducted to validate candidate predictors identified inthis study as rigid predictors for lung adenocarcinomarecurrencebecause a large number of candidate lipid species species relative to the small number of samplesize cases were screened for candidate predictorsone candidate that shows nearperfect discriminationability can be bound to be identified third adjacentnormal lung tissue samples were lacking hence the difference between the abundance of the identified candidate lipid predictors in the normal lung tissue of therecurrent group and that of the nonrecurrent groupwas not able to be compared fourth because the nonrecurrent group in this study included five cases that received adjuvant chemotherapy the nonrecurrent groupmay possibly include the recurrence highrisk casesamong them recurrence might be prevented by adjuvantchemotherapy moreover the nonrecurrent group inthis study included two cases with recurrence predictionpositive for smd351 additional file supplementalfig among the two cases one patient received adjuvant chemotherapy and the other did not the formercase may be considered as highly at risk for recurrencewhich was prevented by adjuvant chemotherapy thelatter may be an exceptional case that cannot be ruledout by smd351 fifth because lcmsms is not auniversal examination in the clinical field examining alarge number of surgical specimens for recurrence prediction using lcmsms is difficult to utilize thefindings of this study in a clinical field lipid predictorsshould be replaced with other molecules that can be examined by universal methods such as immunohistochemistry of sms or smase involved in the smmetabolism additionallyin thisstudy included histopathological type of adenocarcinomaonly as a topic for future study squamous cell carcinoma a major histological subtype behind adenocarcinomarecurrent predictorsshould be explored forthrough the lipidomic approachthe sample cohortswe propose that smd351 is a hopeful candidate predictor for lung adenocarcinoma recurrence after a radical surgery our findings provide novel insights on themechanisms oflung adenocarcinoma recurrence andcan contribute to the development of precise recurrenceprediction and qualified postoperative therapeutic strategy for lung adenocarcinomasupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020073061additional file supplemental table weights of the frozen tissuesamples each weight of the frozen t
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"ChIP assays were performed with anti-acetyl-H3 (panel b) and anti-Sp1 antibodies (panel c). DNA was extracted for PCR with miR-182 and p21 primers. Data were quantified after three independent experiments (panel d). (F) A549 cells were harvested for DAPA with a biotin-conjugated p21 and miR-182 promoter probes and samples were analyzed by Western blotting using anti-Sp1 antibodies (panel a). Data were quantified after three independent experiments (panel b). (G) Plasmids GFP or GFP-Sp1 were co-transfected with pGL2 pGL2-miR-182 WT or mutation plasmids into H1299 cells for 24 h and then cells were harvested for luciferase activity assays. Data are representative of three independent experiments each of which was performed in triplicate and presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01). Because Sp1 is highly expressed in lung cancer we studied the expression of Sp1 and miR-182 in various lung cancer cell lines and patient samples (). Compared with normal human lung cells (BEAS-2B) lung cancer cell lines expressed higher levels of miR-182 (A). We also assessed the correlation between the miR-182 and Sp1 expression patterns. Sp1 levels in clinical lung tissue samples were highly elevated in the tumorous sections of the lung accompanied by increased expression of miR-182 (B). To confirm this result Sp1 and miR-182 levels were measured in 32 lung cancer patients. Sp1 and miR-182 were upregulated by more than 1.3-fold in 59.4% of the lung adenocarcinoma specimens when compared to expression in normal tissue (C). These results indicate that Sp1 expression positively correlates with miR-182 expression (D). The miR-182 level correlates to Sp1 level Total RNA and cell lysates were prepared from indicated cell lines (A) or from clinical lung tissues of lung cancer patients (B). The miR-182 level was determined by stem-loop RT-PCR and Sp1 level was studied by Western blotting with anti-Sp1 antibodies. U6 and tubulin served as the internal control. (C) Total RNA and cell lysates were prepared from 32 paired normal lung tissues and lung adenocarcinoma samples. The miR-182 level was studied by stem-loop RT-PCR and Sp1 levels were studied by RT-PCR. (D) The relationship between Sp1 and the miR-182 level in the 32 lung cancer samples was statistically analyzed using Fisher's exact test. miR-182 increases lung tumor growth The data shown in indicated that Sp1 regulated miR-182 expression during lung tumorigenesis. To identify the specific gene targets of miR-182 we searched public miRNA target prediction databases (miRDB miRWalk and TargetScanHuman) for candidate target genes. By combining the data from these three databases we identified 161 genes potentially regulated by miR-182 (Supplementary Figure S1A). Moreover pathway analysis using Ingenuity software indicated that the cellular growth and proliferation pathway had the highest score when the association of these 161 genes with biological pathways was examined. This suggests that miR-182 may play a functional role in cancer-associated processes (Supplementary Figure S1B). Indeed when miR-182 was knocked down with miRZip-182 shRNA the percentage of cells in G2/M and sub-G1 phases increased suggesting that miR-182 positively regulated cell cycle progression in the lung cancer cells (A). To further elucidate miR-182's effect on the cell cycle cells were synchronized at prometaphase using nocodazole treatment. After removing nocodazole more miRZip-182 than miRZip cells remained in G2/M phase providing further evidence that miR-182 positively regulates cell cycle progression (B). Consistently knockdown of miR-182 expression inhibited cell growth (C). miR-182 increases cancer cell proliferation (A) The miRZip and miRZip-182 stably expressed H1299 cells were fixed with 70% ethanol and stained with propidium iodide for cell cycle analysis by FACS. (B) Mitotic cells were released into growth by removing nocodazole then fixed at indicated time points for cell cycle progression assay by FACS. (C) The growth rates of miRZip and miRZip-182 stably expressed H1299 cells were calculated by cell counting within 5 days. Data are representative of six independent experiments and presented as the mean ± SEM. (D) Bioluminescent imaging was performed on 10 severe combined immunodeficient (SCID) mice implanted with miRZip and miRZip-182 stable expression H1299 cells (106 cells/mouse) at day 14 (panel a) then image signal was analyzed using Living Image software and presented as total flux measurements in photons/second (panel b). (E) Tumors from SCID mice implanted with miRZip and miRZip-182 stable expression H1299 cells for 4 weeks are shown (panel a) and tumor weights were analyzed (panel b). Data are representative of ten independent experiments and are presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01; *** p<0.001). To confirm the effect of miR-182 on tumor formation cells stably expressing with miRZip or miRZip-182 were implanted into SCID mice and tumor growth was monitored in vivo (D). The miRZip lentivector contains a copGFP gene and the GFP signal in miRZip-182-expressing cells was lower than that in miRZip control cells (D). Furthermore tumor volume and tumor weight were also lower in miRZip-182-implanted mice than in miRZip-implanted mice (N = 10 per group) (E). These results suggest that miR-182 overexpression facilitates lung tumor growth in vivo. Sp1 inhibits FOXO3 expression by inducing miR-182 expression To investigate the molecular mechanism underlying miR-182-mediated cancer cell proliferation we studied an important miR-182 target gene FOXO3. FOXO3 expression was higher in cells stably expressing miRZip-182 than in control cells (A). Knockdown of miR-182 expression enhanced the luciferase activity of a pGL3 vector containing the 3?-UTR of FOXO3 (B) indicating that miR-182 downregulated FOXO3 expression. Further to determine whether Sp1 downregulated FOXO3 expression through miR-182 GFP-Sp1 was expressed in cells stably expressing miRZip-182 (C). Overexpression of GFP-Sp1 reduced FOXO3 protein expression in miRZip stable cells but increased FOXO3 levels in miRZip-182-expressing cells implying that different effect of Sp1 is existed on the regulation of FOXO3 expression. Regulation of FOXO3 by miR-182 and Sp1 (A) Lenti-miRZip and lenti-miRZip-182 viruses were infected into H1299 for 96 h individually. FOXO3 level was studied by Western blotting with anti-FOXO3 antibodies and miR-182 level was studied by stem-loop RT-PCR. (B) Plasmids pGL3 and pGL3-FOXO3-3'UTR were transfected into miRZip and miRZip-182 stably expressed H1299 cells for 24 h and then cells were harvested for luciferase activity assays. (C) Different doses of GFP-Sp1 adenovirus were infected into the miRZip and miRZip-182 stably expressed H1299 cells for 48 h. FOXO3 level was studied by Western blotting using anti-FOXO3 antibodies (panel a). Quantitative results from three independent experiments are shown (panel b). The level of statistical significance was determined by t-test (* p<0.05; *** p<0.001). Therefore we further investigated the relationship between Sp1 and miR-182 in the context of FOXO3 regulation. The expression of Sp1 and FOXO3 in patients with lung cancer was examined (A). In normal tissue samples Sp1 levels were low and FOXO3 levels were high. In tumor tissue samples two Sp1 expression patterns i.e. high and low Sp1 expression were identified. Samples with higher Sp1 levels exhibited lower FOXO3 levels whereas samples with lower Sp1 levels exhibited higher FOXO3 levels suggesting that there is an inverse correlation between Sp1 and FOXO3 levels in lung specimen (A). The levels of FOXO3 and Sp1 in the lung cancer cell lines A549 H1299 CL 1-0 and CL 1-5 were studied (B). Higher levels of Sp1 expression were accompanied by lower levels of FOXO3 expression in A549 and CL 1-0 cells and lower levels of Sp1 expression were accompanied by higher levels of FOXO3 expression in H1299 and CL 1-5 cells suggesting that there is an inverse correlation between Sp1 and FOXO3 expression in lung tumorigenesis. Overexpression of GFP-Sp1 decreased FOXO3 mRNA and protein levels in a dose-dependent manner (C panel a) whereas knockdown of Sp1 expression increased FOXO3 mRNA and protein levels (C panel b). These results indicate that Sp1 negatively regulates FOXO3 expression. Sp1 negatively regulates FOXO3 expression through regulating miR-182 (A) The Sp1 and FOXO3 levels in clinical lung tissue samples were studied by IHC staining using antibodies against Sp1 and FOXO3 respectively. (B) Cell lysates were harvested from various cell lines for Western blotting using antibodies against FOXO3 and Sp1 and tubulin as an internal control. (C) Adeno-GFP-Sp1 viruses were infected into IMR-90 cells for 48 h and FOXO3 mRNA and protein were studied by RT-PCR and Western blotting respectively. GAPDH served as the internal control (panel a). Scramble and Sp1 shRNAs were transfected into H1299 for 48 h then FOXO3 mRNA and protein levels were studied by RT-PCR and Western blotting (panel b). (D) Scramble and Sp1 shRNAs were transfected into H1299 for 48 h and then cells were harvested at indicated time points following cycloheximide treatment for studying the Sp1 and FOXO3 levels with Western blotting. The levels of FOXO3 protein from three independent experiments were quantified using tubulin as an internal control. (E) Plasmids pGL2 or pGL2-FOXO3 (-1000/+50) were cotransfected with GFP or GFP-Sp1 into H1299 cells for 24 h then cell lysates were harvested for luciferase activity assays. (F) Adeno-GFP-Sp1 viruses were infected into H1299 cells for 24 h and cells were then transfected with pGL3 or pGL3-FOXO3-3'UTR plasmid for 24 h. Cells lysates were harvested for luciferase activity assays. (G) H1299 cells which were infected with GFP-Sp1 adenovirus for 24 h were then transfected with pGL3 or pGL3-FOXO3-3'UTR plasmid for 24 h. Total RNA was extracted at various time points following actinomycin D treatment. The mRNA levels of luciferase were determined by using quantitative RT-PCR and quantified using GAPDH as an internal control. Data are representative of three independent experiments each of which was performed in triplicate and presented as the mean ± SEM. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01). Next we investigated the mechanism by which Sp1 regulates FOXO3 expression. FOXO3 protein half-life was studied after Sp1 knockdown. Knockdown of Sp1 expression did not affect FOXO3 protein stability (D). We then constructed a luciferase reporter construct containing the FOXO3 promoter (-1000/+50) to study the effect of Sp1 on the promoter-mediated transcription of FOXO3 (E). GFP-Sp1 overexpression significantly enhanced the luciferase activity indicating that Sp1 positively regulated FOXO3 transcription (E). However FOXO3 mRNA and protein levels decreased as shown in C. The data shown in indicated that Sp1 increased miR-182 expression which suggests that post-transcriptional processing contributes to the regulation of FOXO3 expression. Thus the 3'-UTR of FOXO3 might play an important role in stabilizing FOXO3 mRNA and in FOXO3 translation. Consequently a luciferase reporter construct containing the 3?-UTR of FOXO3 was generated. GFP-Sp1 overexpression reduced the luciferase activity (F). Furthermore the stability of the luciferase mRNA containing the 3?-UTR sequence of FOXO3 decreased dramatically upon GFP-Sp1 overexpression (G). These results indicate that Sp1 regulates FOXO3 expression through transcriptional and post-transcriptional regulation with a net negative effect on FOXO3 expression. miR-182 inhibits lung cancer metastasis activity The data shown in indicated that miR-182 positively regulated lung cancer cell growth. Therefore the role of miR-182 in lung cancer metastasis was studied (Figure 6). The morphology of miRZip-182 cells was markedly altered: circular structures of actin filaments were absence and pseudopodia were enriched suggesting that miR-182 decreased the cells' migratory ability (Figure 6A). Indeed knockdown of miR-182 expression increased the migration ability of lung cancer cells suggesting that miR-182 inhibits lung cancer migration (Figure 6B). Moreover transwell migration assays showed that knockdown of miR-182 expression enhanced cell's invasive capacity (Figure 6C). In mice injected with miRZip-182-treated cells the knockdown of miR-182 expression also increased the number of nodules in the lung suggesting that miR-182 represses metastatic ability in vivo (Figure 6D). The effects of miR-182 knockdown were partially reversed by knockdown of FOXO3 suggesting that miR-182 functions as a suppressor of lung cancer metastasis by repressing FOXO3 expression (Figure 6E panel a). The endothelial-mesenchymal transition (EMT) marker N-cadherin increased after miR-182 knockdown but this effect was abolished by FOXO3 knockdown. Thus miR-182 might repress lung cancer metastasis by decreasing the expression of N-cadherin (Figure 6E panel b). However the expression of other genes regulated by miR-182 might also play a role in metastasis (Figure 6F and Supplementary Figure S3). Therefore we generated gene expression profiles using microarray analysis. Functional grouping analysis using DAVID bioinformatics resources showed that 19 of the genes differentially regulated by miR-182 knockdown were related to cell migration. The expression of these genes was increased in miR-182-knockdown cells indicating that they are potential targets of miR-182 (Figure 6F). Many metastasis-related genes such as CD44 CDH9 and ADAM9 were upregulated after the knockdown of miR-182 expression (Figure 6F). Figure 6 miR-182 attenuates lung cancer cell metastasis (A) Immunofluorescent staining of Alexa Fluor 568-conjugated phalloidin that is a high-affinity probe for F-actin (red) in miRZip and miRZip-182 stably expressed H1299 cells. DNA was stained with DAPI (blue). Stained cells were photographed under a fluorescence microscope at x 600 magnification. (B) Confluent monolayers of miRZip or miRZip-182 stably expressed H1299 cells were wounded and incubated for an additional 16 h (panel a). Migratory area was calculated for quantification (panel b). (C) The migration activities of H1299 cells (2 x 104) expressing miRZip or miRZip-182 were studied by Transwell chambers. (D) The miRZip or miRZip-182 stably expressed H1299 cells (4 x 106) were suspended in 100 ?l of PBS and injected into the lateral tail vein of SCID mice. After 8 weeks all mice were killed and the number of pulmonary tumor nodules was calculated after fixation of lungs with 4% formaldehyde for 48 h (panel a) and the number of pulmonary metastatic tumor nodules was counted (panel b). (E) FOXO3 and miR-182 in H1299 cells were knockdown by shFOXO3 and miRZip-182 respectively and then migration of cells (3 x 104) was studies by Transwell chambers (panel a). In addition cell lysates were harvested from FOXO3 and miR-182 knockdown cells for Western blotting using antibodies against N-cadherin ?-catenin vimentin FOXO3 and tubulin (panel b) respectively. (F) Heat map of the 19 of genes from miRZip and miRZip-182 microarray data the red color represents genes that are upregulated and the green color represents genes that are downregulated. The level of statistical significance determined by t-test (* p<0.05; ** p<0.01; *** p<0.001). DISCUSSION Our recent studies showed that Sp1 increased the growth of lung cancer cells but inhibits metastatic activity [23 32]. In the present study we found that Sp1 which accumulated in the early stages of cancer positively regulated miR-182 gene expression to silence FOXO3 expression and thereby promote cancer cell growth. In addition decreased levels of Sp1 in the late stages of cancer increased the expression of FOXO3 and N-cadherin leading to cancer metastasis (Figure 7). Figure 7 (A) Clinical samples from lung cancer patients of stage I and IV were used to study the Sp1 level by IHC staining with anti-Sp1 antibodies (B) Schematic diagram illustrates Sp1 regulates miR-182 to silence FOXO3 expression in early and late stages of lung cancer progression. Sp1 functions as a transcriptional activator by recruiting p300 to its target genes and as a repressor by the recruiting HDACs. Because Sp1 accumulates in several types of cancer including lung cancer [33] understanding the Sp1 transcriptional regulatory network may provide novel insights into the molecular origins and treatment of lung cancer. In our previous studies of lung cancer we found that Sp1 was highly upregulated in the early stages of cancer progression but partially down regulated in the late stages. Our previous studies also showed that regulation of Sp1 protein stability by phosphorylation and sumoylation contributed to its expression in the early and late stages of cancer respectively [32]. Kras activation and the Notch pathway might activate ERK1/2 to phosphorylate Sp1 thus stabilizing Sp1 in the early stages of cancer [32 34]. In the late stages Sp1 could be sumoylated leading to recruitment of its E3-ligase RNF4 followed by polyubiquitination and degradation [32]. To clarify the molecular mechanism underlying gene regulation by Sp1 we used microarray analysis to assess gene expression in KrasG12D-induced lung tumor transgenic mice and identified thousands of genes potentially regulated by Sp1 [23]. However some of the genes do not harbor a conserved Sp1 binding motif within their promoter region suggesting that another regulatory mechanism is involved in Sp1-mediated gene regulation. In this study we identified a novel pathway for Sp1-mediated activation wherein miR-182 expression downregulated the expression of FOXO3 a known miR-182 target gene [35]. Sp1 activated miR-182 and FOXO3 at the transcriptional level; however FOXO3 protein expression decreased. These results suggest that post-transcriptional regulation by miRNAs is a powerful mechanism by which to control the final level of protein expression. Many coding genes with Sp1 binding element(s) in their promoters harbor conserved miRNA target sequences in their 3'-UTR. To our knowledge this is first study to demonstrate that Sp1 regulates the expression of a target gene by regulating promoter activity and post-transcriptional processing in parallel. Few studies have characterized the regulation of miRNA by Sp1. Herein using a bioinformatics approach we identified several miRNAs potentially regulated by Sp1 including miR-182. We then showed that Sp1 specifically targets the miR-182 promoter region and activates miR-182 expression. miR-182 reportedly forms a gene cluster with two adjacent miRNAs (miR-96 and miR-183) [35]. The expression of miR-96 and miR-183 also decreased following Sp1 knockdown (Supplementary Figure S2A). Moreover we also investigated the binding of Sp1 to the miR-212 promoter because the latter contains 13 putative Sp1 binding sites (Supplementary Table S1). We found that Sp1 bound to the miR-212 promoter sequence (Supplementary Figure S2B and S2C). Interestingly a recent study showed that FOXO3 is a direct target of miR-212 in the neurons of patients with Alzheimer's disease [36]. miR-182 and miR-212 might cooperate to downregulate FOXO3 expression upon Sp1 overexpression. We cannot rule out this possibility. However depletion of miR-182 was sufficient to impair the Sp1-mediated reduction of FOXO3 expression in our experiments (C) suggesting that miR-182 is the major regulator of FOXO3 in lung cancer cells. Several studies have shown that miR-182 is upregulated in lung cancer. This suggests that miR-182 plays a positive role in lung tumorigenesis. However in two studies of miR-182 function in lung cancer miR-182 inhibited the proliferation of human lung adenocarcinoma cells [37 38]. Our results in this study provide several pieces of evidence to support the notion that miRNA-182 is a positive regulator of lung cancer cell proliferation. Firstly miR-182 was upregulated in the majority of lung cancer clinical samples and lung cancer cell lines examined. Secondly miR-182 knockdown inhibited cell cycle progression and cell growth. Finally miR-182 knockdown reduced lung tumor growth in vivo. Discrepancies in the role of miRNA-182 in lung cancer cell proliferation might derive from the different experimental designs of the studies. For example because miR-182 expression is upregulated in lung cancer we knocked down its expression and examined the effects on cancer cell proliferation. However other studies that described a negative role of miR-182 in lung cancer used miR-182 overexpression to study miR-182's role in cancer cell proliferation. Overexpression conditions can alter the function of many genes [39]. For example Sp1 accumulates in most of cancers; knockdown of Sp1 expression decreases cell proliferation but Sp1 overexpression also attenuates cancer cell growth [40]. Because post-translational modifications affect protein function overexpressed proteins might not be completely processed which could affect their function. Previous studies in melanoma and hepatocellular carcinoma indicated that miR-182 enhanced tumor metastasis [35 41]. However our data as shown in Figure 6 indicated that miR-182 knockdown altered cell morphology and increased migration and invasion activities. In addition miR-182 knockdown increased N-cadherin levels suggesting that miR-182 promotes the mesenchymal to epithelial transition (MET) [42]. Previous studies have shown that TIMP-2 enhances the E-cadherin/?-catenin complex in A549 lung cancer cells [43]. Whether Sp1 or miR-182 regulates TIMP-1 in lung cancer needs to be addressed in future studies. Finally miR-182 levels were lower in CL1-5 cells then in CL1-0 cells resulting in increased metastatic activity in CL1-5. Collectively our data suggest that miR-182 inhibits lung cancer metastasis. Our previous study indicated that Sp1 is down regulated in the late stages of lung cancer progression [32]. Therefore in the late stages of lung tumorigenesis miR-182 expression was down regulated compared with expression in the early stages which led to tumor metastasis through at least in part an increase in FOXO3 expression. It is still not clear why miR-182 has different roles in different types of cancer; this awaits further study. Although we found that FOXO3 is involved in miR-182-mediated lung cancer progression FOXO3 knockdown did not completely abolish the effects of miR-182 knockdown suggesting that other genes regulated by miR-182 contribute to the inhibition of metastasis by miR-182. With this in mind we determined the expression profile of miR-182-regulated genes. Many metastasis-related genes were induced in miR-182-knockdown cells including CD44 ADAM9 and CDH9. CD44 which localizes to the cell membrane is reportedly involved in cell migration in various cancer types [44]."
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"diagnostic performance of intravoxel incoherent motion diffusionweightedimaging IVIMDWI in the differential diagnosis of pulmonary tumors remained debatable among published studiesThis study aimed to pool and summary the relevant results to provide more robust evidence in this issue using ametaanalysis methodMaterials and methods The researches regarding the differential diagnosis of lung lesions using IVIMDWI weresystemically searched in Pubmed Embase Web of science and Wangfang database without time limitation ReviewManager was used to calculate the standardized mean difference SMD and confidence intervals ofapparent diffusion coefficient ADC tissue diffusivity D pseudodiffusivity D and perfusion fraction f Stata was used to pool the sensitivity specificity and area under the curve AUC as well as publication bias andheterogeneity Fagans nomogram was used to predict the posttest probabilitiesResults Eleven studies with malignant and benign lung lesions were included Most include studies showed alow to unclear risk of bias and low concerns regarding applicability Lung cancer demonstrated a significant lower ADCSMD P D SMD P and f values SMD P than benign lesions except Dvalue SMD P D value demonstrated the best diagnostic performance sensitivity specificity AUC and highest posttest probability and for D ADC f and D values in the differential diagnosisof lung tumors followed by ADC sensitivity specificity AUC f sensitivity specificity AUC and D values sensitivity specificity AUC Continued on next page Correspondence 849049724qqcom wuypsysucccnhenisysucccn Jianye Liang Jing Li and Zhipeng Li contributed equally to this work2Department of Radiology Maoming Peoples Hospital Maoming Guangdong China1Department of Medical Imaging Sun Yatsen University Cancer Center StateKey Laboratory of Oncology in South China Collaborative Innovation Centerfor Cancer Medicine No651 Dongfeng Road East Guangzhou Guangdong China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cLiang BMC Cancer Page of Continued from previous pageConclusion IVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lungtumors based on the tumor cellularity and perfusion characteristics and D value demonstrated better diagnosticperformance compared to monoexponential ADCKeywords IVIMDWI Posttest probability Diagnostic performance Lung neoplasm Magnetic resonance imaging MetaanalysisIntroductionLung cancer is the most commonly diagnosed cancer of the total cases and the leading cause of cancerdeath of the total cancer deaths in aroundthe world [] The incidence and mortality of lung cancer still increased in recent years Accurate and earlydiagnosis is help to select optimal treatment strategy andimprove the outcome of patients with lung cancerlungtumorsandefficacyComputed tomography CT is the main imaging modality for lung lesions largely based on morphologicaland enhanced characteristics However the relativelylow specificity and administration of contrast agent limitits wide use in clinical practice Magnetic resonance imaging MRI was rarely used in detecting lung lesionspreviously due to the obvious cardiac and respiratorymotionlow signaltonoise ratio from the inherentlylow lungproton density and magnetic susceptibilityartifact of airfilled pulmonary tissue subjected to highfield strength [] With the development of MRI hardwares and various rapid imaging technologies such asimproved gradient performance parallel imaging techniques and freebreathing acquisition MRI has been inidentification of benign andcreasingly used formalignantevaluationDiffusionweighted imaging DWI is a radiationfreeand contrastfree functional imaging sequence which allows measurement of water molecular movement usingapparent diffusion coefficient ADC and demonstratespotential to differentiate malignant from benign lung lesions A previous metaanalysis even reported a higherdiagnostic performance with a pooled sensitivity specificity and areas under the curve AUC of and in DWI compared to PETCT whose sensitivityspecificity and AUC were and respectivelyThe monoexponential model is expressed as SI SI0 expb·ADC where SI0 refers to the mean signal intensity SI of the region of interest for b smm2 while SIrefers to the signal intensity for higher b values However the monoexponential model cannot separate thepseudodiffusion from pure molecular diffusion andADC calculated from the monoexponential modelmixesthe conventionalmonoexponential model cannot accurately reflect thetrue diffusivity owing to the influence of microcirculation perfusion []the two effects Thereforechangesthe microenvironmentIntravoxel incoherent motion IVIM is an advancedimaging technique which was first proposed by Le Bihan [] It can separate the incoherent motion of watermolecules within the capillaries from molecular diffusionin the extravascular space [] The true diffusion coefficient D value pseudodiffusion coefficient D valueand perfusion fraction f value were generated using abiexponential model with multiple bvalues expressed asSI SI0 f · expbD f · expbD The IVIMmodel can separate the pseudodiffusion from pure molecular diffusion and independently reflect the microcirculation perfusion D and tumor cellularity D basedon that equation [] This model provides more detailedand accurate information and can make a better interpretation forandcharacterization of tumor grades As such these parameters are important to be analyzed Several studies hadapplied IVIMDWI to discriminate lung cancer from benign lesions and demonstrated better or comparablediagnostic performance compared with traditional ADCvalue [] However the diagnostic performances ofIVIMDWI derived parameters in the differentiation oflung tumors were not consistent and the application stillremained debatable in the lung For example severalstudies indicated that lung cancer had a higher D valuethan benign lesion [] while some studies reportedadverse [ ] or insignificant results [ ] Theoretically the true diffusitivity should have better diagnostic performance than ADC in distinguishing lunglesions but some studies indicated a much lower areaunder the curve AUC or accuracy in D value comparedto ADC [ ] Cancerous tissue generally has activeangiogenesis and rich blood supply compared to benignlesions but most studies indicated a lower f value inlung cancer the results of which should be further confirmed The sample sizes in most studies were still notenough to draw a robust for its performancethe application of IVIMDWI in the lung has not yetformed a clinical guideline or become a routine sequence in the MRI protocol Therefore we attempted topool all the published results about the diagnostic performance of IVIMDWI in the differentiation of malignant and benign lung lesions using a metaanalysismethod Besides the diagnostic performance of IVIMDWI was compared to conventional DWIderived ADC 0cLiang BMC Cancer Page of this study provides additionalvalue to determine the suitability for clinical applicationThe controversialissues between different researcheswill also be addressed with more reliable evidence Furthermoreinformationabout technical feasibility on lung MRI and the functional changes oflung lesions with IVIMDWI Thisstudy may further attract the researchers to perform thelung studies using noninvasive MR imaging by solvingthe technical issues on Lung MRIMaterials and methodsData sourcesThe studies regarding the differential diagnosis of lungtumors using IVIMDWI parameters were systemicallyretrieved by two senior librarians in PubMed EmbaseWeb of science and Wangfang database without timelimitation A searching formula was formed with different combinations of the medical subject headings or keywords from IVIM intravoxel incoherent motion multiple bvalue DWI biexponential and lung or pulmonarylesion cancer carcinoma neoplasm The primarysearches were limited in the titles and abstracts We alsoperformed a manual retrieval of the reference lists fromincluded studiesbStudies selectionStudies met the following criteria were included a theresearch purpose was to differentiate lung cancer frombenign lesions using IVIMDWI parametersthemean and standard deviation SD of each parameterwas provided c their diagnostic performance aboutsensitivity and specificity or truepositive TP falsenegative FN falsepositive FP and truenegative TNwere reported d the lung cancer should be confirmedby pathology after initial MRI examination Exclusioncriteria mainly included a duplication from the sameauthors or institutions b metaanalysis conference abstract review or any unpublished results and c animalexperiments or nonlung researchesData extractionA spreadsheet was used to extract the mean values andSD as well as the diagnostic performance of ADC D Dand f values with threshold value AUC sensitivityand specificity in respective study by one author andreviewed by another one Other information includedthe first author publication years field strength and vendors b values patient ages tumor sizes and numbers ofmalignant and benign lesions TP FN FP and TN canbe calculated when only the amount of malignant andbenign lesions as well as sensitivity and specificity or receiver operating curve was providedQuality assessmentThe quality of studies and likelihood of bias were evaluated using Review Manager software Cochrane Collaboration Oxford UKreferring to the QualityAssessment of Diagnostic Accuracy Studies [] Weassessed the risk of bias and applicability in four domains including patient selection index tests referencestandard flow and timing []Publication bias and heterogeneity evaluationAs two parts of data were pooled in our study includingquantitative values and diagnostic performance of eachparameter funnel plots and Beggs test were used tovisually and quantitatively assess the publication bias forthe continuous variables and Deeks plot assessed thepublication bias of sensitivity and specificity using Stataversion StataCorp LP College Station TX Anasymmetric or skewed funnel plot P of Beggs testor Deeks test indicated the potential of publication bias[] Inconsistency index I2 and Cochrans Q tests wereused to explore the heterogeneity of included studieswith I2 or P for Cochran Q test suggestedstatistically significant heterogeneity and a randomeffect model was applied in subsequent pooling or afixedeffect model when I2 []Evidence synthesisWe constructed the forest plots for continuous variablesand calculated the standardized mean difference SMDbetween lung cancer and benign lesions using ReviewManager software We used the bivariate regressionmodel to pool the diagnostic performance with sensitivity specificity positive likelihood ratio PLR negativelikelihood ratio NLR diagnostic odds ratio DOR andAUC using Stata version The summary receiveroperating characteristic curves and Fagans nomogramswere also plotted to determine the diagnostic values andpredict the posttest probabilities of ADC D D and fvalues in the differential diagnosis of lung tumorsResultsLiterature search and selectionBy searching the key words in the titles and abstracts atotal of potential studies were obtained from multiple databases A total of studies regarding metaanalysis conference abstract case report and reviewwere excluded after screening the titles and abstractsAnimal studies nonlung researches and duplicationfrom the same authors or institutions led to further exclude studies We scrutinized the fulltexts of theremaining studies in detail and excluded an additional studies for the following reasons a lack ofsufficient data to be pooled b low quality assessmentcIVIMDWI was interfered by treatment and d 0cLiang BMC Cancer Page of cancer was not confirmed by pathology Eventually eligible studies with malignant and benign lunglesions were included for analysis The flowchart detailing the process of study selection was provided in Fig Basic information and diagnostic performance for eachincluded study was detailed in Table and Table Inother to include every potential we did not set acriterion on the field strength T or T FromTable there are three studies using T and eightstudies using T for imaging Although field strengthof T is better for image quality the results from Tscanner are also acceptable Therefore studies with either of field strengths are included for analysisQuality assessmentThe distribution of Quality Assessment of DiagnosticAccuracy Studies scores for risk of bias and applicability concerns were shown in Fig The overall qualityof included studies was acceptable Regarding patient selection four studies were marked unclear risk of bias dueto ambiguity for consecutive enrollment and prospectivedesign or not The applicability concerns remainedunclear concern as the tumor types were inconsistentbetween malignant and benign tumors from two studiesTwo studies were marked unclear and high risk of biaswith unclear concern of applicability for index test asthe threshold values for D and f values were not provided Three studies showed unclear risks of bias for reference standard because some of the benign lesionswere diagnosed through a long time followup Threestudies were marked unclear and high risk of bias in patient flow and timing domain because the time intervalbetween MR examination and pathological confirmationwas not reportedQuantitative analysisADC used for diagnosis of lung tumorNine studies regarding ADC used in differentiating lungtumors were included for analysis The Ï2 andP of heterogeneity test with I2 suggestedmoderate heterogeneity among included studies Theforest plot in Fig showed the distribution of ADC between lung cancer and benign lesions A randomeffectsmodel generated a SMD of P Fig Flowchart detailing the study selection process Eleven studies that met the inclusion criteria were included FN false negative FP falsepositive TN true negative TP true positive 0cLiang BMC Cancer Page of Table Basic information for each included studyAuthorDeng []Machine type T PhilipsYearb values smm2Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Koyama []NA Not available T GE T Siemens T GE T Philips T Siemens T Philips T Siemens T GE T GE T PhilipsAge years ± ± ± NA ± ± ± Tumor size cm Malignant ± BenignNA ± NA ± NA ± Table The diagnostic performance for each included studyIndicatorADCAuthorDeng []ThresholdYearHuang []Jiang []Wan []Wang Y []Yuan []Zhou []Huang []Jiang []Jiao []Wan []Wang LL []Wang Y []Yuan []Zhou []Wang XH []Deng []Huang []Jiang []Wan []Yuan []Zhou []Wang XH []Deng []Huang []Wan []Wang LL []Yuan []NANANADDfAUCNANANANANANASensitivitySpecificityTPFPFNTNWang XH []NA Not available ADC Apparent diffusion coefficient D Tissue diffusivity D pseudodiffusivity f Perfusion fraction AUC Area under the curve FNFalse negative FP False positive TN True negative TP True positive Threshold values of ADC D and D are factors of mm2s 0cLiang BMC Cancer Page of Fig The distribution of risk of bias and applicability concerns for each included study using QUADAS2 a and a summary methodologicalquality b between lung cancer and benign lesions forADC A basically symmetric funnel plot in Fig andP of Beggs Test suggested no publication biasin ADCD value used for diagnosis of lung tumorEleven studies regarding D value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studies Theforest plot in Fig showed the distribution of D value between lung cancer and benign lesions A randomeffectsmodel generated a SMD of P between lung cancer and benign lesions for D value A basically symmetric funnel plot in Fig and P of BeggsTest suggested no publication bias in D value 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of apparent diffusion coefficient ADC between lung cancer and benign lesions The standardized meandifferences indicated that lung cancers had a significantly lower ADC than benign lesionsFig Funnel plot of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f Thebasically symmetric funnel plots indicated no publication bias in these parameters 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of tissue diffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower D value than benign lesionsD value used for diagnosis of lung tumorTen studies regarding D value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested obvious heterogeneity among included studiesThe forest plot in Fig showed the distribution of Dbetween lung cancer and benign lesions A randomeffects model generated a SMD of P between lung cancer and benign lesions forD A basically symmetric funnel plot in Fig and P of Beggs Test suggested no publication bias in Df value used for diagnosis of lung tumorEleven studies regarding f value used in differentiatinglung tumors were included for analysis The Ï2 and P of heterogeneity test with I2 suggested moderate heterogeneity among included studiesThe forest plot in Fig showed the distribution off value between lung cancer and benign lesions Arandomeffects model generated a SMD of P between lung cancer andbenign lesions for f value A basically symmetricfunnel plot in Fig and P of Beggs Testsuggested no publication bias in f valueDiagnostic performanceThe Diagnostic performance with pooled sensitivity specificity PLR NLR DOR and AUC of ADC D D and fvalues were listed in Table Deeks funnel plots in Fig and asymmetry tests indicated no obvious publicationbias in ADC D D and f values P and for ADC D D and f values respectively Fig plotted the summary receiver operating characteristiccurves of ADC D D and f values D value demonstrated the best diagnostic performance sensitivity specificity AUC in the differentialdiagnosis of lung tumors followed by ADC sensitivity specificity AUC f sensitivity Fig Forest plot of the mean value of pseudodiffusivity D between lung cancer and benign lesions The standardized mean differencesindicated that the difference of D value between lung cancers and benign lesions were insignificant 0cLiang BMC Cancer Page of Fig Forest plot of the mean value of perfusion fraction f between lung cancer and benign lesions The standardized mean differencesindicated that lung cancer had a significantly lower f value than benign lesionsspecificity AUC and D values sensitivity specificity AUC Posttest probabilitiesLikelihood ratio and posttest probability were also important for diagnosing a disease [] which provided alikelihood that a patient was diagnosed with a certaindisease or not using the MRI parameters Fig plottedthe Fagans nomograms of ADC D D and f values forpredicting posttest probabilities All the pretest probabilities were set at by default We regarded thediagnosis of lung cancer as a positive event corresponding to a lower ADC D and f values Similarly the noncancerous tissues with a higher ADC D and f valueswere regarded as a negative event The posttest probability increased to from a pretest probability of with a PLR of and decreased to with a NLRof with the prompt of ADC This indicated that thediagnostic preference to lung cancer will be obviouslyenhanced with the help of ADC a lower ADC compared with the condition without the prompt of ADCwhose diagnostic probability was set at beforehandIn contrast the probability of diagnosing lung cancerwill significantly drop from to when a negativeevent occurs a higher ADC Similarly the posttestprobability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of using D for guiding The posttest probability of diagnosing lung cancer will reach to with a PLR of and drop to with a NLR of in the help of fvalue These data indicated that both ADC and IVIMparameters helped to enhance the accuracy for diagnosing lung cancerDiscussionIVIMDWI is a noninvasive technique that shows superiority in reflecting tumor cellularity and perfusion without the need of contrast agent It had already beenapplied in the differentiation of thyroid nodules []breast [] liver [] and brain tumors [] with gooddiagnostic performance To our best knowledge there isstill no pulmonary study with large sample size to settledown the value of IVIM for quantitatively distinguishinglung cancer from benign tissues in the background ofIVIM becoming a research hotspot in the wholebodytumors Our study provided a timely summary in thisissue through pooling all published evidence with strictinclusion criteria and quality assessment The resultsdemonstrated IVIM model had a good diagnostic performance in distinguishing lung lesionsTable Pooled estimates and heterogeneity measures for ADC D D and f valuesDORIndexSpecificitySensitivityNLRPLRAUCADC DD I2 SensitivitySpecificity fADC Apparent diffusion coefficient D Tissue diffusivity D Pseudodiffusivity f Perfusion fraction PLR Positive likelihood ratio NLR Negative likelihood ratio DORDiagnostic odds ratio AUC Area under the curve I2 inconsistency index 0cLiang BMC Cancer Page of Fig Deeks funnel plots regarding diagnostic performance for a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivityD and d perfusion fraction f No publication bias was indicated in the four parameters P In this metaanalysis the SMDs suggested that lungcancer demonstrated a lower ADC and D values thanbenign lesions The lung cancer usually has dense cellularity and nucleoplasm ratio with active proliferativecapacity which may reduce the extracellular space andrestrict the movement of water molecules causing a reduction in diffusion coefficient The pooled results alsosuggested an excellent diagnostic performance with ahigh sensitivity specificity AUC and increased posttestprobability in both ADC and D values followed by fvalue Monoexponential modelancannot provideindependent perfusionrelated parameter and may miscalculate the water molecule movement due to a mixwith microcirculation perfusion and therefore resultedin an overestimated ADC value in a certain extent []Therefore the best diagnostic performance was observedin D value instead of ADC valueInterestinglylung cancer demonstrated a significantlower f value but insignificant D value compared withbenign lesions F value refers to vascular volume ratioand reflects the microcirculation perfusion in the capillaries F value increases with increased tissue perfusion 0cLiang BMC Cancer Page of Fig Summary receiver operating characteristic SROC curve of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction f in the diagnosis of lung lesions D value demonstrated the highest area under the curve followed byADC f and D valuesinflammatoryconsistHigher f value is supposed to be observed in malignanttumors due to neovascularization compared to benignlesions However these results are not unreasonable because the benign lesions occurring in the lung are generallyoftuberculosisinfectiongranuloma or bloodrich tumor such as inflammatorypseudotumor They are usually featured by marked vascular changesincreased bloodflow and enhanced vessel permeability which generallyincluding vasodilationinfections whichanic pneumoniafungaloccur at the capillary network [] A perfusion studyusing CT with exogenous contrast indicated active infectious nodules had comparable or even higher perfusionpeak enhancement increment and blood volume withsteeper time to peak than malignant nodules [] Theresults were in good agreement with our study in another aspect However the diagnostic performance of fvalue was relatively low with the sensitivity specificityand AUC of and only F value is also associated with echo time relaxation effects and T2 0cLiang BMC Cancer Page of Fig Fagans nomogram of a apparent diffusion coefficient ADC b tissue diffusivity D c pseudodiffusivity D and d perfusion fraction fD and ADC demonstrated similar and highest posttest probability among the four parameterscontribution [] which may reduce its diagnostic accuracyperformance to a certain extentD value is proportional to the average blood velocityand mean capillary segment length [] D value wasnot statistically significant in differentiating benign andmalignant lung lesions in this metaanalysis A poormeasurement reproducibility of D was indicated by thehuge standard deviations in the included studies Theoretically the more bvalues are selected the higher theaccuracy of model fitting will be Besides measurementat lower bvalue had been reported to be less reproducible and stable compared with measurement at higherbvalue and previous studies suggested measurements ata larger number of lower bvalue should be obtained forreducing measurement errors and signalto noise variation [ ] However a larger number of bvalue applied in IVIM model will significantly prolong thescanning times and introduce obvious motion and susceptibility artifacts especially in the pulmonary MRITherefore D value is still not adequate to differentiatelung lesions due to the low reliability stability and accuracy as indicated in our metaanalysisADC D D and f values all demonstrated moderate toobvious heterogeneity which should be explored Firstboth T and T MR scanners with various combinations of bvalue were used to perform IVIMDWI inthese studies which may influence the accurate calculations of diffusion and perfusion coefficients and decrease the diagnostic performance compared to monoexponential ADC Second the lesion sizes and density oflung cancer such as ground glass opacity on initial CTvaried from studies to studies which may perform different biological characteristics and also lead to themeasurement variability in ADC and IVIM parametersindicated by Weller [] and Jiang []Third the benign lesions consisted of a variety of inflammatory infections and benign tumors which mayintroduce significant heterogeneity in these parameterswhen compared with lung cancer Last most studies delineated the regions of interest on the largest slice instead of the entire tumors which may lead to someselection bias owing to tumor heterogeneity Histogramanalyses for the whole lesions which can reduce themeasurement variability may be a more promisingmethod for assessing lung nodules in the future studyThere were several limitations First as the sensitivityof detecting pure ground glass opacity or small lesionsare quite low on conventional DWI or IVIMDWI theselesions were inevitably excluded from the original studies which may decrease the availability of IVIM in theclinical application to a certain extent Second we hadnot performed a direct comparison with dynamic contrast enhancedCTMRI or Fluorine 18FDG PETCTwhich was also commonly used in the diagnosis of lungcancer The issue about whether IVIMDWI addedvalues to multiparametric MRI or CT in a large samplesize was still not clearConclusionsIVIMDWI parameters show potentially strong diagnostic capabilities in the differential diagnosis of lung tumors and D value demonstrated better diagnosticperformance compared to monoexponential ADC Fvalue can differentiate the perfusion difference betweenlung cancer and benign lesions The application ofIVIMDWI will further help the clinicians make a bettermanagement for cancer treatment and prognosis evaluation based on the tumor cellularity and perfusion characteristics detected by IVIM technique 0cLiang BMC Cancer Page of AbbreviationsAUC Area under the curve ADC Apparent diffusion coefficient D Tissuediffusivity D Pseudodiffusivity IVIMDWI Intravoxel incoherent motiondiffusionweighted imaging SMD Standardized mean differenceI2 Inconsistency index PLR Positive likelihood ratio NLR Negative likelihoodratio DOR Diagnostic odds ratioAcknowledgementsNot applicableAuthors contributionsNH was the guarantor of this metaanalysis and had full access to all the datain the study and took responsibility for the integrity of the data and the accuracy of the data analysis NH YW and XL conceived the study and revisedthe manuscript JL ZL and TM drafted the manuscript JC and WM searchedthe databases and acquired the data WM and SC performed data analysisand interpretation Jing Li substantively revises the manuscript based on thecomments and provides language proofreading for the revised version Allauthors had read and approved the manuscriptAuthors informationNot applicableFundingThe Highlevel Hospital Construction Research Project of Maoming PeoplesHospital supported the consultation fee from a statistician for checking thecorrectness of the statistical methods the National Key Research and Development Program of China grant no 2017YFC0112605 and the Medical Science Research Foundation of Guangdong Province of China grant no supported the fee for language editing and processingcharge for accessAvailability of data and materialsAll the original data were provided in the main document as well as thetables and figures They can also be obtained from the Internet databasesEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsThe authors have stated explicitly that there are no conflicts of interest inconnection with this Received May Accepted August ReferencesBray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancerstatistics GLOBOCAN estimates of incidence and mortality worldwidefor cancers in countries CA Cancer J Clin httpsdoi103322caac21492Koyama H Ohno Y Seki S Nishio M Yoshikawa T Matsumoto S Maniwa YItoh T Nishimura Y Sugimura K Value of diffusionweighted MR imagingusing various parameters for assessment and characterization of solitarypulmonary nodules Eur J Radiol httpsdoi101016jejrad201411024Le Bihan D Turner R The capillary network a link between IVIM andclassical perfusion Magn Reson Med httpsdoi101002mrm1910270116Le Bihan D Breton E Lallemand D Grenier P Cabanis E LavalJeantet MMR imaging of intravoxel incoherent motions application to diffusion andperfusion in neurologic disorders Radiology httpsdoi101148radiology16123763909Liang J Ma R Chen H Zhang D Ye W Shi C Luo L Detection ofHyperacute reactions of Desacetylvinblastine Monohydrazide in a Xenograftmodel using Intravoxel incoherent motion DWI and R2 mapping AJR Am JRoentgenol httpsdoi102214AJR1820517Liang J Cheng Q Huang J Ma M Zhang D Lei X Xiao Z Zhang D Shi CLuo L Monitoring tumour microenvironment changes during antiangiogenesis therapy using functional MRI Angiogenesis httpsdoi101007s10456019096704Deng Y Li X Lei Y Liang C L
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"As one of the DNA repair genes ataxia-telangiectasia mutated (ATM) gene which is responsible for the multisystem autoxomal recessive disorder ataxia-telangiectasia (AT) plays a crucial role in the recognition signaling and repair of DNA damage especially DNA double-strand breaks (DSBs) [4] [5]. The ATM protein is a member of phosphoinositide 3-kinase (PI-3 kinases) and can be activated by DSBs caused by ionizing radiation or reactive oxygen intermediates [6] [7]. Once activated ATM can phosphorylate various downstream substates that function in cell cycle arrest apoptosis and DNA repair such as p53 NBS1 BRCA1 and Chk2 [8] [9]. Therefore genetic variants in ATM gene may lead to the structure and function change of the protein and act as important factors indicating individual susceptibility to cancer. ATM -111G>A (rs189037) resides in the promoter of ATM gene. Increasing studies have shown that variations in the DNA promoter sequence may potentially alter the affinities of multiple regulatory proteins-DNA interactions or the specificity of the transcriptional process [10][13]. Although this polymorphism makes no amino acid change the alleles may have different binding affinity to the transcription factor and exhibit different levels of mRNA expression [14] [15]. Zhang et al. [16]declared that ATM rs189037 AA genotype was associated with a lower ATM mRNA levels than GG genotype in lung tissue samples. Their results showed that the G-to-A change might create a transcriptional inhibitor-binding site for ATM rs189037 A allele promoter and subsequently reduce the ATM mRNA expression. Consequently lower expression of ATM might cause elevated sensitivity to ionizing radiation defects in the activation of cell cycle checkpoints a reduced capacity for DNA repair and abnormal apoptosis. All of these features would contribute to increased individual cancer susceptibility. In recent years a number of studies have evaluated the association between this polymorphism and cancer risk such as thyroid carcinoma [17] oral cancer [18] breast cancer [19] leukemia [20] nasopharyngeal carcinoma [21] glioma [22] and lung caner [23][25]. Previous studies of ATM rs189037 have included cigarette smokers as cases and controls that made it difficult to judge whether this polymorphism were associated with lung cancer or tobacco use. Considering the facts in China the incidence and death rate of lung cancer in women continues to increase and this phenomenon is frequently occurring in those who have never smoked. In order to have a better control of confounding of gender or smoking we performed a case-control study to identify the association between the polymorphism of ATM rs189037 and the risk of lung cancer in the non-smoking females in Chinese Han population. We also investigated the interaction between genetic polymorphism and environmental exposure in lung cancer. Methods Subjects This hospital-based case-control study included 487 lung cancer patients and 516 cancer-free hospital controls. All subjects were female non-smokers and they were from unrelated ethic Han Chinese. The cases were recruited during January 2002 to November 2012 at Liaoning Cancer Hospital & Institute. All patients were histologically confirmed to have lung cancer before any radiotherapy and chemotherapy. During the same time controls were selected from patients with other lung diseases but free of cancer history and symptom. Controls suffered mainly from bronchitis pneumonias fibrosis sarcoidosis chronic obstructive pulmonary disease and emphysema. Controls were all non-smoking females and frequency-matched to case subjects for age (±5 years). This study was approved by the institutional review board of China Medical University and written informed consent was obtained from each participant or each participant's representatives if direct consent could not be obtained. Data Collection A total of 10 ml of venous blood was collected from each patient. Patients were interviewed to collect information for demographics and environmental exposure at the time they were admitted to hospital. Information concerning demographic characteristics passive smoking cooking oil fume exposure fuel smoke exposure family history of cancer occupational exposure and dietary habit was obtained for each case and control by trained interviewers. An individual was defined as a smoker if she had consumed a total of 100 cigarettes in her lifetime; otherwise she was considered as a non-smoker. About fuel smoke exposure participants who used coal-fuel-burning stoves without chimneys were regarded as fuel smoke exposure. For exposure to cooking oil fumes participants were mainly asked about the method of cooking and eyes or throat irritation. For cooking methods participants were asked whether they cooked food in a stir-frying way and how many times a week; for eyes or throat irritation participants were asked how often they felt eyes or throat irritated by the oily smoke. There were four possible responses ranging from never seldom sometimes and frequently. Subjects were considered as cooking oil fume exposure if they met criteria as follows: (1) have cooked for over 15 years; (2) cooked food in a stir-frying way for more than twice a week; (3) felt eyes or throat irritated by oily smoke. Exposure for cooking oil fume was categorized as an indicator variable equal to 1 if participants reported frequently or sometimes and equal to 0 otherwise. Genotype Analysis Genomic DNA was extracted from peripheral blood samples by the conventional phenol-chloroform extraction method. SNP was genotyped by investigators blinded to case-control status in order to avoid any genotyping bias using TaqMan methodology and read with the Sequence Detection Software on an Applied Biosystems 7500 FAST Real-Time PCR System according to the manufacturer's instructions (Applied Biosystems Foster City CA). Amplification was done under the following conditions: 95°C for 10 min followed by 47 cycles of 92°C for 30 s and 60°C for 1 min. In this study 487 lung cancer patients and 516 controls were all genotyped successfully and 5% duplicated samples were randomly selected to assess the reproducibility for quality control with a concordance rate of 100%. Statistical Analysis The x2 test and t test were applied to estimate differences in demographic variables and distributions of genotypes between cases and controls. The association of genotypes of ATM rs189037 with risk of lung cancer was estimated by computing the odds ratios (ORs) and 95% confidence intervals (CIs) in unconditional logistic regression analysis. The Hardy-Weinberg equilibrium (HWE) was tested using goodness-fit x2 test to compare the genotype frequencies in the control subjects from those expected. A logistic regression model was used to evaluate gene-environment interactions. All data were analyzed with Statistical Product and Service Solutions (SPSS) v13.0 for Windows if not otherwise specified. All statistical analysis were two-sided and the significance level was set at P<0.05. Results Population characteristics A total of 487 lung cancer and 516 age-matched cancer-free controls were enrolled in this study. As shown in the mean ages of cases and controls (mean ±S.D.) were almost identical (56.5±11.7 and 56.3±12.5 respectively). All cases were female non-smoking lung cancer patients. No statistically significant difference was found between cases and controls in terms of age (P?=?0.248) and monthly income (P?=?0.084). Cases included 434 non-small cell lung cancer (NSCLC) patients and 53 small cell carcinoma patients. In the NSCLC cases there were 320 adenocarcinomas 73 squamous cell carcinomas and 41 other tumors with a variety of different pathologies (such as large cell carcinomas mixed cell carcinomas or undifferentiated carcinomas). .0096911.t001 Characteristics of lung cancer cases and controls. Variables Cases(%) Controls(%) P value Female 487 516 Mean age (years) 56.5±11.7 56.3±12.5 0.248a Income (yuan/month) 628.9±419.3 563.5±387.6 0.084a Never smoker 487 516 Histological type NSCLC 434(89.1) Adenocarcinoma 320(65.7) Squamous cell carcinoma 73(15.0) Small cell carcinoma 53(10.9) Other 41(8.4) a Student's t-test was used to compare the frequency distributions of demographic variables between the cases and controls. Association analysis The observed genotype frequencies among the control subjects was in agreement with that expected under the Hardy-Weinberg equilibrium (P?=?0.119). The distribution of ATM rs189037 genotypes among subjects were displayed in Table 2. Using subjects with the ATM rs189037 GG genotype as the reference group we calculated the ORs and 95%CIs for heterozygous carriers of GA genotype and homozygous carriers of AA genotype. No significant difference was observed between lung cancer cases and controls in each test (P>0.05). In order to increase the statistical power we combined the GA genotype with the AA genotype to compare with GG genotype as a dominant model and combined the GA genotype with the GG genotype to compare with AA genotype as a recessive model. The results indicated that individuals with AA genotype had a significantly elevated risk of lung adenocarcinoma compared with those carrying the GG or GA genotype (OR?=?1.44 95%CI 1.022.02 P?=?0.039). .0096911.t002 Table 2 Distribution of ATM rs189037 genotypes and ORs for lung cancer cases and controls. Genotype Cases(%) Controls(%) ORc 95%CI P overall (n?=?487) GG 148(30.4) 152(29.5) ref GA 240(49.3) 272(52.7) 0.91 0.681.20 0.494 AA 99(20.3) 92(17.8) 1.11 0.771.59 0.590 dominant modela 0.96 0.731.25 0.742 recessive modelb 1.18 0.861.61 0.313 NSCLC (n?=?434) GG 129(29.7) 152(29.5) ref GA 213(49.1) 272(52.7) 0.92 0.681.24 0.573 AA 92(21.2) 92(17.8) 1.18 0.811.71 0.397 dominant model 0.98 0.741.30 0.906 recessive model 1.24 0.901.71 0.192 Adenocarcinoma (n?=?320) GG 94(29.4) 152(29.5) ref GA 150(46.9) 272(52.7) 0.89 0.641.23 0.485 AA 76(23.7) 92(17.8) 1.33 0.901.99 0.156 dominant model 1.00 0.741.36 0.987 recessive model 1.44 1.022.02 0.039* Squamous cell carcinoma (n?=?73) GG 24(32.9) 152(29.5) ref GA 39(53.4) 272(52.7) 0.90 0.521.56 0.706 AA 10(13.7) 92(17.8) 0.69 0.321.51 0.355 dominant model 0.85 0.501.43 0.537 recessive model 0.74 0.371.50 0.400 *P<0.05. a GA+AA vs GG. b AA vs GA+GG. c adjusted for age and data were calculated by unconditional logistic regression. According to the results above we assumed that ATM rs189037 AA genotype might affect lung adenocarcinoma risk among non-smoking Chinese females. To test this hypothesis and explore the gene-environment interaction we adopted all the lung adenocarcinoma patients and cancer-free controls whose information about environmental risk factors were completely obtained such as fuel smoke exposure cooking oil fume exposure passive smoking and family history of cancer. Cases and controls were not included in the association analysis if any item of their environmental risk factors data was incomplete. After screening we had 242 lung adenocarcinoma cases and 277 cancer-free controls that were eligible. Selected demographic variables and environmental risk factors for the cases and controls were listed in Table 3."
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" fasassociated factor faf1 has been implicated in parkinsons disease pd and activates the celldeath machinery in the cytosol however the presence of extracellular faf1 has not been studiedmethods serumfree conditioned medium cm from faf1transfected shsy5y cells was concentrated andanalyzed by western blotting exosomes were isolated from cm by ultracentrifugation and analyzed by westernblotting electron microscopy and nanop tracking analysis soluble faf1 from cm was immunodepleted usingantifaf1 antibody transmission of secreted faf1 was examined by transwell assay under a confocal microscopecminduced cell death was determined by measuring propidium iodide pi uptake using a flow cytometerresults faf1 was secreted from shsy5y cells via exocytosis and brefeldin a bfaresistant secretory pathwaysfurthermore faf1 was secreted as a vesiclefree form and a genuine exosome cargo in the lumen of exosomes inaddition faf1 increased the number of exosomes suggesting a regulatory role in exosome biogenesis extracellularfaf1 was transmitted via endocytosis to neighboring cells where it induced cell death through apoptotic andnecrotic pathwayss this study presents a novel route by which faf1 induces neuronal death through celltocelltransmissionkeywords faf1 secretion exosome vesiclefree form celltocell transmission cell death cells secrete proteins harboring signal peptides throughthe classical secretory pathway via the endoplasmicreticulum ergolgi complex from which vesicles withcargo proteins move toward and fuse with the plasmamembrane and subsequently export cargos to the extracellular space however proteins lacking signal peptides are secreted via alternative nonclassical secretorypathways these pathways are classified as vesicularand nonvesicular secretory pathways some proteins correspondence eunheecnuackr gyeongrin park and bokseok kim are considered cofirst authorsdepartment of biological sciences chungnam national university daehakro yuseonggu daejeon south koreaare secreted via extracellular vesicles including exosomesand other vesicles of various sizes [ ] alternativelyother proteins are secreted through membrane poresand atpbinding cassette abc transporters althoughthe exact mechanisms of nonvesicular secretory pathways are elusive [ ]exosomes which are nanosized membrane vesicles nm in diameter secreted into the extracellular environment by various cell types are associated with intercellular communication with neighboring cells and play arole in a myriad of pathological functions in diseases including cancer cardiovascular and neurodegenerative diseasestheextracellular matrix and promote metastasis angiogenesisin particular exosomes[]remodel the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpark cell communication and signaling page of thrombosis and tumor cell proliferation in cancer exosomes in cardiovascular disease display proangiogenesis procoagulant and proinflammatory effects on thevessel walls in neurodegenerative diseases exosomesare potential sources of key pathogenic proteins such astau amyloid prion and αsynuclein []inflammation triggersin addition to their vesiclemediated secretion manyproteins are secreted through nonvesicular mechanismsfor instance the proteins such as tau and fibroblastgrowth factor are recruited to the plasma membrane toform lipidic pores and are subsequently secreted [ ]in additionthe secretion ofinterleukin1 and transglutaminase through pores fibroblast growth factor translocates across the plasmamembrane via poorly understood mechanisms includingthe membrane release complex upon stress proteinssuch as hydrophilic acylated surface protein b are secretedby abc transporters proteins secreted via nonvesicular secretory pathways are advantageous over cargo proteins in vesicles as immunotherapeutic targets because ofthe antibody accessibility of the extracellular spacefasassociated factor faf1 is involved in diverse biochemical processes including cell death inflammation cellproliferation and proteostasis [] consistent with itsdiverse functions faf1 has been implicated in certain diseases first faf1 plays a tumor suppressive rolethrough activation of the apoptotic machinery and nfκbsuppression [ ] faf1 also suppresses tumor metastasis via tgf signaling moreover faf1 expressionis downregulated in various cancers including lung colonliver prostate brain ovarian and breast cancers second faf1 is involved in parkinsons disease pd as it iscolocalizes with αoverexpressed in pd patientssynuclein and acts as a substrate of parkin furthermorefaf1 mediates the degeneration of dopaminergic neuronsthrough apoptosis and parthanatos []faf1 is an intracellular protein present mainly in thecytosol to date faf1 secretion has not yet been reportedherein we uncovered for the first time that faf1 is secreted via an ergolgiindependent pathway specificallyfaf1 is secreted through exosomal and nonvesicular pathways in shsy5y cells in addition faf1 augments thenumber of exosomes suggesting the involvement of faf1in exosome biogenesis furthermore extracellular faf1moves into neighboring cells via pinocytosis and clathrinmediated endocytosis transmitted faf1 induces cell deathvia apoptosis and necrosis collectivelythese resultspresent a novel measure by which faf1 propagates itsdeath function through celltocell transmissionmethodsreagents and antibodiesthe following reagents and antibodies used in this studywere purchased commercially tnfα from abfrontierseoul south korea zietdfmk caspase8 inhibitormouse antiha antibody and rabbit antigm130 antibody from abcam cambridge uk ²²dichlorofluorescin diacetate dcfhda hydrogen peroxide h2o21methyl4phenylpyridinium mpp propidium iodidepi polydlysine brefeldin a bfa gw4869 monensin cycloheximide chx necrostatin1 nec1 dpqproteinase k pk dynasore heparin heparinase iiimouse antiactin and mouse antiflag antibody fromsigmaaldrich saint louis mo usa ²6diamidino2phenylindole dapi horseradish peroxidase hrpconjugated antimouse antibody and hrpconjugatedantirabbit antibody from thermo fisher scientific incrockford il usa mouse antiflotillin1 from bd biosciences san jose ca usa bafilomycin a1 mouseantialix antibody mouse antigalactosidase antibody401a rabbit anticalregulin antibody mouse anticd63 antibody mouse antiparkin antibody and mouseantifaf1 antibody from santa cruz biotechnologydallas tx usa mouse antihsc70 antibody andmouse antihsp90 antibody from enzo life sciencesfarmingdale ny usa and zvadfmk zvad fromcalbiochem darmstadt germanycell culture and transfectionshsy5y mef hek293 raw2647 hela panc1mia paca2 and mcf7 cells were maintained in dulbeccos modified eagles medium dmem welgenedaegu korea containing fetal bovine serum fbsatlas biologicals fort collins co usa and antibioticantimycotic gibco brl grand island neusa unless otherwise specified cells were transfectedwith the indicated plasmids using bio t manvillescientific inc manville nj usa following the manufacturers protocol rat midbrain cultures derived frompostnatal day were prepared using standard procedures briefly material dissected form the ventral portion of the midbrain was cleaned free of meningealtissue minced and enzymatically dissociated in a mixture of papaindnase sigmaaldrich dissociated cellswere plated onto aminecoated 6well plates bd biosciences cells were maintained in neurobasal mediumgibco with b27 serumfree supplements gibco mm lglutamine after days of culture the cells wereinfected using aav1 adenoassociated virus 1hfaf1viral vectors moi of and sitedirected mutagenesisthe sirnaresistant parkin construct was generatedusing quikchange sitedirected mutagenesis kit stratagene la jolla ca usa the primers were as follows²gagctgagaaacgactggactgtgcagaattgtg3²²gggaaggagctgagaaacgattgcactgtgcaga ² 0cpark cell communication and signaling page of rna interferenceall small interfering rnas sirnas against parkin andscrambled rna scrna were purchased from bioneerdaejeon south korea the sequences of the sirnasused in this study were as follows sirna against parkin²ugaggaaugggacugu3² thescrna orsirna were transfected into shsy5y cells using lipofectamine rnai max thermo fisher scientific according to the manufacturers instructionspreparation of conditioned mediumto prepare conditioned medium cm cells transfectedwith the indicated plasmids were cultured in mmdiameter dishes in dmem containing fbs after h the cells were switched to serumfree dmem forthe indicated times here we used serumfree mediumto avoid interference from albuminenriched fbs thenthe cm was collected and centrifuged at ¨¯g for min and ¨¯g for min to remove cellular debrisfor western blot analysis the cm was concentratedusing kda or kda cutoff amicon ultra filtersmillipore billerica ma usa at ¨¯g for min or minwestern blot analysiscells were harvested washed twice with pbs and lysedwith mammalian lysis buffer [ mm triscl ph mm nacl mm edta nonidet p40 mmphenylmethylsulfonylfluoride] then the protein concentrations were quantified by using a biorad proteinassay kit biorad hercules ca usa after quantification samples were boiled in à protein sample buffer[ mm triscl ph glycerol sds mercaptoethanol] then samples were electrophoresedby sdspage and transferred to nitrocellulose membranes ge healthcare maidstone uk the membranes were blocked with skim milk in pbs with tween20 pbst and incubated with the indicatedprimary antibodies overnight after their washing withpbst the membranes were incubated with secondaryantibodies immunoblot signals were measured by usingchemiluminescent detection lab frontier anyangkoreachx chase assayshsy5y cells were transiently transfected with the indicated plasmids for h after transfection the cells wereswitched to serumfree dmem containing chx sigmaaldrich μgml for the indicated of times subsequently the medium was concentrated with kda cutoff amicon ultra filters millipore and analyzed bywestern blot analysispurification of exosomeswe followed a previously described protocol with somemodifications briefly cells were transfected withthe indicated plasmids for h and incubated in dmemcontaining exosomedepleted fbs system biosciences palo alto ca usa for h the cm was thencollected and subjected to sequential centrifugation at¨¯g for min ¨¯g for min and ¨¯g for min at °c to remove cellular debris using a beckmancoulter optima l90 k ultracentrifuge with a type 41tirotor the supernatant was then spun down at ¨¯gfor min the pellet was resuspended in pbs and thenspun again at ¨¯g for min at °c finally thepellet was resuspended in pbs or radioimmunoprecipitation assay ripa buffer sigmaaldrichin addition to their isolation via ultracentrifugationwe isolated exosomes using exoquicktc system biosciences according to the manufacturers instructionstreatment of vesicles with na2co3to separate integral membrane proteins and luminalproteins purified exosomes were treated with mmna2co3 ph for min at °c as previously described after centrifugation at ¨¯g for minintegral proteins remained in the pellet fraction whileluminal proteins remained in the supernatant fractionthe pellet fractions were resuspended in ripa buffersigmaaldrich and the supernatants were collected ina separate tube for western blot analysisproteinase k digestionpk sigmaaldrich was added to the samples at a finalconcentration of μgml then the samples were incubated at °c for min and mm phenylmethylsulfonyl fluoride was added to inhibit the activation of pkfollowed by the addition of protein sample bufferimmunodepletion of cmfifty microliters of protein gconjugated dynabeadsthermofisher scientific was incubated overnight withmouse monoclonal antibody against faf1 final concentration of or μgml before its addition to cm theantibodydynabeads complex was incubated with mlof cm at °c overnight after the complex was removedusing a magnet the immunodepleted cm was concentrated using a kda cutoff amicon ultra filter andused for western blot analysis for flow cytometryunconcentrated immunodepleted cm was applied to recipient cellsflow cytometryto evaluate cell death we measured pipositive cellstaining by using a guava easycyte flow cytometermillipore briefly cells were switched to serumfree 0cpark cell communication and signaling page of dmem or neurobasal medium for the indicated timeadditionally to measure recipient cell death shsy5yand rat primary neuronal cells were treated with cmfrom donor cells for the indicated times the cells were μgml and evaluatedharvested stained with piusing a guava easycyte flow cytometer following whichthe results were quantified using incyte softwaremilliporeconcentrated cm treatmentshsy5y cells donor cells were plated on mm tissueculture dishes and transfected with gfpvector or gfpfaf1 plasmid at h after transfection the cells wereincubated in serumfree medium for h after the cmwas concentrated by using kda cutoff amicon ultrafiltersthe concentrated cm was dissolved in newserumfree medium that was applied to recipient cellson polyllysinecoated coverslips in 12well plates for hpropagation assayshsy5y cells donor cells were plated on mm tissueculture dishes donor cells were transfected with gfpvector or gfpfaf1 plasmids at h after transfectionthe cells were collected and replated in cell culture inserts polycarbonate membrane μm pore size corning kennebunk me usa at a density of ¨¯ cellsshsy5y cells recipient cells were plated at a densityof ¨¯ cells on polydlysinecoated coverslips in well plates after h the cultures were combined suchthat the donor cells were in the insert and separatedfrom recipient cells plated on a coverslipconfocal microscopycells were fixed with paraformaldehyde for minthe cisgolgi were stained with gm130 after the nucleiwere stained with dapi for min the coverslips weremounted onto microscope slides using fluorescencemounting medium dako carpinteria ca usa andanalyzed using a zeiss lsm laser scanning confocalmicroscope carl zeiss oberkochen germanyelectron microscopyfor transmission electron microscopy tem sampleswere prepared using the exosometemeasy kit containing a formvarcarboncoated em mesh gridwash buffer and em solution bio mountain viewca usa the pellets from the ml of cm vector orfaf1transfected cells obtained by ultracentrifugationwere resuspended in μl of pbs μl of which was applied to the grid all samples were prepared followingthe manufacturers instructions for immunoem thepellets were first fixed with μl of paraformaldehyde and glutaraldehyde sigmaaldrich overnightat °c then the fixed exosome solution was transferredto grids and subsequently treated with m glycinefor min to quench free aldehyde groups after blocking with pbs containing bsa for min the gridswere incubated for h with the indicated antibodies diluted in pbs containing bsa atroomtemperature after three washes with pbs containing bsa the grids were incubated for h with the secondary antibody antimouse igg conjugated to nmgoldroomtemperature three washes to eliminate secondary antibody were followed by incubation with em solution anda wash step samples were viewed under a talos f200xtransmission electron microscope fei hillsboro orusa operated at kv and images were capturedwith a ceta m pixel cmos camera feisigmaaldrichatpsnanop tracking analysisfollowing isolation by differential ultracentrifugation orexoquicktc system biosciences the exosome pelletswere resuspended in μl of pbs then μl of theexosome solution was diluted in pbs to a total volumeof ml the samples were analyzed by nanoptracking analysis using a nanosight ns300 malvernpanalytical ltd malvern uk equipped with a nmlaser to accurately identify the vesicles the detectionthreshold was set at the number of vesicles in eachsample represents the number of ps per ml ofmedium cells were counted using a muse count viability kit millipore on a muse cell analyzer milliporecaspase3 activity assayshsy5y cells were treated with cm from faf1transfected cells plus caspase8 inhibitor or tnfα abfrontier plus chx sigmaaldrich at the indicated concentrations for the indicated times then caspase3 activity was measured by using a caspase3 colorimetricassay kit biovision milpitas ca usa in accordancewith the manufacturers protocol the absorbance at nm was measured with the use of a victor microplate reader perkinelmer norwalk ct usasignalp41we investigated the presence of a signal peptide in faf1using signalp41 httpwwwcbsdtudkservicessignalp41 with secretogranin1 used as a positive controlas it contains a signal peptidestatistical analysesexperiments were independently carried out three timesn all the data are expressed as the mean ± standarddeviation sd statistical comparisons were performedusing students ttest or oneway analysis of varianceanova followed by tukeys hsd post hoc analysis 0cpark cell communication and signaling page of using spss software statistics version ibm incchicago il usa statistical significance was established when the pvalue was lower than resultsfaf1 is secreted via nonclassical exocytosisaccording to the csf proteome resource faf1 is detected in the cerebrospinal fluid and plasma additionalfile this study aimed to determine whether faf1 isalso secreted at the cellular level and investigate faf1secretion in shsy5y human neuroblastoma cellsbecause faf1 is a deathpromoting protein sublethal experimental faf1 transfection conditions wereused to exclude cellular debris due to death additionalfile fig s1a and b the faf1 transfection conditionsin shsy5y cells under which faf1 secretion but notcell death occurs were determined by pi stainingfollowed by flow cytometry cells were transfected with3xflagfaf1 plasmid at a sublethal dose for h andsubsequently moved to serumfree medium faf1 wasdetected in the serumfree medium at h and accumulated henceforth indicating that faf1 is secreted in atimedependent manner fig 1a this finding suggeststhat faf1 is constitutively released from shsy5y cellsto exclude a tagging artifact another epitope taghemagglutinin ha was used hafaf1 was alsodetected in the cm conditioned medium in a dosedependent manner additional file fig s1c furthermore we examined the faf1 secretion with rat primaryneurons using aav1 adenoassociated virus 1mediated faf1 overexpression faf1 overexpression in ratprimary neuronal cells demonstrated consistent resultsin shsy5y cells fig 1b moreover with thatgalactosidase was not detected in the cm of galactosidasetransfected cells demonstrating that therelease of faf1 is not a transfection artifact fig 1c next a pulsechase experiment using chx to inhibit de novo protein synthesis was performed consistently faf1 was secreted and accumulated over timefurther confirming that faf1 is constitutively secretedfrom shsy5y cells fig 1d next we examinedwhether faf1 is also secreted by other cells faf1 wasdetected in the extracellular space of mefs and hek293cells furthermore faf1 was present in the cm of eachof a number of various cancer cell types mcf7 helapanc1 and mia paca2 cells this shows that faf1secretion is notadditional file fig s2afspecific to shsy5y cellsbecause faf1 has been implicated in pd pathogenesisfaf1transfected shsy5y cells were treated with thepdassociated stressors such as mpp h2o2 bafilomycin a1 and αsynuclein overexpression at sublethal dosesadditional file fig s3 there were no significantchanges of faf1 expression in clslysatescelldependent on various pdassociated stressortypeshowever faf1 secretion increased in cms upon allstressors used in this study implying that pdassociatedstressors positively regulate faf1 secretion fig 1eto elucidate the mechanism by which faf1 is secreted two sets of experiments were performed as follows first we examined whether faf1 is released viaexocytosis or passive diffusion as exocytosis is affectedby temperature faf1transfected shsy5y cellswere incubated at either °c or °c faf1 secretionat °c was significantly reduced compared to that at °cindicating that faf1 is released via exocytosisfig 1f second we examined whether faf1 is secretedvia the classical ergolgidependent secretory pathwayusing bfa which generates ros and disassembles golgithrough ergolgi pathway inhibition bfa did not affectfaf1 release fig 1g additional file fig s1d furthermore a signal peptide was not found in faf1when its sequence was analyzed using singalp41 furtherexcluding the possibility of ergolgimediated secretionof faf1 additionalfile fig s4 taken togetherthese data demonstrate that faf1 is released via nonclassical exocytosisfaf1 is secreted via exosomal and nonvesicular pathwaysto determine the mechanism by which faf1 is secreted exosomes were isolated from cm using a differential ultracentrifugation procedure as previouslydescribed and exoquicktc following the manufacturers protocol western blot analysis of exosomesisolated by ultracentrifugation revealed the presenceofthe exosome markers alix cd63 hsc70 andhsp90 but not calregulin an er resident protein a negative exosome control fig 2a exosomes isolated from shsy5y cells by exoquicktc showed anexosome marker profile consistent with that of exosomes purified by ultracentrifugation additional file fig s5a moreover nanop tracking analysisand tem data further confirmed that our purifiedexosomes exhibited a typical size distribution with adiameter ranging from to nm and a typicalmorphology of exosomes fig 2b and cendogenous as well as overexpressed faf1 proteinswere detected in the exosomal fractions isolated by bothmethods indicating that faf1 is secreted as a genuineexosomal cargo fig 2a similarly faf1 was also foundin exosomes isolated from the various indicated cell linesby exoquicktc additional file fig s5bh next weinvestigated whether faf1 is present on the membraneor in the lumen of exosomes exosomes were treatedwith mm na2co3 ph to distinguish betweenthe exosomal membrane and the lumen both alix andflotillin1 were present in the exosomal membrane positive controls whereas faf1 was mainly present in the 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is secreted via nonclassical exocytosis a shsy5y cells were transfected with vector control vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium for the indicated times se short exposure le long exposure b ratprimary neuronal cells were transduced with aav1hfaf1 viral vectors at days after transduction the culture medium was replaced withserumfree neurobasal medium for h c cells were transfected with lacz or 3xflagfaf1 plasmid at h after transfection the culture mediumwas replaced with serumfree medium and cells were cultured for h d cells were transfected with vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium containing chx μgml for the indicated times e left panel cellswere transfected with vc or 3xflagfaf1 plus αsyn plasmid at h after transfection the culture medium was replaced with serumfreemedium containing dmso vehicle mpp mm h2o2 μm or baf a1 nm for h right panel the graph shows the densitometricanalysis of immunoblotting of faf1 in conditioned medium cm shown in the left panel n f upper panel cells were transfected with3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium and the cells were cultured at °cor °c for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting in cm shown in the upper paneln g upper panel cells were transfected with 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium containing bfa μgml for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting incm shown in the upper panel n cell lysate cl and concentrated cm were analyzed by western blotting with the indicated antibodies alllanes were loaded with the same amount of total protein the data are expressed as the mean ± sd of three independent experimentsstatistical comparisons were performed using using anova followed by tukeys hsd post hoc analysis e and students ttest f and g p p p and ns not significanttheexosomeabolishedstructureslumen of the exosomes fig 2d the topology of exosomal faf1 was further examined using pk a nonspecificprotease to digest proteins outside of the exosomesexosomal faf1 but not cytosolic faf1 was protectedfrom pk treatment in the absence of triton x100 txfig 2e tx in contrast pk treatment with tx disintegratingtheexosomemediated protection of faf1 fig 2e txour data demonstrated the presence of faf1 in thelumen of exosomes furthermore the presence of faf1in theconfirmed with theimmunogoldlabeled faf1 under anvisualization ofelectron microscope as shown in fig 2f immunogoldlabeled cd63 was mainly present outside exosomeswhereas immunogoldlabeled faf1 was present in thelumen of exosomes collectively these results consistently showed that faf1 is located in the lumen ofexosomeslumen wasexosomalbecause certain exosomal cargo proteins are alsosecreted via the nonvesicular secretory pathway [] the possibility that faf1 is also secreted viaa nonvesicular route was investigated to this endthe cm was fractionated into exosomal pellet andnonexosomal supernatant fractions by ultracentrifugation faf1 from the cm was present in nonexosomal as well as exosomalfractions fig 2gimplying the presence of a soluble form of faf1 tofurther investigate this cm from faf1transfectedshsy5y cells was treated with antifaf1 antibodyone microgram of antifaf1 antibody almost eliminated faf1 from the cmindicating that faf1 ispredominantly secreted in a soluble form fig 2htaken together these results demonstrate that faf1is concurrently released as a bona fide cargo of exosomes and in a soluble form this new finding addsfaf1 to the list of proteins secreted by nonvesicularas well as exosomal routesrespectivelytransfection offaf1 positively regulates exosome numberthe exosomal cargos such as hsp20 hsp90 andstat3 increase exosome number [] here weexamined whether faf1 also participates in regulating exosome number the exosome markers hsc70alix and cd63 were increased by ± 009fold ± 013fold and ± 022foldinthe cm of faf1transfected shsy5y cells compared with control cells fig 3a additional file fig s6a next we further studied exosome numberchanges using sirnamediated depletion of parkina e3ubiquitin ligase of faf1 siparkin treatment elevated secretion as well as expression of faf1 inshsy5y cells moreoversirnaresistant parkin construct reverted the increased secretion and expression of faf1 by siparkin in shsy5y cells the expression levels of alix and cd63in the cm of shsy5y cells in which parkin hadbeen depleted were elevated by ± and ± 040fold respectively fig 3b collectivelythese data imply that faf1 positively controls exosome number for the direct quantification of exosome number nanop tracking analysis wasapplied the vesicle size distribution profile showinga diverse range of sizes showed that exosomes werepresent predominantly fig 3c the exosome numbers were normalized by cell number additionalfile fig s6b the exosomes of faf1transfectedcells were increased by 25fold compared to thoseof control cells hence these data robustly demonstrate that faf1 augments exosome numberinaddition gw4869 an exosome release inhibitor interfered with the ability of faf1 to increase exosome number while monensin an exosome releasepromoter enhanced this ability implying that faf1functions upstream of the exosome release processfig 3a [ ] 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is released to the extracellular space via both exosomal and nonvesicular pathways a shsy5y cells plated on mm dishes weretransfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with exosomedepleted medium andthe cells were cultured for h then exosomes were isolated from the cm by ultracentrifugation cl and isolated exosomes exos wereanalyzed by western blotting with the indicated antibodies calr calregulin b purified exosomes were characterized using nanop trackinganalysis c representative tem images of exosomes are shown scale bar nm left or nm right d the purified exosomes were treatedwith na2co3 after centrifugation at ¨¯g the integral membrane proteins were pelleted memb and nonintegral and luminal proteinsremained in the supernatant sol these fractions were analyzed by western blotting with the indicated antibodies e the purified exosomeswere incubated with pk μgml in the absence or presence of triton x100 tx tx with tx tx without tx f immunogold labelingof purified exosomes with anticd63 antibodies left and antifaf1 antibodies from vctransfected middle or 3xflagfaf1transfected rightcells scale bar nm g cells were transfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replacedwith serumfree medium and the cells were cultured for h after the cm was isolated by ultracentrifugation the supernatant sup and pelletexo were analyzed by western blot with the indicated antibodies h after immunoprecipitation of faf1 from cm with antifaf1 monoclonalantibody the immunodepleted cm was concentrated and analyzed by western blotting with the indicated antibodies all lanes were loaded withthe same amount of total proteinsecreted faf1 is transmitted to adjacent cellswe wondered whether extracellular faf1 is internalizedby neighboring cells donor cells were transfected withgfp or gfpfaf1 plasmid for h subsequently thecm which contained gfp or gfpfaf1 was concentrated and nontransfected recipient cells were treatedwith the concentrated cm for h confocal microscopic images showed the presence of gfpfaf1 in thecytoplasm of recipient cells indicating that gfpfaf1had moved from donor cells to recipient cells in contrast gfp from the cm of donor cells was not detectedin recipient cells excluding the effect of tagging ontransmission fig 4a similarly donor cells were transfected with 3xflagfaf1 plasmid as described abovedonor cellderived faf1 also moved into recipient cellsas shown by western blotting fig 4b corroborating theimmunofluorescence results in fig 4a in these data consistently demonstrate that secreted faf1can be internalized by neighboring cellsto further validate the celltocell transfer of faf1 anin vitro coculture system in which donor cells expressinggfpfaf1 were incubated in upper transwellinsertswhile nontransfected recipient cells were incubated inlower compartments was used consistent with theabove data confocal microscopy of recipient cells revealed the presence of gfpfaf1 indicating that gfpfaf1 moved from cells in the upper transwell inserts tocells in the lower compartments fig 4c consequentlythese results show that extracellular faf1 was transferred to neighboring cells without celltocell contactwe investigated the type of extracellular faf1 thatmoves into recipient cells to this end donor cells weretreated with gw4869 an exosome release inhibitorafter which recipient cells were analyzed by confocal microscopy gw4869 failed to inhibit faf1 transmissionto recipient cells to eliminate vesiclefree faf1 cm ofdonor cells was immunodepleted wit
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"elucidate the molecular mechanism underlying the involvement of abnormal DNA methylation in the development of glioma and identify potential newtargets for glioma therapyMethods The GSE79122 chip achieved from the Gene Expression Omnibus GEOdatabase containing glioma samples and normal samples was analyzed Methylationspeciï¬c polymerase chain reaction MSPCR or MSP reverse transcriptionPCR andWestern blot analysis were used to conï¬rm the methylation level and expression level ofthe interleukin receptorassociated kinase IRAK3 gene in glioma cells glioma samplesand the corresponding normal samples In vitro the proliferation apoptosis rate migrationand invasion abilities of glioma cells were detected by Cell Counting Kit8 assay Transwellassay enzymelinked immunosorbent assay and ï¬ow cytometry respectively Besides thexenograft assay of nude mice was used to conï¬rm the effect of the IRAK3 on glioma in vivoResults Microarray analysis showed that the IRAK3 was one of the most hypermethylated genesin glioma and the related mitogenactivated protein kinase MAPK signaling pathway wasactivated More experiments supported the higher methylation level and lower expression levelof the IRAK3 in glioma tissues and cell lines The viability migration and invasion ability ofglioma cells significantly reduced and the apoptosis rate increased with the overexpression anddemethylation of the IRAK3 in vitro Besides treatment with the MAPK signaling pathwayinhibitor PD325901 alone or the overexpression or demethylation of the IRAK3 had a similareffect as the overexpression or demethylation of the IRAK3 alone in glioma cells In vivoxenotransplantation experiments in nude mice conï¬rmed that the overexpression and demethylation of the IRAK3 and suppression of the MAPK signaling pathway inhibited the development ofgliomaConclusion IRAK3 inhibited the development of glioma progression through the MAPKsignaling pathwayKeywords glioma IRAK3 MAPK signaling pathway methylationIntroductionGlioma also known as glioblastoma is one of the most common primary malignant braintumors The average incidence rate of glioma is in individuals1 Despiteimprovements in neurosurgery and radiotherapychemotherapy most patients die within months of diagnosis1 and less than patients survive for years or even more2Recently the molecular mechanisms of glioma have gained increasing attention so as toï¬nd some better methods to defeat this disease Previous studies evaluated that longtermsurvivors of glioma often displayed some favorable molecular characteristics such as thesubmit your manuscript wwwdovepresscomDovePresshttp102147CMARS252772Cancer Management and Research Wu This work is published and licensed by Dove Medical Press Limited The full terms of this license are available at wwwdovepresscomtermsphpand incorporate the Creative Commons Attribution Non Commercial unported v30 License httpcreativecommonslicensesbync30 By accessing the workyou hereby accept the Terms Noncommercial uses of the work are permitted without any further permission from Dove Medical Press Limited provided the work is properly attributed Forpermission for commercial use of this work please see paragraphs and of our Terms wwwdovepresscomtermsphp 0cWu et alDovepresshypermethylation of O6methylguanine DNA methyltransferase MGMT promoter3 which is known as a meaningfulpredictive biomarker for the favorable prognosis of the chemotherapeutic drugs4 Therefore this study proposed that epigenetic regulation might play a key role in the development ofglioma which deserves further research for understanding thiscancerDNA methylation isan epigenetic modiï¬cationinvolved in many biological processes especially geneexpression regulation5 The DNA methylation patterns ofnormal cells are controlled well by a balance betweenmethylation and demethylation However this balance isalways disturbed in cancer cells through ectopic deï¬cientor excessive methylation leading to abnormal biologicalactivities6 Hypermethylation of CpG islands on speciï¬cpromoters inhibiting the transcription of downstreamtumor suppressor genes has been discovered in manycancers which may provide clinicians a new strategy forpatients with cancer7 For instance the promoter region ofSEPT9 is hypermethylated in colorectal cancer Hence theSEPT9 gene methylation assay might become a potentialoption for the early detection and screening of colorectalcancer8 CpG islands also display aberrant hypermethylation at a large number of loci and deï¬ne the subgroup ofglioma910 However the underlying molecular events ofDNA methylation and glioma development remain poorlyunderstoodRecently technical advances in genomewide expression analysis have enabled an improved understanding ofthe diagnosis and prognosis of many types of tumors11Genomewide DNA methylation analysis allows comprehensive DNA methylation proï¬ling of the whole genomehelping in the effective identiï¬cation of novel genes thathave a potential value in clinical practice12 Previous studies have reported many speciï¬c methylation signaturegenes in differenttypes of cancers such as thyroidcancer13 lung squamous cell carcinoma14 hepatocellularcarcinoma15 prostate cancer16 and so on using DNAmethylation analysisThis study aimed to examine the genomewide DNAmethylation proï¬ling of glioma tissuesrevealing thehypermethylation of several genes in glioma Then oneof these hypermethylated genes IRAK3 was selected toconductSubsequentlya relationship between IRAK3 and MAPK signaling pathway was demonstrated by using DNA methyltransferaseinhibitor overexpression of IRAK3 and MAPK signalingpathway inhibitor which can disrupt the development ofcomprehensiveaanalysisgliomas in vitro and in vivo The ï¬ndings might providesome clues for the regulatory role of DNA methylationand the underlying application of targeted treatment ingliomaMethodsTissue SamplesGlioma tissues and adjacent normal tissues were collectedfrom patients with primary glioma n admitted to theZhangye Peoples Hospital Afï¬liated to Hexi UniversitySample collection and use was performed according to theapproval of the ethics committee of the Afï¬liated Hospitalof Shandong University Written informed consent wasprovided by every patient with glioma All samples werefrozen in liquid nitrogen and stored at °C All humanspecimens were obtained with the approval by theInstitutional Ethics Committee of Zhangye PeoplesHospital Afï¬liated to Hexi UniversityCell CultureHuman gliocyte cell line HEB and glioma cell linesSHG44 U251 GOS3 HS683 and SF539 wereobtained from Bena Culture Collection Beijing ChinaHEB U251 HS683 and SF539 cells were grown in highsugar Dulbeccos modiï¬ed Eagles medium DMEMwith fetal bovine serum FBS SHG44 cells weregrown in RPMI1640 medium containing NaHCO3 gL glucose gL and sodium pyruvate gLwith FBS The GOS3 cells were grown with minimum essential medium with Earles Balanced Saltswith FBS and mML glucose All cells were cultured at °C under a humidiï¬ed atmosphere with CO2Cell TransfectionpcDNA31interleukin receptorassociated kinase pcDNA31IRAK3 was obtained from GenePharma ShanghaiChina and 5azadC 5aza2ʹdeoxycytidine 5azadCand signaling pathway inhibitor PD325901 were obtainedfrom SigmaAldrich MO USA U251 cells were seeded insixwell plates at cellswell and cultured at °C andthe cell conï¬uence reached CO2 untilTransfections were executed using Lipofectamine Invitrogen CA USA following the manufacturers protocols The medium was changed with the complete mediumafter h of transfectionsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressTable qRTPCR PrimersWu et alAccession NumberSequence 5ʹ3ʹForwardReverseForwardReverseForwardReverseForwardReverseACCCAAACTAACTGATTTTGCCAAGAGAAATTCCGAGGGCAGGCGACCTCCCATGGCAATTTTAACAGAGACAGGCATGGGAAGCCATCTCGACCAGTCCGTCTAGTTGGTCTGTCTCCGCTAAATACGGACTGCAGCCCTGAGGTCAATGAAGGGGTCGTGeneIRAK3MEK1CfosNM_007199NM_002755NM_005252GAPDHNM_008084GenomeWide DNA MethylationAnalysisFor DNA methylation proï¬ling Inï¬nium HumanMethylation450K BeadChip illuminaga_rnaseqv2100 was obtainedfrom the Gene Expression Omnibus GEO database TheDNA methylation data of chip number GSE79122 whichcontained glioma tissues glioblastomas diffuseastrocytomas and anaplastic astrocytomas and controlbrain tissues were analyzed The Inï¬nium MethylationAbbiotec CA USA and Illumina BeadStudio softwareGenetech Biotech Taipei Taiwan were used to measurethe methylation proï¬les of modiï¬ed DNA and loci CpGThe methylated signal intensity at particular CpG loci and450K BeadChip assay were presented as β values and percentage respectivelyQuantitative RTPCRTotal cellular RNA was extracted using PureLink Invitrogenfollowing the manufacturers protocol RT was performed on µg total cellular RNA using a HighCapacity cDNA ReverseTranscription Kit with RNase Inhibitor Applied Biosystemspurchased from Thermo Fisher Scientiï¬c MA USASubsequently quantitative reverse transcriptionpolymerasechain reaction RTPCR was performed using ArcturusParadise Plus qRTPCR Kit Applied Biosystems ThermoFisher Scientiï¬c Comparative expression values were calculated by the ÎÎCt method GAPDH was used for internalreference All primer sequences involved are listed in Table converted into uracil without changing the state of methylated cytosine The IRAK3 methylation level in glioma wasidentiï¬ed using methylationspeciï¬c PCR MSP The MSPprimers are listed in Table EnzymeLinked Immunosorbent AssayThe human interleukin IL6 enzymelinked immunosorbent assay kit Sangon Biotech Shanghai China was usedto test the IL6 level in the glioma cell culture medium Theglioma cell culture medium was centrifuged at rpm for min to remove cells and polymers The supernatant ï¬uidstored at °C was preserved in the supernatant ï¬uid at °C A normal glioma cell culture medium was used as thecontrolWestern Blot AnalysisLysis buffer RIPA Thermo Fisher Scientiï¬c and NPERThermo Fisher Scientiï¬c were used to extract proteinsfrom glioma cells and tissues respectively Then μg totalprotein per sample was separated using SDSPAGE andtransferred to polyvinylidene ï¬uoride membranes ThermoFisher Scientiï¬c The membranes were probed with primaryantiIRAK3 antibody ab8116 Abcam MA USA antiMEK1 phospho S298 antibody [EPR3338] ab96379 antiERK1 ERK2 antibody [ERK7D8] ab54230 anticFosphospho T232 antibody ab17933 and antiGAPDH antibody [6C5] ab8245 Abcam as control The number ofTable MethylationSpeciï¬c PrimersDNA Methylation AssayGenomic DNA in glioma tissues and cells was extracted andtreated with bisulï¬te using CpGenome DNA Modiï¬cationKit Chemicon CA USA following the manufacturersprotocol All unmethylated cytosine residues in DNA wereGeneIRAK3 forwardIRAK3 reverseIRAK3 forwardIRAK3 reverseSequence 5ʹ3ʹ5ʹTCGGGATAGTGGTTAATATTTC3ʹ5ʹTTTTTTTCGTTTTTCGTAAAA3ʹ5ʹ AGTTTGGGATAGTGGTTAATATTTT 3ʹ5ʹ TTTTTTTCATTTTTCATAAAAAAA 3ʹCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Genomewide methylation data were obtained from the GEO database for available glioma9 adjacent mucosa A Density of methylated DNA intensity byeach sample B Type I and Type II assays showed variant β value distributions The differences between probe types were regulated by normalization procedures whichshowed that represented unmethylated sites while represented fully methylated sites C Multidimensional scaling plot showing differential clustering of control versustumor tissues D Dendrogram produced for probes in normal and tumor tissues E Heatmap of top differentially methylated imprinted CpG sitesAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Distribution of top differentially methylated imprinted CpG sites A Distribution of top differentially methylated imprinted CpG sites according toCpG islands island sea shelf and shore B Distribution of top differentially methylated imprinted CpG sites according to the position relative to genes 1stexon ² UTRs or ² UTRs body IGR TSS1500 and TSS200 C Combined genetic and epigenetic annotation information revealed the distribution of the top differentially methylated imprinted CpG sitesbinding proteins was measured using AlphaEaseFC softwareGenetic Technologies FL USACell Counting Kit8 AssayU251 cells were seeded in 96well plates and allowed toadhere for h at °C Then these cells were transfectedwith pcDNA31IRAK3 and treated with 5azadC orPD325901 After h Cell Counting Kit8 DojindoKumamoto Japan was used to test the cell viability at respective time points To summarize mL of the triazoliumsubstrate was added to each well and coincubated with cellsat °C for h The absorbance was measured at nm andCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure DNA methylation analysis of tumor tissues and normal tissues and analysis of methylation degree for seven CpG sites of IRAK3 A The expression of the top candidate genes was analyzed IRAK3 was hypermethylated significantly in tumor tissues compared with normal tissues B The number of IRAK3methylated CpG islands ineach isosite CI Seven CpG sites for IRAK3 which are presented in the boxplot displayed a decreased methylation in the tumor group The boxplot for cg Ccg D cg E cg F cg G cg H and cg IAbbreviation MG malignant gliomasubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related signaling pathway A The top signaling pathways with the highest and lowest correlation with glioma B The top signalingpathwayrelated genes enriched in the glioma the MAPK signaling pathway was highly expressed C The MAPK signaling pathway was activated in glioma D The status ofthe top enriched signaling pathways in gliomaAbbreviation MG malignant gliomaCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressthe results were normalized to untreated cells at respectivetime pointsCell Migration and Invasion AssayBoth cell migration and invasion were performed using theTranswell assay For cell migration assay U251cells were seeded in a serumfree medium in the upperchamber which contained a polyethylene terephthalatemembrane with mm in diameter and μm in poresize The bottom chamber was prepared with FBS asa chemoattractant After incubating at °C for hnonmigrated cells were scraped from the upper surfaceof the membrane with a cotton swab and the cells migrating to the bottom chamber were ï¬xed with paraformaldehyde and stained with crystal violet Finally the stainedcells were counted under a microscope at magniï¬cation in ï¬ve randomly selected ï¬elds for quantiï¬cationinvasion assay U251 cells weresuspended in mL of serumfree DMEM and thentreated using the same procedure as for the migrationassay following the manufacturers protocol but the chambers mm BD Biosciences San Jose CA USA wereplated with BD BioCoat MatrigelFor cellFlow CytometryEach treatment of U251 cells was washed with phosphatebuffered saline and resuspended in μL of AnnexinV binding buffer After staining with Alexa Fluor Annexin V and 7AAD Viability Staining Solution for min in the dark at room temperature these cells were analyzed using multicolor ï¬ow cytometer Data were analyzedusing Kaluza softwarethe mice were keptXenograft StudiesMale BALBc nude mice weeks were maintained underpathogenfree conditions Allina temperaturecontrolled airconditioned conventional animal house with a h lightdark cycle and given free accessto food and water Besides animal health and behaviour weremonitored every two days All experiments were approvedby the Ethics Committee of Zhangye Peoples Hospital toguarantee that all studies involving experimental animalswere performed in full compliance with National Institutesof Health Guidelines for the Care and Use of LaboratoryAnimals The U251 cells100 μL cells were transfected with pcDNA31IRAK3 and then transferred to micen12 Normal U251 cells were transferred to mice in the5azadC and PD325901 groups and then 5azadC andPD325901 were subcutaneously injected respectively intothe posterior ï¬ank of nude mice After and daysculture the mice were sacriï¬ced and the tumor size wasmeasured The tumor volume was measured using a caliperfollowing the formula length width22Statistical AnalysisAll data were collected from three independent experiments and presented as mean ± standard deviationStatistical analyses were performed using GraphPad software Differences between groups were analyzed usingStudent' ttest or chisquare test Statistical signiï¬cancewas set at P ResultsGenome Methylation Proï¬le in GliomaA total of glioma tissues and adjacent normal sampledata publicly available at GEO were analyzed to revealthe global DNA methylation patterns of glioma Firstdensity plots of β values generated from each samplewere used identifying a poor performance of methylatedsignals for raw data Figure 1A Inï¬nium Type I and TypeII probe assays also showed somewhat different distribution of β value ranging from unmethylated sites to fully methylated sites Figure 1B Therefore these datawere regulated by normalization procedures to reduce thepotential impact later Multidimensional scaling MDSplot and dendrogram exhibited a differential clusteringbetween tumor and normal groups which distinguishedglioma from adjacent normal tissues Figure 1C and DFurther heatmap of the top differentially methylatedCpG sites indicated a visible difference in DNA methylation proï¬les between the tumor and normaltissueand general hypermethylation occurred insamplesglioma Figure 1EDistribution of Genomic Regions forDifferentially Methylated CpG SitesBased on the position relative to gene [1st exon ² untranslated region UTR ² UTRs body transcription start sites bp TSS1500 and TSS200] as well as CpG islands andneighborhood content shores shelves islands and sea the distribution of genomic regions for the differentiallymethylated CpG sites was exhibited More than and methylation differences occurred in bodyIGR and CpGisland sea respectively which was obviously higherthan that in other regions Figure 2A and B From thesubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Analysis of the IRAK3related gene expression level A Based on the differential genes in the disease data the distribution of upregulated genes anddownregulated genes in the GOrelated pathway was enriched B The expression level of top MAPK signaling pathwayrelated genesAbbreviation MG malignant gliomaperspective of both genetics and epigenetics gene and CpGcontent regions were combined for more analyses whichshowed that CpG sites in genetic bodyisland sea andIGRisland sea were differentially methylated betweenglioma and adjacent normal samples Figure 2CIRAK3 Was Hypermethylated in GliomaThe heatmap was used to present the top hypermethylationgenes in glioma compared with normal tissues so as to ï¬gureout the most characteristic gene with methylation change intumors indicating that the IRAK3 was hypermethylated theCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressmost in glioma Figure 3A Then the methylation differentialdistribution of the IRAK3 was upregulated at each siteFigure 3B Furthermore the β value of CpG sitesIRAK3such as cg cg cg cg cg cg cg andso on on the IRAK3 was significantly higher in the tumorindicating that IRAK3group than in the normal groupwas hypermethylated in the tumor group due to CpGsFigure 3CI The aforementioned results showed that theIRAK3 CpG sites were highly methylated in glioma tissuesthan in normal tissuestissues and the top signaling pathwayrelated genesenriched in the glioma were listed Figure 4A and B Theresults suggested that the MAPK signaling pathway washighly expressed and activated Figure 4C and D Based onthe differential genes in the disease data the distribution ofupregulated genes and downregulated genes in the GOrelatedpathway was enriched Figure 5A The expression level ofMAPK signaling pathwayrelated genes in glioma is shown inFigure 5B The results of Figures and manifested thatIRAK3related MAPK signaling pathway was highly activatedin glioma However whether any correction existed with thedevelopment of glioma remained to be veriï¬edMAPK Signaling Pathway Expression Leveland State in GliomaFor the identiï¬cation of gliomarelated signaling pathwaysthat might be inï¬uenced by aberrant DNA methylation thetop IRAK3related signaling pathways in glioma and normalMethylation and Expression Level of theIRAK3 in Glioma Tissues and CellsThe impact of hypermethylation on the expression of IRAK3in glioma tissues and cells was elucidated The IRAK3 wasFigure Methylation level and expression level of the IRAK3 in glioma tissues and cells A The IRAK3 was highly methylated in glioma tissues compared with adjacent tissues BThe IRAK3 was less expressed in glioma tissues than in adjacent tissues C The IRAK3 in the ï¬ve glioma cells SHG44 U251 HS683 SF539 and GOS3 was hypermethylatedcompared with U251 glioma cells D The IRAK3 was less expressed in the ï¬ve glioma cells than in normal glioma cells The difference was significant P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alFigure Effect on glioma cells after the overexpression and demethylation of the IRAK3 A The expression level of the IRAK3 was significantly higher in the glioma U251 cells of thepcDNA31IRAK3 and 5azadC groups than in the control group B and C The protein expression level of the IRAK3 significantly increased in the pcDNA31IRAK3 and 5azadC groupscompared with the control group D The expression level of the inï¬ammatory factor IL6 significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with thecontrol group E The activity significantly decreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantlyincreased in the pcDNA31IRAK3 and 5azadC groups compared with the control group H and I The migration capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups compared with the control group J and K The invasiveness capability was significantly lower in the pcDNA31IRAK3 and 5azadC groups than in the the control group Allthe mentioned differences were significant P The IRAK3 had a suppressive effect on glioma cells in vitroCancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepresshighly methylated and less expressed in glioma tissues than inadjacent normal tissues Figure 6A and B Next the IRAK3 inï¬ve glioma cell lines SHG44 U251 HS683 SF539 andGOS3 also showed hypermethylation and less expressioncompared with normal gliocytes Figure 6C and D U251cells were selected for subsequent experiments because themethylation level of the IRAK3 in U251 cells was the highestFigure 6COverexpression or Demethylation of theIRAK3 Inhibited the Development of GliomaCellsA systematic test with the overexpression or demethylation ofthe IRAK3 was conducted to understand whether the correctionof abnormal IRAK3 methylation and expression affected thedevelopment of glioma After using pcDNA31IRAK3 tooverexpress or 5azadC to demethylate the IRAK3 the proteinexpression level of the IRAK3 in glioma U251 cells increasedin the tumor group compared with the normal group Figure7AC IL6 an inï¬ammatory factor secreted by the MAPKsignaling pathway [PMID ] so that its level in theculture medium could be used to represent the status ofMAPK signaling pathway activation decreased in thepcDNA31IRAK3 and 5azadC groups compared with thecontrol groups Figure 7D Surprisingly the viability ofglioma cells significantly decreased while the apoptosis rateincreased Figure 7EG and the migration and invasivenesscapability decreased in the pcDNA31IRAK3 and 5azadCFigure Effect on glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The expression level of MAPKsignaling pathwayrelated genes expression level was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group B and C Theexpression level of MAPK signaling pathwayrelated proteins significantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the controlgroup D The level of inï¬ammatory factor IL6 was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group E The activitysignificantly decreased in the PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group F and G The apoptosis rate significantly increased inthe PD325901 pcDNA31IRAK3 and 5azadC groups compared with the control group All the mentioned differences were significant P P submit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et algroups Figure 7HK which elucidated the associationbetween IRAK3 and the development of glioma The aforementioned results veriï¬ed that the overexpression or demethylation of the IRAK3 inhibited the development of glioma cellsin vitroMAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited the Development ofGlioma CellsThe MAPK signaling pathway inhibitors PD325901pcDNA31IRAK3 and 5azadC were used alone or incombination in vitro to conï¬rm the inï¬uence of the IRAK3and MAPK signaling pathways on glioma In PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901groups the mRNA and protein expression levels of theMAPK signaling pathwayrelated genes such as MEK1ERK and cFos as well as the level of IL6 decreased inglioma Figure 8AD The cell viability significantlythe apoptosis rate increased Figure 8EGdecreasedand the migration and invasiveness capability significantlydecreased in PD325901 pIRAK3PD325901 and 5azadCPD325901 groups Figure 9AC The aforementioned results veriï¬ed that the MAPK signaling pathwaysuppression alone or combined with the overexpression ordemethylation of IRAK3MAPK Signaling Pathway SuppressionAlone or Combined with theOverexpression or Demethylation of theIRAK3 Inhibited Glioma in vivoFinally whether MAPK signaling pathway suppression aloneor combined with the overexpression or demethylation of theFigure Effect on the migration and invasiveness capability of glioma cells after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and5azadC A and B The migration capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group A and C Theinvasiveness capability was significantly lower in the PD325901 pcDNA31IRAK3 and 5azadC groups than in the control group All the mentioned differences weresignificant P Cancer Management and Research submit your manuscript wwwdovepresscomDovePress 0cWu et alDovepressFigure Effect on mice after treatment with the MAPK signaling pathway inhibitor PD325901 pcDNA31IRAK3 and 5azadC A The tumor in the transplantationPD325901 p IRAK3 PD325901 and 5azadC PD325901 treatment groups after and days B The tumor volume was smaller in the PD325901pcDNA31IRAK3PD325901 and 5azadCPD325901 groups compared with the control group C The tumor weight followed the same trend as the volume D Theexpression level of the IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups compared with thetransplantation control group E and F The protein expression level of IRAK3 was lower in the transplantation PD325901 pcDNA31IRAK3 PD325901 and 5azadC PD325901 treatment groups than in the transplantation control group All the mentioned differences were significant P P IRAK3 had a suppressive effect on glioma in vivo was studiedAfter transplanting glioma U251 cells treated with PD325901pcDNA31IRAK3 PD325901 or 5azadC PD325901the tumorigenesis significantly decreased compared with thatin the control group mice Figure 10AC Finally mRNAand protein expression levels ofthe MAPK signalingsubmit your manuscript wwwdovepresscomDovePressCancer Management and Research 0cDovepressWu et alpathwayrelated genes in xenograft glioma were detectedthey were found to be significantly decreased in thePD325901 pcDNA31IRAK3 PD325901 or 5azadC PD325901 group Figure 10DFDiscussionAccording to the World Health anization the overallsurvival of patients with cancer has increased significantlyover the years with the improvement in diagnosis and treatment However glioma is still a highly fatal disease1Therefore this study aimed to elucidate the detail of itsprogression mechanisms and search for new therapeutic strategies One recent study reported that the use of MGMTpromoter methylation test in hospitals had a strong inï¬uenceon the prognosis of glioma17 suggesting a significant clinicalapplication prospect of DNA epigenetic regulation Hencethe main focus of the present study was on the relationshipbetween DNA methylation and glioma IRAK3IRAK3 belongs to the IL receptorassociated kinaseIRAK family involved in inhibiting Tolllike receptorsignaling by changing the activity of other members ofthe IRAK family to decrease inï¬ammatory response1819Increasing evidence supported thatthe expression ofIRAK3 in tumorassociated macrophages led to compromised immune surveillance of cancer cells when it proï¬tably prevented excessive inï¬ammation contributing tocancer development Therefore the growth of transplantable cancer cells could be inhibited by enhancing hostimmune responses in IRAK3deï¬cient mice20 A smallnumber oftumor cellintrinsicIRAK3 could also support the progression of tumor cellsin colorectal and lung cancers depending on the dysfunctional innate immune system indirectly Interestingly theexpression of IRAKM in colorectal and lung cancers correlated with poor prognosisin patients with thesecancers2122 However little is known about the molecularmechanism of the IRAK3 in glioma This study revealedthe downregulation of IRAK3 glioma tissues and cellsthrough DNA methylation analysis which seemed to becontrary to the previous ï¬ndings on other cancers Thisstudy investigated whether a special signaling pathwayexisted that connected IRAK3 and glioma progressionshowed thatstudiesAn ongoing study has validated that all members of theIRAK family mediate the activation of MAPK and nuclearfactorκB NFκB signaling pathways23 MeanwhileIRAK3 a general negative regulator was conï¬rmed toinhibit MAPK and NFκB activation2425 Likewisethese results identiï¬ed that the MAPK signaling pathwaywas highly activated in glioma and its related geneexpression level was downregulated after overexpressionof the IRAK3 In fact upstream genomic events andordifferent extracellular stimuli could activate the MAPKsignaling pathway mediating a wide range of cellularprocesses26 The activation of the MAPK signaling pathway often led to abnormal cell growth and tumorigenesisThe predominant effect relied on the signal intensity andthe context or tissue in which the signal occurred2728Zhang revealed that aberrant DNA methylation inthe promoters of MMPTIMP axis genes upregulated theMAPK signaling pathway promoting the progression oftumor cells Correction of the abnormal DNA epigenotypes attenuated the migration and invasion of tumorcells in vitro and reduced tumorigenicity in vivo29Intriguingly these results were consistent with those ofthe present studyConsidering the reverse expression pattern of IRAK3between glioma and other cancers2122 different epigeneticmodiï¬cation was regarded as the major reason causing thisdistinction In many cancers the DNA methylation patternsbecame aberrant with tissue speciï¬city serving as diagnosticmarkers and therapeutic targets30 Hence based on thelower expression and the tumorigenic function of the IRAK3in glioma its epigen
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" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity []in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but its raw reads without coding sequences prediction and functional annotation third generation sequencing tgs is known for itskilobasesized long reads and is an outstanding strategyfor better understanding rna processing for exampleit can be used to analyze different transcript isoformsregulated by alternative splicing which greatly increasesthe repertoire of proteins lead to genetic and functionaldiversity and is prevalent in most eukaryotic anisms the long reads could also provide sequence information on genecoding regions for functional analysis atthe transcriptional level and thus can be applied to refine an assembled genome for better annotation however tgs could not quantify gene expression forthe moment and have a relatively high error rate thansgs the combination of tgs and sgs are able to solvethis problem and are highly recommended by most researchers with the transcriptome and genome data availablefunctional genomics research is being performed whichrelied heavily on gene expression analysis reversetranscription quantitative real time pcr rtqpcr hasbeen reported to be a very sensitive and accurate technique to analyze gene expression level but it requiresappropriate reference gene as an internal control tonormalize mrna levels between different samples foran exact comparison of gene expression [ ] anideal reference gene should be expressed at a constantlevel across various experimental conditions howeverstudies have shown that no single reference gene is universal for all experimental conditions [] therefore its necessary to estimate the stability of referencegenes under particular experimental condition beforeusing them for gene expression analysisin the present study to provide highquality and morecomplete assemblies in genome and transcriptome studies of phytolaccaceae a hybrid sequencing approachcombining the sgs and tgs technologies was carriedout first fulllength transcriptome of the invasive plantspecies of pameracana and an noninvasive exotic conicosandra was generated by singlemoleculegener prealtimesmrtsplicingevents simple sequence repeats ssr coding sequencesprotein annotations and long noncoding sequenceswere analyzed respectively at transcription level furtherthe stability of reference genes was evaluated in two phytolaccaceae species mentioned above and one nativecongener p acinosa by rtqpcr in order to facilitatefuture research on functional gene expressionsequencing alternativeresultsto classify the plant species these three phytolaccaceaemembers p americana p icosandra and pacinosa wereidentified by pcr and followed by sequences alignmentbased on sequences of second internal transcribed spacer its2 and the intergenic spacer of photosystem iiprotein d1 gene and trnahis gene of chloroplast genome psbatrnh table s1 the sequences of its2 andpsbatrnh in p americana that we employed had theidentity of with the sequences reported by chen in p icosandra the sequences of its2 had identity and the sequences of psbatrnh had identity with the results of chens in pacinosa 0cliu bmc plant biology page of similarity of its2 and identify of psbatrnh werefound isoforms afterremoving redundantsmrt sequencing data outputusing the pacific biosciences smrt sequencing protocol gb clean reads of invasive species p americana were obtained after preprocessing on the basis offull passes and sequence quality circular consensus sequences ccs with fulllength rate were obtained including fulllengthnonchimeric flnc sequences and highqualityconsensussequences from the high quality consensus isoforms transcripts alternative splicing events ssr complete coding sequences lnc rnasand annotated transcripts in p americana wereachieved similarly gb clean reads in p icosandrawere identified and ccs with fulllength rate flnc sequences as well as highquality consensus isoforms were filtered subsequently transcripts and alternative splicingevents were obtained whats more ssrs complete coding sequences lnc rnas and annotated transcripts were identified in picosandratable transcriptome analysisbased on the structure of achieved transcripts and alternative splicing events were identified in pamericana and p icosandra respectively transcripts of bp in total in p americana wereemployed for ssr analysis based on the sequence lengththat was more than bp including ssrs and ssrcontaining sequences similarly transcripts bp in total in picosandra wereemployed for ssr analysis and ssrs togetherwith ssrcontaining sequences were identifiedtable summary of fulllength transcriptome sequencingclean reads gbccsflncflnc flncccsconsensus isoformhigh quality consensus isoformtranscriptsalternative splicingssrcomplete coding sequenceslncrnaannotated transcriptsp americanap icosandrathe detail information about the number of sequencescontaining more than one ssr the number of ssrspresent in compound formation and the number of different types of ssrs were shown in table in additiontotal of complete coding sequences cds in pamericana and cds in p icosandra were identified by using transdecoder the length distribution ofpredicted proteins was shown in fig s1in pdatabasespecifically nrwith the eight protein databases sequence alignmentswere performed to annotate predicted proteins in total transcripts in p americana and tranicosandra were annotated separatelyscriptstable the number of annotated protein sequencesin p americana was similar with p icosandra under aparticularncbi nonredundant protein analysis revealed that approximately transcripts in p americana and transcripts in picosandra showed the highest sequencesimilarity with beta vulgaris fig go gene ontology assignment also suggested that similar amount ofsequences in the two plant species belonged to the sameterm and many were classified into cell part and cellterm of cellular component catalytic activity and binding of molecular function and metabolic process andcellular process of biological process fig cogclusters of orthologous groups of proteins annotationshowed that a large number of predicted proteins in thetwo plant species were linked to functional class r general function prediction only j translation ribosomalstructure and biogenesis t signal transduction mechanisms g carbohydrate transport and metabolism ando posttranslational modification protein turnoverchaperones fig s2 the result of eggnog evolutionary genealogy of genes nonsupervised orthologousgroup annotation indicated that most of the annotatedproteins in the two plant species were belonging to thefunctional class s function unknown fig s3 kogeukaryotic ortholog groups functional classificationsuggested that r general function prediction only ando posttranslational modification protein turnover andchaperones were the most abundant functional categories in the two plant species fig s4 these results indicated that most of the sequences obtained were trulyfunctional proteins and had a similar functional classification in p americana and its congener picosandraeven though more work is needed to identify sequencesthat regulated or involved in the invasion success of pamericana the annotation of predicted proteins provided necessary information for further studiesbesides the transcripts encoding proteins long noncoding rnas lncrnas were achieved lncrnas arereported to be key regulators in plant biological prolncrna in pameracana andcesses the number ofpicosandra was predicted by cpc coding potential 0cliu bmc plant biology page of table ssrs obtained from transcripts with more than bpsearching itemtotal number of sequences examinedtotal size of examined sequences bptotal number of identified ssrsnumber of ssr containing sequencesnumber of sequences containing more than ssrnumber of ssrs present in compound formationnumber of mono nucleotide ssrnumber of di nucleotide ssrnumber of tri nucleotide ssrnumber of tetra nucleotide ssrnumber of penta nucleotide ssrnumber of hexa nucleotide ssrcalculator cnci codingnoncoding index pfamand cpat coding potential assessment tool respectively in total lncrna in pamericana and lncrna in picosandra were predicted by all these fourmethods fig subsequentlytranscription factorstfs that are key components involved in the transcriptional regulatory system were predicted in p americana tfs of types were filtered and in picosandra tfs of types were predicted thesetwo plant species shared the first types of tfs butthe number of each type tf was not similar especiallyrlkpelle_dlsv c3h snf2 and camk_camklchk1 indicating the particular functions on transcriptregulation fig amplification performance of rtqpcrprimers designed for rtqpcr were evaluated by pcrfirst the primers which produced single ampliconwithout primer dimer were chosen for melting curveanalysis only primers which produced a single fragmentefficiencywereqpcr amplificationchosenforp americanap icosandraassessment the qpcr efficiency of each primer pair wasgenerated from a 10fold serial dilution of pooled cdnaand was shown in table the threshold cycle ct values of each reference genewere employed to evaluate expression level under different experimental conditions fig average ct valuesfor all the seven candidate reference genes ranged from to in which ef1α showed the highest expression level and 28s rrna had the lowest expression levelit was also suggested that ct values of βactin and tubulin fluctuated significantly across all the experimentalsamplesstability of candidate reference genesforto determine the appropriate reference genesnormalization in different experimental conditions theexpression data was analyzed by genorm normfinderand bestkeeper respectively table s2when expression stability of reference genes wereanalyzed in different tissues leaves stems roots andflowers of p americana 18s rrna and ef2 oftable number of proteins annotated via differential protein databasedatabasesp americanaannotated number ¤ length length ¥ p icosandraannotated number ¤ length length ¥ coggokeggkogpfamswissproteggnognrall 0cliu bmc plant biology page of fig homologous species distribution of p americana and p icosandra annotated based on the nr database a p americana b p icosandrapamericana were identified as the most suitable reference genes by genorm and normfinder and 18s rrnawas also suggested by bestkeeper pairwise variation valueof v23 was below the cutoff value of which meansthe combination of two reference genes were most suitable for gene expression normalization fig whentested in picosandra ef1α was recommended for normalizing gene expression analysis not only by genorm butalso by normfinder ef2 was also suggested by genormand bestkeeper in pacinosa ef1α was the best reference gene suggested by genorm and bestkeeper but 18srrna was recommended by normfinder the use of tworeference genes was suitable because pairwise variationvalue of v23 was below when pooled the data of different tissues from pamericana and picosandra togetheref2 was shown to be the most stable gene by all the threemethods when investigated the expression stability ofreference genes in different tissues of pamericana andpacinosa 18s rrna showed the best expression stabilityby genorm and normfinder while ef2 was referred asthe most stable one by bestkeeper however the combination of five reference genes was recommended bygenorm for v56 which was less than when thedata of different tissues from picosandra and pacinosawas put together ef1α was identified as the best oneby genorm and normfinder whereas ef2 was suggested to be the best stability reference gene by bestkeeper when set the data of these three plant speciesas a pool ef1α was suggested to be the most stableone by genorm and normfinder while ef2 was alsorecommended by genorm and bestkeeper accordingto these results it is very important to select the appropriate reference gene when analyze the gene expressionlevel among plant species 0cliu bmc plant biology page of fig classification of the transcripts annotated by the gene ontology gowhen analyzed the data among germinating seeds28s rrna and ef1α were identified as the best reference genes by genorm while 18s rrna was recommended by normfinder and gapdh was suggested bybestkeeper three reference genes were sufficient tonormalize gene expression for v34 was below inflowers only of these three plant species ef1α was confirmed by all the three methods the genorm analysisshowed that the value of v45 was below so fourreference genes in combination were suggested thesefig venn diagram of the number of lncrnas predicted by cpc cnci cpat and pfam a p americana b p icosandra 0cliu bmc plant biology page of fig classification of predicted transcription factorsresults indicated that when focusing on particular tissuesof different plant species the selection of reference genewas also very essentialwhen plants were infested by slitura 18s rrnashowed the most expression stability suggested by genorm and normfinder in different tissues of pamericanawhile ef1α wasby bestkeeper therevealedcombination of two reference genes was suggested bygenorm due to the value of v23 was less than 18srrna was also recommended by genorm in s liturainfested picosandra and ef1α was shown to be themost stable one by normfinder and bestkeeper fourreference genes in combination were recommended bygenorm 18s rrna was also identified as the besttable primers for rtqpcr analysisgene nametubulingene descriptiontubulin ef1αelongation factor 1alphaprimer sequence ²²f gtaaggaagccgagaattgr tcaacaacagtgtcagagaf tgaagaaggtcggatacaatr gtagacatcctggagtgggaphdglyceraldehyde3phosphate dehydrogenasef tggtgctaagaaggttattatcef2elongation factor 18s rrna18s rrnaβactinactin728s rrna28s rrnar2 linear regression coefficientr gagtgaacggtggtcataf gtatcaccatcaagtcaactgr acaatcaaccacaacaaggf acttcctcttctcgtatcattr tgttcagcatagactgtgaf atgctatccttcgtctggr tactcttggctgtctctgf tacgattggttacggacatr ttctcatcaacaacagcatatlength bppcr efficiency r2 0cliu bmc plant biology page of together 18s rrna showed the best stability in genormand normfinder while the expression stability of ef1αwas suggested by bestkeeper in pamericana and picosandra 18s rrna was identified as the best referencegene by all the three algorithms in pamericana andpacinosa 18s rrna and βactin were suggested bygenorm in picosandra and pacinosa while gapdhand ef2 were recommended by normfinder and bestkeeper respectively when take all the data of s liturainfested plant species into account 18s rrna exhibitedthe most stable expression suggested by genorm andnormfinder while ef2 was the gene with the most constant expression identified by bestkeeperdiscussionfulllength transcripts are fundamental resources forstructuralfunctional and comparative genomics research [ ] smrt sequencing has been acknowledged by enabling the generation of multikilobasesequences to improve genome and transcriptome assembly the fulllength cdna sequences generated areable to characterize the posttranscriptional processsuch as alternative splicing lncrna prediction and coding sequences for further gene functional studies basedon the fulllength transcriptome data generated about gb of clean data were obtained for pamericana andpicosandra respectively table accordinglythenumber of ccs flnc consensus isoforms highqualityfig rna transcription levels of seven candidate reference genesin p americana picosandra and p acinosa the expression level ofcandidate reference genes in total samples n was presentedas cycle threshold number ctvalue and explained by box andwhisker plots the asterisks represented the minimum and maximumct value the squares indicated the 25th and 75th percentiles andthe median was represented by a bar across the squarereference gene by genorm in plant species pacinosawhile tubulin was suggested by normfinder and 28srrna was recommended by bestkeeper the combination of three reference genes was appropriate by genorm when analyzed the data oftwo plant speciesfig pairwise variation analyzed by genorm to determine the optimal number of reference genes for accurate normalization a threshold valueof was suggested for valid normalization if the value of vnn pairwise variation is less than then n reference genes in combinationare recommended for gene normalization if the value of vnn is more than then vn 1n should be taken into account pam pamericana pic p icosandra pac p acinosa lsrf different tissues of leaves stems roots and flowers gs germinating seeds of these three plantspecies f flowers of these three plant species lsr different tissues of leaves stems and roots i infested by s litura of third instar 0cliu bmc plant biology page of isoforms transcripts alternative splicing events ssrscomplete coding sequeces lncrnas and annotated transcripts were analyzed providing basic transcriptomic information for further studiesreports have showed that fulllength transcriptome ofzea mays have greatly helped in refining gene annotation and revealed the complexity of gene expression inmaize similar analysis has also been conducted inshum bicolor whats more the world expansioncapability of cydia pomonella has been informed according to its genome information molecularmechanism of rapid growth and invasive adaptation ofan invasive species mikania micrantha has also been investigated according to itsreference genome therefore the fulllength transcriptome data of pamericana and picosandra will contribute to the genomic research and provide insights into invasive mechanism ofpamericana through comparative genomics study inphytolaccaceae speciesgenereliesonanalysisexpressionaccurate relative quantification of rtqpcr for furtherrobustnormalization by stably expressed reference genes tominimize error in the experimental process therefore suitable reference genes for the normalization ofrelative gene expression data in three phytolaccaceaespecies pamericana picosandra and pacinosa weresought under a diverse set of conditions these resultsdemonstrated the importance of validating referencegenes under the relevant experimental conditions forexamplein different tissues leaves stems roots andflowers of pamericana 18s rrna and ef2 were recommended to be the bestsuited reference genes and similar results were found in s liturainfested pamericanahowever even though the appropriate reference genesin picosandra were ranked according to the analyzed results of the three methods all the pairwise variationvalues were above the cutoff value of while thecombination of 18s rrna βactin ef1α and ef2 weremost suitable in s liturainfested picosandra ef2 andef1α have been considered as the ideal reference genesin pacinosa whereas the combination of 18s rrna βactin and gapdh were recommended after s litura infestation researches have also showed that no singlereference gene is stably expressed among different tissues of an anism such as the reference gene selectionin amygdalus persica solanum lycopersicum and glycine max [ ] whats more our results alsosuggested that reference genes identified based on transcriptome data should be confirmed by experimentalevidence in jainduced transcriptome of p americana28s rrna showed stable expression between exogenousjatreated and control plants ja signal pathway ofplants can be induced by lepidopteran herbivores infestation however 18s rrna and ef2 were identifiedas the most stable expression reference genes in pamericana after s litura infestationin order to conductthe gene expression analysisamong different plant species of phytolaccaceae the dataof the three plant species were also compared togetherwhen compared the data in germinating seeds of threeplant species various genes were recommended by thethree methods the combination of plant species underother experimental conditions showed that the pairwisevalues of almost all the combination were higher thanthe cutoff value of exceptthe combination ofpamericana and pacinosa where five reference geneswere recommended for data normalization as well as thecombination of sliturainfested pamericana and sliturainfested picosandra where three reference geneswere suggested these results indicated that no particular gene was expressed constantly across different plantspecies even though these plants are congeners therefore reference genes should be employed appropriatelyunder the relevant experimental conditionsthe research has provided transcriptomewide fulllength isoforms of pamericana and picosandraproviding insights into invasive success of pamericanaguidelines for selecting appropriate reference genesunder different tissues in one plant species or amongvaried plant species were recommended further no particular gene was expressed constantly under differentexperimental conditions indicating the necessity of reference gene identification these results would facilitatethe exploration of functional and comparative genomicsstudies in phytolaccaceae to better understand plantbiologymethodsplant and insect materialsplants of p americana °²n °²e p icosandra°²n °²e and p acinosa °²n °²eused in this study which was named m k and q firstwere collected in yunnan china sampling was permitted when conducted complying with locallegislationthe formal identification of the samples were conductedby chao chen botany major of laboratory of ecologyand evolutionary biology state key laboratory for conservation and utilization of bioresources in yunnanyunnan university according to flora of china vol5 flora of north america vol43 chinese virtual herbarium httpwwwcvhaccn and global plants on jstor httpplantsjstor dna identification was also employed according tothe its2 region of nuclear ribosomal dna one of themost widely used dna fragments in plant molecularsystematics at the generic and species levels and the 0cliu bmc plant biology page of chloroplast psbatrnh intergenic region all voucher specimens were maintained at an experimental fieldof laboratory of ecology and evolutionary biology statekey laboratory for conservation and utilization of bioresources in yunnan yunnan universitytissues of leaves stems roots and flowers from oneindividual plant of p americana or p icosandra werecollected individually from the wild in yunnan provinceand no permission is needed for collecting theses samples each sample was flash frozen in liquid nitrogen andstored at °c for further experimentsshop101732681taobaocomthird instar larvae of spodoptera litura were purchased from henan jiyuan baiyun industry co ltdchinaand then werereared on artificial diet in a climate chamber h at °c with light and h at °c without light for further usefor reference gene evaluation seeds of p americanap icosandra and p acinosa were collected first from thewild in yunnan province and no permission is neededthe seeds were sown separately in agar plates andcultivated in the climate chamber after d five germinating seeds of one plant species were collected togetheras one sample for subsequent experiments each plantspecies have three replications two weeks later othergerminating seeds of each species were transplanted intoplastic pots cm diameter and cm height withsoil jiangsu peilei matrix technology development coltd china and cultivated with adequate water in artificial chambers with same conditions as described abovefour months later leaves stems roots and flowers ofeach plant species were collected individually simultaneously six larvae s litura of third instar were employedto infest on p americana p icosandra or p acinosawith one insect per leaf control treatments were herbivore free after h infestation leaves stems and rootsof these three plant species were harvested individuallyall samples collected were flash frozen in liquid nitrogenand stored in °c for subsequent assays and threereplicates were conducted for each treatmentnucleic acid extraction and assaysgenomic dna was isolated from the leaves of differentplant species following protocols provided by dnaquickplant system tiangen biotech co ltd beijing chinathen it was employed as the pcr template for plantspecies identificationpurekitplanttotal rnas from different tissues was prepared usingrnapreppolysaccharides polyphenolicsrich tiangen biotech co ltd beijingchina according to the manufacturers instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number
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Cabozantinib is an oral multikinase inhibitor whose targets include vascular endothelial growth factor receptors MET and the TAM family of kinases TYRO3 AXL MER Cabozantinib is approved for patients with advanced hepatocellular carcinoma who have been previously treated with sorafenib based on improved overall survival and progressionfree survival relative to placebo in the phase III CELESTIAL study During CELESTIAL the most common adverse events AEs experienced by patients receiving cabozantinib included palmarplantar erythrodysesthesia fatigue gastrointestinalrelated events and hypertension These AEs can significantly impact treatment tolerability and patient quality of life However AEs can be effectively managed with supportive care and dose modifications During CELESTIAL more than half of the patients receiving cabozantinib required a dose reduction while the rate of treatment discontinuation due to AEs was low Here we review the safety profile of cabozantinib and provide guidance on the prevention and management of the more common AEs based on current evidence from the literature as well as our clinical experience We consider the specific challenges faced by clinicians in treating this patient population and discuss factors that may affect exposure and tolerability to cabozantinib IntroductionThere has been a marked increase in liver cancer deaths in recent years In there were a0 new cases of liver cancer worldwide and liver cancer accounted for almost deaths making it the sixth most prevalent cancer worldwide [] The most common primary malignancy of the liver is hepatocellular carcinoma HCC [] The frequency burden and etiology of HCC vary across geographic regions and populations but are linked to prevalence of predisposing chronic hepatic conditions such as Electronic supplementary material The online version of this s1152 contains supplementary material which is available to authorized usersKey Points Cabozantinib represents a treatment option for patients with advanced hepatocellular carcinoma who progress after sorafenibAdverse events associated with cabozantinib may be effectively managed with supportive care and dose modifications thereby allowing patients to continue treatment at the appropriate dose with minimum interruptionStudies of cabozantinib in the firstline setting are ongoing by understanding the safety profile of this drug clinicians will be able to balance efficacy with tolerability for each patient Gabriel Schwartz GabrielSchwartzucsfedu Gastrointestinal Medical Oncology Clinic University of a0California San Francisco Fourth St Fourth Floor San a0Francisco CA a0 USAIndiana University Health Simon Cancer Center Indianapolis IN USA Department of a0Medicine University of a0California San Francisco San a0Francisco CA USAIRCCS Istituto Clinico Humanitas Rozzano Milan Italy viral hepatitis and nonalcoholic fatty liver disease NAFLD or nonalcoholic steatohepatitis NASH which generally develop in the setting of cirrhosis [ ] In recent years the incidence of nonviral HCC has increased while the proportion of HCC cases related to viral hepatitis has declined [ ] Additional risk factors for HCC include alcohol consumption smoking obesity and diabetes [] As the epidemiology of these conditions has evolved so too has the etiology of HCC []Vol0123456789 0c G a0Schwartz et alFor patients with advanced HCC the vascular endothelial growth factor receptor VEGFRtargeting tyrosine kinase inhibitor TKI sorafenib has been a standard of care [] however the treatment landscape has been transformed in recent years with the introduction of newer TKIs immunotherapies and monoclonal antibody therapies [] This provides clinicians and patients with a variety of treatment options based on mechanism of action and safety profileCabozantinib is a multikinase inhibitor that targets VEGFR MET the TAM family of kinases TYRO3 AXL MER RET ROS1 KIT TRKB FLT3 and TIE2 [ ] several of which are implicated in tumor growth angiogenesis and immune regulation [] VEGFR MET and AXL have been implicated in the pathogenesis of HCC [] A capsule formulation of cabozantinib was first approved in for treatment of progressive metastatic medullary thyroid carcinoma MTC [] The tablet formulation not bioequivalent or interchangeable with the capsule [] was subsequently approved for patients with advanced renal cell carcinoma RCC [ ] and more recently for patients with advanced HCC who have received prior sorafenib [ ] The approval in HCC was based on outcomes from the pivotal phase III CELESTIAL trial which showed significantly improved overall survival OS and progressionfree survival PFS with cabozantinib relative to placebo in patients who received prior sorafenib [] The safety profile of cabozantinib was manageable nearly all patients receiving cabozantinib experienced an adverse event AE but these were effectively managed with dose modification and supportive care measuresClinicians treating patients with advanced HCC can face significant challenges as many patients present with cirrhosis and comorbidities that can impact treatment tolerability Adequate assessment of liver function and management of comorbidities are therefore essential before and during HCC treatment [] Here we provide guidance on the management of AEs associated with cabozantinib in patients with advanced HCC We briefly review outcomes from CELESTIAL and focus on managing some of the more common AEs experienced by patients based on current evidence from the literature as well as our own clinical experience Cabozantinib in a0Hepatocellular Carcinoma CELESTIALIn the phase III CELESTIAL study patients with advanced HCC were randomized to treatment with cabozantinib a0mg daily or placebo [] Patients were required to have had prior treatment with sorafenib and could have received up to two prior systemic regimens for HCC Eastern Cooperative Oncology Group ECOG performance status PS of or and ChildPugh class A liver function see Electronic Supplementary Table a0 for definition were also required At the second planned interim analysis patients had been randomized The study met its primary endpoint with significantly improved OS with cabozantinib relative to placebo median OS was versus months hazard ratio confidence interval [CI] p a0 a0 Cabozantinib also improved PFS with a median of versus months hazard ratio CI p a0 a0 as well the objective response rate per Response Evaluation Criteria In Solid Tumors RECIST v11 vs a0 p a0 a0 Safety and a0TolerabilityAllcause AE rates were generally higher in the cabozantinib arm than in the placebo arm some of the more common AEs experienced by patients in the cabozantinib a0 versus placebo arms included diarrhea vs decreased appetite vs palmarplantar erythrodysesthesia PPE vs fatigue vs nausea vs hypertension vs vomiting vs asthenia vs and increased aspartate aminotransferase AST vs Fig a0 The most common grade AEs in the cabozantinib versus placebo arms were PPE vs hypertension vs increased AST vs fatigue vs and diarrhea vs Overall the safety profile of cabozantinib was consistent with those from the phase III studies in RCC and MTC with gastrointestinal GI events PPE fatigue and hypertension being the most common AEs experienced by patients across studies [ ]In addition to supportive care measures protocolspecified dose modification including dose interruption and reduction was utilized to manage AEs [] Eightyfour percent of patients in the cabozantinib arm had an AE that led to dose interruption and had a dose interruption due to a grade AE [] Sixtytwo percent of patients had at least one dose reduction due to an AE [] and dose reduced due to a grade AE [] Thirtythree percent of patients had a second dose reduction [] Median time to first and second dose reduction in the cabozantinib arm was a0days and a0days respectively PPE was the event that most commonly led to dose interruption and dose reduction followed by diarrhea and and fatigue and [] Although most patients receiving cabozantinib required a dose interruption the rate of discontinuation due to treatmentrelated AEs was relatively low in the cabozantinib arm vs in the placebo arm indicating that the majority of AEs were adequately managed with dose modification and supportive care In the cabozantinib group AEs that led to treatment discontinuation in ¥ a0 of patients were PPE fatigue decreased appetite diarrhea and nausea In a subgroup analysis of patients 0cAE Any grade [Grade Grade ]Fatigue [ ]Hypertension [ ]Increased AST [ ]Increased ALT [ ]Asthenia [ ]Nausea [ ]Vomiting [ ]Decrease appetite [ ]Weight loss [ ]Diarrhea [ ]Constipation [ ]Abdominal pain [ ]PPE [ ]Fig Incidence rates for select AEs experienced by patients with HCC receiving cabozantinib during the CELESTIAL trial [] AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders AE adverse event ALT alanine aminotransferase AST aspartate aminotransferase HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesiawho received sorafenib as the only prior treatment for HCC duration of prior sorafenib did not appear to impact the types or rates of grade AEs []Generally the more common AEs emerged in the first weeks of treatment Fig a0 However clinicians should be aware of infrequent or serious events that can occur in the later phases of treatment Hemorrhagic events of grade or higher were reported in of patients in the cabozantinib arm including five patients with a grade event Bleeding complications are associated with antiangiogenic therapies and may arise as a result of reduced vascular integrity [] Median time to onset of hemorrhagic events was a0weeks in CELESTIAL Other grade or higher rare but serious AEs in patients receiving cabozantinib included fistulas of patients GI perforations and arterial and venous or mixed thrombotic events [] Median time to first occurrence was approximately a0weeks for GI perforations weeks for venous and arterial thromboembolisms and weeks for fistulas [] Two patients in the cabozantinib arm had developed ChildPugh C ie decompensated cirrhosis by the week assessment []Reversible posterior leukoencephalopathy syndrome RPLS a syndrome of subcortical vasogenic edema diagnosed by magnetic resonance imaging has been reported with cabozantinib and other TKIs [ ] Although there were no RPLS events during CELESTIAL [] clinicians should be aware of the symptoms which include headaches seizures confusion changes to vision or altered mental function [ ] Osteonecrosis of the jaw ONJ whereby necrotic jaw bone becomes exposed is another rare but serious AE associated with TKIs including cabozantinib [] although again there were no ONJ events reported during this study [] The use of antiresorptive drugs in patients with bone metastases is also associated with development of ONJ []A post hoc analysis estimated the incremental qualityadjusted lifeyears accrued with cabozantinib compared with placebo using the fivedimension fivelevel EuroQol questionnaire [] Cabozantinib treatment was associated with an initial decline in mean total qualityadjusted lifeyears during the first a0months relative to placebo followed by longterm improvement that was significantly greater than that observed with placebo p a0 a0Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c Fig Rates and timing of select AEs in patients with HCC receiving cabozantinib during the CELESTIAL trial The size of the circle is proportional to the AE rate AEs are color coded by system blue gastrointestinal purple skin and subcutaneous tissue green constitutional orange hepatic disorders red cardiovascularhematological disorders black generalother AE adverse event ATE arterial thrombotic event GI gastrointestinal GR grade HCC hepatocellular carcinoma PPE palmarplantar erythrodysesthesia VTE venous thrombotic eventMedian time to first dosereduction to mg weeksG a0Schwartz et alMedian time to seconddose reduction to mg weeksFistulas Hemorrhage GR ATEs VTE Wound complication GI perforations Hepatic encephalopathy Diarrhea PPE Hypertension Median time to first occurrence weeks Factors Affecting Tolerability of a0Cabozantinib ComorbiditiesHCC emerges primarily in older adults [] In addition to the underlying HCC etiology older adults with HCC are likely to have additional comorbidities such as cardiovascular or pulmonary disease [] and it is not uncommon for patients with HCC to have multiple comorbidities [] Liver cirrhosis with compromised liver function and decreased hepatic reserve is a major risk factor for HCC development Other HCCrelated comorbidities include hepatitis B virushepatitis C virus infection alcoholic liver disease NASH and diabetes [] In addition metabolic syndrome characterized by hyperlipidemia and hypertension is linked to development of NAFLD which may progress to NASH cirrhosis and finally HCC [] For patients with HCC assessment of liver function is a key step in treatment decisionmaking [] Patients with moderate or severe hepatic impairment are predominantly excluded from clinical trials in HCC therefore treatment of these patients is complicated by a lack of prospective clinical data as well as competing comorbidities []Although the number of patients with HCC and prior an transplant is limited these patients are generally excluded from clinical trials and treatment is complicated by the need for immunosuppression TKIs may be used to treat posttransplant HCC recurrence although supporting data are limited The use of TKIs in these patients is complex so treatment decisions should involve collaboration between the oncology and transplant medicine care teams The use of sorafenib in patients receiving mammalian target of rapamycin inhibitorbased immunosuppression has been associated with an increased risk of fatal bleeding [ ] Immunotherapies are associated with an increased risk of an rejection in posttransplant patients [] Cabozantinib Clearance and a0ExposureTKIs are associated with high interpatient variability in clearance and exposure which may affect both efficacy and tolerability This variability may be due to a variety of factors including genetic background drugdrug interactions drugfood interactions and renal or hepatic impairment [] As evidenced by exposureresponse modeling patients with low clearance of cabozantinib may have higher exposure and an increased risk of developing certain AEs [ ] Awareness of these nuances may help clinicians to mitigate their effects thereby balancing efficacy with tolerability Hepatic and a0Renal ImpairmentAccording to pharmacokinetic analyses of patients with HCC and other tumor types mild hepatic impairment is predicted to have a minimal effect on cabozantinib exposure [] therefore adjustment of the recommended 60mg starting dose is not necessary for patients with ChildPugh A 0cliver function [ ] Data on the pharmacokinetics of cabozantinib in patients with moderate ChildPugh B or severe ChildPugh C hepatic impairment are limited [] As per the US Food and Drug Administration FDA prescribing information the starting dose of cabozantinib should be reduced to mg in patients with moderate hepatic impairment while cabozantinib is not recommended for patients with severe hepatic impairment [] Note that the European Summary of Product Characteristics SmPC does not recommend dose adjustments for moderate hepatic impairment owing to limited data [] For patients with HCC increased exposure due to hepatic impairment should be considered if intolerable AEs develop and dose modification undertaken as recommended Fig a0 [ ] Cabozantinib should be used with caution in patients with mild or moderate renal impairment owing to the potential for increased exposure although no dose adjustments are necessary Cabozantinib is not recommended for use in patients with severe renal impairment owing to lack of data on safety and efficacy in this population [ ] DrugDrug and a0DrugFood InteractionsGiven the range of comorbidities that may exist in patients with advanced HCC it is important to review all concomitant medications for potential interactions prior to initiation of treatment with cabozantinib Certain medications and foods have been shown to modulate the pharmacokinetics of cabozantinib which may in turn impact exposure levels efficacy and risk of AEs Cabozantinib is metabolized in the liver primarily by the enzyme cytochrome P450 3A4 CYP3A4 [] therefore CYP3A4 inhibitors or inducers may impact exposure examples of CYP3A4 inducersinhibitors are shown in Electronic Supplementary Table a0 Strong CYP3A4 inhibitorsinducers should be avoided in patients receiving cabozantinib If concomitant administration of a strong CYP3A4 inhibitor is necessary then the cabozantinib Recommended dose at initiation mg Except for¢ Patients with moderate hepatic impairment or coadministration of a strong CYP3A4 inhibitor initiate cabozantinib at mg ¢ Patients with coadministration of a strong CYP3A4 inducer initiate cabozantinib at mg Safety assessmentNo AEsGrade Grade AE or ONJSupportive caresee Tables DosemodificationImprovementtolerableelbarelotnIlitnu esod dloHgrade ¤Continue at tolerated doseReduce dose by mg and restart mg mg mg mg mg mg mg mg or discontinueImmediate Discontinuation¢ Severe hemorrhage¢ Development of GI perforation or unmanageable fistula¢ Serious thromboembolic event eg myocardial infarction cerebral infarction¢ Hypertensive crisis or severe hypertension despite optimal medical management¢ Nephrotic syndrome¢ Reversible posterior leukoencephalopathy syndromeFig Cabozantinib dosing algorithm [ ] AE adverse event CYP3A4 cytochrome P450 3A4 GI gastrointestinal ONJ osteonecrosis of the jawManagement of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et aldose should be reduced by a0mg for example from to a0mg [] Conversely the cabozantinib dose should be increased by a0mg if strong CYP3A4 inducers need to be coadministered [ ]Cabozantinib should not be taken with any food as this may affect absorption [] The label recommends that cabozantinib be taken at least h before or at least h after eating [] Grapefruit and grapefruit juice are strong CYP3A4 inhibitors and should be avoided [ ]Cabozantinib may be used with caution in patients who are receiving concurrent antiarrhythmics or other QTprolonging agents [] This is based on a study of patients with MTC who received a daily 140mg capsule dose of cabozantinib recommended for this indication in which the mean deltadelta QT interval was increased by approximately a0ms with upper CIs not exceeding ms [] Such an increase is within the range considered to be acceptable for oncology drugs in this setting [] No patient in the aforementioned study or in CELESTIAL had a confirmed QTcF QT corrected using Fridericias method a0 ms [] which is considered clinically significant [] For patients receiving cabozantinib monitoring with periodic electrocardiogram and electrolyte measurements may be advisable particularly in patients with risk factors such as cardiac disease or a prior history of QT prolongation [] Concomitant use of proton pump inhibitors PPIs such as esomeprazole does not affect cabozantinib exposure levels [] However PPIs may cause hypomagnesemia which is linked to an increased risk of QT prolongation [] Therefore coadministration of PPIs and cabozantinib should be undertaken with caution following an individualized assessment of the patients baseline magnesium levels and concomitant medications that may also influence QT Pretreatment AssessmentsGiven the heterogeneity of the HCC patient population and the complexity associated with comorbidities and concomitant medications all patients should undergo a comprehensive assessment of medical history prior to initiation of treatment with cabozantinib Ideally the multidisciplinary care team should include an oncology pharmacist [] A brown bag medication review should be carried out prior to treatment initiation [] whereby the patient brings in all current medications including overthecounter medicines vitamins herbal remedies etc Therapeutic duplications should be eliminated for example concomitant PPIs and histamine H2 antagonists H2 blockers Switching and deprescribing should be considered where possible to minimize the risk of drugdrug interactions Adverse Event ManagementThe AE profile of cabozantinib is generally similar to that of other VEGFRtargeting TKIs with GIrelated AEs fatigue PPE and hypertension being the most common AEs [] Other AEs that occur less frequently can also have a significant impact on quality of life QoL and treatment adherence such as mucosal inflammation [] Hepatobiliary AEs such as elevated AST alanine aminotransferase ALT and bilirubin are particularly relevant in the context of advanced HCC and need to be carefully monitoredProphylactic and supportive care measures for the more common cabozantinibassociated AEs grade or tolerable grade are outlined in Tables a0 and discussed in the upcoming sections Symptom gradings are summarized in Electronic Supplementary Table a0 Dose interruption is recommended for management of intolerable grade AEs not resolved with supportive care measures or for any grade AEs Fig a0 [ ] Cabozantinib may be reinitiated at a reduced dose once the event resolves to grade ¤ a0 PalmarPlantar ErythrodysesthesiaPPE is one of the more common events associated with anticancer therapies including VEGFRtargeting multikinase inhibitors [] PPE is characterized by pain redness tingling and swelling of hands and feet [] Presentation may vary according to the etiologic agent PPE induced by TKIs is typically localized to pressurebearing areas in contrast to that caused by chemotherapy which has a more diffuse pattern It has been hypothesized that inhibition of multiple angiogenic pathways by TKIs may compromise repair of capillary microtrauma in areas exposed to mechanical stress such as the hands and feet [ ] Although not lifethreatening PPE can rapidly progress to a debilitating condition negatively impacting QoL [ ]Prophylaxis and prompt management of emerging symptoms may help to minimize the impact of PPE on QoL and adherence Table a0 Prophylactic measures predominantly involve skin care practices to remove hyperkeratotic areas and to minimize friction and damage prior to the start of treatment [ ] Recommendations include use of thick cotton gloves and socks padded insoles in shoes and avoidance of heat or friction on the hands and feet [ ] Patients with potentially predisposing comorbidities such as peripheral neuropathy [ ] as well as patients with persistent symptoms may benefit from involvement of a podiatrist andor dermatologist within their multidisciplinary care team [] Treatment strategies involve moisturization prevention of infection and analgesia [ ] Monitoring is crucial so that emerging symptoms can be proactively managed Patients should be assessed at baseline 0cTable Adverse event management strategiespalmarplantar erythrodysesthesia PPE PPEProphylaxisProvide education on prophylactic skin care before starting treatment []Advise manicure and pedicure before and during treatment to remove hyperkeratotic areas [ ]Protect sensitive areas recommend sunscreen with SPF protection ¥ a0 thick cotton gloves and socks padded insoles and wellfitting shoes avoid heat sources and use cooling aids and avoid activities that may cause force or rubbing on the hands and feet eg heavy lifting dish washing [ ] delegate such tasks to caregiversAdvise on optimal hand cleaning avoid fragrancedfoaming soaps and hand sanitizers containing alcohol ensure hands are dried thoroughly after cleaning []Prophylactically administer keratolytic cream eg urea [ ]Monitor regularly in order to proactively manage skin toxicities evaluate at baseline monitor up to weekly for the first months and monthly thereafter [ ]Supportive careContinue prophylactic measures []Maintain moisture of skin using emollients [ ]Consider topical treatment with salicylic acid urea cream either alone or with tazarotene cream or fluorouracil cream andor clobetasol cream topical analgesics may be added for pain control [ ]Topical cortisone and clobetasol may also be used consider oral analgesics eg NSAIDs pregabalin cautious use of opioids [ ]Consult with a dermatologist to drain blisters and remove hyperkeratotic areas []To prevent infection of cracked skin soak in equal parts vinegar and water for min per day [] a0Antibiotics should be prescribed only if there is evidence of infection [] a0There is limited evidence for the use of pyridoxine vitamin B6 []NSAID nonsteroidal antiinflammatory drug SPF sun protection factorTable Adverse event management strategiesfatigue FatigueProphylaxisProvide patient education about fatigue management tools and available support []Establish baseline fatigue levels with a fatigue scale and remeasure regularly during patient visits []Ensure adequate fluid and nutritional intake []Advise behavioral modifications balancing rest with physical activity recommendations include relaxation massage yoga aerobic or resistance exercise programs and energy conservation strategies []Assess thyroid function prior to treatment and monitor during treatment [ ]Supportive careRule out alternative causes of fatigue eg anemia endocrine disorders such as hypothyroidism pain dehydration hypercalcemia or depressionanxiety [ ]Advise patient to increase activity consider referral to a physical therapist []Consider referral to nutritional counselor for nutritional therapy []Incorporate psychosocial measures including cognitive therapy social support biofeedback and sleep therapy []Incorporate management with psychostimulants eg methylphenidate [ ] or corticosteroids eg methylprednisolone []Owing to effects on CYP3A45 substrates including cabozantinib longterm use of modafinil should be avoided []CYP3A4 cytochrome P450 3A4 CYP2C19 cytochrome P450 2C19Management of CabozantinibAssociated Adverse Events in Patients with Hepatocellular Carcinoma 0c G a0Schwartz et alTable Adverse event management strategiesgastrointestinal GastrointestinalDiarrheaProphylaxisInstruct patients to monitor food and fluid intake [] a0Recommended water intake per day from all beverages and food [] L oz for women L oz for men a0Advise patients to keep a stool diary and to promptly report diarrhea to their healthcare provider [ ]Advise patients to avoid foods that may cause GI events such as lactosecontaining foods caffeine highfat or highfiber food eg nuts seeds legumes and raw fruit and vegetables [ ]Implement dehydration prevention management through oral rehydration with electrolytes []Supportive careAdminister loperamide at the first sign of diarrhea [ ] a0 mg orally followed by mg every h until h after last bowel movement maximum of mg in h a0For chronic diarrhea mg twice daily titrated as needed a0Alternatives to loperamide include diphenoxylate and tincture of opium []Implement supportive dietary modifications continuous oral hydration correction of fluid and electrolytes small frequent meals avoid lactosecontaining food and drink [ ] a0The BRAT bananas rice applesauce toast diet may help to alleviate mild diarrhea []If there are signs of severe dehydration administer IV fluid replacement isotonic saline or balanced salt solution []Rule out nontreatmentrelated causes eg infectious diarrhea []Decreased appetiteProphylaxisAdvise patients to monitor their appetite and weight []Encourage patients to consume highprotein calorierich food fruit and vegetables nutritional supplements that they may snack on throughout the day [ ]Advise patients to preprepare and freeze nutritional preferred food []Supportive careTreat underlying nausea []Consider involving a dietitian who may recommend scheduled eating times []Recommend a highcalorie diet []Provide dietary education alongside dietary modifications andor nutritionalvitamin supplements []Use a pharmacologic agent to stimulate appetite such as a CB1 receptor agonist dronabinol [ ] systemic corticosteroid methylprednisolone [ ] progestin megestrol acetate [ ] or mirtazapine [ ]NauseavomitingProphylaxisAssess risk factors for nauseavomiting prior to treatment []Metoclopramide may be administered prophylactically []Advise patients to avoid foods that are overly sweet greasy fried or spicy []Supportive careAntiemetic agents such as dopamine receptor antagonists eg metoclopramide prochlorperazine or 5HT3 receptor agonists eg ondansetron are recommended for management of nausea or vomiting [ ] a0Certain NK1 receptor agonists eg aprepitant and netupitant and dexamethasone are inducers inhibitors andor substrates of CYP3A4 and thus could alter cabozantinib exposure [ ] however the potential for ondansetron to prolong the QT interval must also be considered [] There is moderate evidence for olanzapine an antipsychotic drug that blocks multiple neurotransmitters as an antiemetic in this setting [] 0cTable continuedMucosal inflammationstomatitisProphylaxisA comprehensive dental examination should be conducted prior to treatment to identify potential complications []Mitigation of potential risk factors [ ] a0Modification of illfitting dentures a0Appropriate care for preexisting dental problems such as caries ulcers etcRegular oral assessments should be conducted throughout treatment [ ]Educate patients on good oral hygiene and oral care protocols including written instructions [] a0The oral cavity should be washed using salinecontaining mouthwash up to four times daily and dentures should be regularly cleaned []Painful stimuli eg smoking alcohol hot fooddrink sharp or spicy food should be avoided [ ]Supportive careTreat pain with doxepin mouthwash or viscous lidocaine [ ]Lactobacillus lozenges may be used to reduce inflammation []Obtain bacterialviral culture if oral infection is suspected and treat infection as clinically indicated []5HT3 5hydroxytryptamine CB1 cannabinoid CYP3A4 cytochrome P450 3A4 GI gastrointestinal IV intravenous NK neurokininTable Adverse event management strategieshypertension HypertensionProphylaxisMonitor BP before initiation of cabozantinib using a minimum of two standardized BP measurements alongside patient history physical assessment directed laboratory evaluation and an instrument test to determine cardiovascular risk factors [ ]Educate patients on BP selfmonitoring and advise they keep a BP log []BP should be well controlled prior to initiating cabozantinib ensure patients who have already been prescribed antihypertensive therapy are adherent and that therapy has been titrated to effective doses [ ]Check for potential drugdrug interactions of existing antihypertensive agents with cabozantinib Supplementary Table a0Consider effects of concomitant medications on BP eg antiinflammatory drugs can increase BP opiates can lower BP []Monitor BP during cabozantinib treatment weekly during first cycle every ¥ a0 weeks thereafter []Supportive careAdd antihypertensive medications or increase dose of existing medication as indicated [ ]Patients with portal hypertension should be treated with nonselective betablockers []The antihypertensive agent should be carefully considered owing to potential inhibition of CYP3A4 [ ] Supplementary Table a0 a0Thiazides angiotensinconverting enzyme inhibitors and angiotensin receptor blockers may be used to treat hypertension and are not known CYP3A4 substrates [ ] a0Thiazide diuretics should be prescribed with caution owing to the associated risk of diarrhea [] a0Diltiazem and verapamil are moderate inhibitors of CYP3A4 [] a0Amlodipine felodipine lercanidipine nisoldipine and nifedipine are not considered to be CYP3A4 inhibitors []BP blood pressure CYP3A4 cytochrome P450 3A4and monitored at least weekly for the first months of treatment and monthly thereafter [ ] Close monitoring in the early stages of treatment need not involve weekly visitsphone calls from a clinician nurse or pharmacist may facilitate monitoring in between scheduled appointments [] Patients should be encouraged to report early signs of PPE to their healthcare provider [] it may also be reassuring for patients to know that early reporting and management of AEs
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"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh ï¬avor and health beneï¬ts and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneï¬cial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneï¬cial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health beneï¬ts and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2
egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg ï¬nally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals
egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe ï¬eld of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientiï¬c community and the general public indeedegcg has shown both prophylactic and therapeutic eï¬cacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table []apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoho2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site speciï¬city of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpspparð¾ cebpð¼aco pparð¼ cpt1acad pgc1ð¼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis []accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by ï¬ow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form bioï¬lms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibioï¬lm eï¬cacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results veriï¬ed the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the eï¬cacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microï¬orawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ ] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are ï¬nal products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for ï¬ve days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these ï¬ndings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o
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purpose to explore a new therapeutic option for patients with hepatocellular carcinoma hcc the efficacy and safety of a group of traditional chinese medicines banxia xiexin recipe as monotherapy for patients with advanced hcc was studied materials and methods the study included patients with advanced hcc from august to august for analysis these eligible patients received treatment with banxia xiexin recipe for at least month the primary endpoints were progressionfree survival pfs and overall survival os the secondary efficacy endpoints included objective response rate orr and disease control rate dcr in addition safety was also assessed results the median treatment duration of these patients was months range months and followup is still ongoing the median pfs was months confidence interval [ci] months and the median os was months ci months the orr was and the dcr was in the subgroup analysis the median os in the transcatheter arterial chemoembolization tace group was not reached and the median os in the no tace group was months ci months in addition no drugrelated serious adverse events were observed during the study this is the first clinical analysis of traditional chinese medicine as a single treatment for advanced hcc the obtained results are encouraging as they suggest that this panel of chinese herbs is safe and it may be effective for patients with advanced hcc in a realworld clinical settingkeywordshepatocellular carcinoma herbs tace banxia xiexin recipe clinical researchsubmitted december revised june accepted june introductionliver cancer is one of the most frequent malignant tumors and the third leading cause of cancerrelated deaths worldwide both the incidence and mortality rates for liver cancer are increasing while the overall incidence and mortality for all cancers combined tend to decline12 hepatocellular carcinoma hcc is the most common and deadly form of liver cancer in china chronic hepatitis b virus hbv infection and subsequent liver fibrosis and cirrhosis are the 1affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province chinathese authors contributed to the work equally and should be regarded as cofirst authorscorresponding authorshaoquan xiong department of medical oncology affiliated hospital of chengdu university of traditional chinese medicine chengdu sichuan province china email xsquan106163comcreative commons non commercial cc bync this is distributed under the terms of the creative commons attributionnoncommercial license creativecommonslicensesbync40 which permits noncommercial use reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages ussagepubcomenusnamopenaccessatsage 0c integrative cancer therapies leading causes of liver cancer due to the lack of effective treatment options as well as the high recurrence and metastasis rates hcc is associated with an extremely poor prognosis at present surgical resection and liver transplantation are effective treatments for liver cancer but only a few patients are surgical candidates due to tumor extension poor hepatic functional reserve or underlying liver cirrhosis34 even for the few lucky patients who have completed the surgery the recurrence rates at years following liver resection have been reported to exceed for patients with advanced liver cancer it is essential to improve general wellbeing promote quality of life and prolong survival time it is well known that chemotherapy effects in patients with advanced liver cancer are not outstanding67 some patients can consider local treatment such as transarterial chemoembolization tace the progressionfree survival pfs of patients receiving tace treatment is reported to be months8 molecular targeted drugs are also one of the options for patients with advanced liver cancer such as sorafenib and lenvatinib however according to reports on sorafenib the only approved molecular targeted therapy for advanced hcc the median pfs of patients treated with sorafenib was only months in the asiapacific region9 in recent years immunotherapy which has been attracting worldwide attention is also becoming an important treatment method for example nivolumab and pabolizumab which have been approved by the food and drug administration for secondline treatment of advanced hcc can bring certain benefits to patients with advanced liver cancer but are expensive and difficult to afford for many patientsin china traditional chinese medicine tcm therapies which can date back more than years are widely used in cancer treatment10 banxia xiexin decoction was created by the ancient doctor zhang zhongjing it is mainly used to treat digestive diseases such as bloating and abdominal pain it has a history of more than years in a previous retrospective study it was found that banxia xiexin decoction can bring survival benefits to patients with advanced liver cancer and one of them achieved complete response cr11 as a tcm hospital many patients with advanced liver cancer come to our hospital to undergo chinese medicine for treatment therefore we designed a prospective study of banxia xiexin decoction as monotherapy for advanced liver cancer and analyzed its treatment effect and safetymethodsstudy design and patient enrollmentin this prospective openlabel study patients from the oncology department of the affiliated hospital of the chengdu university of traditional chinese medicine chengdu china were recruitedaccording to the american association for the study of liver diseases criteria eligible patients were adults with advanced hcc confirmed by pathological assessment or noninvasive assessment12 these patients had not received any cancerrelated treatment within weeks before the start of study treatment and the disease had progressed after the prior treatment in addition these patients had to have at least one measurable lesion defined by modified response evaluation criteria in solid tumors for hcc mrecist and recist version inclusion criteria also included eastern cooperative oncology group ecog performance status of to an estimated life expectancy of months childpugh liver function class a or b adequate an function such as white blood cell count ¥ cellsl neutrophils ¥ cellsl and platelets ¥ cellsl and the absence of any serious comorbidities such as significant cardiac cerebrovascular hepatic or nephritic abnormities or other important an dysfunctionskey exclusion criteria included patients with low compliance or use of other antitumor treatments at the same time diagnosis of other malignant tumors and hcc with brain metastasisthe protocol was approved by the ethics committee of the affiliated hospital of chengdu university of traditional chinese medicine no 2015bl003 all patients provided written informed consent before study entrystudy treatment and proceduresthe enrolled patients received treatment with banxia xiexin recipe in the form of decoction the herbal components and their doses in the banxia xiexin recipe decoction are listed in the appendix with the use of an automatic herbboiling machine the decoction volume of each prescription was about ml patients were instructed to take ml each time times dailytreatment was continued until disease progression death development of unacceptable toxicity patients refusal to continue or at the discretion of the investigator when the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do sofollowing an initial baseline assessment within days of the start of study treatment the investigator assessed tumor response by using computed tomography ct scan after to months of treatment subsequently followup assessments were done every months until the patient died adverse events were recorded continuously from enrollment to the end of the final study visitoutcomesthe primary endpoints were pfs time from enrollment to radiological disease progression or death and overall survival os time from enrollment to death due to any cause the secondary efficacy endpoints included the proportion 0cwang et al of patients who had an objective response complete or partial response [pr] according to recist version namely objective response rate orr and disease control rate dcr the proportion of patients who had a cr pr or stable disease besides safety was also assessed by the incidence and nature of adverse eventsstatistical analysisthe pfs and os were estimated using the kaplanmeier method all statistical analyses were performed using graphpad prism software version graphpad software inc and logrank testresultsfrom august to august a total of patients with advanced hcc were enrolled from the oncology department of the affiliated hospital of chengdu university of traditional chinese medicine however only the results of the mediumterm analysis are presented here since patients were still on treatment and had not yet reached the assessment time point therefore patients were finally included in the current analysis meaningful results were found to report in this midterm analysis of course we will update the results according to the latest followup data in the future the baseline characteristics of the included patients are shown in table notably patients were without any treatment in this study all patients who were previously treated with surgery or tace had progressive disease which was similar to the clinical trials of sorafenib9 the number of patients receiving chemotherapy and molecular targeted therapy is and respectively patients who had received previous systemic therapy were excluded in the clinical trials of sorafenib but these patients had progressive disease after the prior treatment according to recist version in this studythe data cutoff date for the current analysis was august the median treatment duration of these patients was months range months and followup is still ongoing since if the disease progressed treatment could also be continued for month to assess tumor response as long as the patient agreed to do so or if thepatient would like to use it as an adjuvant treatment to other treatment such as tace moleculartargeted therapy and so onin the primary analysis the median pfs was months confidence interval [ci] figure and the median os was months ci figure thirtysix patients died all due to disease progression the 1year survival rate was notably most of these patients received prior treatments such as tace and targeted molecular therapy before enrollment in order to explore the potential effect of previous treatment we performed a subgroup analysis the median pfs months ci was longer in the subgroup of patients who had previously received tace compared with the median pfs months ci who had not previously received tace thus the treatment effect in this type of patient seems to be better p figure the median os of advanced hcc patients who had previously received tace was not reached and the median os of subgroups who had not received tace was months ci as shown in figure in terms of tumor response after treatment with banxia xiexin recipe patients achieved a pr had stable disease the orr was and the dcr was table particularly the imaging data of typical cases were presented as follows the first patient was a 58yearold male who was diagnosed with advanced hcc with metastasis to the right kidney on august at baseline the ct scan showed that the size of the target lesion in the liver was cm cm cm figure 5a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was this patient had not received any anticancer treatment previously after months treatment with banxia xiexin recipe the ct scan showed that the target lesion in the liver shrank to cm cm figure 5b and reached a pr according to recist version the ecog performance status score improved from to the patient is still currently in treatment and has not yet had progressive diseaseone 54yearold male patient was first diagnosed to have hcc in december he was treated by surgical resection on january no antineoplastic treatment was done thereafter when a regular review was conducted on september it showed intrahepatic recurrence and right lung metastasis and then he received treatment with chinese herbs at baseline the ct scan showed that the size of the target lesion in the liver was cm cm cm figure 6a blood tests revealed elevated αfetoprotein µgml and the ecog performance status score was after a 32month treatment with modified xiexin recipe the ct scan showed that the target lesion in the liver shrank to cm figure 6b and reached a pr according to recist version the duration of the pr was months he continued to insist on treatment after the disease progressed and he died in october concerning safety no adverse events were observed except for the occurrence of mild nausea three patients reported mild nausea after taking pills but the symptom was relieved spontaneously after week without any 0c integrative cancer therapies table the clinical characteristics of the patients with advanced hepatocellular carcinomavariablesnumber of patientspercentageage years ¥sex male femaleecogps childspugh class a bhepatitis b virus infection yes noliver cirrhosis yes noafp µgl ¥ tumor size cm ¥pvtt yes noextrahepatic spread yes noinvolved disease sites per patient ¥previous treatment no sur tace rf che mttajcc stage iiibivaivbabbreviations ecogps eastern cooperative oncology groupperformance status pvtt portal vein tumor thrombus sur surgical resection tace transcatheter arterial chemoembolization rf radiofrequency che chemotherapy mtt molecular targeted therapy ajcc american joint committee on cancerfigure kaplanmeier analysis of progressionfree survivalfigure kaplanmeier analysis of overall survivalfigure kaplanmeier subgroup analysis of progressionfree survival stratified by prior tace treatmenttreatment in addition no serious adverse events such as liver or kidney damage were detected during the followup period thus the tcm treatment was safe and welltolerated in these patients with advanced hccaccording to the international tumor chemotherapy adverse drug evaluation systemctcae v4014 patients included in this study were evaluated for safety during the followup the adverse reactions were nausea 0cwang et al control effect of tcm as monotherapy for patients with liver cancer banxia xiexin decoction has been used for treating digestive diseases for more than years in our previous retrospective analysis it was found that banxia xiexin decoction can relieve the symptoms of patients with advanced liver cancer inhibit tumor growth and prolong survival one of the patients tumors bulk was significantly reduced to reach cr status this state has been maintained for up to months11 therefore it is necessary to carry out further scientific researchto the authors best knowledge this is the first prospective study of chinese herbs alone for the treatment of advanced hcc that used the widely accepted endpoints of pfs os orr and dcr according to recist version in the present study the median pfs was months in clinical studies of sorafenib an oral multikinase inhibitor which is the first molecularly targeted drug approved by multiple countries around the world for firstline treatment of advanced hcc the median pfs was months in western countries19 and only months in the asiapacific region9 in current research work all patients were from china and in particular of patients had been diagnosed with liver cirrhosis when enrolled table the manifestations of liver decompensation such as splenomegaly and portal hypertension had appeared however the childpugh class was a or b it had been reported that hcc patients with liver cirrhosis showed significantly worse os than noncirrhotic hcc patients months vs months p even so the median pfs reached months and the median os reached months in a realworld clinical setting these findings suggest that the efficacy of the banxia xiexin recipe may be superior to that of sorafenib in patients with advanced hcc although this was not a headtohead comparison a further study directly comparing chinese herbs versus sorafenib for advanced hcc will be conducted in the futurein the reflect trial21 a subgroup analysis of the chinese population found that the os of the lenvatinib group was significantly longer than that of the sorafenib group by months with statistical differences in addition when combined with hbv infection the survival benefit was significantly different between the lenvatinib group and the sorafenib group os months vs months therefore it was approved by the china food and drug administration on september and approved for domestic listing it is important to note that the experimental conditions for entering the reflect trial were strict excluding ecog performance status patients with a score of or and a large liver mass however in patients enrolled in the present study had liver masses cm and had ecog scores of to nevertheless the median os of this study was figure kaplanmeier subgroup analysis of overall survival stratified by prior tace treatmenttable tumor response according to recist version variablesnumber of patientspercentagecrprsdpdorrdcrabbreviations cr complete response pr partial response sd stable disease pd progressive disease orr objective response rate dcr disease control ratevomiting diarrhea abdominal distension oral ulcers the painless mass of the scalp and so on the overall adverse reaction rate was all of which were grade i to ii adverse reactions and responded to symptomatic treatment there was no adverse reaction of grade iii or above and no drugrelated lethal events occurred specific adverse reaction events and their incidence rates are shown in table discussionit is wellknown that hcc is one of the leading causes of cancerrelated mortality worldwide the longterm survival rate remains unsatisfactory due to the high recurrence and metastasis rates after conventional treatment to improve the outcome of patients with hcc there is an urgent need for more effective therapies at present tcm is often used as an adjuvant therapy to conventional treatments such as tace in treating patients with liver cancer1518 to reduce the toxicities of other treatments relieving symptoms and improving quality of life tcm has only been considered as a complementary or alternative treatment for cancer patients and there is a paucity of data regarding the tumor 0c integrative cancer therapies figure a b the imaging data of one partial response patient diagnosed as advanced hepatocellular carcinoma with metastasis to the right kidneyfigure a b the imaging data of another partial response patient diagnosed as advanced hepatocellular carcinoma with intrahepatic recurrence and right lung metastasismonths therefore for patients with worse economic and physical conditions banxia xiexin decoction may be a better choice for many patients with advanced liver cancer in addition some other new drugs have been tried for the treatment of hcc unfortunately all phase trials assessing novel systemic drugs2224 have failed to improve outcomes over sorafenibin immunotherapy immune checkpoint inhibitors represented by pd1pdl1 monoclonal antibodies have made rapid progress the results of the checkmate040 study25 showed that nivolumab achieved an os of months in newly diagnosed patients and a 150month survival benefit in patients who had undergone sorafenib treatment pabrizumab which is also a pd1 immunosuppressant used in the keynote224 study26 for patients with advanced hcc who had previously progressed with sorafenib achieved a pfs of months median os of months compared with the enrollment conditions for keynote224 the condition of the patients enrolled in the present study are more complicated the largest diameter of the mass in of patients is ¥ cm of patients are in stage iv of patients had hbv infection and of patients had hepatic cirrhosis even so this study still achieved a pfs of months higher than the above chemotherapy and immune drugs it can be seen that the efficacy of banxia xiexin decoction for the treatment of advanced liver cancer may be better than the abovementioned immune drugs and it has the potential to become one of the important methods for liver cancer treatmentfurther subgroup analysis revealed that previous treatment might affect the efficacy of chinese herbs in the current analysis patients had been treated with tace previously whose outcome was slightly better compared with those who had not been treated with tace previously pfs vs months p in terms of os the difference is more obvious os undefined vs months p subsequent treatment with banxia xiexin decoction seems to have a greater benefit for patients with advanced liver cancer who have progressed with interventional therapyoverall the orr was and the dcr was in this study in comparison with the data reported in 0cwang et al table adverse reactionsgrade igrade iigrade iiigrade ivany grade grade iii nauseavomitingdiarrheabloatingulcersuperficial painless mass related literature the orr and dcr were respectively and in the orient trial of sorafenib919 it should be noted that the ecogps score of the included patients was to in our study but to in the orient trial indicating that tcm treatment may have a wider scope of application and may benefit more patients compared with targeted therapyin terms of safety no serious adverse events such as liver or kidney damage were observed during the followup period thus banxia xiexin recipe was generally safe and welltolerated in patients with advanced hccas for the antitumor mechanism of tcm it has been reported that tcm or its extracts have direct antitumor effects such as pinellia27 ginseng2829 coptis30 glycyrrhiza31 atractylodes macrocephala32 tangerine peel33 and scutellaria34 the effect of inducing apoptosis is also noted such as with berberine contained in coptis3537 the effect of regulating immunity is noted such as with pinellia27 ginseng3839 atractylodes macrocephala40 and milkvetch root41 antitumor vascular growth such as that of scutellarin4243 can inhibit protein kinase b akt phosphorylation downregulate vegf and inhibit tumor angiogenesis thus playing a role in inhibiting tumor growth and metastasis also it has been reported that the imbalance of intestinal flora not only affects the occurrence and development of intestinal cancer and irritable bowel syndrome but also is closely related to the occurrence of breast cancer lung cancer liver cancer and other malignant tumors4446 however atractylodes macrocephala scutellaria baicalensis and coptis4047 can indirectly inhibit tumors by regulating intestinal flora it is worth mentioning that much basic research has found that pinellia48 coptis4950 ginseng51 scutellaria52 atractylodes macrocephala53 and astragalus54 have antihepatoma effects for example berberine4950 a constituent of the extract of coptis in banxia xiexin decoction can inhibit the pi3kakt pathway of liver cancer downregulate mhcc97h and hepg2 cells phosphorylate the expression of akt and pi3k and inhibit the growth of liver cancer cells in a dosedependent manner besides it can induce cell cycle arrest and promote apoptosis to treat hccthere are only a few patients with tumor shrinkage in the present work it is believed that the direct antitumor effect of banxia xiexin decoction is not outstanding and there may be other mechanisms after analysis it was observed that the more prominent effect of banxia xiexin decoction on liver cancer is reflected in os which is similar to the efficacy characteristics of pd1 immune checkpoint inhibitors banxia xiexin decoction contains pinellia27 ginseng3839 atractylodes40 and astragalus41 medicinal herbs with immunomodulatory effects for example ginseng38 could relieve immunosuppression by increased viability of natural killer cells enhanced immune an index improved cellmediated immune response increased content of cd4 and ratio of cd4cd8 and recovery of macrophage function therefore it was speculated that liver cancer patients in this study may benefit from indirect antitumor mechanisms such as antivascular immunomodulation and others in addition some studies5556 confirmed that some ingredients of banxia xiexin decoction could improve liver function and regulate gastrointestinal function these comprehensive effects can also partially explain why liver cancer patients in this study seem to obtain longer pfs and osin china most patients with hcc have liver diseases such as hepatitis and cirrhosis among the patients enrolled in this study patients had a history of hepatitis b and patients had liver cirrhosis hcc and basal liver disease often affect each other and form a vicious circle the scutellaria baicalensis ginseng licorice tangerine peel and astragalus in banxia xiexin decoction all have liverprotective effects and licorice has a direct antihbv and hepatitis c virus effects5758 from this point of view this is also one of the reasons why banxia xiexin decoction may be effective in treating liver cancer although much basic research has confirmed the antitumor effects of single herbal extracts in banxia xiexin decoction a single herb may not have a good antitumor effect in clinical use even though the decoction affects liver cancer after being prescribed still each herb may play its respective role whether it causes a new antitumor effect or through compatibility with the effects of other herbs the antitumor mechanism is unclear and needs further studylimitationsthe report is limited by its singlearm research design and cannot be directly compared with standard chemotherapy 0c integrative cancer therapies targeted therapy or immunotherapy at the same time the small number of cases in this study is also one of the disadvantages but this study is still continuing and there will be more data analysis in the future due to the willingness of patients to choose tcm for treatment in tcm hospitals it is difficult to avoid certain limitations when we adopt the single treatment of tcm although oral chinese medicine decoction is difficult to take according to scientific equal dosage and concentration it is in line with the use habits of tcmsin this is the first prospective study of chinese herbs as monotherapy for the treatment of advanced hcc that used the internationally accepted endpoints of pfs os orr and dcr the findings are encouraging as they suggest that this panel of chinese herbs is safe and may be effective for patients with advanced hcc in a realworld clinical setting further studies are required to assess the comparative efficacy of tcm treatment versus other antitumor therapies in this patient populationappendixcomponents and their doses in the modified xiexin recipe decoction chinese latin and english nameschinese namelatin nameenglish nameplace of origin production batch dose gbanxiahuangqinganjiangrenshenhuangliandazaogancaobaizhuchenpihuangqirhizoma pinelliaeradix scutellariaerhizoma zingiberisradix ginsengrhizoma coptidisfructus jujubaeradix glycyrrhizaerhizoma atractylodis macrocephalaepericarpium citri reticulataeradix astragalipinellia tuberbaikal skullcap rootdried gingerginsengcoptisjujubae chinese datelicorice rootlargehead atractylodes rhizometangerine peelmilkvetch rootsichuanshanxisichuanjilinsichuanxinjiangsichuanzhejiangsichuangansuacknowledgmentsthe authors acknowledge the contributions of the other investigators in this trialauthor contributionslijuan wang jianlong ke and shaoquan xiong conceived and designed the study lijuan wang jianlong ke cui wang yaling li qiuyue luo rui cai qian ding panpan lv and tingting song collected the data lijuan wang jianlong ke and cui wang processed the data lijuan wang and jianlong ke wrote the manuscript all authors have participated in the drafting review and approval of the report and the decision to submit for publicationdeclaration of conflicting intereststhe authors declared no potential conflicts of interest with respect to the research authorship andor publication of this fundingthe authors disclosed receipt of the following financial support for the research authorship andor publication of this this research was supported by the national natural science foundation of china no and sichuan provincial science and technology department project no2017jy0327orcid idslijuan wang jianlong ke panpan lv orcid0000000317048847 orcid0000000150587581 orcid0000000271727659references torre la bray f siegel rl ferlay j lortettieulent j jemal a global cancer statistics ca cancer j clin doi103322caac21262 chen w zheng r baade pd et al cancer statistics in china ca cancer j clin doi103322caac21338 hung h treatment modalities for hepatocellular carcinoma curr cancer drug targets doi102174 vitale a trevisani f farinati f cillo u treatment of hepatocellular carcinoma in the precision medicine era from treatment stage migration to therapeutic hierarchy hepatology published online february doi101002hep31187 bruix j sherman m practice guidelines committee american association for the study of liver diseases management of hepatocellular carcinoma hepatology doi101002hep20933 lee dw lee kh kim hj et al a phase ii trial of s1 and oxaliplatin in patients with advanced hepatocellular carcinoma bmc cancer doi101186s1288501840399 0cwang et al zaanan a williet n hebbar m et al gemcitabine plus oxaliplatin in advanced hepatocellular carcinoma a large multicenter ageo study j hepatol doi101016jjhep201209006 wang y cui w raltitrexed based transcatheter arterial chemoembolization tace for unresectable hepatocellular carcinoma a singlecenter randomized controlled study chemotherapy doi104172216777001000192 cheng al kang yk chen z et al efficacy and safety of sorafenib in patients in the asiapacific region with advanced hepatocellular carcinoma a phase iii randomised doubleblind placebocontrolled trial lancet oncol doi101016s1470204508702857 anonymous miraculous pivot ling shu jing originally published during warring states period to bc peoples health press shaoquan x lijian w qiuyue l et al retrospective analysis of modified xiexin recipe combined retrospective analysis of modified xiexin recipe combined chin j integr tradit western med bruix j sherman m american association for the study of liver diseases management of hepatocellular carcinoma an update hepatology doi101002hep24199 lencioni r llovet jm modified recist mrecist assessment for hepatocellular carcinoma semin liver dis doi101055s00301247132 gao wj liu yy yuan cr international evaluation system for adverse events of chemotherapeutic drugs in cancer treatment ctcae v40 tumor meng mb cui yl guan ys et al traditional chinese medicine plus transcatheter arterial chemoembolization for unresectable hepatocellular carcinoma j altern complement med doi101089acm20080060 xu h deng y zhou z huang y chinese herbal medicine chaihuhuaji decoction alleviates postem
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"Ovarian cancer is the second most common gynecologic cancer with high mortality rate andgenerally diagnosed in advanced stages The 5year diseasefree survival is below MicroRNAs subset of thenoncoding RNA molecules regulate the translation in post transcriptional level by binding to specific mRNAs topromote or degrade the target oncogenes or tumor suppressor genes Abnormal expression of miRNAs were foundin numerous human cancer including ovarian cancer Investigating the miRNAs derived from the peripheral bloodsamples can be used as a marker in the diagnose treatment and prognosis of ovarian cancer We aimed to findbiological markers for early diagnosis of ovarian cancer by investigating BRCA1 gene mutation carrier monozygoticdiscordant twins and their high risk healthy family individuals miRNAsMethods The study was conducted on monozygotic twins discordant for ovarian cancer and the liquid biopsyexploration of miRNAs was performed on mononuclear cells that were isolated from the peripheral blood samplesThe miRNA expression profile changes in the study were found by using microarray analysis miRNA isolationprocedure performed from the lymphocyte in accordance with the kit protocol The presence and quality of theisolated miRNAs screened by electrophoresis Raw data logarithmic analysis was studied by identifying thethreshold normalization correlation mean and median values Target proteins were detected for each miRNA byusing different algorithmsResults After the comparison of monozygotic discordant twins for epithelial ovarian carcinoma upregulation of the miRNAs miR6131 miR1305 miR1973p miR3651 and downregulation of miRNAs miR3135b miR4430 miR664b5p miR7663p were found statically significantContinued on next page Correspondence hy2188istanbuledutrDepartment of Cancer Genetics Istanbul Faculty of Medicine OncologyInstitute Istanbul University Istanbul Turkey The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cTuncer Journal of Ovarian Research Page of Continued from previous pageConclusions The detected miRNAs out of miRNAs might be used in the clinic as new biological indicatorsin the diagnosis and follow up of epithelial ovarian cancer with complementary studies The miRNA expressionprofiles were identified to be statistically significant in the evaluation of ovarian cancer etiology BRCA1 mutationstatus and ovarian cancer risk in accordance with the obtained dataThere is a need for validation of the miRNAs which were particularly detected between monozygotic twins and itsassociation with ovarian cancer was emphasized in our study in wider cohorts including ovarian cancer patientsand healthy individualsKeywords Monozygotic twins miRNA expression profiles BRCA1 and BRCA2 BiomarkersBackgroundOvarian cancer is a significant cause of mortality in gynecologic cancers and one ofthe leading cause ofcancerassociated mortality [] In Turkey ovarian cancer is the 7th most common type of cancer in women inaccordance with worldwide Globocan datas showthat each year more than women are diagnosedwith ovarian cancer OC worldwide and approximately women die from it The data of Globocan for Turkey shows that annually women are diagnosed with ovarian cancer and women die fromthis malignancy The 5year survival rate was given as These data revealed that ovarian cancer is an important reason of gynecologic cancer associated mortality rate [] The epithelial ovarian cancersEOCoriginating from the ovarian surface epithelium constitutes approximately of ovarian malignancy [] Themajority of EOC patients are diagnosed in advanced stages Stage III and IV and year freesurvivalrate is below [] The standard treatment for newlydiagnosed ovarian cancer is the combination of cytoreductive surgery and platinbased chemotherapy Significant advances in radical surgery and chemotherapystrategies have improved clinical outcomes but unfortunately no progress has been made with relapse andtreatment resistance [] Ninety percent of ovarian cancer occurs sporadically in the population whereas hereditary type appears of ovarian cancer patientsBRCA1 and BRCA2 genes are the most common breastovarian cancer syndrome associated genes Both BRCA1and BRCA2 have roles in the control of the genomic stability cell cycle and apoptosis The mutations occurringin these genes result with the inability of DNA repairand therefore results in the accumulation of the mutations in the cell The rate of the breast cancer susceptibility of women with BRCA1 gene mutation until theage of years was and the rate of ovarian cancersusceptibility rate is breast cancer susceptibilitywomen with BRCA2 gene mutation until the age of years is and ovarian cancer development risk is [] Twin studies became important on genetics by theendandcentury Geneticnineteenthofthethe differentiated genesepidemiologic studies with monozygotic twins were accepted as highly useful investigation models in the pastdecades and have been used recently [] When a similarity for a disease or a quantitative feature betweenmonozygotic and dizygotic twins is compared variationsare excluded according to studies conducted in thepopulation and thereforeit is easier to identify andmake etiological differences visible via twin studies Because the affected siblings and dizygotic twins share theapproximately ofthephenotypic differences between twins are known to beassociated with the genetic variation In addition diversity may be revealed with a very limited patient population Therefore the results of the twin studies can beapplied to the population and can make valuable contributions to the genetic studies Monozygotic twins aregenetically similar and generally expected to be compatible for congenital malformations chromosomal abnormalities and Mendelian disorders There are numerousstudies conducted via discordant monozygotic twins revealing the genetic contribution [] Therefore investigating the genetic variability in monozygotic twins ishighly important and the majority of the human geneticsassociated research focus on finding genetic variability indiscordant monozygotic twins Phenotypically discordantmonozygotic twins are used as the model systems inidentification of the variable in understanding the pathogenesis of a disease The most striking study is the oneconducted with monozygotic twins in Canada and evidencing that multiple sclerosis MS was a genetic disease [] MicroRNAs are one of the subset of the noncoding RNAs generally consisting of single strand in nucleotide length not transformed to protein havingroles in post transcriptional regulation or suppression oftranslation of the target mRNAs [ ] The regulatoryroles of miRNAs were demonstrated to occur in tumorigenesis cell differentiation proliferation and apoptosis[] miRNA genes are known to locate in thechromosomal breaks This DNA breaks cause chromosomal abnormalities frequently associated with cancersusceptibility and tumor development [] The noninvasive biologicalindicators have been used for the 0cTuncer Journal of Ovarian Research Page of treatment resistance of ovarian cancer The most common of this indicators are the cancer antigen125 CA and cancer antigen153 CA153 These biological indicators can be used in the followup of thetreatment response in the diagnosed patients but cannotbe used in the early diagnosis and in differentiation ofthe malignant disease [] Therefore there is a need forspecial therapeutic agents customized for patients thatmay be used target specific therapies and in the earlydiagnosis of the ovarian cancer in identification of theefficacy of therapy and in the follow up period Thusstudies investigating the target molecules and biologicalindicators are required that will enable the early diagnosis and in the development of the better therapy optionsDifferentially synthesize miRNAs such as miR miR141 miR125b miR2223p or let7 family has beenshown in studies with ovarian cancer patients [] However the use of these miRNAs as a biomarker in ovariancancer is not yet available In order to clearly define therole of miRNAs in the pathogenesis of ovarian cancerwe planned to investigate the BRCA mutant monozygotic twins with the same genetic profile but with discordant for ovarian malignant transformation In thisstudy miRNAs which are thought to have the potential biological indicator role were studied from bloodsamples of both discordant monozygotic twins andBRCA wild type healthy siblingsMethodsPatients recruitmentThe peripheral blood lymphocytes of monozygotic twinsdiscordant for ovarian cancer and healthy individuals inthe same family were used in the study The patient diagnosed with ovarian cancer and all family members applied to the Cancer Genetics Clinic of OncologyInstitute of Istanbul University for BRCA breast cancersusceptibility gene testing were examined for BRCAgene mutation All family members in the study consisted of highrisk individuals with Hereditary Breast andOvarian Cancer HBOC syndrome and the people included in the study were given as BR codes according topatient file number The monozygotic ovarian cancer patient healthy monozygotic twin healthy sisters and niece were found to have BRCA1 gene mutation c5266dupC pGln1756Profs74 rs397507247 on exon Thepatients brother and daughter were found negative forBRCA1BRCA2 gene mutations In this study lymphocyte cells separated from peripheral blood belonging tototal of cases including younger age ovarian cancer patient and healthy monozygotic twin a patients daughter elder sisters a younger sister a nephew and a brotherwere examined by miRNA microarray method Thepedigree of the family included in the study and theirhierarchical cluster analaysis via Euclidean method isshown on Fig The study was approved by the Ethics Committee ofthe Istanbul Faculty of Medicine Following InstitutionalEthics Committee approval informed consents were obtained from all participants before enrollment into thestudy Ethics Committee Approval Number atstoredthey wereLymphocyte and miRNA isolationFicoll SigmaAldrich Darmstadt Germany density gradient was used to separate white blood cells mononuclear cells from other blood components miRNAisolation procedure was performed from the lymphocytein accordance with the kit protocol using the miRNeasyMini Kit Qiagen cat NoID The proceduresteps in accordance with the protocol are as follows μL QIAzol solution was included on the cells storedin nitrogen tank Cell fractionation was enabled by mixturing using the vortex For complete nucleoproteinfractionation °C roomtemperature for min By adding μL chloroformthey were shacked and mixed on hand The tubes wereincubated for min at °C room temperature thenwere centrifuged for min at °C and g Thesupernatant formed after centrifugation was transferredto collection tube using a pipette and was mixed usingvortex by inclusion of μL ethanol The supernatant formed after the ethanol centrifuging was transferred to collection tube was removed with a pipettewas mixed with vortex by including μL ethanol Seven hundred microliters was taken from the obtained mixture and was transferred to the RNeasyMiniElute spin colon placed on mL collection tubeThe tubes were centrifuged for s at g at °Croom temperature Seven hundred microliters RWT buffer was added to spin colons and the colons werewashed by centrifuging at g for sThe centrifuging procedure was repeated by including μL RPE buffer twice consecutively to colons The colons placed into clean tubes with mL were dried bycentrifuging min in maximum speed The colonsplaced in mL sterile tubes were included μL distilled water by centrifuging at g in min and themiRNAs were collectThe quality control of the miRNAsThe presence and quality of the isolated miRNAs werescreened by electrophoresis at V on Agarose gelThen the purity and concentrations of the miRNAswere measured on Thermo Scientific NanoDrop spectrophotometer NanoDrop Technologies Wilmington DE USA device The miRNA purity for each persondevicemeasurement result were obtained with the comparisonaccordance withthe NanoDropin 0cTuncer Journal of Ovarian Research Page of Fig The pedigree of the family included in the study and their hierarchical cluster analysis Legend The pedigree of the family and using thecorrelations between samples plotted a dendrogram for sample grouped by Hierarchical clustering Euclidean distance Complete Linkage BR Healthy Brother BRCA1 negative nonBRCA1 mutation carrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR Healthy Monozygotic twin BRCA1 positive BRCA1 mutation carrier BR Monozygotic twindiagnosed with ovarian cancer BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrier BR Healthy Sister BRCA1 positive BRCA1 mutation carrierof the measurements at spectrophotometrically at nm and nm wave lengths The measurement rates at nm wave lengths is a sign of quality of the purity of the samples therefore the samples in the idealvalue interval of and for RNA measurementswere included in the study The purity of miRNAs wereevaluated using a Bioanalyser device BioanalyserAgilent Technologies Santa Clara CA USA AgilentRNA Nano Kit Agilent Technologies Santa ClaraCA USA for confirming whether the miRNAs were appropriate and in adequate level for microarray analysisThe evaluated sample concentrations and results wereanalyzed The samples with RNA concentrations between ngμL and rRNA rate over and RNA integrity number values between and were evaluated asthe appropriate samples for array study 0cTuncer Journal of Ovarian Research Page of Microarray trial protocolMicroarray protocol was performed by preparing theSpikein solution sample marking hybridization sampledephosphorylation sample denaturation sample ligationhybridization of the samples slide loading preparationof the hybridization unit and elution and scanning ofslides The slide scanning procedure was performedusing the Agilent Microarray Scanner Agilent Microarray Scanner with Surescan High Resolution Technology Agilent Technologies Santa Clara CA USAdevice The scanning procedure of the slides were performed on SurePrint G3 Human miRNA MicroarrayRelease 8x60K Agilent Inc Santa Clara CA platform and using the Agilent Technologies G2600D scanning protocol The analysis of the TIFF Tagged ImageFile Format extensioned files obtained after scanningprocedure was performed using the Agilent Feature Extraction v11011 programThe success levels of stages developed in all experiment process with this analysis program the quality ofthe levels the process were monitored and evaluatedThen Bioinformatic Analysis procedure was performedData analysisRaw data logarithmic analysis was studied by identifyingthe threshold normalization correlation mean and median values Then the miRNAs demonstrating differentexpression profile among the samples were filteredUsing the Fold change rates and independent twosample T test the possible difference between the compared groups were evaluated All evaluations were performed to enable the cutoff values as the foldchangerates FC ¥ and pvalue Hierarchical clusteranalysis was performed using the Euclidean method Fig and Complete Linkage cluster method The control ofthe experimental errors and the detection of the erroneous finding rate were identified using the HochbergmethodBioinformatic analysisTarget proteins were detected for each miRNA by usingtwo algorithms Targetscan71 httpswwwtargetscanvert_71 and MirdbV5 httpsmirdbmiRDBThe targeted genes thought for each miRNA wereconfirmed by also both algorithms and the miRNAtarget relations were also experimentally confirmed mirTarbase70httpsmirtarbasembcnctuedutwphpindexphp databaseComparison groupsIn the study miRNA analysis was performed at the genome level withwithout mutation in cases withwithoutovarian cancer The miRNA data was evaluated by comparing different groups in order to investigate the effectof BRCA mutation in ovarian malignancy developmentand determine the miRNAs that can be important in theovarian cancer pathogenesis In Group the monozygotic twins discordant for ovarian cancer were comparedin order to find the effects of miRNAs in the formationof ovarian cancer In Group the family members withBRCA1 mutation were compared with family memberswithout BRCA1 mutation to identify the changes ofmiRNAs expression levels according to BRCA positivityIn Group the monozygotic ovarian cancer patient withBRCA1 mutation carrier and the other healthy familymembers with mutation carrier were compared for investigate the effects of both ovarian cancer developmentand BRCA positivity on miRNAs expression level InGroup all family members were compared with ovarian cancer monozygotic twin in order to find the miRNAs that might be important in the predisposition ofovarian cancer The comparison groups also showed inTable ResultsWe identified differentially expressed comparisonof miRNAs between the groups The raw data obtainedafter experimental studies were filtered before the comparisons between the groups The upregulated or downregulated miRNAs expression levels more than foldFC and smaller than the p value p wereconsidered in evaluation and the comparisons betweenthe groups were performed based on these values Allthese comparisons were evaluated for ovarian cancer etiology BRCA1 mutation carriage and the ovarian cancerrisk Hierarchical cluster analysis of the expression of miRNAs represents sharp separations of upregulatedyellow from downregulated blue in Fig miRNAs total of miRNAs were found statistically different after the comparison of phenotypicallydiscordant monozygotic twin siblings The miRNAsmiR1273 g3p miR1305 miR1973p miR3651 miR and miR92a3p expressions were found to haveupregulated and the other miRNAs let7i 5p miR125a5p miR15b5p miR22 3p miR3135b miRTable Comparison groups and cases in the groupsCaseGroup BR1639ControlBR1447Group BR1639BR1447BR1547BR2030BR1546BR1861BR1850BR2028Group BR1639Group BR1639BR1447BR1447BR1547BR2030BR1546BR1861BR1547BR2030BR1546BR1861BR1850BR2028 0cTuncer Journal of Ovarian Research Page of Fig Hierarchical cluster analysis of the expression of miRNAs Legend BR Healthy Brother BRCA1 negative nonBRCA1 mutationcarrier BR Healthy Niece BRCA1 positive BRCA1 mutation carrier BR Healthy Daughter BRCA1 negative BR HealthyMonozygotic twin BRCA1 positive BR Monozygotic twin diagnosed with ovarian cancer BRCA1 positive BR Healthy Sister BRCA1positive BR Healthy Sister BRCA1 positive BR Healthy Sister BRCA1 positive The miRNAs that may be effective in the etiology ofovarian cancer were identified after the comparison of monozygotic twins who were phenotypically discordant for ovarian cancer diagnosis ingroup 320d miR3423p miR4430 miR451a miR664b5pand miR7663p expressions were found to have downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are given in Table and Fig Different miRNAs level were compared between group in order to determine the effect of BRCA1 gene mutation Group was consisted after the comparison of theBRCA1 gene mutation carrier family members and individuals not carrying BRCA12 gene mutation accordingto miRNAs expression profiles After the comparisonsdownregulated and upregulated miRNAsrelated toBRCA1 gene mutation carrier were determined The expression of a total of miRNAs including miR4449miR46533p miR4865p miR5739 miR6165 andmiR 8743p associated with the BRCA1 gene mutationcarrying were upregulated and the expression of a totalof miRNAs including miR1263p miR320a miR320b miR320c miR320d miR320e miR3243p miR miR miR4428 miR4516 miR4741 miR miR564 miR6089 miR68695p miR68915pmiR71075p and miR78473p were found downregulated After the bioinformatic analysis a total of upregulated and downregulated statistically significantmiRNAs and their target molecules are shown in Table and Fig AftercomparisonDifferent miRNA levels were compared between group in order to determine the relation with ovarian cancerdevelopment and BRCA positivity Group consists ofcomparison of miRNAs of BRCA1 positive ovarian cancer patient with all other BRCA1 positive healthy individualsanddownregulated miRNAs related to mutation carriage inBRCA1 gene and epithelial ovarian cancer etiology weredetermined The expression of miRNAs includingmiR1260a miR1260b miR165p miR175p miR181b5p miR 26b5p miR4281 miR4286 miR5100miR68403p miR71145p miR7975 and miR7977were found to have upregulated and the expression of miRNAs including miR12255p miR1423p miR 26a5p miR miR29a3p miR30d5p miR3196upregulated 0cTuncer Journal of Ovarian Research Page of Table Ovarian cancer etiology related upregulated and downregulated miRNAs and target proteins in monozygotic twinsmiRNAsSequence of miRNAmiRNAStatusTarget genesFold change FCvaluesACCACUGCACUCCAGCCUGAGUpregulatedZNF138 TMEM239 BMP3UUUUCAACUCUAAUGGGAGAGAUpregulatedPTPN4 PRKAA1 PAPD7FRAT2 DEPDC1 FBXO41UUCACCACCUUCUCCACCCAGCUpregulatedCD82 PMAIP1 MTHFD1 CHECK1 AGO1 CASP10CAUAGCCCGGUCGCUGGUACAUGA UpregulatedGGCUGGUCAGAUGGGAGUGUpregulatedRACGAP1 OLA1 TEX261PTGS1 NFIC ZNF200PAGR1 IGF2BP1 CACNG8UAUUGCACUUGUCCCGGCCUGUUGAGGUAGUAGUUUGUGCUGUUUCCCUGAGACCCUUUAACCUGUGA Downregulated ERBB3 CDKN1A TP53 ERBB2 EGFR STAT3MYCSTAT3 PTEN ATM NOTCH2 CDH1 NFKB1UpregulatedDownregulated TLR4 BMP4 EIF2C1 NEUROG1 SOCS1 IGF1VEGFAmiR1273 g3pmiR1305miR1973pmiR3651miR6131miR92a3plet7i5pmiR125a5pmiR15b5pUAGCAGCACAUCAUGGUUUACAmiR223pAAGCUGCCAGUUGAAGAACUGUDownregulated BCL2 VEGFA CCND1 CCNE1 CDK1 CDK4 CDK6 E2F3MAPK1Downregulated CDKN1A WNT1 ERBB3 MYCBP HMGB1 E2F2 PTENPOTED SOD2miR3135bmiR320dmiR3423pmiR4430miR451amiR664b5pGGCUGGAGCGAGUGCAGUGGUGAAAAGCUGGGUUGAGAGGAUCUCACACAGAAAUCGCACCCGUAGGCUGGAGUGAGCGGAGAAACCGUUACCAUUACUGAGUUUGGGCUAAGGGAGAUGAUUGGGUADownregulated BIRC5 ABL2 MAPK1 MYCNDownregulated DCTN5 SYNCRIP FBXO28Downregulated GEMIN4 DNMT1 ID4 SREBF1SREBF2 BMP7Downregulated ZNF485ABL2 MAPK1 MSH5 PTENDownregulated CPNE3 RAB5A IL6R AKT1 MMP2Downregulated CD55MSN RHOBTB3 PLAG1miR7663pACUCCAGCCCCACAGCCUCAGCDownregulated COX1 MAPK1 NF2 RAD51 STK4 STK24 VEGFCFig The upregulated and downregulated miRNAs and foldchanges associated with ovarian cancer etiology in monozygotic twins 0cTuncer Journal of Ovarian Research Page of Table BRCA1 gene mutation positivity related upregulated and downregulated miRNAs and target molecules An additional tablefile shows this in more detail [see Additional file ]miRNAsSequence of miRNATarget genesmiRNAStatusFold changeFC valuesmiR4449miR46533pmiR4865pmiR5739miR6165miR8743pmiR1263pmiR320amiR320bmiR320cmiR320dmiR320emiR3243pmiR3656miR4284miR4428miR miR4741miR484miR564miR miR6869 5pmiR68915pmiR71075pmiR7847 3pCGUCCCGGGGCUGCGCGAGGCAUpregulatedZFHX3UGGAGUUAAGGGUUGCUUGGAGAUpregulatedATG2A CREBL2 MAT2A FRS2 TMED4 UBN2UCCUGUACUGAGCUGCCCCGAGGCGGAGAGAGAAUGGGGAGCCAGCAGGAGGUGAGGGGAGCUGCCCUGGCCCGAGGGACCGAUpregulatedUpregulatedUpregulatedUpregulatedOLFM4CD40ARHGAP5 IGF1R DOCK3 CADM1DLX6CD207CHIC1PPL2A PLXDC1PER1TFAP2AFADS1 AMER1LUZP1 COX6B1HDAC1 AQP3 STAT3 CDK9UCGUACCGUGAGUAAUAAUGCGDownregulatedTOM1 CRK VEGFA SOX2 TWF1 PITPNC1 IGFBP2 KRASAAAAGCUGGGUUGAGAGGGCGADownregulatedMCL1 BANP ITGB3 BMI1 NRP1 NFATC3 TRPC5AAAAGCUGGGUUGAGAGGGCAADownregulatedCDK6DCTN5SYNCRIPARF1 BCL9L ZNF600AAAAGCUGGGUUGAGAGGGUAAAAGCUGGGUUGAGAGGAAAAGCUGGGUUGAGAAGGACUGCCCCAGGUGCUGCUGGGGCGGGUGCGGGGGUGGDownregulatedDownregulatedSYNCRIPFBXO28SMARCC NPM3DCTN5 SYNCRIP FBXO28DCTN5 NPM3 ZNF275 DDX19A NCAPD2 TXNL1DownregulatedDownregulated WNT9B CREBBP DVL2 WNT2BDownregulatedMRPL12 LSP1 MNT PRDM2ZNF770 CECR1GGGCUCACAUCACCCCAUDownregulatedBCL2L11RBBP5HNRNPA1 ZNF264 TRIB3 CRTAPCAAGGAGACGGGAACAUGGAGCDownregulatedMSL1MAPRE3MYH14CASP2 CCND2 CDK14TP63GGGAGAAGGGUCGGGGCDownregulatedSTAT3M6PRGPR137CCCND2 CCNT1 CDKN1A SCOC TP53CGGGCUGUCCGGAGGGGUCGGCUDownregulatedDDX39BMAPK1 ZBTB39 HMGA1UCAGGCUCAGUCCCCUCCCGAUDownregulatedFIS1 PAGR1 ZEB1 SLC11A2SMAD2 ANAPC7 TBRG1AGGCACGGUGUCAGCAGGCDownregulatedGID4 CNBP E2F3 RCAN3 AKT2 APPL1 SLC1A2 GPR155GGAGGCCGGGGUGGGGCGGGGCGGDownregulatedNKX2 TPT1 KCTD5 BBX SGCD CDH7 CCNB1GUGAGUAGUGGCGCGCGGCGGCDownregulatedTUBB2AMAPK1NRBF2 WEE1HMGA2 MAPK1 STAG2UAAGGAGGGGGAUGAGGGGDownregulatedCHD4 CD207 DDX6 CHRDL1CCND2 TP63UCGGCCUGGGGAGGAGGAAGGGDownregulatedVAV3 CASP16 CCND1 CASP16 MAPK14CGUGGAGGACGAGGAGGAGGCDownregulatedHAVCR1 POTED DNAJC10 SOD2 M6PR CDK19miR3423p miR3665 miR3960 miR4466 miR4530miR46873p miR47875p miR4943p miR50015pmiR50065p miR5787 miR6068 miR6087 miR miR6090 miR6124 miR6125 miR638 miR65105p miR68005p miR7704 miR8063 and miR were found to have downregulated After bioinformatic analysis a total of upregulated and downregulated statistically significant miRNAs and the targetmolecules are given in Table and Fig For identifying the ovarian cancer predisposition allfamily members were compared with ovarian cancermonozygotic twins in group The upregulated ordownregulated miRNAs in association with the epithelialovarian cancer risk were identified after the comparisonThe expression of the miRNAs consisting of let7a5plet7b5p miR181a5p miR1973p miR215pmiR2233p miR23a3p miR27a3p miR36533pmiR4255p miR572 miR5745p miR6127 and miR were detected to be upregulated The expression ofthe miRNAs consisting of let7i5p miR 125a5pmiR15b5p miR1505p miR22 3p miR3283pmiR4430 miR451a miR46975p miR664b5p andmiR7663p were detected to have downregulated Atotal of upregulated and downregulated statisticallysignificant miRNAs and the target molecules are givenin Table and Fig DiscussionWomen are diagnosed with ovarian cancer at an advanced stage due to limited number of biologicalmarkers for ovarian cancer patients Although existingovarian cancer biomarkers cancer antigen125 and cancer antigen153 CA125 CA15 are sensitive in thefollowup of diagnosed gynecological cancers they have 0cTuncer Journal of Ovarian Research Page of Fig The upregulated and downregulated miRNAs and foldchanges associated with BRCA1 gene mutation positivityofearlysensitivityin the diagnosislessstagegynecological cancers and separation of malignant tumorformations from benign formations [] Therefore tounderstand the underlying mechanisms of ovarian cancer and to explore targeting drugs and to improve newtreatment protocols for ovarian malignancy revealingsignificant genetic changes is necessary The genetic andepidemiologic studies conducted on monozygotic twinsare known to provide accurate and direct informationabout the gene and environment interaction with thedisease occurrence mechanism [] The changes in genesthat result in the occurrence of tumors such as miRNAexpression level among the monozygotic twins providesinformation on the etiology of disease and may have arole as a biological indicator in identifying the early stagedisease and in the follow up of the prognosis We aimedto identify the noninvasive biological markers that maybe used in the early diagnosis of ovarian cancer throughinvestigating the miRNAs in the peripheral blood ofmonozygotic twin siblings discordant for ovarian cancerwith the miRNA molecules of the other healthy members in the family Thus that may cause less bias thanthe controls to be selected from the population Ninetynine different miRNA molecules presented in the studywere detected after the comparison of monozygotic twinsiblings who were discordant for ovarian cancer andwith the other healthy individuals Seventeen differentmiRNAs were found that could be used for detectingearly diagnosis and prognosis of ovarian cancer betweenthe monozygotic twin siblings who were discordant forovarian cancer in our study The association between out of miRNAs and ovarian carcinoma is beingreported for the first time in this study Due to the highnumber of newly detected miRNAs in our study the discussion and comparison were only made between thecandidate miRNAs Although miR1973p miR1305miR6131 miR3651 miR3135b miR4430 miR664b5p and miR7663p have not been shown to be associated with ovarian cancer in literature but limited number of studies have suggested the association with othercancersWang found the elevated level of miR1973pinthe same way as we do The upregulated miR1973p expression level was shown to promote the cellular invasion and metastasis in bladder cancer in that studyResearchers reported that LINC00312 gene was responsible for invasion and metastasis mechanisms and thisgene inhibited the cellular migration and invasion bysuppressing the miR1973p expression Similar resultswere detected in thyroid cancer in the study of Liu et al[ ] Jin reported that increased expressionlevel of miR1305 caused pluripotent stem cells to accelerate the cell cycle G1S transfer in addition to causingthe cellular differentiation with the increased miR1305expression [] The expression levels ofreducedmiRNA125a5p and let7i5p found in the scope of ourstudy have been shown to parallel with other studies inthe literature Langhe suggested thatlet7i5pmight be described as a diagnostic indicator in ovariancancer [] The miRNA125a5p expression was upregulated to inhibit the cancer proliferation and migrationin the in vitro study of Qin in human cervical carcinomas [] and miR125a5p upregulated expressionlevel was demonstrated to inhibit the cervical cancer 0cTuncer Journal of Ovarian Research Page of Table BRCA1 mutation carriage and epithelial ovarian cancer etiology related upregulated and downregulated miRNAs targetmolecules An additional table file shows this in more detail [see Additional file ]miRNAsSequence of miRNAmiRNAStatusTarget genesAUCCCACCUCUGCCACCAAUCCCACCACUGCCACCAUUAGCAGCACGUAAAUAUUGGCGUpregulatedUpregulatedUpregulatedCAAAGUGCUUACAGUGCAGGUAGUpregulatedPSAT1UNC13A RPS27 BRD7SFRP1 DKK2 SMAD4 PSAT1UNC13A RPS27CCNE1 ARL2 BCL2 HMGA1 CDK6 CCND1VEGFA RECK PRDM4TGFBR2 PTEN CDKN1A BCL2L11E2F1TP53STAT3AACAUUCAUUGCUGUCGGUGGGUUpregulatedTCL1A TIMP3 PLAG1 BCL2RNF2VSNL1 ATMFold changeFC valuesmiR1260amiR1260bmiR16 5pmiR175pmiR181b5pmiR26b5pmiR4281miR4286miR5100UUCAAGUAAUUCAGGAUAGGUUpregulatedGGGUCCCGGGGAGGGGGGACCCCACUCCUGGUACCUpregulatedUpregulatedUUCAGAUCCCAGCGGUGCCUCUUpregulatedPTGS2 EPHA2 CHORDC1 EZH2CCNE1ABCA1 GATA4NCDN CDKN1A BCL3LDLR ZNF354B NSD1 RABGAP1TAOK1 MKNK2COX10 DEK KCNN3 RAB11FIP1DYNLT1 NOTCH2SLFN12L CTC1 GXYLT2GDE1FADS1PER1 ATG9AmiR68403pGCCCAGGACUUUGUGCGGGGUGUpregulatedmiR71145pUCUGUGGAGUGGGGUGCCUGUUpregulatedM6PR HNRNPUL1 SHMT1 ZNF529ACVR2B PAICS TAF8miR7975miR7977AUCCUAGUCACGGCACCAUUCCCAGCCAACGCACCAUpregulatedUpregulatedmiR12255pGUGGGUACGGCCCAGUGGGGGGDownregulatedKBTBD8GULP1 CASZ1 RAD51HSPA1B ZNF703 TMEM185BSF3B3COX6B1 CCDC9 CDH7ORC4 ODF2L MTRNR2L7PSMG2 MTRNR2L3miR1423pmiR26a5pmiR2861miR29a3pmiR30d5pmiR3196miR 3423pmiR3665miR3960miR4466miR4530miR46873pmiR47875pmiR4943pmiR50015pmiR50065pmiR miR6068miR6087miR6088miR6090miR6124UGUAGUGUUUCCUACUUUAUGGADownregulatedARNTLTGFBR1 RAC1 ROCK2 CCNT2 TAB2 PTPN23UUCAAGUAAUCCAGGAUAGGCUDownregulatedEZH2RB1ADAM17 HMGA2CCND2 CPEB3 DNMT3BGGGGCCUGGCGGUGGGCGGUAGCACCAUCUGAAAUCGGUUAUGUAAACAUCCCCGAC
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"trial in patients with advanced non-small-cell lung cancer. J Clin Oncol 2010 28 3076 3083 20479403 29. Miller VA Hirsh V Cadranel J Afatinib versus placebo for patients with advanced metastatic non-small-cell lung cancer after failure of erlotinib gefitinib or both and one or two lines of chemotherapy (LUX-Lung 1): a phase 2b/3 randomised trial. Lancet Oncol 2012 13 528 538 22452896 Clin Orthop Relat Res Clin. Orthop. Relat. Res Clinical Orthopaedics and Related Research 0009-921X 1528-1132 Springer US Boston 24249538 3971232 3385 10.1007/s11999-013-3385-9 Clinical Research Does Intensity of Surveillance Affect Survival After Surgery for Sarcomas? Results of a Randomized Noninferiority Trial Puri Ajay MS docpurigmail.com Gulia Ashish MS Hawaldar Rohini PhD Ranganathan Priya MD Badwe Rajendra A. MS Orthopaedic Oncology Tata Memorial Hospital Room No. 45 E Bes Road Mumbai India Tata Memorial Hospital Mumbai India Anaesthesiology Critical Care and Pain Tata Memorial Hospital Mumbai India Surgical Oncology Tata Memorial Hospital Mumbai India 19 11 2013 5 2014 472 5 1568 1575 30 7 2013 8 11 2013 The Association of Bone and Joint Surgeons® 2013 Background Whether current postoperative surveillance regimes result in improved overall survival (OS) of patients with extremity sarcomas is unknown. Questions/purposes We hypothesized that a less intensive followup protocol would not be inferior to the conventional followup protocol in terms of OS. We (1) assessed OS of patients to determine if less intensive followup regimens led to worsened survival and asked (2) whether chest radiograph followup group was inferior to CT scan followup group in detecting pulmonary metastasis; and (3) whether less frequent (6-monthly) followup interval was inferior to more frequent (3-monthly) followup in detecting pulmonary metastasis and local recurrence. Methods A prospective randomized single-center noninferiority trial was conducted between January 2006 and June 2010. On the basis of 3-year survival of 60% with intensive more frequent followup 500 nonmetastatic patients were randomized to demonstrate noninferiority by a margin (delta) of 10% (hazard ratio [HR] 1.36). The primary end point was OS at 3 years. The secondary objective was to compare disease-free survival (DFS) (time to recurrence) at 3 years. At minimum followup of 30 months (median 42 months; range 3081 months) 178 deaths were documented. Results Three-year OS and DFS for all patients was 67% and 52% respectively. Three-year OS was 67% and 66% in chest radiography and CT groups respectively (HR 0.9; upper 90% confidence interval [CI] 1.13). DFS rate was 54% and 49% in chest radiography and CT groups respectively (HR 0.82; upper 90% CI 0.97). Three-year OS was 64% and 69% in 6-monthly and 3-monthly groups respectively (HR 1.2; upper 90% CI 1.47). DFS was 51% and 52% in 6-monthly and 3-monthly groups respectively (HR 1.01; upper 90% CI 1.2). Almost 90% of local recurrences were identified by patients themselves. Conclusions Inexpensive imaging detects the vast majority of recurrent disease in patients with sarcoma without deleterious effects on eventual outcomes. Patient education regarding self-examination will detect most instances of local recurrence although this was not directly assessed in this study. Although less frequent visits adequately detected metastasis and local recurrence this trial could not conclusively demonstrate noninferiority in OS for a 6-monthly interval of followup visits against 3-monthly visits."
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" atopic dermatitis ad is a worldwide chronic skin disease which burden public health sea buckthornsbt hippophae rhamnoides l elaeagnaceae oil as a traditional herbal medicine has been used for diseasetreatment for many years the effects of sbt oil on ad mouse model induced by repeated administration of dinitrochlorobenzene dncb in balbc mice was evaluated in this studymethods mice were divided into four groups including the normal control group ad model group ad modelgroup treated with sbt oil mlkg and ad model group treated with sbt oil mlkg same volume at differentconcentrations of sbt oil was applied daily on the latter two groups by gavage for days following ad modelinduction the function of skin barrier and the production of il4 ifnÎ tnfα and tslp were examined afteranimal sacrifice the migration and mature of langerhans cell lcs in lymph node was further assessed by flowcytometryresults sbt oil alleviated dermatitis scores decreased ear thickness prevented infiltration of mast cell reducedlymph node weight and depressed activity of th2 cells sbt oil also reduced the expression of il4 ifnÎ tnfα andtslp in ear tissue ige level in serum and mrna relative expression of il4 ifnÎ tnfα in lymph node moreoversbt oil inhibited the migration of lcs cells from local lesions to lymph node and its mature in lymph nodes these results suggest sbt oil had a beneficial effect either systemic or regional on dncbinduced admice via maintain the balance of th1th2 and may be a potential complementary candidate for ad treatmentkeywords atopic dermatitis sea buckthorn oil 24dinitrochlorobenzene cytokine ad is a chronic inflammatory skin disease characterizedwith eczematous pruritic rash which has high morbidityin children and could be recurrent in adulthood [ ]as a general public health disease the prevalence of adhas increased in recent years [ ] ad affects nearly correspondence hongquanguansinacom houdiandong163com xinxin wang and sijia li contributed equally to this work5college of integrated traditional chinese and western medicine liaoninguniversity of traditional chinese medicine chongshan road no79shenyang liaoning pr chinafull list of author information is available at the end of the of children and of adults worldwide and the incidents become higher and higher although thepathogenesis of ad is not explicit utterly genetic riskenvironmental factors skin barrier dysfunction and immune dysregulation are thought to play important rolesduring the pathogenesis of ad [] as for immunedysregulation th2 skewing seems to be the key point ofad pathogenesis [ ] immunological disorder ofth1th2 balance due to strong type immune responses characterized by over infiltration of mast cellincreased production of th2 cytokines and ige level in the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc complementary medicine and therapies page of serum plays crucial role in the onset and process of adthese th2 cytokines subsequently induce the release ofother proinflammatory cytokines such as ifnÎ throughactivating of th1 cells in clinical practicedue to the heavy burden ad placed on society and patients [ ] treatment approaches are needed imperativelyregional emollient andsystemic corticosteroids were generally used to cure ad[ ] however experts ofinternational eczemacouncil reached on a that although the useof corticosteroids for ad is widespread it is also discouraged due to the side effects and the risk of reboundin consideration of potential fearful side effect of topicalsteroid and immunosuppressive application [ ]there is a strong enthusiasm in seeking alternative andcomplementary medication to treat ad recently seeking new potential candidate from natural materials forad management attracted greatly attention [ ]sbt is a wild deciduous shrub or dwarf tree belongingto the elaeagnaceae family which has been used in tibetan mongolian and chinese traditional medicine extensively for disease management [] according tomany researchers sbt has various medicinal effects suchas antitumor antistress antiinflammatory antiulcerantioxidant healing regulation of cardiovascular andimmune system [] sbt oil which contains richfatty acids tocopherols Ï3 and Ï6 etc is a main bioactive part of sbt and it has been proved to have beneficial effect on skin inflammation conditions and have theability to improve the composition of fatty acid in skin[ ] therefore this study carried out to explore thebeneficial effects of sbt oil on dncbinduced admouse model and its possible mechanismmethodssbt oilsbt oil was provided by liaoning dongning pharmceutical co ltd the oil was extracted from thedried press residue including berry flesh seeds andpeel of sbt juice processing by aseptic supercriticalcarbon dioxide process analysis of samples was performed using a hp5ms capillary column m mm μm agilentinc santaclara ca usa in a gcms 5975c agilent technologies inc sample was injected into the columnand ran using split mode split ratio the helium carrier gas was programmed to maintain atechnologiesconstantflow rate of mlmin oven was initially °c for min then finally raised to °c at °cmin fatty acids were identified by a reference standard mixture fame supelco bellefonte pa usa analyzed under the same operating conditions as thoseemployed for fame of the samples the componentsin sbt oil are exhibited in table animals and animal treatmentfemale healthy specific pathogenfree balbc miceaged weeks weighted ± g were provided byliaoning changsheng biotechnology co ltd benxichina all mice were housed in groups of mice percage waiting to be grouped in a specific pathogenfreeenvironment in h lightdark cycle and allowed free towater and food mice were acclimatized for week before ad model induction mice were randomly dividedinto groups the normal control group ad modelgroup ad model group treated with sbt oil mlkgand ad model group treated with sbt oil mlkgeach group with mice the dorsal skin of each mousewas shaved following dncb μl1 sensitizationthree times in total from day to day and the skin ofleft ear was challenged by dncb μl05 seventimes in total from day to day ad model grouptreated with sbt oil mlkg was given ml sbt oilplus ml olive oil per mice ad model group treatedwith sbt oil mlkg was given ml sbt oil permice oil was applied by intragastric administration oncea day from day15 to day olive oil ml was givenfor the normal control group and ad model group atthe same time at the same time the thickness of left earwas measured every days all animals were sacrificedat day and samples including blood left ear tissuesand submaxillary lymph nodes were collected the micewere anesthetized with avertin solution g tribromoethanol powder dissolved into ml 2methyl2butanol and ml pbs at °c which was filtered usinga nalgene μm filter thermo fisher scientific incwaltham ma usa and sacrificed via exsanguination the fullscale procedures of ad model inductionand treatment are shown in fig all experimental procedures performed were approved by the ethical committee of experimental animal careat liaoninguniversity of traditional chinese medicine shenyangpr chinatable major fatty acids and contents of sitosterol and βcarotene in sbt oilfatty acids myristic acidc140palmitic acidc160palmitoleicacidc161stearic acidc180oleic acidc181linoleic acidc182linolenic acidc183sitosterolmggβcarotenemgg 0cwang bmc complementary medicine and therapies page of fig general schematic diagram for ad model induction and sbt oil treatmentevaluation of ad severitydermatitis severity was assessed by ear thickness anddermatitis scores through the method of blind ear thickness was measured every days since day with a digitalthickness gauge mitutoyo co kanagawa japan dermatitis scores was calculated according to main characteristics drynesscrusting hemorrhageerythema erosionexcoriation and edema each one was marked on ascale from none mild moderate to severethe overall dermatitis score was consist of the sum of individual scores which range from to μm thick werehistopathological analysisthe left ear samples of mice were collected on day then fixed in formalin and embeded in paraffin thesectionseither withhematoxylin and eosin he for visualizing dermal andepidermal thickness or with toluidine blue tb for visualizing mast cell numbers the mast cells were countedin sections of power fields at magnificationstainedimmunohistochemistryin short after deparaffinization and rehydration the eartissue slides were treated with hydrogen peroxidemethanol for inhibiting endogenous peroxidase and withhigh pressure for antigen retrieval then the slides wereincubated with sheep serum for min at °c withprimary antibodies abcam for overnight at °c withsecondary antibodies provided by zhongshanjinqiaobeijing china for h at °c at last the slides werestained with diaminobenzidine dab provided byzhongshanjinqiao beijing china for coloration resultwas analyzed by imagejrealtime polymerase chain reaction rtpcrmouse submaxillary lymph nodes were collected andweighted while sacrifice and total rna was extractedfrom lymph node tissues using trizol reagent invitrogen thermo fisher scientificfollowing theincmanufacturers protocols and reverse transcribed withprime scripttmrt reagent kit takara biotechnologyco ltd dalian china realtime polymerase chain reaction analyses were performed under the protocols ofsybr®premix ex taqtm ii takara biotech co ltddalian china and the primers used in this study weredesigned as shown in table relative quantities of alltargets in test samples were normalized to their corresponding gapdh levels the 2δδct method was usedto calculate relative expression quantifyflow cytometrythe antibodies used for flow cytometry were providedby bd usa and the scheme was performed follow induction about cells of submaxillarylymph node was collected in ep tube centrifuged at rpm and °c for min one hundred μl pbs wasmixed with cells after discarding the supernatant thenanother μl pbs containing fluorescent antibodieswas added for staining at °c for min and the processwas kept out of the sun five hundred μl pbs was addedand blended repeatedly for washing then centrifuged at rpm and °c for min the supernatant was discarded carefully and another μl pbs was added andmixed finally the mixture was transferred to flow tubefor flow cytometrytable primers used for rtpcrgeneil4ifnÎtnfαgapdhprimer sequenceforward ²acaggagaagggacgccat3²reverse ²gaagccctacagacgagctca3²forward ² tgagctcattgaatgcttgg ²reverse ² ggccatcagcaacaacataa ²forward ²ggaaaggacggactggtgta3²reverse ² tgccactggtctgtaatcca ²forward ²tggtgaaggtcggtgtgaac3²reverse ²actgtgccgttgaatttgcc3² 0cwang bmc complementary medicine and therapies page of statistical analysisthe data is presented as mean ± sd the significance ofdifferences of different groups was evaluated by oneway analysis of variance anova followed by the dunnett t test p005 was considered statistically significantresultssbt oil has a beneficial effect on skin against thedevelopment of dncbinduced ad models in balbcmiceto investigate the effect of sbt oil on adlike skinlesions in our model sbt oil mlkg10 mlkg wasapplied by gastric administration once a day followingthe ad model induction by dncb showed in fig topical application of dncb including sensitizationand challenge induced adlike skin lesions presenting as erythemaitching and hemorrhage companiedby abnormal scratching marks and dryness dermatitisseverity was assessed by ear thickness and dermatitisscores ear thickness was measured every days sinceday and the dermatitis scores was evaluated according to main characteristics as described previously after sbt oil administration for days theear thickness and dermatitis scores were significantlydecreased in a dosedependent manner compared tothe ad model group fig fig effects of sbt oil on ad model skin induced by dncb a examples of characteristic of adlike skin lesions b the ear thickness of the micec the dermatitis scores were summarized by the sum of scores according to various ad symtoms p p 005vs the control group p p vs the ad model group 0cwang bmc complementary medicine and therapies page of sbt oil contributed to the skin barrier repair anddecreased infiltration of mast cell in ad model miceinduced by dncbto evaluate the effect of sbt oil on adlike skin lesions histologically he and tb staining were performed on tissue slides repetitive application ofdncb induced dermal thickening epidermal hyperplasia and increased mast cellinfiltration in admodel group while according to he staining slidesthe dermal and epidermalthickness were both decreased and the epidermal hyperplasia was suppressedafter sbt oil administration for days which relatedto dosage fig 3a b according to tb staining slidesthe infiltration numbers of mast cell were also decreased in mice treated with sbt oil fig 3b csbt oil decreased the lymph node weights in ad modelmice induced by dncbthe submaxillary lymph nodes were collected andweighted after mice sacrifice to estimate whether sbtoil play a part in the process of ad induction the results indicated an increase in submaxillary lymph nodeweights in ad model group which was decreased bysbt oil administration fig sbt oil inhibited the expression of il4 ifnÎ tnfα andtslp in ear tissue in ad model mice induced by dncbin order to evaluate the effects of sbt oil on regulationof th1th2 cytokines the expression of il4 ifnÎtnfα and tslp in ad model mice was examined byimmunohistochemistry staining on ear tissue slides results demonstrated an upregulation expression of il4fig effects of sbt oil on histological ear skin tissue a he staining thickness in hestained tissue mast cell number in tb stained tissue c tb staining epidermis mast cell dermis b dermal and epidermal 0cwang bmc complementary medicine and therapies page of fig weights of submaxillary lymph node a submaxillary lymph node b lymph node weight a normal control group b ad model groupc treated with sbt oil mlkg d treated with sbt oil mlkg p p vs the control group p p vs the admodel groupifnÎ tnfα and tslp in ad model group which wasinhibited dosedependent by application of sbt oilfig sbt oil downregulated ige level in serum and mrnarelative expression of il4 ifnÎ and tnfα in lymphnodewe next measured ige levelin serum by elisa andmrna relative expression of il4 ifnÎ and tnfα inlymph node by rtpcr we found that ige levelinserum was increased in ad model mice induced bydncb the increase was suppressed significantly ingroups treated with sbt oil in a dosedependent manner the same as above mrna relative expression ofil4 ifnÎ and tnfα in lymph node was increased inad model group and decreased in groups treated withsbt oil fig sbt oil decreased numbers of lcs in draining lymph nodeand the expressions of cd86 ox40l and mhcii on lcsin order to assess the effect of sbt oil on the maturityand migration of lcs cell in submaxillary lymph nodewe detected cell numbers expressing cd207cd326cd86 cd80 ox40l and mhc ii by flow cytometryresults as shown in fig indicated that the expressionsof cd207cd326 cd86 ox40l and mhciion lcs cellin submaxillary lymph node were all increased in admodel groups induced by dncb and decreased ingroups treated with sbt oildiscussionad is a skin inflammatory disease induced by haptenand mediated by t cells clinically the main characteristics of ad are erythema edema papule blisterbleb bullous reaction and even necrosis the pathological changes of ad were infiltration of inflammatorystudy weand tissuecellsedemain thisemployed dncbinduced ad model using balbcmice which has been proposed as an appropriate representative of human ad because of similar symptoms including skin erosion hemorrhage epidermalhyperplasia mast cellinfiltration and increased igelevel in serum etc sbt oil as a traditional herbal extracts have been proved diversified pharmacologicalactions such as antiinflammatory relieve the pressure protecting vascular endothelial cell and immunomodulatory effects [ ] the main constituents ofsbt oilinclude fatty acids such as myristic acidpalmitic acid palmitoleic acid oleic acid etc sitosterol and βcarotene fatty acids are crucial components of cell membranes and play important role inbiologicalfunction of cells some of the fattyacids are required for innate immune activation andpathogen defense sitosterol is the main constituent of many plants and vegetables and has the abilityto modulate the functions of macrophages and antiinflammation [ ] βcarotenealso has beenshown to suppress the cellular and tissue response toinflammation [ ] in view of the immunoregulation and antiinflammatory actions of sbt oil weassessed the antiad effects of sbt oil in vivotopical application of dncb followed schedule including sensitization and challenge induced adlikeskin lesions presenting as erythemaitching andhemorrhage companied by abnormal scratching marksand dryness the ear thickness and dermatitis scoreswere all significantly increased in ad model groupcompared to control group after sbt oil administration for daysthickness and dermatitisscores in groups treated with sbt oil were significantly decreased in a dosedependent manner compared to the ad model group which indicate thatsbt oil administration suppressed the development ofadlike skin lesionsthe ear 0cwang bmc complementary medicine and therapies page of fig effects of sbt oil on expression of il4 ifnÎ tnfα and tslp in ear tissue a immunohistochemical staining of il4 ifnÎ tnfα and tslpin ear tissue b aod analysis of il4 ifnÎ tnfα and tslp p vs the control group p vs the ad model groupad is recognized as a th2midiated allergic disease withover expression of th2 cytokines and increased serum igelevel being the antibody synthesized by plasma cellsige plays an essential role in some hypersensitivity suchas ad allergic asthma and allergic rhinitis ige has thecapability of elevating the production of th2 cytokinesth2 cytokines il4 induced immunoglobulin switching inplasma cells and resulting in upregulation of serum igelevel mast cell as one of granular leukocytes can releasemany cytokines to mediate inflammatory reaction and immune regulation these cytokines also participate inpathological manifestations of many allergic disorders including ad the infiltration of mast cell which wasactivated by ige is one of the key features of adlike skin 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on ige level in serum and mrna relative expression of il4 ifnÎ and tnfαin lymph node a ige level in serum b mrnarelative expression of il4 ifnÎ and tnfα in lymph node p p vs the control group p p vs the ad model grouplesions [ ] cytokines released from activated mastcells attract eosinophils into the skin which give rise toskin tissue damage histologically according to tb staining slides the numbers of mast cell infiltration in ear skintissue of ad model mice were increased by application ofdncb and were inhibited remarkably by sbt oil the results indicated that sbt oil has beneficial effects on suppression of skin tissue mast cell accumulation in dncbinduced ad mice our tb staining results indicated thatmast cells in skin tissue were scarce in control group whileabundance in ad model group which highly conform tothe pathological changes of ad the mast cell numberwas reduced remarkably after sbt oil administration insbt oil treated group compared with ad model groupthese results suggest that sbt oil has inhibiting effect ofmast cell infiltrationaccording to studies published the over expression ofth2 cytokines go hand in hand with tslp tslp whichcan strongly promote the differentiation of th0 cells toth2 phenotype through activation of dendritic celldcs was determined as a crucial factor in the induction of th2 skewing in ad the expression of tslphas been shown to be enhanced markedly in keratinocytes of ad lesions both in ad patients and in mousemodels il4 can in turn induce the synthesis oftslp by keratinocytes importantly the migration ofdcs to draining lymph node was triggered by tslpmore interesting th1 cytokines ifnÎ which can activate keratinocytes was found also elevated in ad ifnÎand tnfα can synergistically stimulate the release ofcytokines and chemokines in chronic stage of ad inour study sbt oiltreatment reduced the increasedserum ige level which was induced by dncb application moreover sbt oil treatment also reduced dncbinduced increases in expression of il4 ifnÎ tnfαand tslp in ear tissue and mrna relative expression ofil4 ifnÎ and tnfα in lymph node these resultssuggest that sbt oil ameliorated ad symptoms partlythrough the activity suppression of th1th2 cells according to the downregulated effects sbt oil did to thetslp expression in ear tissue we speculated that sbtoil may have the ability to suppress both the activationand migration of dcs cell in order to clarify our speculationflow cytometry was used to do further studyabout lcsdraining lymph node plays an important role in thepathogenesis of ad the weight and volume of lymphnode will increase company with strengthened functionwhen it is active we investigated the local lymph nodesthrough different means first of all the submaxillarylymph node weights of dncbinduced ad model micewere increased significantly which were markedly reducedafter intragastric application of sbt oil for days in adosedependent manner we further assessed the expressions of cd207cd326 mhc class ii cd80 cd86 onlcs and ox40l on cd4 t cells in lymph node by flowcytometry because the complex immune reaction of adwas mainly taken place in lymph node langerin cd207a type ii transmembrane protein is a ctype lection oflcs lcs are virtual mediators of immune surveillance and tolerance which resided at epidermis as dc subpopulation antigens both external and internal werecaptured by lcs and presented to th0 cells within theskin draining lymph node cd207 is the only surface antigen just restricted to lcs epithelial cell adhesionmolecule epcamcd326 a cell surface protein is highlyexpressed on lcs and appears to stimulate lcs migration since cd207cd326 as the main symbol of lcs 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on the maturity and migration of lc cell a the scatter diagram which indicate the proportion of cells in lymph nodeexpressing cd207cd326 mhc ii cd86 cd80 and ox40l b the histogram of abovementioned results p p 005vs the controlgroup p p vs the ad model groupmigrated into lymph nodethe proportion changesshowed that sbt oil suppressed the migration of lcs cellfrom skin lesion to draining lymph node after degradingproteins derive from extracellular environment were takenup by endocytosis or phagocytosis and captured by mhcclass ii molecules then result in peptideloaded mhc iiand migrate to the surface of antigen presenting cell waiting for recognition by cd4 t cells finally activate adaptive immune response the increased cell proportionof mhc class ii indicated the uptrend tendency of mature 0cwang bmc complementary medicine and therapies page of lcs in lymph node in dncb induced ad model micecompared with normal control mice while this uptrendtendency was inhibited by sbt oil application in micetreated with sbt oil constant epidermal lcs are immature normally and barely express costimulatory moleculessuch as cd80 and cd86 while upon lcsmaturation the expression of these costimulatory molecules was enhanced in this study sbt oil down regulatedthe expression of cd86 on lcs in lymph node which wasenhanced by dncb in ad model mice it suggested theeffect of sbt oil on inhibiting lcs maturationox40cd134 is a transmembrane protein of tumor necrosis factor receptor superfamily member which mainlyexpressed on activated cd4 t cells and upregulatedwithin inflammatory lesions on the antigenactivated tcells [ ] tslp can stimulate the expression of ox40ligand ox40l on lcs lcs expressing ox40l migratefrom skin lesion to local lymph node and induce the transformation of th0 cells to th2 cells on one hand the increased proportion of ox40l cells in lymph node ofdncb induced ad mice was suppressed by sbt oil administration which confirmed the effect of sbt oil on activation of cd4 t cells on the other hand sbt oilconduced to the normal function of lcs through renovating the keratinocyte and suppressing tslp release as aresultthe abnormal th2 skewing inflammation wasinhibited by sbt oil administrationall in all our results suggest that sbt oil inhibitedboth the migration of lcs to lymph node and its maturation in lymph node thereby inhibited the transformation of th0 cells to th2 cells and finally limited theoccurrence of th2 type inflammatory responsein summary our study results attested that sbt oil application suppressed dncbinduced adlike symptomsby downregulating serum ige level and the productionof cytokines and chemokines and regulated th1th2balance in addition our results also indicated that sbtoil treatment inhibited the migration of lcs to draininglymph node and its maturation taken together sbt oilhas excellent therapeutic effect on inflammatory skindiseases and might be a potential complementary candidate for ad treatment in further studiesit will beworthwhile to explore the mechanism of sbt oil and itsactive constituent in the treatment of adabbreviationsad atopic dermatitis dab diaminobenzidine dcs dendritic cell dncb dinitrochlorobenzene he hematoxylin and eosin ifnÎ interferonÎige immunoglobulin e il4 interleukin4 lcs langerhans cell mhcii majorhistocompatibility complex ii rtpcr realtime polymerase chain reactionsbt sea buckthorn tb toluidine blue th1 thelper th2 thelper tnfα tumor necrosis factorα tslp thymic stromal lymphopoietinacknowledgementsnot applicableauthors contributionsxxw carried out the experiment and drafting of the manuscript sjl and jplassisted to accomplish the experiment dnk and xwh carried out statisticalanalysis pl and mx did the interpretation work hqg and ddh revised theresearch and manuscript ddh designed the experiment and submitted themanuscript all the authors read and approved the final manuscriptfundingthis project was supported by the national natural science foundation ofchina grant no which was granted to diandong hou the resultsindicated in this manuscript were main achievements of the projectavailability of data and materialsall data and materials are contained and described within the manuscriptethics approval and consent to participateall experimental procedures were conducted according to the guidelinesprovided by the ethical committee of experimental animal care at liaoninguniversity of traditional chinese medicine shenyang pr chinaconsent for publicationnot applicablecompeting intereststhe authors state no potential conflict of interestauthor details1liaoning university of traditional chinese medicine shenyang liaoning prchina 2basic medical and forensic medicine baotou medical collegebaotou inner mongolia pr china 3neurosurgery department northernhospital of inner mongolia baotou inner mongolia pr china 4liaoningdongning pharmceutical co ltd fuxin liaoning pr china 5college ofintegrated traditional chinese and western medicine liaoning university oftraditional chinese medicine chongshan road no79 shenyang liaoning pr chinareceived december accepted june referencesklonowska j new cytokines in the pathogenesis of atopic dermatitisnew therapeutic targets int j mol sci choopani r treatment of atopic dermatitis from the perspective oftraditional persian medicine presentation of a novel therapeutic approach jevid based complementary altern med brunner pm guttmanyassky e leung dy the immunology of atopicdermatitis and its reversibility with broadspectrum and targeted therapiesj allergy clin immunol 20171394ss65leung dym new insights into atopic dermatitis j clin investig kim je molecular mechanisms of cutaneous inflammatory disorderatopic dermatitis int j mol sci kim j molecular mechanism of atopic dermatitis induction followingsensitization and challenge with 24dinitrochlorobenzene in mouse skintissue toxicol res hou dd sea buckthorn hippophae rhamnoides l oil improvesatopic dermatitislike skin lesions via inhibition of nfkappab and stat1activation skin pharmacol physiol campana r molecular aspects of allergens in atopic dermatitis curropin allergy clin immunol brandt eb sivaprasad u th2 cytokines and atopic dermatitis j clin cellimmunol de vuyst e atopic dermatitis studies through in vitro models frontmed lausanne park jg tabetri tabebuia avellanedae ethanol extract amelioratesatopic dermatitis symptoms in mice mediat inflamm 0cwang bmc complementary medicine and therapies page of liu r βsitosterol modulates macrophage polarization and attenuatesrheumatoid inflammation in mice pharm biol vollmer d west v lephart e enhancing skin health by oral administrationof natural compounds and minerals with implications to the dermalmicrobiome int j mol sci cicero afg colletti a effects of carotenoids on health are all the sameresults from clinical trials curr pharm des ito t il33 promotes mhc class ii expression in murine mast cellsimmun inflamm dis dubois a regulation of th2 responses and allergic inflammationthrough bystander activation of cd8 t lymphocytes in early life jimmunol girtsman t natural foxp3 regulatory t cells inhibit th2 polarizationbut are biased toward suppression of th17driven lung inflammation jleukoc biol wallmeyer l tslp is a direct trigger for t cell migration in filaggrindeficient skin equivalents sci rep leyvacastillo jm tslp produced by keratinocytes promotes allergensensitization through skin and thereby triggers atopic march in mice jinvest dermatol feinberg h trimeric structure of langerin j biol chem zhang x tim4 is differentially expressed in the distinct subsets ofdendritic cells in skin and skindraining lymph nodes and controls skinlangerhans cell homeostasis oncotarget kumkate s cd207 langerhans cells constitute a minor population ofskinderived antigenpresenting cells in the draining lymph node followingexposure to schistosoma mansoni int j parasitol gaiser mr cancerassociated epithelial cell adhesion moleculeepcam cd326 enables epidermal langerhans cell motility and migrationin vivo proc natl acad sci u s a 201210915e889 natarajan k the role of molecular flexibility in antigen presentationand t cell receptormediated signaling front immunol weinberg ad science gone translational the ox40 agonist storyimmunol rev kinnear g a diametric role for ox40 in the response of effectormemory cd4 t cells and regulatory t cells to alloantigen j immunolpublishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsshrestha s burden of atopic dermatitis in the united states analysis ofhealthcare claims data in the commercial medicare and medicaldatabases adv ther arima k burden of atopic dermatitis in japanese adults analysis ofdata from the national health and wellness survey j dermatol megna m systemic treatment of adult atopic dermatitis a reviewdermatol ther heidelb glatz m emollient use alters skin barrier and microbes in infants at riskfor developing atopic dermatitis plos one 2018132e0192443 drucker a
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"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as San ye qing is a kind of folk plant Because of its slow growth it usually takes years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang Peoples Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as natural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new eight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiangs dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our teams preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally cm long and cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are cm long and cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4 on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of 4αDGalAp1 4DGalp1 and 4αDGlcp1 residues as backbones and DManp1 36DManp1 and αDAraf1residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52 Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a
| 2 |
"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh ï¬avor and health beneï¬ts and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneï¬cial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneï¬cial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health beneï¬ts and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2
egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg ï¬nally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals
egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe ï¬eld of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientiï¬c community and the general public indeedegcg has shown both prophylactic and therapeutic eï¬cacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table []apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoho2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site speciï¬city of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpspparð¾ cebpð¼aco pparð¼ cpt1acad pgc1ð¼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis []accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by ï¬ow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form bioï¬lms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibioï¬lm eï¬cacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results veriï¬ed the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the eï¬cacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microï¬orawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ ] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are ï¬nal products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for ï¬ve days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these ï¬ndings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o
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