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"At present the relationship between hypothyroidism and the risk of breast cancer is still inconclusive Thismetaanalysis was used to systematically assess the relationship between hypothyroidism and breast cancer riskand to assess whether thyroid hormone replacement therapy can increase breast cancer riskMethods The relevant s about hypothyroidism and the risk of breast cancer were obtained on the electronicdatabase platform Relevant data were extracted and odd ratios OR with corresponding confidence intervalsCI were merged using Stata SE softwareResults A total of related studies were included in the metaanalysis including cohort studies and casecontrol studies The results show that hypothyroidism was not related to the risk of breast cancer odd ratios CI In the European subgroup we observed that patients with hypothyroidism have a lower risk ofbreast cancerodd ratios CI Furthermore no significant correlation was observed betweenthyroid hormone replacement therapy and the risk of breast cancer odd ratios CI Conclusion Hypothyroidism may reduce the risk of breast cancer in the European population and no significantcorrelation was observed between hypothyroidism and breast cancer risk in nonEuropean populations Due to thelimited number of studies included more largescale highquality longterm prospective cohort studies areneededKeywords Hypothyroidism Thyroid hormone replacement therapy Breast cancer MetaanalysisBackgroundAs a global public health problem breast cancer has anincreasing incidence on a global scale [] According tothe US cancer statistics breast cancer has becomethe most common malignant tumour in women withabout new cases each year accounting for of new malignant tumours in women [] Therefore theidentification of risk factors for breast cancer and the Correspondence Yanhuangdr163com Ruobaolidr163com2Department of Oncology Affiliated Hospital of Weifang Medical UniversityWeifang China3School of Basic Medicine Weifang Medical University Weifang ChinaFull list of author information is available at the end of the adoption of effective early prevention and interventionmeasures are of great significance for patients withbreast cancerThe physiology and pathology of the breast are closelyrelated to the endocrine of the body [] As the largestendocrine an in the human body the thyroid glandhas specific regulation effects on various hormone levelsand cell growth and development in the body whichbrings new enlightenment to the research of breast cancer [] In Kapdi et alfirst proposed thathypothyroidism maybe increase the risk of breast cancer[] Since then many scholars have studied the relationship between hypothyroidism and the risk of breast 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 0cWang BMC Cancer Page of []cancer However the relationship between the two diseases remains controversial [] Some studies haveshown that hypothyroidism increases the risk of breastcancerthathypothyroidism reduces the risk of breast cancer []Besides some studies have found no correlation betweenthyroid disease and breast cancer risk [] Thereforewhether hypothyroidism can increase the risk of breastcancer is worthy of further studystudiesshownSomehaveTwo metaanalyses have previously been studied forhypothyroidism and breast cancer risk [ ] Based onprevious research we have included more prospectivestudies and Asian population studies to assess the relationship between hypothyroidism and breast cancer risksystematically Besides the impact of thyroid hormonereplacement therapy on breast cancer risk was exploredin this metaanalysisMethodsSearch strategyRelevant clinical literature was extracted by systematicretrieval of PubMed Medline EMBASE Springer Webof Science and Cochrane Library electronic databasesup to date to October Our search strategy includedorhypothyroidism or HT and thyroid diseases orbreast cancer or BC or breast neoplasms or mammarmy cancer and risk orincidence At the sametime we manually screened out the relevant potentialliterature in the references extracteddysfunctionthyroidtermsforInclusion and exclusion criteria The inclusion criteria Types of studies Published studies exploring therelationship between hypothyroidism and breastcancer risk Subject Female Exposure factors Primary hypothyroidism thediagnosis needs to be based on the detection ofthyroid function Outcome indicators the occurrence of primarybreast cancerThe exclusion criteria Nonprimary hypothyroidism due to other causes Non observational studiesInsufficient information was provided or no fulltext Unable to obtain full text or quality assessment ofthe literature Studies were repeated or publications overlappedData extraction and quality assessmentTwo researchers separately conducted literature screening data extraction and literature quality evaluationand any differences could be resolved through discussionor a third inspector Information secured from the enrolled literature included first authors surname year ofpublication country ofthe population sample sizefollowup time and data on the relationship betweenhypothyroidism and the risk of breast cancerThe NewcastleOttawa Scale NOS was used to assessthe quality of the study from three aspects cohort selection cohort comparability and outcome evaluation []NOS scores of at least six were considered highqualityliterature Higher NOS scores showed higher literaturequalityStatistical analysisAll data analysis was performed using Stata120 softwareMetaanalysis was performed according to the PRISMAguidelines The OR and 95CI from included studieswere treated with the combined effect size After thatthe heterogeneity test was conducted When P ¥ orI2 was performed it mean that there was no apparent heterogeneity and the fixedeffect model shouldbe applied for a merger When P or I2 ¥ indicated high heterogeneity the randomeffect model wasapplied Combined effect size if OR indicates thathypothyroidism is an unfavorable factor for breast cancer If OR is the opposite Publication bias Begg funnel plot and Egger test linear regression test were usedto research publication bias detection of the literatureincluded If P indicates obvious publication biasResultsProcess of study selection and description of qualifiedstudiesA total of studies were identified on our online databases After exclusion of duplicate references129 s were considered After screening the andtitle s were excluded After careful review ofthe full texts studies have been excluded because ofthem did not provide relevant data and s didnot have fulltext Nineteen s published between and met the inclusion criteria Fig A total of samples from studies involvingwere enrolled in this metaanalysis [ ] Sixcohort studies and casecontrol studies were includedin the study Twelve s were studied in the European population five in the North American populationand two in the Asian population All s are of highquality because of NOS score no less than The chiefcharacteristics of the enrolled materials are detailed inTable 0cWang BMC Cancer Page of Fig Flow chart of search strategy and study selectionRelationship between hypothyroidism and breast cancerriskThere were studies reported the relationship betweenhypothyroidism and breast cancer risk With obviousheterogeneity I p among these studies so a random effect model was used for assessmentThe pooled analysis suggested that was not related tothe risk of breast cancer OR CI P 0001Fig explorethefurtherrelationshipSubgroup analysis of hypothyroidism and risk of breastcancerTobetweenhypothyroidism and breast cancer risk subgroup analysis was conducted from three aspects study typepopulation distribution and followup time The resultsof subgroup analysis were shown in Table In theEuropean subgroup we observed that patients withhypothyroidism have a lower risk of breast cancer OR CI P In the subgroup witha followup date of more than four years patients withhypothyroidism can reduce the risk of breast cancerwith borderline significance OR CI In otherP found thathypothyroidism was not related to the risk of breastcancersubgroups weRelationship between thyroid hormone replacementtherapy and breast cancer riskA total of studies reported the relationship betweenthe use of thyroid hormone replacement therapy and therisk of breast cancer [ ] Asobvious heterogeneity observed the fixedeffect modelwas usedI p The result suggestedthat patients who received thyroid hormone replacementtherapy was not related to the risk of breast cancerOR CI 109P Fig Publication biasFigure 4a shows the results of publication bias for the relationship between hypothyroidism and breast cancerrisk which were evaluated by funnel plots and Eggerstest The Begg test Pr and the Egger testP were used to detecting publication bias showedthat there was no possibility of publication bias Asshown in Fig 4b there were no publication biases in the 0cWang BMC Cancer Table Main characteristics of the included studies in ouranalysisStudySampleYearRegionAdamiKalacheHoffmanBrintonMosesonSmythSheringTalaminiSimonTurkenKuijpensCristofanilliSandhuHellevikDitschGraniSøgaardWengKimSwedenUKSwedenUSACanadaIrelandIrelandItalyUSAPragueNetherlandsUSACanadaNorwegianGermanyItalyDanishUSAKoreaMedianMean ageyearsNANA ± NANA ± ± ¥ ± ¥Page of NOSFollowupyearsNANANAStudydesignCasecontrolCasecontrolCohortCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCohortCasecontrolCohortCohortCasecontrolCasecontrolCohortCasecontrolCohortStudyIDAdami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Overall Isquared p ES CIES CI WeightWeightNOTE Weights are from random effects analysisFig Relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Table Stratiedanalysis of the relationship between hypothyroidism and breast cancer riskVariableOR95CINoofstudiesPHeterogeneityI2RegionEur orth AmericaAsiaStudy designCasecontrolCohortFollowup date ¤ Page of ModelusedFixedRandomedFixedRandomedFixedFixedRandomedPhStudyIDHoffman Kuijpens Sandhu Ditsch Cristofanilli Simon Moseson Brinton Adami Weng ES CIES CI WeightWeightOverall Isquared p NOTE Weights are from random effects analysisFig Relationship between thyroid hormone replacement therapy and breast cancer risk 0cWang BMC Cancer Page of A ]rr[golB ]rh[golBegg's funnel plot with pseudo confidence limitsEgger's publication bias plotse of log[rr]Begg's funnel plot with pseudo confidence limitstceffe idezdradnatstceffi edezdradnatsprecisionEgger's publication bias plotse of log[hr]precisionFig Publication bias assessment a hypothyroidism b thyroid hormone replacement therapy Metaanalysis estimates given named study is omitted Lower CI Limit Estimate Upper CI Limit Adami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Fig Sensitivity analysis for relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Page of s on the study of thyroid hormone replacementtherapy The Egger test was P and the Begg testwas Pr Sensitivity analysisThe results of sensitivity analysis are generally stableand the primary source of heterogeneity is in the research of Cristofanilli []Fig So we excludedthe literature of Cristofanilli and analyzed the otherstudies The results revealed that the hypothyroidismcould reduce the risk of breast cancer was borderlineOR096 95CI092 P andsignificantthere was no heterogeneityI2 P cohortstudy ofDiscussionMore than years ago Beatson used thyroid extracts to treat patients with metastatic advanced breastcancer The condition was significantly alleviated sparkinginterest in exploring the relationship between thyroid andbreast cancer [] Subsequently a prospective study enrolled women and women with earlier diagnosisof hypothyroidism observed the occurrence of breast cancer during followup showed that low serum free thyroxine levels increased the risk of breast cancer [] In aprospective women withhypothyroidism and hyperthyroidism found thathypothyroidism slightly reduced the risk of breast cancer[] However a prospective cohort study of women with autoimmune hypothyroidism and women with normal thyroid function indicated that autoimmune hypothyroidism was not associated with breastcancer risk [] Besides some animal experiments alsoreflect the relationship between the two [ ] Animalexperiments by López Fontanafound thathypothyroidism mice inhibit the development of breastcancer and promote the apoptosis of breast cancer cellsdue to the low expression of βchain protein and activation of the apoptotic pathway on the tumour cell membrane [] Due to the inconsistency ofthe aboveconclusions we performed a metaanalysis to evaluate therelationship between hypothyroidism and breast cancerrisketalA total of studies were included in this metaanalysis and the results showed that patients withhypothyroidism not related to the risk of breast cancerHowever there was significant heterogeneity among theincluded studies After subgroup analysis and sensitivityanalysis we found that Cristofanillis research may causeheterogeneity [] Cristofanillis research is a retrospective study and the diagnosis of hypothyroidism patientswas based on the information recorded in the medicalrecords which may lead to the bias risk of misclassification and have a positive impact on the positive results ofthis study [] After excluding Cristofanillis researchwe found that patients with hypothyroidism had a lowerrisk of breast cancer with borderline significance [] Theresults of the metaanalysis are inconsistent with the findings of Hardefeldt and Angelousi [ ] Perhaps because our study included more prospective studiesand Asian population cohort study In addition we evaluated the risk of breast cancer in thyroid hormone replacement therapy and show that patients who received thyroidhormone replacement therapy was not related to the riskof breast cancerIn the analysis of the European population the resultsshow that hypothyroidism may reduce the risk of breastcancer We also found that patients with hypothyroidismcan reduce the risk of breast cancer was borderline significance in the subgroup with more longer followupdate However the relationship between the two was notobserved in North American and Asian populationsThe possible reasons for these disparities may be as follows First followup time may be the main contributorsto this difference A longer followup is required to demonstrate the relationship between hypothyroidism andbreast cancer risk In the metaanalysis five studies provided North American population data and two reported Asian population data However only one ofseven nonEuropean studies followup time for morethan years Second the differences may be attributedto different ethnicities sharing different genegene andgeneenvironmental backgrounds Third social and environmental factors are another critical cause for thisdifference With these in mind our findings suggest thathypothyroidism may reduce the risk of breast canceronly in the European population and more largescalehighqualitylongterm prospective cohort studies arestill needed to study on different human populationsThe following may explain the potential relationshipbetween hypothyroidism and the risk of breast cancerHealthy mammary epithelial cells can express a largenumber of T3 receptors and breast cancer cells have asimilar ability to bind to T3 [] T3 has an estrogenlike effect that promotes the growth of mammary glandlobes and stimulates normal breast tissue differentiation[ ] Therefore T3 can mimic the effect of estrogenon the proliferation of breast cancer cells When theconcentration of T3 is low in vivo it may inhibit theproliferation of breast cancer cells Hypothyroidism mayreduce the risk of breast cancer by affecting T3concentrationSome basic experiments support this theory In GonzalezSancho studied the relationship betweenT3 and breast cancer [] It was found that there is anoverexpressed T1 gene in human breast cancer cellsand T3 inhibits the proliferation of mammary epithelialcells by inhibiting the expression of cyclin D1 and T1thereby inhibiting the proliferation of breast cancer cells 0cWang BMC Cancer Page of Author details1School of Clinical Medicine Weifang Medical University Weifang China 2Department of Oncology Affiliated Hospital of Weifang MedicalUniversity Weifang China 3School of Basic Medicine WeifangMedical University Weifang ChinaReceived December Accepted July foundthat MartinezIglesias[] Afterthathypothyroidism can inhibit the growth of breast cancercells [] In Tosovic conducted a prospectivestudy of T3 levels associated with breast cancer risk andfound that T3 levels in postmenopausal women werepositively correlated with breast cancer risk in a doseresponse mannerthathypothyroidism through lower levels of T3 could reducethe incidence of breast cancer Our metaanalysis resultsalso confirm the above conjecture[] Therefore we suspectHowever this conclusion needs to be taken with caution as this study has several limitations First the studies that have been included do not adjust for importantrisk factors for breast cancer Second in subgroup analysis for example there are only two s in Asianstudies and we should be cautious about the results ofAsian analysis Third the results of this metaanalysis indicate that there is a large heterogeneity between studiesFourth followup time at different endpoints cannot beuniform Finally publication bias cannot be avoidedentirelyConclusionHypothyroidism may reduce the risk of breast cancer inthe European population and no significant correlationwas observed between hypothyroidism and breast cancerrisk in nonEuropean populations Furthermore therewas no obvious correlation between thyroid hormone replacement therapy and breast cancer risk It is necessaryto conduct a large sample size strictly controlled prospective study of hypothyroidism patients further todemonstrate the relationship between hypothyroidismand breast cancer riskAbbreviationsOR Odd ratios CI Confidence intervals NOS NewcastleOttawa ScaleAcknowledgementsNot applicableAuthors contributionsStudy design BW ZL RLYH and TL Data extraction BW ZL TL and YH Dataanalysis BW ZL RLand YH Manuscript writing BW and RL Manuscriptedition RL and YH All authors have read and approved the manuscriptFundingNo sources of funding were used to conduct this study or prepare this letterAvailability of data and materialsAll the published s and data were available onlineEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsNoneReferencesSiegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin httpsdoi103322caac21442Praestegaard C Kjaer SK Andersson M StedingJensen M Frederiksen KMellemkjaer L Risk of skin cancer following tamoxifen treatment in morethan breast cancer patients a cohort study Breast cancer httpsdoi101007s1228201506605 Mittra I Hayward JL Hypothalamicpituitarythyroid axis in breast cancerLancet httpsdoi101016s0140673674903444Adami HO Rimsten A Thoren L Vegelius J Wide L Thyroid disease andfunction in breast cancer patients and nonhospitalized controls evaluatedby determination of TSH T3 rT3 and T4 levels in serum Acta Chir ScandDargent M Berger M Lahneche B Thyroid function in patients with Cancerof the breast Acta Mustacchi P Greenspan F Thyroid supplementation for hypothyroidism Anlatrogenic cause of breast cancer JAMA Kapdi CC Wolfe JN Breast cancer Relationship to thyroid supplements forhypothyroidism JAMA httpsdoi101001jamaKuijpens JL Nyklictek I Louwman MW Weetman TA Pop VJ Coebergh JWHypothyroidism might be related to breast cancer in postmenopausalwomen Thyroid httpsdoi101089thy200515 Weng CH Chen YH Lin CH Luo X Lin TH Thyroid disorders and breastcancer risk in Asian population a nationwide populationbased casecontrolstudy in Taiwan BMJ 201883e020194 httpsdoi101136bmj 2017020194Sogaard M Farkas DK Ehrenstein V Jensen JO Dekkers OM SorensenHT Hypothyroidism and hyperthyroidism and breast cancer risk anationwide cohort study Eur J Endocrinol httpsdoi101530EJE150989 Angelousi AG Anagnostou VK Stamatakos MK Geiopoulos GAKontzoglou KC Mechanisms in endocrinology primary HT and risk forbreast cancer a systematic review and metaanalysis Eur J Endocrinol httpsdoi101530EJE110838 Hardefeldt PJ Eslick GD Edirimanne S Benign thyroid disease is associatedwith breast cancer a metaanalysis Breast Cancer Res Treat httpsdoi101007s1054901220193Stang A Critical evaluation of the NewcastleOttawa scale for theassessment of the quality of nonrandomized studies in metaanalyses Eur JEpidemiol httpsdoi101007s106540109491zKalache A Vessey MP McPherson K Thyroid disease and breast cancerfindings in a large casecontrol study Br J Surg httpsdoi101002bjs1800690731 Hoffman DA McConahey WM Brinton LA Fraumeni JF Jr Breast cancer inhypothyroid women using thyroid supplements JAMA Brinton LA Hoffman DA Hoover R Fraumeni JF Jr Relationship of thyroiddisease and use of thyroid supplements to breast cancer risk J Chronic Dis httpsdoi1010160021968184900626 Moseson M Koenig KL Shore RE Pasternack BS The influence of medicalconditions associated with hormones on the risk of breast cancer Int JEpidemiol httpsdoi101093ije2261000Shering SG Zbar AP Moriarty M McDermott EW O'Higgins NJ Smyth PPThyroid disorders and breast cancer Eur J Cancer Prevent Smyth PP Smith DF McDermott EW Murray MJ Geraghty JG O'Higgins NJA direct relationship between thyroid enlargement and breast cancer J ClinEndocrinol Metab httpsdoi101210jcem813Talamini R Franceschi S Favero A Negri E Parazzini F La Vecchia CSelected medical conditions and risk of breast cancer Br J Cancer httpsdoi101038bjc1997289 0cWang BMC Cancer Page of Simon MS Tang MT Bernstein L Norman SA Weiss L Burkman RT DalingJR Deapen D Folger SG Malone K Marchbanks PA McDonald JA Strom BLWilson HG Spirtas R Do thyroid disorders increase the risk of breast cancerCancer Epidemiol Biomarkers Prevent Turken O NarIn Y DemIrbas S Onde ME Sayan O KandemIr EG Yaylac IMOzturk A Breast cancer in association with thyroid disorders Breast CancerRes 200355R110 httpsdoi101186bcr609 Cristofanilli M Yamamura Y Kau SW Bevers T Strom S Patangan M Hsu LKrishnamurthy S Theriault RL Hortobagyi GN Thyroid hormone and breastcarcinoma Primary hypothyroidism is associated with a reduced incidenceof primary breast carcinoma Cancer httpsdoi101002cncr20881 Hellevik LR Vierendeels J Kiserud T Stergiopulos N Irgens F Dick ERiemslagh K Verdonck P An assessment of ductus venosus tapering andwave transmission from the fetal heart Biomech Model Mechanobiol httpsdoi101007s1023700901554Sandhu MK BrezdenMasley C Lipscombe LL Zagorski B Booth GLAutoimmune hypothyroidism and breast cancer in the elderly BreastCancer Res Treat httpsdoi101007s10549008 Ditsch N Liebhardt S Von Koch F Lenhard M Vogeser M Spitzweg CGallwas J Toth B Thyroid function in breast cancer patients Anticancer Res Grani G Dicorato P Dainelli M Coletta I Calvanese A Del Sordo M DeCesare A Di Matteo FM D'Andrea V Fumarola A Thyroid diseases inwomen with breast cancer La Clin Terapeut 20121636e401Kim EY Chang Y Lee KH Yun JS Park YL Park CH Ahn J Shin H Ryu SSerum concentration of thyroid hormones in abnormal and euthyroidranges and breast cancer risk a cohort study Int J Cancer httpsdoi101002ijc32283 Beatson GT On The Treatment Of Inoperable Cases Of Carcinoma Of TheMamma Suggestions For A New Method Of Treatment With IllustrativeCases1 Lancet Lopez Fontana CM Zyla LE Santiano FE Sasso CV CuelloCarrion FDPistone Creydt V Fanelli MA Caron RW Hypothyroidism reduces mammarytumor progression via Betacateninactivated intrinsic apoptotic pathway inrats Histochem Cell Biol httpsdoi101007s004180171544x MartinezIglesias O GarciaSilva S Regadera J Aranda A Hypothyroidismenhances tumor invasiveness and metastasis development PLoS One 47e6428 httpsdoi101371journalpone0006428 Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast cancer cell lines J Steroid Biochem Mol Biol httpsdoi101016s0960076096001173 Alyusuf RH Matouq JA Taha S Wazir JF The pattern of expression and roleof triiodothyronine T3 receptors and type I ²deiodinase in breastcarcinomas benign breast diseases lactational change and normal breastepithelium Appl Immunohistochem Mol Morphol httpsdoi101097PAI0b013e3182a20917 Pereira B Rosa LF Safi DA Bechara EJ Curi R Control of superoxidedismutase catalase and glutathione peroxidase activities in rat lymphoidans by thyroid hormones J Endocrinol httpsdoi101677joe01400073 GonzalezSancho JM Figueroa A LopezBarahona M Lopez E Beug HMunoz A Inhibition of proliferation and expression of T1 and cyclin D1genes by thyroid hormone in mammary epithelial cells Mol Carcinog httpsdoi101002mc10046Tosovic A Bondeson AG Bondeson L Ericsson UB Malm J Manjer JProspectively measured triiodothyronine 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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 | 0 |
Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedObjective To investigate whether preventive administration of a proton pump inhibitor PPI can reduce the occurrence anddevelopment of traumatic granuloma TG following type IVVI cordectomy Methods We retrospectively analyzed the statusof postoperative granulomas in patients who underwent type IVVI cordectomy due to glottic cancer and determinedwhether postoperative administration of a PPI had any impact on granuloma formation and development Results Thepercentage and number of patients with granuloma in the PPI treatment group experimental group at the 1st 2nd 3rd and6th month following surgery were and respectively The percentageand number of patients with granuloma in the noPPI group control group were and respectively The granuloma percentage of the PPI treatment group was lower than that of thecontrol group at all postoperative time points assessed The diï¬erences were not statistically significant at the 1st monthp but were statistically significant at the 2nd and 3rd months after surgery p p ConclusionPreventive use of a PPI in patients after type IVVI cordectomy can shorten the TG recovery duration and may reduce theseverity of TG but it cannot prevent TG from occurring Our results should be conï¬rmed by prospective randomized controlledtrials with large sample sizes IntroductionLaryngeal squamous cell carcinoma LSCC is a commonhead and neck cancer It had an incidence of approximately in China between and [] and new cases were reported worldwide in [] Itis estimated that there will be new cases worldwidein [] Approximately twothirds of LSCCs originatein the glottic area The presence of hoarseness in patientswith earlystage glottic cancer GC prompts the patients toseek medical treatment Anatomically the larynx is surrounded by cartilage and has sparse lymphatic tissue As aresult patients with GC are mostly diagnosed at an earlystage which is clinically deï¬ned as T12N0M0 []In recent years surgery has been gradually abandoned in the treatment of earlystage GC and has beenreplaced with transoral microsurgery TM or radiotherapyTM has the advantages of being minimally invasive and having a high laryngeal preservation rate and high costeï¬ectiveness Transoral laser microsurgery TLM is the mostcommon surgical method used for GC although a few studies in the literature have adopted transoral coblation microsurgery TCM Although TM methods diï¬erthermalinstruments are often needed for the surgery []Traumatic granuloma TG is a common complication ofTM especially after type IVV cordectomy Mild TG mayonly manifest as hoarseness foreign body sensation and frequent throat clearing The severe granuloma may cause or becomplicated by perichondritis which leads to severe symptoms such as dyspnea Additionally this granulation isbelieved to be an important factor in the formation of glotticweb and larynx stenosis [] Reï¬ux is an important 0cBioMed Research Internationalfactor that aï¬ects granuloma formation Proton pump inhibitors PPI are often used empirically in the treatment ofpatients with postoperative granulomas [ ]We found in our previous clinical practice that patientswith a wide range of resections such as type IVVI cordectomies have a higher risk of postoperative granuloma Thiscan sometimes be very severe even requiring temporary tracheotomy to alleviate dyspnea which greatly and adverselyimpacts patients quality of life PPIs have been empiricallyused for the treatment of patients with postoperative granuloma and evidence of its eï¬ectiveness has been published[] However there has not been any research on the prevention of postoperative granuloma formation with PPIsTherefore we initiated PPI treatment for patients who hadundergone type IVVI cordectomies and compared theresults with those of patients who had also undergone thistype of surgery but were not treated with PPIs to evaluatethe eï¬ect of PPI treatment for preventing postoperative granuloma formation in this patient population Materials and Methods Patients This was a retrospective study Patient inclusioncriteria i patients with vocal cord cancer who agreed toundergo type IV V and VI cordectomies ii patients whodid not undergo preoperative and postoperative radiotherapy Exclusion criteria i patients who were complicatedwith diabetes and were not treated routinely ii patientswho used PPIs regularly iii patients who continued tosmoke and drink after surgeryA total of patients between January and December were recruited as the control group who did not use PPIsimmediately after surgery and did not take PPIs persistentlyA total of patients between January and June were recruited as the experimental group PPI treatment group who took PPIs immediately after the surgeryand routinely Since this was a retrospective study only thepatients in the experimental group underwent preoperativereï¬ux symptom index RSI and reï¬ux ï¬nding score RFSassessments Patients with an RSI score points andoran RFS ¥ points were considered to have laryngopharyngeal reï¬ux disease LPRD [ ]All patients signed an informed consent form prior to thesurgery All clinical experiments conformed to the guidelinesissued by the committee on clinical research of Peking UnionMedical College Hospital PUMCH Ethics Committeeapproval was obtained at PUMCH and all patients providedspeciï¬c written informed consent Surgical Procedure According to the European Laryngological Society classiï¬cation endoscopic cordectomies includethe following types type IV total cordectomy type Vaextended cordectomy encompassing the contralateral cordtype Vb extended cordectomy encompassing the arytenoidtype Vc extended cordectomy encompassing the ventricularband type Vd extended cordectomies encompassing thesubglottis and type VI extended cordectomies encompassing the anterior commissure [ ] During the surgerythe larynx was fully exposed with a selfretaining laryngo°scope Karl Storz Tuttlingen Germany and the tumor°was resected en bloc under direct visualization of a orlaryngoscope Karl Storz Tuttlingen Germany using amodel coblator ArthroCare Corp Sunnyvale CA witha coblation level of and a coagulation level of Postoperative Treatment Procedure and FollowUp Thepatients in the experimental group were treated with intravenous omeprazole mg per day before the recovery of oralfeeding cases recovered oral feeding on the ï¬rst day aftersurgery and cases recovered oral feeding on the third dayThen they were given mg oral omeprazole twice daily minutes before breakfast and dinner for consecutiveweeks The patients in the control group were not treatedwith PPIs but if severe granulomas formed during followup and required intervention they were also given PPIs routinely patients in the control group developedgranulomas But only patients developed severe granulomas on the 2nd 3rd and 3rd month after surgery respectively and began to be given PPIs for weeks the same asthe experimental group while the other cases in the control group were not given PPIs throughoutthe wholefollowup period All patients were treated with cefuroximeat mg twice daily for week to prevent infection Afterthe surgery the patients were required to quit smoking anddrinking and engage in reasonable vocal useThe followup procedure included regular checkups at and months after surgery If a granuloma was detectedduring the 3month checkup the patients were followedmonthly until the granuloma disappeared Postoperativegranuloma was deï¬ned as relatively smooth tissue in the surgery region that protruded from the surrounding area thatmay be attached to a pseudomembrane The granulomaand the surrounding area did not have obvious vascularhyperplasia The proportion of patients with postoperativegranuloma was used as an observation indicator Additionally the number of unscheduled visits and the rate of reoperationincluding tracheotomy and granulectomy wererecorded as indicators of granuloma severityDuring followup if the granuloma was found to severelyimpact vocalization andor breathing or if patients were suspected of having a recurrent tumor the granuloma was surgically resected and the specimen was sent for examination Statistical Analysis Data were statistically analyzed withSPSS software SPSS Chicago IL Nonnormally distributed quantitative data are represented as median and interquartile range and were subjected to the Wilcoxon ranksumtest Normally distributed measurement data are representedas mean ± standard deviation and were subjected to theindependent sample t test Count data were subjected to thechisquared test Diï¬erences with p were consideredstatistically significant Results Baseline Characteristics The baseline data of the patientsin the PPI treatment group and the control group are shownin Table Among the patients in the PPI treatment 0cBioMed Research InternationalTable General information DiscussionVariableSex MFAge yearsTumor staging T1aT1bT2Surgery type IVVVIPPIn ± Controln ± p valuegroup patients were male and patient was female withan average age of ± years patients were at theT1 stage and patients were at the T2 stage patients underwent type IV surgery patients underwent type V surgeryand patients underwent type VI surgery Among the patients in the control group patients were male and patient was female with an average age of ± years patients were at the T1 stage and patients were at theT2 stage patients underwent type IV surgery patientsunderwent type V surgery and patients underwent typeVI surgery The two groups of patients did not diï¬er significantly in the baseline conditions Table Postoperative Granulation The numbers and percentageof patients with granuloma in the PPI treatment group atthe 1st 2nd 3rd and 6thmonth followup were and respectively The numbers and percentage of patients with granuloma in the control group at those time points were and respectively Although the percentage of granuloma in the PPItreatment group was lower than that of the control group ateach stage only the diï¬erences at the 2nd and 3rd monthsafter surgery were statistically significant p and p respectively Only one patient in the PPItreatment group required a second surgery due to persistentgranulation patients in the control group underwent a second surgery among whom patients had granuloma complicated with chondronecrosis and required totracheotomy due to dyspnea Figure However the diï¬erence between the two groups was not statistically significantp In the PPI treatment group only patients hadtwo unscheduled visits In the control group patients had unscheduled visits among them one patient had visitsdue to dyspnea The diï¬erence in the number of unscheduledvisits was not statistically significant p Table Eï¬ects of PPI Treatment in Patients with LPRD in theExperimental Group Among the patients in the experimental group were diagnosed with LPRD according to preoperative RSI and RFS scores and patients did not have LPRD The numbers of LPRDpatients with granuloma at the 1st 2nd 3rd and 6th monthsafter surgery were and respectively and the numbers of LPRD patients with granuloma at these time pointswere and While the data showed that the percentageof granuloma in the LPRD patients was higher than that inthe LPRD patients only the diï¬erence at the 3rd month aftersurgery was statistically significant p Table Transoral microsurgery TM for earlystage glottic cancerGC can achieve oncological therapeutic eï¬ects similar tothose of radiotherapy TM has the advantages of being minimally invasive and having a high laryngeal preservation rateand a low tracheotomy rate its disadvantage includes postoperative complications such as bleeding infection airwayburns and granuloma formation Therefore currently thetreatment selected for patients with earlystage GC is determined by the disease conditions as well as patient needs[ ] Postoperative traumatic granuloma TG is a common complication during the healing process after TLM surgery Severe TG may cause or be complicated by chondritis orchondronecrosis leading to severe complications includingdyspnea With the increase in the range of cordectomy theincidence of TG is also significantly increased Therefore itis necessary to investigate ways to reduce the incidence andseverity of postoperative TG [ ] Like TLMthecoblationassisted endoscopic cordectomy or TCM used inour study is also based on thermal damage whose working°C much lower than lasers workingtemperature is °°temperature which is C1000C and as a result its healing process and the mechanism of TG formation are alsosimilar to those of TLM Current literature indicates thatreï¬ux may be an important factor in the occurrence of postoperative TG [ ] Therefore we aimed to investigatewhether antiacid therapy could reduce TG through an analysis of the eï¬ects of PPI treatment in patients who underwenttype IVVI cordectomy which has rarely been reported°C70Our study showed that the incidences of TG after transoral surgery were as high as and in the twogroups which were much higher than the incidences of reported by Nerurkar and Shah and reported by Wang The reason for this discrepancy may be that the patients in our study all underwent typeIV or higher surgeries Enlarged wounds and damage to theperichondrium or cartilage may lead to increased TG andchondronecrosis Nerurkar and Shah reported that the incidence of TG in patients who have undergone type IV TLMwas and the study by Wang showed that the incidences of TG in patients who have undergone type IV andtype V TLM were and respectively [ ]Meanwhile the incidence of chondronecrosis was not rare especially on whom the surgeon had to expose thethyroid cartilage during tumor resection in TLM [] Thesestudies suggest that type IV or higher surgeries lead to a highlikelihood of TG occurrenceOur study showed that PPI treatment did not suppressthe formation of TG at the 1st month after surgery but thepercentage of TG in the PPI treatment group graduallybecame lower than that of the control group and the diï¬erences were statistically significant at the 2nd and 3rd monthsafter surgery At the 6th month granulomas disappeared inboth groups Our ï¬ndings suggest that although PPI treatment cannot reduce the incidence of TG it can significantlyshorten the duration of granulomas a ï¬nding that is similarto the study by Wang [] Additionally we did notobserve any severe cases of TG that were complicated with 0cBioMed Research InternationalabcdefghiFigure af Shows a typical case in the control group who was a male patient for years old with glottic cancer T2N0M0 at the left sidefollowed by type V cordectomy a One month after surgery a granuloma was found in the left vocal cord under a ï¬brolaryngoscope b months after surgery the granuloma enlarged and the right vocal cord become edema obviously c months after surgery d monthsafter surgery e Computerized tomography CT scan taken before surgery f months after surgery chondronecrosis was found in the CTscan where the white arrow points out gi Shows a typical case in the experimental group who was a male patient for years old withglottic cancer T2N0M0 at the right side followed by type V cordectomy g h and i were taken under a ï¬brolaryngoscope in month months and months after surgery respectively A granuloma could be found in g but disappeared in h and idyspnea in the PPI treatment group however TG in patients in the control group caused or was complicated bychondritis or chondronecrosis which led to severe dyspneaThere were only unscheduled visits among the PPI treatment group compared to unscheduled visits in the controlgroup Unfortunately the diï¬erences in the two indicators ofTG severity between the two groups were not statistically significant although we believe PPI treatment did alleviate the 0cBioMed Research InternationalTable Percentage and severity of granuloma in the two groups1st month2nd month3rd month6th monthResurgeryNumber of unscheduled visits asmedian and interquartile rangePPI treatment groupControl groupp valueNote aindicates that the diï¬erence is statistically significant0005a0037a Table Percentage of granuloma in patients with or without LPRDin the experimental group1st month 2nd month 3rd month 6th monthLPRD n LPRD n p valueNote aindicates that the diï¬erence is statistically significant0029aseverity of TG In this study postoperative PPI was used for weeks referring to the antiacid duration weeks in amedical routine of vocal process granulation and laryngopharyngeal reï¬ux disease J R Lechiens report in andChinse experts consensus on diagnosis and treatment of laryngopharyngeal and reï¬ux disease in [] The resultsshowed that PPI could shorten the recovery time and potentially prevent severe complicationsOur study showed that the percentage of TG did not differ significantly between the LPRD and LPRD patientsp However it took longer for granulomas to disappear in LPRD patients than in LPRD patients and the difference in the number of patients with granulomas wasstatistically significant between the two groups at monthsafter surgery p The reason for this phenomenonmay be that during the early stage of recovery after vocalcord injury changes in the extracellular matrix are the mainmanifestation and injury impacts a lot while acid reï¬ux hasa little eï¬ect Acid reï¬ux may impact the repair process in themiddle and late stages [ ]This was a retrospective nonrandomized controlledstudy with a small sample size We only analyzed granulomaformation in the patients and did not assess their oncologicaloutcome We based the diagnosis of LPRD on RSI and RFSscores and did not perform dualprobe 24hour pH monitoring Therefore we were unable to determine whethernonacid reï¬ux had an impact on the formation and development of postoperative granuloma ConclusionThe preventive use of PPI in patients who have undergonetype IVVI cordectomy cannot reduce the incidence of TGwhile it can shorten the TG recovery duration and may alsoreduce the severity of TG Our ï¬ndings should be conï¬rmedby prospective randomized controlled studies with largersample sizesData AvailabilityAccess to these anonymized data will be made available bythe corresponding author Dr Jian Wang upon reasonablerequestConflicts of InterestThe authors declare that they have no conï¬icts of interestï¬nancial or nonï¬nancial to discloseAuthors ContributionsXiaofeng Jin and Yanyan Niu contributed equally to thisstudyAcknowledgmentsThis study was funded by the Nature Science Foundation ofBeijing China Grant No References[] L B Du W M Mao W Q Chen Incidence and mortality of larynx cancer in China during ZhonghuaLiu Xing Bing Xue Za Zhi vol no pp [] F Bray J Ferlay I Soerjomataram R L Siegel L A Torreand A Jemal Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA A Cancer Journal for Clinicians vol no pp [] B Gupta N W Johnson and N Kumar Global epidemiology of head and neck cancers a continuing challenge Oncology vol no pp [] M F Vaculik C A MacKay S M Taylor J R B Trites R DHart and M H Rigby Systematic review and metaanalysisof T1 glottic cancer outcomes comparing CO2 transoral lasermicrosurgery and radiotherapy Journal of Otolaryngology Head and Neck Surgery vol no p [] W Steiner Results of curative laser microsurgery of laryngealcarcinomas American Journal of Otolaryngology vol no pp [] M S Strong Laser excision of carcinoma of the larynxLaryngoscope vol no pp [] A S Carney M S Timms C N Marnane S KrishnanG Rees and S Mirza Radiofrequency coblation for the resection of head and neck malignancies Otolaryngology and Headand Neck Surgery vol no pp [] M Lee M A Buchanan F Riï¬at and C E Palme Complications after CO2 laser surgery for early glottic cancer an 0cBioMed Research Internationalinstitutional experience Head Neck vol no S1 pp E987E990 [] B Liu L Cheng H Ming and C Zhong Treatment of theearlystage glottic cancer using lowtemperature radiofrequency coblation Journal of Cancer Research and Therapeutics vol no pp [] Y Zhang B Wang G Sun G Zhang L Lu and G LiangCarbon dioxide laser microsurgery versus lowtemperatureplasma radiofrequency ablation for T1a glottic cancer asingleblind randomized clinical trial BioMed Research International vol Article ID pages [] M Remacle H E Eckel A Antonelli Endoscopic cordectomy A proposal for a classiï¬cation by the Working Committee European Laryngological Society European Archivesof OtoRhinoLaryngology vol no pp [] L Wang S Sun S Wang D Liang and W Ji Clinical observation of traumatic granuloma after CO laser cordectomy andlaryngopharyngeal reï¬ux Zhonghua Er Bi Yan Hou Tou JingWai Ke Za Zhi vol no pp [] M Canis F Ihler A Martin C Matthias and W SteinerTransoral laser microsurgery for T1a glottic cancer reviewof cases Head Neck vol no pp [] A Galli L Giordano D Sarandria D di Santo and M BussiOncological and complication assessment of CO2 laserassisted endoscopic surgery for T1T2 glottic tumours clinicalexperience Acta Otorhinolaryngologica Italica vol no pp [] N K Nerurkar and R Shah Factors responsible for the development of carbon granuloma post transoral laser cordectomy Lasers in Medical Science vol no pp [] P C Belafsky G N Postma and J A Koufman The validityand reliability of the reï¬ux ï¬nding score RFS Laryngoscopevol no pp [] P C Belafsky G N Postma and J A Koufman Validity andreliability of the reï¬ux symptom index RSI Journal of Voicevol no pp [] M Remacle C van Haverbeke H Eckel Proposal forrevision of the European Laryngological Society classiï¬cationof endoscopic cordectomies European Archives of OtoRhinoLaryngology vol no pp [] J Yoo C Lacchetti J A Hammond R W Gilbert and Headand Neck Cancer Disease Site Group Role of endolaryngealsurgery with or without laser versus radiotherapy in themanagement of early T1 glottic cancer a systematic reviewHead Neck vol no pp [] C M Chiesa Estomba F A Reinoso A O Velasquez J LFernandez J L Conde and C S Hidalgo Complications inCO2 laser transoral microsurgery for larynx carcinomas IntArch Otorhinolaryngol vol no pp [] J R Lechien F Mouawad M R Barillari Treatment oflaryngopharyngeal reï¬ux disease a systematic review WorldJournal of Clinical Cases vol no pp [] M K Wani and G E Woodson Laryngeal contact granuloma Laryngoscope vol no pp [] C Ling M Yamashita J Zhang D M Bless and N V Welham Reactive response of ï¬brocytes to vocal fold mucosalinjury in rat Wound Repair and Regeneration vol no pp 0c' | 2 |
"what clinicians know about the diagnosis treatment and prevention of disease originates from studies mostly done on male cells male mice and men1 historically for multiple reasons including the purported safety of women and their offspring women of childbearing age were excluded from clinical trials as a result medical research and care have been centred on male physiology the assumption was that male and female cells and animals were biologically identical and evidencebased medicine was defined by clinical trials done predominantly in men1 in the us national institutes of health nih mandated the inclusion of women in nihfunded clinical trials but many investigators did not follow this mandate and many of those who did include women did not analyse the results by sex23 minimising the effectiveness of this policy preclinical research and drug development studies have also predominantly used male animal models and cells4 it is not surprising that a us government accountability office report found that eight of the ten prescription drugs withdrawn from the market between and posed greater health risks for women than for men most funding agencies from europe and north america have implemented policies to support and mandate researchers to consider sex and gender at all levels of medical research8 still the field of sexbased biology and medicine is often viewed as a specialised area of interest rather than a central consideration in medical research essential for the success of clinical care and translational science is awareness by clinicians and researchers that the diseases they are treating and studying are characterised by differences between women and men in epidemiology pathophysiology clinical manifestations psychological effects disease progression and response to treatmentthis review explores the role of sex biological constructs and gender social constructs as modifiers of the most common causes of death and morbidity and articulates the genetic biological and environmental determinants that underlie these differences we aim to guide clinicians and researchers to better understand and harness the importance of sex and gender as genetic wwwthelancetcom vol august biological and environmental modifiers of chronic disease ultimately it is a necessary and fundamental step towards precision medicine that will benefit women and mensex as a genetic modifier of biology and diseasesex differences in disease prevalence manifestation and response to treatment are rooted in the genetic differences between men and women genetic sex differences start at conception when the ovum fuses with a sperm cell carrying an x or a y chromosome resulting in an embryo carrying either xx or xy chromosomes this fundamental difference in chromosome complement eg genes outside the testisdetermining sry gene generates ubiquitous sex differences in the molecular makeup of all male and female cells9 first the y chromosome carries genes that exhibit subtle functional differences from their xlinked homologues eg zfy vs zfx and uty vs utx and also carries genes with no homologue at all eg sry in addition in men the x chromosome carries only maternal imprints ie epigenetic modifications made by the parent in generating the sex cellswhich alter the expression of genes in the offspring as women have x chromosomes from both parents they carry maternal and paternal imprints which target a different set of genes random inactivation of one of the x chromosomes in female cells which prevents sex differences in x chromosome gene dosage causes another degree of sex difference in gene expression as some of these xlinked genes escape inactivation in women those genes are often expressed at higher levels in women than in men9 sexspecific gene expression due to genomic search strategy and selection criteriawe searched pubmed for papers published in english between jan and june using sex or gender and the name of the disease of interest as search terms although we tried to cite seminal studies when necessary because of space limitation representative reviews were often selectedlancet diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine and southeast louisiana veterans health care system medical center new orleans la usa prof f mauvaisjarvis md barbra streisand womens heart center cedarssinai smidt heart institute los angeles ca usa prof n bairey merz md national heart lung institute imperial college london london uk prof p j barnes md department of pharmacology and department of neurology college of medicine center for innovation in brain science university of arizona tucson az usa prof r d brinton phd department of medical epidemiology and biostatistics and center for gender medicine karolinska institutet stockholm sweden prof jj carrero phd channing division of network medicine and the division of pulmonary and critical care medicine department of medicine brigham and womens hospital harvard medical school boston ma usa d l demeo md neuroscience institute and department of biology geia state university atlanta ga usa prof g j devries phd department of psychiatry university of colorado school of medicine anschutz medical campus aurora co usa prof c n epperson md division of oncology department of medicine washington university school of medicine st louis mo usa prof r govindan md w harry feinstone department of molecular microbiology and immunology the johns hopkins bloomberg school of public health baltimore md usa prof s l klein phd department of biomedical metabolic and neural sciences university of modena andreview 0creggio emilia azienda ospedalierouniversitaria di modena ospedale civile di baggiovara modena italy prof a lonardo md department of psychiatry department of psychology and department of obstetrics gynecology university of illinois at chicago chicago il usa prof p m maki phd department of neurology mcgovern medical school university of texas health science center houston tx usa prof l d mccullough md berlin institute of gender medicine charituniversittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for womens health research divisions of general internal medicine and cardiology university of colorado school of medicine aurora co usaimprinting extends to autosomes as well and these imprinted genes exhibit sexspecific and tissuespecific expression in humans10 thus fundamental sex differences deriving directly from genetic heterogeneity between the x and y chromosome complements and parentoforigin inher itance exist at the molecular level in all human cells these sex differences persist throughout life and are independent of sex hormones figure arguably the greatest source of differences between men and women comes from the y chromosomal sry gene which directs the development of a testis in men the ensuing developmental surge of testicular testosterone permanently masculinises the reproductive tract and the anisation of brain circuits affecting male behaviour at puberty1112 in humans the first surge occurs at the end of the first trimester of pregnancy because it alters cellular gene expression and tissue structure in multiple ans of men via epigenetic mechanisms this testosterone surge is also paramount in programming sex differences in physiology and susceptibility to diseases that will manifest in adulthood after this initial testicular testosterone surge gonadal hormone concentrations remain low until puberty which triggers lasting sex differences in circulating oestrogens and testosterone concentrations after puberty cells with androgen or afemale sexbrandom x chromosomeinactivation and escapexxxxxxxxxxxxxxxxxxxy chromosome complementmale sexsryctesticular testosterone surgefetal testistestosteroneohogenetic diï¬erences of male and female cellsepigenetic programming of male cellsfigure genetic causes of sex differencesa genetic sex differences start with cells carrying either xx or xy chromosome complement eg genes outside the testisdetermining sry gene which generates ubiquitous sex differences in the molecular makeup of all male and female cells b random inactivation of one x chromosome in female cells causes another level of sex differences in gene expression some xlinked genes escape inactivation in female individuals and have a higher expression in female than male individuals c the y chromosomal sry gene directs the development of a testis in male individuals which produces a surge of testicular testosterone at the end of pregnancy the testosterone surge programmes cellular gene expression and tissue structure in multiple ans of male individuals via epigenetic remodelling the combination of these genetic and developmental events programmes sex differences in physiology and susceptibility to diseases that will manifest in adulthoodoestrogen receptors will be affected differ ently in men and women the combination of all genetic and hormonal causes of sex differences aforementioned culminates in two different biological systems in men and women that translate into differences in disease predisposition manifestation and response to treatment therefore sex is an important modifier of physiology and disease via genetic epigenetic and hormonal regulations figure gender as a determinant of patients and doctors behaviour and as a modifier of health disease and medicinegender according to the global health definition refers to the socially constructed norms that impose and determine roles relationships and positional power for all people across their lifetime13 gender interacts with sex the biological and physical characteristics that define women men and those with intersex identities13 gender is not a binary term it includes the understanding that in many people traits of masculinity or femininity coexist and are expressed to different degrees gender attributes are fluid more than two thirds of women and men report genderrelated characteristics traditionally attributed to the opposite sex1415 in transgender people gender identity differs with the sex they were assigned at birth so far transgender people have generally been underrepresented in clinical studies to date although this underrepresentation is changing gender is an equally important variable as biological sex in human health and influences the behaviour of communities clinicians and patients1415 gender roles represent the behavioural norms applied to men and women in society which influence individuals everyday actions expectations and experiences including diet perceived stress smoking and physical activity and affect health and disease susceptibility gender identity describes the fluidity of how a person perceives oneself as a woman or a man which affects feelings and behaviours gender relations refer to how we interact with or are treated by people on the basis of our ascribed gender institutionalised gender reflects the distribution of power between men and women in the political educational and social institutions in society and shapes social norms that define perpetuate and often justify different expectations and opportunities for women and men1617 as such the distribution of genderrelated charac teristics within populations of men and women can influence health differently than biological sex together these gender constructs determine access to health care helpseeking behaviours and individual use of the healthcare system being perceived as a man or a woman triggers different responses from clinicians who might diagnose and suggest interventions differently according to gender as such gender largely determines the use of preventive measures and referral for or acceptance of invasive therapeutic strategies genderrelated behaviours contribute to risk exposure and preventive behaviour in several wwwthelancetcom vol august review 0cdiseases this postulation is well exemplified in the cardiovascular field in which women often underestimate their risk compared with men and seek consultation later than men in the clinic for treatment of myocardial infarction1819 in the genesispraxy prospective study mortality year after an acute coronary event was more strongly associated with gender than with biological sex1415 similarly control of cardio vascular risk factors hypertension diabetes depres sive symptoms was better predicted by gender than by biological sex1415 therefore including a gender dimension in clinical studies and practice will contribute to the understanding of different clinical manifestations and outcomes of diseases in women and men1617 although beyond the scope of this review it is also important to consider that regarding health and disease gender intersect with race or ethnicity and age20 sex and gender are fundamentally and frequently reciprocally interrelated in biology and disease24 sex influences behaviours eg towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and pain and nutritional habits might produce epigenetic modifications that modulate gene expression and biological phenotypes figure summarises how sex and gender are interrelated in biology and diseasesex and gender differences in major chronic diseaseshaving established the importance of sex and gender in disease we will summarise their influences on the most common causes of death and debilitating diseases in the usa as an example figure note that these sex and gender disparities are relevant to other highincome countries as well as lowincome and middleincome countries where the burden of these diseases becomes increasingly like those in highincome countries26 in most diseases efforts to separate the effects of sex and gender are still incomplete so that we just refer to the differences among women and men because current knowledge about pathophysiology diagnosis and treatment of disease is primarily based on men as representative of the human species this review focuses on how women differ from men we discuss some key aspects regarding the dimensions of men in a dedicated sectionheart diseaseepidemiology pathogenesis manifestations and diagnosisheart disease is the leading cause of death in the usa in heart disease accounted for · of all deaths for men and for · of all deaths for women figure ischaemic heart disease and heart failure are major contributors to heart disease mortality and have important sex and gender differences for example heart failure disproportionately contributes to coronary heart disease mortality in women27 potentially due to undiagnosed ischaemic heart disease in women the strength of the association with cardiovascular risk factors differ by sex biological sexsex chromosomesepigeneticeï¬ectssex hormonesohohohobehaviour of patients and doctorssocietygender constructslifestylenutritional habitsexerciseperceived stresssmokingdiseasepathophysiologymanifestationresponse to treatmentdisease perceptionhelpseeking behaviouruse of health caredecision makingtherapeutic responsesex and gender diï¬erences in health disease and medicinefigure interrelation between sex and gender in health diseases and medicinebiological sex causes sex differences through genetic and hormonal influences in disease pathophysiology clinical manifestations and response to treatment sex also influences behaviours towards more aggressive or caring phenotypes on the other hand genderrelated behaviours eg smoking lifestyle perceived stress and nutritional habits produce epigenetic modifications that modulate the expression of biological sex gender constructs determine patients perception of disease helpseeking behaviour and individual use of health care gender constructs also influence decision making and trigger different therapeutic responses from providers biased by gendersystolic blood pressure and hypertension smoking and diabetes are associated with higher hazard ratios for myocardial infarction in women than in men28ischaemic heart disease is the most recognised example for integrating the concept of gender and sex which shape divergent or distinct disease outcomes compared with men women suffering from ischaemic heart disease are older this difference is historically believed to be due to the protection of endogenous oestrogens29 although contemporary study refutes this simplistic explanation30 and associations cannot be inferred to be causation still women suffering from ischaemic heart disease are underdiagnosed3132 and less likely to have a prehospital diagnosis of myocardial infarction33 the reasons for this disparity reflect the intersection between sex and gender first biological sex differences exist in the pathogenesis of ischaemic heart disease whereas men are more likely to be affected by obstructive coronary artery disease of large vessels than women coronary microvascular dysfunction36 leading to chronic myocardial ischaemia without obstructive coronary artery disease has a higher prevalence in women than men37 a metaanalysis reported that following acute myocardial infarction both sexes most often presented with chest pain but compared with men women were more likely to present with pain between the shoulder blades nausea or vomiting and shortness of breathprof j g regensteiner phd department of medicine department of paediatrics and department of neuroscience washington university school of medicine st louis mo usa prof j b rubin md center for the study of sex differences in health aging and disease geetown university washington dc usa prof k sandberg phd division of gastroenterology duke university medical center durham nc usa a suzuki md and durham va medical center durham nc usa a suzukicorrespondence to prof franck mauvaisjarvis diabetes discovery sexbased medicine laboratory section of endocrinology john w deming department of medicine tulane university school of medicine new orleans la usa fmauvaistulaneeduwwwthelancetcom vol august review 0cmale individualsother·heart disease·protection might disappear after menopause45 by contrast testosterone induces adverse cardiac remod elling in the male heart44chronic liver disease ·inï¬uenza and pneumonia ·suicide ·alzheimers disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·inï¬uenza and pneumonia ·type diabetes ·injuries ·alzheimers disease ·stroke ·cpd ·cancer·heart disease·cancer·figure percent distribution of the ten leading causes of death by sex usa adapted from heron25 cpdchronic pulmonary diseasesecond a gender bias appears to be responsible for the absence of recognition of ischaemic heart disease presentation in women38 men and women with ischaemic heart disease who score high on feminine roles and personality traits on questionnaires designed to ascertain aspects of gender are at an increased risk of recurrent ischaemic heart disease independent of female sex39heart failure affects of adults aged years and older and more women than men in absolute numbers4041 heart failure occurs at an older age and with less ischaemic causes in women than in men however hypertension and diabetes predispose older women to heart failure to a greater extent than men heart failure with preserved ejection fraction a form of heart failure with normal systolic function is twice as prevalent in women as in men by contrast heart failure with reduced ejection fraction affects more men than women women who have heart failure with preserved ejection fraction have smaller and stiffer hearts than men inflammation and the resulting fibrosis play a sexspecific role in the pathogenesis of heart failure with preserved ejection fraction under stress premenopausal womens hearts develop less inflammation resulting in less fibrosis than mens hearts4243 this difference is partially driven by sex hormones as oestrogens produce antiinflammatory actions on endothelial and immune cells and promote cardioprotective effects in premenopausal women44 this response to treatmentcompared with men women suffering from ischaemic heart disease are less likely to receive evidencebased treatment3132 and when suffering from acute myocardial infarction they are less likely to receive reperfusion33 an stelevation myocardial infarction registry revealed that compared with men women exhibit delayed reperfusion leading to higher mortality46 women suffering from acute myocardial infarction treated by male emergency physicians have a higher mortality rate than those treated by female physicians38 additionally male physicians are more effective at treating female patients with acute myocardial infarction when they work with female colleagues and when they have experience in treating female patients38 this treatment disparity between women and men can be corrected by improving emergency recognition of stelevation myocardial infarction in women and acceleration of percutaneous coronary intervention which equilibrates gender mortality47guidelines for the treatment of heart failure are similar for women and men24 however evidence suggests that optimal survival in women occurs with lower doses of β blockers angiotensin receptor blockers and angiotensin converting enzyme inhibitors than in men48 finally fewer women undergo heart transplantation than men although women are more frequently donors suggesting a referral bias could exist41cancersepidemiology pathogenesis manifestations and diagnosiscancers are the second leading cause of death dominated by lung cancer accounting for · of deaths in men and · of deaths in women figure more men develop cancer than women49 with few exceptions eg meningioma thyroid cancer lung cancer in nonsmokers nonreproductive cancers exhibit a male predominance though for some cancers oropharynx larynx oesophagus and bladder the male versus female incidence ratios can be higher than a male predominance in cancers that affect both sexes is evident around the world in all races and at all ages50 survival is also shorter for men than women across multiple cancer types the higher cancer risk in men is partially explained by gender constructs like dietary habits or risk behaviours such as smoking and alcohol consump tion51 it is unlikely to be the only cause after appropriate adjustment for these risk factors adult men still have a higher cancer risk than women52 moreover a similar male bias in incidence and survival is seen in paediatric cancers before puberty and the adoption of highrisk cancerpromoting behaviours eg smoking53the universal male predominance in cancer incidence and differential outcomes argues for a fundamental role wwwthelancetcom vol august review 0cin inutero tumour suppressors of sex in addition to gender in cancer biology sexspecific biology includes genetic differences xx vs xy chromosomes the incomplete xinactivation in female individuals which results in biallelic expression of xencoded female cells54 y chromosomeencoded oncogenes such as the rnabinding motif on y chromosome in male cells55 and the chromatin remodelling effects of testicular testosterone in male cells56 these mechanisms have an influence on several of the hallmarks of cancer57 including metabolism growth regulation58 angiogenesis and immunity which all contribute to cancer predisposition59 a crucial example is the male predisposition to glioblastoma which is the most common form of brain cancer in glioblastoma there is a cellintrinsic predisposition of male astrocytes a subtype of glial cell to malignant transformation60 after puberty sex hormones produce additional epigenetic and acute effects on cells that further influence sex disparities in cancer for example the increased frequency and aggressive phenotype of hepatocellular carcinoma in male individuals has been linked to the stimulatory effects of androgens in male individuals and the protective effects of oestrogens in female individuals61 importantly the biology of cancer is not the same across histological and genetic diagnostic groups or even within single histol ogical subtypes thus the interaction between sex gender and cancer mechanisms cannot be expected to be constant take colon cancer the second leading cause of cancerrelated death for example although women have a lower overall incidence of colon cancer than men they have a higher incidence of rightsided colon cancers which have the worst outcomes62 tumours from women with rightsided colon cancers exhibit a distinct molecular signature of energy metabolism compared with those of women with leftsided colon cancers63 this molecular signature is not observed when comparing tumours from men with rightsided colon cancers to men with leftsided colon cancers thus overall the male predisposition to cancer is probably the consequence of genetic program ming of male cells and the effect of sex hormones after puberty interacting with genderspecific behaviours to establish cancer risksresponse to treatmentin the future cancer prevention and treatment will be improved by sexspecific and genderspecific approaches for example immune checkpoint inhibitors can improve survival for men with advanced melanomas and nonsmallcell lung cancers more than for women64 the molecular subtyping of glioblastomas based on sexspecific transcriptomes has the potential to enhance chemotherapy in a sexspecific manner65 in colon cancer sex differences in xenobiotic metabolism regulatory networks might underlie greater treatment response in women than men and require modification of approaches for men with colon cancer66 other elements of cancer metabolism are also ripe for novel sexspecific targeting in treatment cellular nutrient partitioning is sexually dimorphic so approaches such as ketogenic diets or glutaminase inhibition might be associated with substantial sexspecific responses67 furthermore data from models of development ageing and cancer all indicate that molecular pathways such as the enzyme phosphatidylinositol 3kinase and tumour suppressors such as p53 and retinoblastoma protein are sexually dimorphic and require sexspecific targeting545968chronic pulmonary diseaseepidemiology pathogenesis manifestations and diagnosischronic pulmonary disease is the third leading cause of death for women · of deaths and the fourth for men · figure it is mostly accounted for by chronic obstructive pulmonary disease and to a lesser extent by asthma chronic obstructive pulmonary disease is characterised by irreversible airflow limitation and is associated with previous exposure to smoking or air pollutants women are overrepresented among individuals with chronic obstructive pulmonary disease especially among those with earlyonset disease or those who have never smoked69 women are also twice as likely to have chronic obstructive pulmonary disease with chronic bronchitis and women with severe chronic obstructive pulmonary disease have as much emphysema as men countering the misconception of emphysema as a male form of chronic obstructive pulmonary disease70 the female lung is more susceptible to chronic obstructive pulmonary disease than the male lung and women develop symptoms of the disease at a younger age with less tobacco exposure than men71 the genetic epidemiology of chronic obstructive pulmonary disease copdgene study72 suggests that earlyonset chronic obstructive pulmonary disease might originate in utero in susceptible women from alterations in lung development potentiated by maternal asthma and smoking genetic factors or hormonal influences future studies should focus on the contribution of maternally inherited factors such as mitochondrial and x chromosome genes to understand disease pathogenesis it is important to consider gender constructs as well smoking advertising campaigns targeting women rose in the 1960s and the resulting higher smoking rates influenced womens risk for developing chronic obstructive pulmonary disease73 from a disease severity vantage point chronic obstructive pulmonary disease exacerbation rates are also higher in women than men especially at a younger age74 additionally despite the burden of symptomatology and increased rates of hospitalisations and deaths women with chronic obstructive pulmonary disease are often misdiagnosed and disproportionally suffer from comorbid conditions including anxiety and depression therefore physicians should consider chronic obstructive pulmonary disease in the differential diagnosis of women with pulmonary symptoms regardless of tobacco or pollutant exposure historieswwwthelancetcom vol august review 0casthma characterised by variable airflow obstruction and chronic airway inflammation also affects men and women differently asthma is more prevalent in prepubertal boys than girls regarding asthma both male biological sex lung development and atopy and male gender constructs related to outdoor play and indoor pet exposure are factors contributing to the devel opment of asthma and sex versus gender contributions could be difficult to separate75 from puberty onwards more female than male individuals have asthma with an increased severity in middleaged women and a higher mortality rate76 this phe nomenon could be secondary to gender differences eg symptoms perception or healthseeking behaviours however biological sex plays a crucial role in asthma and sex hormones have a major impact on female asthma symptoms and severity after puberty75 worsening of asthma occurs in women before menstruation and is known as premenstrual asthma premenstrual asthma is more common in women with severe rather than mild asthma obesity rather than normal weight and a long rather than a short duration of asthma77 premenstrual asthma is hypoth esised to be due to a fall in progesterone and patients with a severe disease respond to progestogens78 during pregnancy approximately a third of asthmatic women exhibit a worsening of asthma a third show an improvement and the remainder are unaffected79response to treatmentthe menopausal transition represents a pivotal time of accelerated decline in lung function in women with chronic obstructive pulmonary disease and thus represents a sexspecific window for treatment intensification these observations also suggest that oestrogens protect from chronic obstructive pulmonary disease women exhibit greater expression of m2 over m3 muscarinic receptors and accordingly show greater improvements in lung function than men in response to the muscarinic anticholinergic bronchodilator ipatroprium80 pooled analyses of drug studies also suggest that women experience a greater improvement in quality of life than men after treatment of chronic obstructive pulmonary disease with a β2adrenoreceptor agonist combined with an anti cholinergic drug eg indacaterol and glycopyrronium81unlike chronic obstructiv | 0 |
" 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 | 0 |
" recently copy number alteration cna of 9p241 were demonstrated in of diffuse large bcelllymphoma dlbcl with gene expression and mutation profiles that were similar to those of primary mediastinallarge bcell lymphoma pmbcl however their cnabased profile and clinical impact still remain unclearmethods multiplex ligationdependent probe amplification were employed to investigate the prevalence of jak2pdl2 amplification in dlbcl and their cnabased pattern of driver genes the clinical outcome and characteristicswere also analyzedresults using unsupervised hierarchical clustering a small group of dlbcl was clustered togetherwith pmbcl as cluster_2 demonstrating amplification of jak2 and pdl2 this subgroups ofdlbcl demonstrated significant higher expression of pdl1 than those with myd88 l265p mutationp andthey exhibited dismal os and pfs as compared with dlbcl_othersp and respectively which issimilar to dlbcl with myd88 l265p mutations dlbcl with amplification of jak2pdl2 exhibits cna pattern that is similar to pmbcl anddemonstrates unfavorable clinical outcome that resembles those with myd88 l265p mutation it is essential toidentify this subgroup of dlbcl who may acquire more benefits from the jak2 and pdl1 signaling inhibitionkeywords diffuse large bcell lymphoma jak2 pdl2 amplification prognosis diffuse large bcell lymphoma dlbcl is a highly heterogeneous disease recently several distinctive geneticsubtypes were identified including schmitz r studymcd bn2 n1 and ezb subtypes and chapuy b study c0 c5 clusters [ ] godfrey j study also correspondence jmyingcicamsaccn lvningcicamsaccn xuemin xue and wenting huang are cofirst authors jianming ying andning lv are cosenior authors1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing chinafull list of author information is available at the end of the identified an unique biological subset of dlbcl withpdl1 gene alterations having high risk features thus the genetics of dlbcl relating to potential therapeutic targets for immune checkpoint inhibitors shouldbe paid much more attention tojanus kinase jak2 programmed cell death ligand pdl1cd274pdcd1lg1 and programmed celldeath ligand pdl2cd273pdcd1lg2 are adjacent to each other on chromosome 9p241 playing keyroles in host immune surveillance amplification of9p241 were frequently seen in celllines of classical and primaryhodgkin lymphoma chl 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 0cxue bmc cancer page of mediastinal large bcell lymphoma pmbcl but much less in dlbcl cell lines ] correspondingly pd1 ligands pdl1 and pdl2transcripts and proteins were more abundant in chl andpmbcl cell lines than that in dlbcl cell lines recently y wang study demonstrated that ofdlbcl had copy number alteration cna of 9p241with a gene expression and mutation profile similar tothose of pmbcl however their cnabased profileand clinical impact still remain unclearin thisthereforestudy we employed multiplexligationdependent probe amplification mlpa to investigate the prevalence of jak2pdl2 amplification indlbcl and their cnabased pattern of driver genesincluding bcl2 cdkn2a and tp53 and we analyzed their longterm survival outcome after treatmentof rchoplike regimemethodscase selectionwe collected consecutive cases of dlbcl and pmbclin our clinical ffpe archives of excisional biopsy database between jan and oct and cases ofdlbcl and cases of pmbcl were found after confirmation one case of dlbcl was diagnosed as pmbclthus cases of dlbcl and cases of pmbcl wereacquired finally see additional file all patients werediagnosed at national cancer centernational clinicalresearch center for cancercancer hospital chineseacademy of medical sciences and peking union medicalcollege according to the revised 4th edition ofthewho classification of tumours of haematopoietic andlymphoid tissues the data regarding treatment andprognosis were acquired by means of medical recordconsultation and telephone conversationmultiplex ligationdependent probe amplification mlpagenomic dna were extracted from formalinfixedparaffinembedded ffpe blocks using qiaamp dnaffpe tissue kit qiagen valencia ca then dnacopy number quantification and myd88 l265p mutation were detected using mlpa kitmrchollandnetherlands the pcr products were detected on anabi genetic analyzer applied biosystems usaand the final result were analyzed using coffalyser software the relative peak ratio prr of probe largerthan was defined as amplification and less than was defined as deletion see additional file geneswhich had two or more probes covering two differentexomes were put into final analysis including jak2 pdl2 mdm2 rel pus10 bcl2 nfatc1 spib foxp1nfkbiz bcl6 prdm1 tnfaip3 cdkn2a ptening1 and tp53 the details of mlpa probes of drivergenes in dlbcl are shown in the online supportingmaterial see additional file true amplification of onegene was regarded only when all probes of this gene exhibited amplification and vice versa see additional file myd88 l265p mutation was identified when theprobe had a high peak myd88 wildtype didnt show anypeak see additional file immunohistochemistry ihc staining of pdl122c3ihc staining was performed on dako autostainer link asl48 platform each ffpe block were cut at athickness of 4μm and then deparaffinized antigen retrieval were performed using the envision¢ flex targetretrieval solution at low ph monoclonal pdl1clone 22c3 dako were used as primary antibodyfollowed by incubation with envision¢ flex mouselinker and then envision¢ flex hrp reagent finally the ihc was visualized by envision¢ flex dabeach ihc slide contained a positive controllungcarcinomaihc score of pdl1 were calculated by multiplyingthe percentage of positive cells with mean intensity no staining weak staining moderate staining strong staining which was reported in previous study the results were evaluated by an experienced hematopathologist xueminstatistical analysisthe differences of clinicopathological characteristicsamong different groups were analyzed using chisquaretest fisher exact test or kruskalwallis rank sum testpdl1 ihc score between different groups was analyzedusing wilcoxon test overall survival os and progressfree survival pfs times were defined from the date ofpathologic diagnosis to the date of the event or the lastfollowup the hazard ratio hr of each parameter wascalculated by univariate cox proportional regressionanalysis firstly in which parameters with p wereevaluated together using multivariate cox proportionalregression analysis the survival curve were made according to kaplanmeier procedure the day oflastfollowup was march 1st all statistical analysiswere two sided and p was defined as significanceunsupervised hierarchical clustering was carried outusing euclidean distance and complete method heatmap was plot using pheatmap packageall above statistical analyses were run in r statistic softwareresultsunsupervised hierarchical clustering of cnas of drivergenes and its survival analysis in dlbcl and pmbclpatientsbased on array cgh lenz g study previouslyidentified specific cnas in pmbcl which were different 0cxue bmc cancer page of from abc and gcb of dlbcl abc dlbcls oftenhave cnas in foxp1 nfkbiz cdkn2a cdkn2binf4a bcl2 nfatc1 and spib while gcb dlbclsfrequently harbor cnas in rel pten mdm2 mihg1and ing1 pmbcl often demonstrate cnas of jak2 andpdl2 using unsupervised hierarchical clustering we explored the cnabased pattern of these genes in dlbcl andpmbcl the result showed that a small group of dlbcl was clustered together with pmbcl as cluster_2 with amplification of jak2 and pdl275068fig 1a this subgroup of dlbcl occurred atthe site of cervical lymph node cases gastrointestinal tract cases nasal cavity case and spleen cases fig 1atable 1additional file the frequency of jak2 and pdl2 amplification in the whole cohort of dlbcl were and while both of them were inpmbcl fig 1a see additional file meanwhile all casesin cluster_3 harbored amplification of nfkbiz which is essential for nfκb activation in abc dlbcl but noamplification of nfkbiz was found in cluster_1as to survival dlbcl in cluster_2 demonstrated significant worse os p and pfs p as compared with dlbcl in cluster_1fig 1b howevercluster_1 and cluster_3 didnt reveal significant differencein survival fig 1b we also analyzed the os and pfs between dlbcl with and without jak2pdl2 amplification and got statistical significance see additional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table fig heatmap and survival analysis based on unsupervised hierarchical clustering and status of jak2pdl1 amplification and myd88 mutationin tcga dataset a heatmap of cnabased profiles of driver genes in dlbcl and pmbcl by using unsupervised hierarchical clustering b survivalcurves and coxregression analysis of os and pfs among three cnabased clusters after rchoplike treatment c status of amplifications of jak2pdl1cd274 and pdl2pdcd1lg2 and mutation of myd88 in dlbcl tcga pancancer atlas from cbioportal [ ] 0ccervical lymphnodefemale high_intermediatehigh_intermediatedlbclnasal cavitymalegcbbreak_apartdlbcldlbcldlbcldlbcldlbcldlbclcervical lymphnodestomachstomachcolondlbclpmbclpmbclpmbclpmbclcervical lymphnodecervical lymphnodemediastinummediastinummediastinummalelow_intermediate non_gcbnormalfemale low_intermediate non_gcbmalemalelowhighnon_gcbgcbnon_gcbnormalnormalnormalnormalfemale low_intermediate non_gcbnormalmalelowfemale female female lowlowhigh_intermediatenanananananormalnormalnormalnormalnormaljak2_amppdl2_ampxue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265pmyc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didnt show any significant differences table jak2pdl2 amplification identify a distinctive cnabasedpattern of dlbcl similar to that of pmbclsince dlbcl with jak2pdl2 amplification had less frequency of myd88 l265p mutation our study separateddlbcl patients into three subgroups dlbcl with jak2pdl2 amplification dlbcl_jak2pdl2_amp dlbclwith myd88 l265p mutation dlbcl_myd88_l265pjak2pdl2 amplification norand dlbcl withoutmyd88_l265p mutation dlbcl_others fig 2a basedon the unsupervised cluster result fig 1a one patientwho had both jak2pdl2 amplification and myd88l265p mutation was clustered into cluster_2 thereforethis patient was put into dlbcl_jak2pdl2_amp subgroup accordingly we also analyzed the data when thiscase was included in dlbcl_myd88_l265p subgroupand got the similar result see additional file unlike dlbcl_myd88_l265p and dlbcl_othersdlbcl_jak2pdl2_amp showed a distinctive pattern similar to that of pmbcl with high frequencyof rel and nfkbiz amplifications but no amplification of bcl2 and nfatc1 and no deletion ofprdm1 was found fig 2awith respectto clinicopathologicdlbcl_jak2pdl2_amp tend to be youngerdlbcl_myd88_l265p p hans modelcharacteristicsthantable whileinternational prognostic index ipi riskpdl1 expression in dlbcl with jak2pdl2 amplificationwas significantly higher than that in dlbcl with myd88l265p mutationtotally cases were performed pdl1 22c3 ihc detection including dlbcl_myd88_l265p cases dlbcl_jak2pdl2_amp cases dlbcl_others cases andpmbcl cases the result showed that pdl1 expressionin dlbcl_jak2pdl2_amp was significantly higher thanthat in dlbcl_myd88_l265p p and dlbcl_others p fig 2b and d while no significant difference was found between dlbcl_jak2pdl2_amp andpmbcl p fig 2bjak2pdl2 amplification identify a subgroup of dlbclwith unfavorable survival outcome similar to that ofmyd88 l265p mutationtrying to explore the survival indication of jak2pdl2 amplification and myd88 l265p mutation cases of dlbcls who received rchoplike regimentwith or without surgical resection were enrolled toperformed cox proportional regression analysis of osand pfs the median followup time was monthsrange monthsin the univariatecompared withdlbcl_others dlbcls with myd88 l265p mutationhad significantly worse os and pfs p andanalysisas 0cxue bmc cancer page of fig comparison of cnabased pattern pdl1 expression and survival analysis among pmbcl and three subgroups of dlbcl a comparison ofcnabased patterns of driver genes among pmbcl and three subgroups of dlbcl according to the status of jak2pdl2 amplification andmyd88 l265p mutation b comparison of pdl1 expression ihc score among pmbcl and three subgroups of dlbcl c survival curves and coxregression analysis of os and pfs among three subgroups of dlbcl after rchoplike treatment d representative images of heà and pdl1à ihc in dlbcl_jak2pdl2_amp and dlbcl_ myd88_l265p respectively and the same to dlbcls withjak2pdl2 amplification p and respectively meanwhile ipi risk category were significantly associated with os and pfs fig 2c tables and in the multivariate analysis ipi risk category andthree subgroups of dlbcl were put into analysis ascompared with dlbcl_others dlbcl with myd88l265p mutation still showed poor os and pfs p and respectively and the same todlbcl with jak2pdl2 amplification for pfs andos p and respectively meanwhile ipirisk category was still an independent risk predictorsfor os and pfs fig 2c tables and either jak2pdl2 amplification or myd88 l265pmutation are frequently seen in relapserefractory dlbclwith pfs less than yearsdlbcl with pfs less than years was defined as primaryrelapserefractory cases among these cases who treated byrchoplike regime the frequency of jak2 and pdl2amplification were and meanwhilethe frequency of myd88 l265p mutation were dlbcl with either jak2pdl2 amplification ormyd88 l265p accounted for discussiondlbcl presents with a wide spectrum of genetic aberration recently shi study exhibited pdl2 amplification in pmbcl and of dlbcl chapuy demonstrated of 9p241 amplification indlbcl meanwhile dlbcl with pdl1 gene alterations was identified as a unique biological subgrouphaving high risk features y wang study demonstrated that of dlbcl had cna of 9p241 withgene expression and mutation profiles that were similarto those of pmbcl in our study by using unsupervised hierarchical clustering cases ofdlbcl were clustered together with pmbcl as cluster_2 indicating that they shared recurrent cnas theywere enriched for jak2 amplification and pdl2 amplification fig 1a 0cxue bmc cancer page of table comparison of characteristics among pmbl and three subgroups of dlbcldlbclothersmyd88_l265pjak2pdl2_amppmbclpatientsage median range bmnegativepositiveihc hans algorithmgcbnongcbipilowrisklow_intermediatehigh_intermediatehighmyc breakapartnegativepositive p_valueÏ2test kruskalwallis rank sum testusing hans model most of dlbcl in cluster_2 werenongcb and tend to be younger than othergroups of dlbcl table which was consistent withprior study therefore coupled with y wang study we confirmed that dlbcl with jak2pdl2 amplification is a unique subgroup resembling the pmbclwith respect to cna patternwith regard to survival increasingly data exhibited thatthe suppression of immune surveillance in dlbcl was associated with poor survival godfrey j study hasdemonstrated that dlbcl with pdl1 gene alterationsshowed high risk features metaanalysis also showedthat pdl1 expression was associated with poor os andadverse clinicopathologic features in dlbcl in y wang study of dlbcl harbored cnaof 9p241 of which were gains and were amplifications and as compared with those who have nogain of 9p241 dlbcl with 9p24 amplification had atrend of better efs while patients with only gain tend tothey didnthave worse prognosis unfortunatelyshow any statistical significance in our study of dlbcl were found that had cna of jak2when jak2 cna was separated into gain mlpa valuebetween and amplification mlpa value as described cases in dlbcl_jak2pdl2_amp group were found that had jak2 gain whichwas slightly lower than that in wang j study asshown in additional file and both dlbcl withjak2 gain and with amplification demonstrated significant poor prognosis as compared with rest of dlbclas shown in additional file more interesting unlikey wang study cases of pmbcl were included in our study as control all of which demonstrating jak2 gains rather than amplifications as shown inadditional file of note we found that dlbcl in cluster_2 enrichedfor jak2pdl2 amplification had less frequency ofmyd88_l265p mutation fig 1a table which was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1cmyd88 l265p is a poor indicator of survival for dlbcl which may lead to primary refractoryrelapsed diseasethis is a gainoffunction driver mutation occurring in of dlbcl but absent in pmbcl [] inour study the frequency of myd88 l265p in dlbcl andpmbcl were and which were in linewith prior studies [] of greatinterest myd88l265p mutation occurred less frequently in cluster_2 which was supported by the data tcga pancancer atlas from cbioportal [ ] thus when we divided dlbcl patients into three subgroups dlbcl_jak2pdl2_amp dlbcl_myd88_l265p and dlbcl_others both dlbcl_jak2pdl2_amp and dlbcl_myd88_l265p demonstrated dismal os and pfs with amedian followup of years as compared with dlbcl_others therefore dlbcl with jak2pdl2 amplification 0cxue bmc cancer page of table os in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc fish breakapartnegativepositiveipi risk categorylowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clustercluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_jak2pdl2_ampdlbcl_myd88_l265poshr_u95ci p_valueoshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysiswas identified as a poor survival subgroup that is similar todlbcl with myd88 l265p mutationmeanwhile we also compared the cna patterns ofdriver genes among dlbcl_jak2pdl2_amp dlbcl_myd88_l265p dlbcl_others and pmbcl dlbcl_jak2pdl2_amp showed a distinctive pattern similarto pmbcl with high frequency of rel and nfkbizamplifications but no amplification of bcl2 and nfatc1 and no deletion of prdm1 was found the profile ofdlbcl_myd88_l265p was closed to dlbcl_othersshowing relatively high frequency of cdkn2a deletionnfatc1 amplification and bcl2 amplificationin our study of dlbcl_jak2pdl2_ampharbored both jak2 and pdl2 amplifications simultaneouslyindicating that they may also have the pdl1amplification because pdl1 located in the middle ofjak2 and pdl2 at 9p241 thus we hypothesized thatpdl1 expression would be upregulated in this subgroup as what we expected using pdl1 22c3 ihcdetection pdl1 expression in dlbcl_jak2pdl2_in dlbcl_amp was significantly higher than thatmyd88_l265p p and dlbcl_othersp fig 2b and d but not in pmbcl p fig 2b meanwhile pdl1 expression could be 0cxue bmc cancer page of table pfs in dlbcl treated by rchoplike regimeage ¥ bmnegativepositivesiteextranodalnodalihc hans algorithmgcbnongcbmyc breakapartnegativepositiveipilowlow_intermediatehigh_intermediatehightherapyrchopresection rchopcna_based_clusterscluster_1cluster_2cluster_3three subgroups of dlbcldlbcl_othersdlbcl_ jak2pdl2_ampdlbcl_ myd88_l265ppfshr_u95ci p_value pfshr_m95cip_value hr_u hazard ratio by univariate analysis hr_m hazard ratio by multivariate analysis because age was contained in the ipi thus it wasnt put into multivariate analysisenhanced not only by pdl1 amplification but also byjak2 activation [ ] therefore dlbcl with jak2pdl2 amplification was confirmed as an unique subtype that is different from dlbcl with myd88 l265pand othersobjective response rates orr of pd1 blockade therapy was in unselected patients with relapsedrefractory dlbcl [ ] the wide spectrum of orrmay be due to high heterogeneity of this subgroupansell sm study demonstrated patients with9p241 alteration in relapsedrefractory dlbcl inour cohort the frequency of jak2 and pdl2 amplification in relapsedrefractory dlbcl were and which were within the range of orr in the prior studies[ ] while patients were found thathad myd88 l265p mutation who may not be suitablefor antipd1 therapy thus the genetic analysis in refractoryrelapsed dlbcl is required for future therapyselection to increase the orr of immune checkpointinhibitorsjak2 amplification could augment the expression of itself and pd1 ligands pdl1 and pdl2 enhancing the 0cxue bmc cancer page of sensitivity to jak2 kinase inhibitor chemical jak2inhibition could reduce the rna transcription and protein expression of pdl1 thus selective inhibitionof jak2 would be a valuable complementary therapy forpdl1 blockadeauthors contributionsxx contributed to pdl1 ihc staining clinical followup data analysis andmanuscript writing wh contributed to ffpe tissues collection mlpa detection and clinical followup tq and lg provided experiment guidance anddata interpretation jy and nl contributed to study design coordination discussion and manuscript editing all authors read and approved the finalmanuscriptsjak2pdl2 exhibitsdlbcl with amplification ofpmbcllike cnas pattern and demonstrates unfavorable outcome resembling those with myd88l265p mutation thusit is essential to identify thissubgroup of dlbcl who may acquire more benefitsfrom the jak2 and pdl1 signaling inhibition andjak2 amplification detection by mlpa would be feasible in routine practice meanwhile the difference ofsurvival outcome between our study and wang j study indicated that pmbcllike dlbcl suggested by 9p241 cna could be an intermixed subgroup which required further explorationsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020072933additional file mlpa results and clinical followup data the clinicopathological characteristics clinical followup data and mlpa results areshowed in this fileadditional file figure s1 representative results of mlparepresentative results of mlpa are showed in this figureadditional file table s1 the details of mlpa probes of genes indlbcl the locations and lengths of mlpa probes of genes are showedin this tableadditional file the detailed information of dlbcl with jak2pdl2amplification the detailed data about clinicopathological characteristicsmorphology immunohistochemistry and treatments of dlbcl with jak2pdl2 amplification are showed in this fileadditional file figure s2 the os and pfs of dlbcl with or withoutjak2pdl2_amp the os and pfs of dlbcl with or without jak2pdl2_ampadditional file figure s3 comparison of cnabased pattern andtheir survival outcome among pmbcl and three subgroups of dlbclone case of dlbcl with jak2pdl2 amplification and myd88 l265p mutation were included in dlbcl_myd88_l265p group a comparison ofcnabased patterns of driver genes among pmbcl and three subgroupsof dlbcl according to the status of jak2pdl2 amplification and myd88l265p mutation b survival curves and coxregression analysis of os andpfs among three subgroups of dlbcl after rchoplike treatmentadditional file figure s4 the frequencies of jak2 gain andamplification and their survival analysis a the frequencies of jak2 gainand amplification in dlbcl_jak2pdl2_amp and pmbcl b the os andpfs of dlbcl with jak2 gain or with jak2 amplificationabbreviationsdlbcl diffuse large bcell lymphoma pmbcl primary mediastinal large bcell lymphoma mlpa multiplex ligationdependent probe amplificationtcga the cancer genome atlas ipi international prognostic indexffpe formalinfixed paraffinembedded os overall survival pfs progressfree survival hr hazard ratioacknowledgementsnot applicablefundingthis study was partly supported by the beijing municipal science technology commission grant number z151100004015121 the cancerfoundation of china grant number lc2014l13 and cams innovation fundfor medical sciences grant number 2016i2m1001 to perform ffpe tissuescollection and mlpa detection and was partly supported by the cancerfoundation of china grant number lc2018b10 and pumc youth fundand the fundamental research funds for the central universities grantnumber to conduct pdl1 ihc staining and clinical followupand collect fish data of cmycavailability of data and materialsall data generated or analyzed during this study are included in thispublished and its supplementary information filesethics approval and consent to participatethis is a retrospective study that was launched in november the casesenrolled in this project were diagnosed between jan and oct whose ffpe samples were used the data regarding treatment andprognosis were acquired by means of medical record consultation andtelephone conversation thus the need for consent was waived by theindependent ethics committee of cancer hospital chinese academy ofmedical sciences national gcp center for anticancer drugs ncc2015st05consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1department of pathology national cancer centernational clinical researchcenter for cancercancer hospital chinese academy of medical sciencesand peking union medical college beijing china 2department ofpathology national cancer centernational clinical research center forcancercancer hospital shenzhen hospital chinese academy of medicalsciences and peking union medical college shenzhen chinareceived march accepted august referencesschmitz r wright gw huang dw johnson ca phelan jd wang jqroulland s kasbekar m young rm shaffer al genetics andpathogenesis of diffuse large bcell lymphoma n engl j med chapuy b stewart c dunford aj kim j kamburov a redd ra lawrencems roemer mgm li aj ziepert m molecular subtypes of diffuse largeb cell lymphoma are associated with distinct pathogenic mechanisms andoutcomes nat med godfrey j tumuluru s bao r leukam m venkataraman g phillip jfitzpatrick c mcelherne j macnabb bw orlowski r pdl1 genealterations identify a subset of diffuse large bcell lymphoma harboring a tcellinflamed phenotype blood green mr monti s rodig sj juszczynski p currie t o'donnell e chapuy btakeyama k neuberg d golub tr integrative analysis reveals selective9p241 amplification increased pd1 ligand expression and furtherinduction via jak2 in nodular sclerosing hodgkin lymphoma and primarymediastinal large bcell lymphoma blood wang y wenzl k manske mk asmann yw sarangi v greipp pt krull jehartert k he r feldman al amplification of 9p241 in diffuse large bcell lymphoma identifies a unique subset of cases that resemble primarymediastinal large bcell lymphoma blood cancer j 0cxue bmc cancer page of lenz g wright gw emre nc kohlhammer h dave ss davis re carty slam lt shaffer al xiao w molecular subtypes of diffuse large bcelllymphoma arise by distinct genetic pathways proc natl acad sci u s aswerdlow sh campo e harris nl jaffe es pileri sa stein h thiele j whoclassification of tumours of haematopoietic and lymphoid tissues revised4th edn lyon iarc cerami e gao j dogrusoz u gross be sumer so aksoy ba jacobsen abyrne cj heuer ml larsson e the cbio cancer genomics portal anopen platform for exploring multidimensional cancer genomics datacancer discov gao j aksoy ba dogrusoz u dresdner g gross b sumer so sun yjacobsen a sinha r larsson e integrative analysis of complex cancergenomics and clinical profiles using the cbioportal sci signal pl1 nogai h wenzel ss hailfinger s grau m kaergel e seitz v wollertwulf bpfeifer m wolf a frick m ikappabzeta controls the constitutive nfkappab target gene network and survival of abc dlbcl blood shi m roemer mg chapuy b liao x sun h pinkus gs shipp ma freemangj rodig sj expression of programmed cell death ligand pdl2 is adistinguishing feature of primary mediastinal thymic large bcelllymphoma and associated with pdcd1lg2 copy gain am j surg pathol qiu l zheng h zhao x the prognostic and clinicopathological significanceof pdl1 expression in patients with diffuse large bcell lymphoma a metaanalysis bmc cancer moelans cb monsuur hn de pinth jh radersma rd de weger ra vandiest pj esr1 amplification is rare in breast cancer and is associated withhigh grade and high proliferation a multiplex ligationdependent probeamplification study anal cell pathol amst fernandezrodriguez c bellosillo b garciagarcia m sanchezgonzalez bgimeno e vela mc serrano s besses c salar a myd88 l265p mutation isan independent prognostic factor for outcome in patients with diffuse largebcell lymphoma leukemia ngo vn young rm schmitz r jhavar s xiao w lim kh kohlhammer h xuw yang y zhao h oncogenically active myd88 mutations in humanlymphoma nature dubois s viailly pj bohers e bertrand p ruminy p marchand vmaingonnat c mareschal s picquenot jm penther d biological andclinical relevance of associated genomic alterations in myd88 l265p andnonl265pmutated diffuse large bcell lymphoma analysis of casesclin cancer res gupta s cheville jc jungbluth aa zhang y zhang l chen yb tickoo skfine sw gopalan a alahmadie ha jak2pdl1pdl2 9p241amplifications in renal cell carcinomas with sarcomatoid transformationimplications for clinical management mod pathol ansell sm minnema mc johnson p timmerman jm armand p shipp marodig sj ligon ah roemer mgm reddy n nivolumab for relapsedrefractory diffuse large bcell lymphoma in patients ineligible for or havingfailed autologous transplantation a singlearm phase ii study j clin oncollesokhin am ansell sm armand p scott ec halwani a gutierrez mmillenson mm cohen ad schuster sj lebovic d nivolumab inpatients with relapsed or refractory hematologic malignancy preliminaryresults of a phase ib study j clin oncol hao y chapuy b monti s sun hh rodig sj shipp ma selective jak2inhibition specifically decreases hodgkin lymphoma and mediastinal largebcell lymphoma growth in vitro and in vivo clin cancer res publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
" tumor mutational burden tmb has both prognostic value in resected nonsmall cell lung cancernsclc patients and predictive value for immunotherapy response however tmb evaluation by wholeexomesequencing wes is expensive and timeconsuming hampering its application in clinical practice in our study weaimed to construct a mutational burden estimation model with a small set of genes that could precisely estimatewestmb and at the same time has prognostic and predictive value for nsclc patientsmethods tmb estimation model was trained based on genomic data from nsclc samples from the cancergenome atlas tcga validation was performed using three independent cohorts including rizvi cohort and ourown asian cohorts including earlystage and n latestage asian nsclc patients respectively tcga data wereobtained on september the two asian cohort studies were performed from september to march pearsons correlation coefficient was used to assess the performance of estimated tmb with westmb thekaplanmeier survival analysis was applied to evaluate the association of estimated tmb with diseasefree survivaldfs overall survival os and response to antiprogrammed death1 pd1 and antiprogrammed deathligand pdl1 therapyresults the estimation model consisted of only genes correlated well with westmb both in the training setof tcga cohort and validation set of rizvi cohort and our own asian cohort estimated tmb by the 23gene panelwas significantly associated with dfs and os in patients with earlystage nsclc and could serve as a predictivebiomarker for antipd1 and antipdl1 treatment responsecontinued on next page correspondence zhangli6mailsysueducn jie_969163comzlhuxi163com yanhua tian jiachen xu and qian chu contributed equally to this work5state key laboratory of oncology in south china collaborative innovationcenter for cancer medicine sun yatsen university cancer center eastdong feng road guangzhou guangdong china1state key laboratory of molecular oncology department of medicaloncology national cancer centernational clinical research center forcancercancer hospital chinese academy of medical sciences and pekingunion medical college panjiayuan south lane chaoyang districtbeijing 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 0ctian bmc medicine page of continued from previous pages the 23gene panel instead of wes or the currently used panelbased methods could be used toassess the westmb with a high relevance this customized targeted sequencing panel could be easily applied intoclinical practice to predict the immunotherapy response and prognosis of nsclckeywords tmb estimation 23gene panel prognostic and predictive value nonsmall cell lung cancertoimmuneinhibitorscheckpoint tumor mutational burden tmb commonly defined asthe number of nonsynonymous mutations has been proposed as a promising predictive biomarker for the responseicisimportantly this metric tightly correlates with overallsurvival os in resected nonsmall celllung cancernsclc patients in rizvi demonstratedthat an increased number of nonsynonymous mutationswere associated with improved objective response durable clinical benefit dcb and progressionfree survivalpfs in nsclc patients who received antiprogrameddeath pd1 therapy clinical studies have also revealed a significant correlation between tmb and objective response rate orr to icis in multiple tumortypes [] in addition devarakonda recently reported that high tmb was associated with a better survival prognosis in patients with resected nsclc andthe benefit of adjuvant chemotherapy was more pronounced in patients with low tmb the gold standards for tmb calculation are throughwholegenome sequencing wgs or wholeexome sequencing wes however several obstacles such as thehigh demand for quality and quantity of tissue samplesthe cost and time consumption and the unavailabilityfor translation to tmb evaluation by circulating tumordna ctdna in blood btmb hinder the clinicalapplication of these techniques as a result targetednext generation sequencing ngs of cancerrelatedgene panels cgp has been developed serving as surrogates for wes for tmb estimation to date the foodand drug administration fda has approved severalngs panels for tmb estimation eg foundationonecdx f1cdx and memorial sloan kettering cancercenters integrated mutation profiling of actionablecancer targets mskimpact which include about genes and cover over one megabase of codingdna [ ] recently many new ngs panels consistingof different numbers of genes have been developed andvalidated most of which were designed initially for guiding the use of target therapies these panels mainly include cancerrelated oncogenes and tumor suppressenes many of which do not contribute to or even negatively correlate with tmb thus are not accurate fortmb evaluation besides inclusion of these genes in anngs panel enlarges the panel size used for tmbis importantestimation and can lead to an inferior costeffective consequence itto note that cancer typespecific mutation load estimation models have proven tobe necessary because of the different mutation landscapes among varying tumor types although dnadamage repair ddr genes negatively predictive genesstk11 and keap1 and tmbassociated genes such asmuc16 pole pold1 and ttn have been included inthe ngs panels for tmb evaluation [] with theburgeoning developments in immunotherapy there is aneed for more specific panels that focus on tmb estimation for nsclcherein by using the cancer genome atlas tcgadatabase as a training set and multiple realworld cohorts as a validation set we constructed an optimizedtmb estimation model with the smallest number ofcarefully selected tmbassociated genes that could beused as both predictive markers for immunotherapy andprognosis biomarkers for resected nsclc patientsmethodspatient cohortsgenomic and clinical data for nsclc samples including lung adenocarcinoma luad and lungsquamous cell carcinoma lusc samples were downloaded from tcga database for the model constructionfor the validation of the model three independent cohorts were used including a previously published studythe rizvi cohort a surgery cohort composing of earlystage nsclc patients who underwent surgicaltreatment and a zs immunotherapy cohort composingof advanced nsclc patients who received ici treatment all the patients in the zs immunotherapy coreceived either antipd1 nivolumab n hortpembrolizumab n shr1210 n or antipdl1 atezolizumab n monotherapy agents there are patients who received durable clinical benefit dcbantipd1 n antipdl1 n and patientswith no durable benefit ndb antipd1 n antipdl1 n all three validation cohorts were used toevaluate the performance of the tmb estimation modeladditionally the surgery cohort was also used for survival validation in resected nsclc patients both therizvi and immunotherapy cohorts were also used forvalidation of ici outcome predictability in advancednsclc patients the clinical details for all enrolled 0ctian bmc medicine page of patients were collected the treatment efficacy for thosetreated with immunotherapy was assessed using response evaluation criteria in solid tumors recistversion with durable clinical benefit dcb definedas partial or stable disease lasting over months allprocedures were approved by the ethics committees ofthe national cancer center all patients provided written informed consentwholeexome sequencing and data processingwe performed wholeexome sequencing of samplesfrom two cohorts in the validation setincluding earlystage nsclc patients who underwent surgicaltreatment and advanced nsclc patients who received ici treatment for those earlystage nsclcpatients both tumor and matched normal samples wereobtained and subjected to wes briefly dna librarieswere prepared using the mgieasy exome capture v4probe set capture kit cat no with a capture region size of mb bgiseq instruments wereused for pairend sequencing à bp the data wereprocessed according to the manufacturers protocol the mean coverage was à and à in tumor andnormal samples respectivelyfor those advanced nsclc patients biopsy specimens were available for wes the genomic dna wasextracted using the qiaamp dna ffpe tissue kit andquantified using the dsdna hs assay kit thermofisher scientific usa libraries were constructed withthe kapa hyper prep kit kapa biosystems usa anillumina hiseq4000 platform was used for sequencingwith pe150 sequencing chemistry illumina usa the average coverage depth was Ãcandidate gene selectiongenomic data for nsclc samples from tcgawere used for candidate gene selection which were usedto construct the mutation load estimation model thecandidate genes were selected based on two criteria mutation frequency higher than or equal to and significant association with mutation load the mutationfrequency of a gene was calculated as the percentage ofpatients with mutation in the gene mutation loadassociated genes were defined as where the westmbwas significantly different between the patients with themutated gene and those with wildtype counterpartsadditional file table s1mutation estimation model constructionthe mutation estimation model construction was basedon tcga data in the training set in detail the first stepwas to build a mutation estimation model using thefewest genes which tightly associated with westmbin our study we constructed the estimation model bysimply randomly selecting a specified number of genesfrom allthe genes or tmbassociated genes andsummed the mutational number as the estimated tmbunder every given number of genes the procedure wasrepeated times resulting in random modelswe then calculated the pearson correlation coefficientr between the estimated and actual mutation load ofwestmb the results allowed us to select the modelwith highest r under the specified number of genes thenext step was to identify which of those best modelsunder the specified number of genes correlated with theclinical outcomes of overall survival os and diseasefree survival dfs the final step was to select a modelusing the fewest genes that tightly associate with thewestmb and have both prognostic value for thoseearlystage nsclc patients and predictive value forthose latestage nsclc patients who received icitreatmentrna expression difference between tmb high and lowgroupsto compare gene expression patterns we downloadedan mrna data set of nsclc patients from tcgadatabase mrna expression was analyzed using gene setenrichment analysis gsea httpsoftwarebroadinstitutegseaindexjsp we divided these patientsinto estimated high ¥ mutational counts and lowtmb groups mutational counts and identifiedwhether immunerelated gene signatures associated withtumor mutation status the genes found to be on theleading edge of the enrichment profile were subjected topathway analysis genes with expression over in morethan of the samples were included in the gseathe normalized enrichment score nes is generally theprimary statistic for examining gene set enrichmentresultsstatistical analysisthe mannwhitney u test was used to assess thedifferences in the mutation load between the twogroups the genes with kruskalwalliscorrected pvalues lower than were identified as the mutationloadassociated genes and selected as potential candidate genes survival analysis was performed using thekaplanmeier curves with a p value determined by alogrank test and the statistical tests were twosidedand considered statistically significant at p unless otherwise stated the analyses were performedusing graphpad prism version graphpad prismcorrelations between estimated mutation burden andwholeexome sequencingcalculated tmb were determinedcorrelation coefficient theanalyses were performed using r353by pearsons 0ctian bmc medicine page of resultscandidate gene selection for model constructionthe flowchart of the construction of estimation model isshown in fig s1 in additional file the somatic mutation data of cases of nsclc were downloaded fromtcga database as the training set tcga cohort including adenocarcinoma and squamous cell carcinoma subtypes of nsclc additional file table s2subsequently a mutation matrix including screened nonsynonymous mutations in genes was generatedfurthermore we identified genetic alterations in genes with mutation frequency ¥ in general nsclcpatients and significantly correlating with westmb pvalue range 695e to 452e these genes werethen used as candidate genes for the construction of thetmb estimation model additional file table s3construction of the tmb estimation modelgenes used for the tmb estimation model were randomly selected from the candidate genes and theserialrandom models theestimated tmb was defined as the sum of all nonsynonymous mutation counts of the selected genes undereach specified number of abstracted genes the procedure was repeated times thus resulting in separatecorrelations ofestimated tmb by these random models and westmbwere evaluated using the pearson correlation coefficientr as expected the correlations between the estimationmodels and westmb increased with the number ofgenes fig 1a b additional file fig s2a b compared with unselected genes in the range of genomicgenes the estimated tmb based on selected geneswas significantly more closely associated with westmb in terms of either the mean or the maximum rfig 1c d additional file fig s2c d the maximumr increased from with one gene included to greaterthan with genes included and then reached aplateau when the included gene number exceeded the r values were comparable though increased slowlyas the number increased fig 1b we asserted that rfig the correlation of westmb and tmb as estimated by different gene panels a b correlation is represented by the pearson correlationcoefficient r genes used for the mutation model construction were either from unselected genes a or from selected genes b that correlatewith westmb c d comparisons of mean c and maximum d r of estimated tmb and westmb using unselected genes or selected genes 0ctian bmc medicine page of greater than in the estimation models was acceptable as such we considered a model with this effectbut including the least number of genes an ideal modelfor clinical applicationin reference to previous reports that tmb is associated with prognosis in patients with resected nsclcsthe optimal tmb estimation model was further evaluated based on the correlation of estimated tmb with osand dfs in models with r over ultimately we constructed an estimated tmb model with only genesand r of p fig 2a additional file which was significantly associated with both os anddfs fig 2b c the cutoff value of the estimated tmbby the 23gene panel was defined as mutational countsthe median value of estimated tmb based on tcgadatabase additional file fig s3a b that were equalor over mutational counts as tmbhigh cases and lessthan mutational counts as tmblow ones these genesincluded unc13c hmcn1 znf536 kmt2d ush2axirp2 pcdh15 ahnak2 adgrl3 reln nf1 ttnadgrg4 cubn cacna1e mrc1 col11a1 nav3csmd1 apob csmd3 col22a1and epha5additional file table s4 the model yielded goodperformances in both subtypes of nsclc with correlations of for luad additional file fig s4aand for lusc additional file fig s4b theaverage cds length of these genes was 12k nucleotides 3k80k additional file table s4 and the totallength was 028m nucleotides which was considered tobe a great reduction of sequencing cost for mutationload estimation we concluded that the 23gene panel isthe ideal model based on tcga training setanalytic validation of the 23gene panel in asian resectednsclc patientsto validate the performance of the estimation model weconducted wes on chinese stage iaiiia nsclcpatients after radical pneumonectomy surgery cohortadditional file table s1 the correlation of 23genetmb with wes was r p fig 3a asshown in fig 3b tmbhigh ¥ mutational counts according to the 23gene panel associated with a betterdfs compared with those with tmblow logrank p besides a tendency towards improved os wasobserved in the patients with higher estimated tmbthough a statistical difference was not reached due tothe fact that most patients were still alive fig 3cperformance verification by comparing the 23gene panelwith other commercial panelsnext we compared the 23gene panel with two commercial panels based the earlystage nsclc data including f1cdx genes and mskimpact genes there are two overlap genes between the gene panel with f1cdx and mskimpact namelynf1 and epha5 the 23gene tmb has a tight correlation with the tmb estimated by f1cdx f1cdxtmbor mskimpact msktmb r and respectively both p fig 4a b in additionwhen the genes were added to the two commercialpanels the correlation of the incorporated panels withwestmb significantly increased from ci to ci p for f1cdx fig 4c d and from ci to ci p for mskimpact fig 4e f to further verify thespecificity of these 23gene panels we compared themwith other randomly selected gene panels from the genes the procedure was repeated timesresulting in the random pearson correlation coefficientsfrom to of f1cdx plus random genesand from to of msk plus random genes the performance of our 23gene model was better than of random models which indicated the irreplaceability of these genesfig tmb estimation model construction based on tcga data in the training set a the correlation of 23gene tmb with westmb is with an empirical p value of r of p b the overall survival is significantly higher in the tmbhigh group ¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the diseasefree survival is significantly higher in thetmbhigh group than in the tmblow group with logrank test p 0ctian bmc medicine page of fig validation of the tmb estimation model based on the earlystage nsclc patients in the validation set a the pearson correlationcoefficient of estimated tmb by the 23gene panel and westmb is with an empirical p value of r of p b the diseasefree survivalis higher in the estimated tmbhigh group ¥ mutational counts n than in the tmblow group mutational counts n with logrank test p c the overall survival is comparable in the two groups with logrank test p fig performance evaluation of the 23gene panel against commercially used gene panels a b the pearson correlation coefficient of 23genetmb with f1cdxtmb a and msktmb b c d the pearson correlation coefficient of westmb with f1cdxtmb c and incorporated panel of cancerassociated genes in f1cdx with 23gene panel f1cdx genetmb d e f the pearson correlation coefficient of westmb withmsktmb e and incorporated panel of cancerassociated genes in mskimpact with genepanel msk genetmb f 0ctian bmc medicine page of f1cdxbased on the survival data from our earlystagensclc patients significant correlations were observedbetween survival outcomes dfs and the tmb levelstratified withor mskimpact paneladditional file fig s5a c interestingly the genescould improve the association of these two commercialpanels with dfs additional file fig s5b d if the incorporated panels were used for analysis tmbhigh estimated by both of the two new panels f1cdx 23genepanel or mskimpact 23gene panel demonstratedimproved dfs compared with those of estimated tmblow under the cutoff values indicated in fig s3c and s5of additional file immuneregulatory gene expression signatures stratifiedby tmb level based on the 23gene panelto investigate the difference in immune status betweentmbhigh and tmblow estimated by the 23genepanel we analyzed immuneregulatory gene expressionsignatures based on the rnaseq data of nsclccases from tcga the gsea revealed a prominent enrichment of mrna signatures involved in the inflammainterferonα γ ifnα γtoryresponse tnfαresponse il6jakstat3 signaling and allograft rejection fig immunotherapy response prediction by the established23gene panelfinally we analyzed the performance of tmb estimatedby the 23gene panel in the prediction of response toicis using two independent nsclc cohorts in therizvi cohort the correlation between the tmb estimatedby the 23gene panel and wes was empirical pvalue of r fig 6a the estimated tmb was significantly different between the patients with durableclinical benefit dcb a partial or stable response lastingover months and no durable benefit ndb mannwhitney p fig 6b survival analysis was thenapplied for the comparison of the pfs between the patients n with tmbhigh ¥ counts and tmblow counts by the 23gene panel patients withtmbhigh demonstrated significantly improved pfscompared with those with tmblow vs months logrank p fig 6cto further validate the performance of the estimationmodel for response to icis we performed wes of fig gene expression differences between the estimated high tmb and low tmb groups af tmbassociated pathways such as inflammatoryresponse tnfα signaling via nfκb interferon α response il6jakstat3 signaling interferon γ response and allograft rejection nes normalizedenrichment score fdr false discovery rate 0ctian bmc medicine page of fig immunotherapy response estimation by the 23gene panel a the correlation of the estimated tmb with westmb using the rizvi datan b estimated tmb in tumors from patients with dcb n or with ndb n mannwhitney p c pfs in tumors withestimated tmbhigh n compared to tumors with tmblow n in patients in the rizvi cohort hr ci to logrank p d the correlation of estimated tmb with westmb using the latestage nsclc patient cohort n e estimated tmb in tumors frompatients with dcb n or with ndb n mannwhitney p f pfs in tumors with estimated tmbhigh n compared totumors with tmblow n in patients in the latestage nsclc patient cohort hr ci to logrank p advanced stage iiibiv nsclcs in another asian cohort zs immunotherapy cohort all of these patients received with antipd1 or antipdl1 treatmentthe r between the estimated and actual mutation burden was calculated to be empirical p value of r fig 6d the estimated tmb was significantlydifferent between the patients with dcb and ndbmannwhitney p fig 6e the pfs was associated with estimated tmb logrank p fig 6fdemonstrating that the estimated mutation burden derived from caucasian nsclcs from tcga could predict the immunotherapy treatment response quite wellin asian patients we further calculated the hr at different cutoff values in the zs immunotherapy cohort andfound the mutational counts in this cohort resultedthe best hr value additional file fig s6 as a resultwhen applied in clinical practice the cutoff value stillneeds to be further evaluated accordinglycomparison of the 23gene panel with previouslyreported tmbrelated genesmutations in ttn muc16 pole and pold1 havebeen previously reported to correlate with elevated tmblevels [] the frequencies ofthese genes innsclc based on cases from tcga were and respectively westmb was significantly different between the patients with these mutatedandthose with wildtypegenescounterpartsadditional file fig s7 however only muc16 mutations exhibit significant correlation with os and dfs intcga cohort additional file fig s7ac while theyfailed to confirm the results in our surgery cohortadditional file fig s8 notably none of these genemutations could predict the response or pfs in eitherthe rizvicohortadditional file fig s9cohort or ourimmunotherapydiscussionin the present study we developed a novel and optimaltmb estimation model composed of only geneswhich allowed precise estimation ofthe wesbasedtmb both in earlystage and latestage nsclc patientsimportantly our established 23gene panel can successfully predict the survival outcomes in both resectednsclcs and patients receiving icis in multiple validation cohorts to the best of our knowledge our tmbestimation model is both the first and the smallest paneldescribed to date which can be used as a biomarker tostratify patients not only after radical pneumonectomybut also with advanced nsclc receiving icisthe total cds length of the 23gene panel was 028mnucleotides with an average of 12k 3k80k the ttnis also included in our panel although it has the longestcds length of 81k the total length was acceptable when 0ctian bmc medicine page of ttn is included besides in a recent study ttn mutation was reported to be associated with tmb in solid tumors including nsclc and correlated with response toicis as a result the 23gene panel was consideredto be a great reduction of sequencing cost for mutationload estimationseveral cancerrelated genes have been previously reported to be associated with westmb in some cancertypes for example melanoma patients with lrp1b mutations exhibited a higher mutationalload than thosewith the wildtype gene li reported that mutations in muc16 are associated with tmb and survivaloutcomes in patients with gastric cancer twoddrrelated genes pole and pold1 were also shownto correlate well with westmb in pancancer types undoubtedly it would be ideal to utilize a singlegene to estimate tmb and effectively predict responseto immunotherapy however we found that singly allthese genes failed to correlate well with the efficacy oficis or survival outcomes after resection the correlationof any individual gene with westmb was moderatemean r these results indicate thatusing a single gene to estimate tmb is insufficienttheoretically the larger a ngs gene panel the closerthe estimated tmb is to the actual amount howeverthe costeffective balance for clinical usage must be considered in particular when tmb is detected using peripheral blood super sequencing depth eg à due to the low abundance of circulating tumordna will significantly drive up the cost to datetwo commercial gene panels f1cdx and mskimpact have been widely used for tmb estimation thesetwo panels demonstrate good performance in correlationwith westmb our established gene panel whichincludes a very limited number of genes demonstratedcomparable correlation coefficients with these two largepanelsindicating the promising reliability of a smallpanel as a surrogate for westmb notably the majority of genes used in our model were not included in thecurrently used commercial gene panels if the genes inour panel were incorporated into the big commercialgene panels the correlation coefficients with westmbincreased these results demonstrate that the geneswe have selected here may be used independently or ascomplement to the currently used gene panels specificfor nsclc inclusion of the genes should be considered in future ngs gene panelsrecently lyu developed a small gene panel with genes to estimate actual tmb derived from luads in tcga database the construction and validation cohorts used for lyu s 24gene panel weremainly from caucasian patients however our 23genepanel though also derived from tcga database wassuccessfully validated in multiple asian patient cohortsthese results suggest that our 23gene panel may bemore suitable to nsclc and applicably potent regardless of race and subtypes additional file fig s10similar with the findings of devarakonda weobserved that high tmb associated with improved os inresected nsclc patients in colon cancer patients withresected stage ii mismatch repair deficiency high tmbhas been utilized as a good prognostic biomarker indeed these results possess internal rationality both highneoepitope burden and intense til infiltration have been associated with favorable survival outcomes inearlystage lung cancer high tmb may reflect the immunogenicity in some degree which could mediate theshaping of tumorhost immune interactions taken together these and our findings suggest that quantifyinggenomic instability through tmb estimation can be usedto stratify patients so as to guide adjuvant treatmentowing to the lack of information on hlai it is difficultto judge whether the predictive value of our gene panel isdue to neoantigen generation derived from the includedgene mutations or if the estimated tmb based on the gene panel is simply a representative reflection of genomicinstability as an accompanying passenger the otherlimitation of our study is the small number of patientswho received the immunotherapy treatment thus a larger number of cases from a multicenter study are requiredfor the validation of the performance of the treatment response prediction in addition our validation cohorts wereretrospective a prospective study is necessary to translateour estimation model into clinical practice in addition totmb other features such as pdl1 expression microsatellite instability and neoantigen burden have emerged aspotential predictive biomarkers for icis [] howeverchallenges in defining cutoff valuesintertumoral andintratumoral heterogeneity and test platform uniformitieshave limited their clinical applications therefore future strategies that combine different predictive featuresmay be more effective biomarkers for the accurate prediction of cancer immunotherapy response but need tobe carefully integratedsin summary we have successfully constructed a noveltmb estimation model using only genes that can beused to estimate the westmb and stratify survivalprognosis after radical surgery and clinical outcomes ofici therapy in nsclc patients thus a customized panelfor the targeted sequencing of these selected genes instead of wholeexome sequencing can be designed or utilized as complementary genes included in the currentngs panels consequently by using our model the costeffectiveness may be considerably improved makingrealization of cancer immunotherapy response more accessible in standard clinical settings 0ctian bmc medicine page of supplementary informationsupplementary information accompanies this paper at httpsdoi101186s12916020016948competing interestsno potential conflicts of interest were disclosed by the authorsadditional file table s1 data sets used to calculate westmb forthe study cohorts table s2 characteristics of the patients included inthis study table s3 candidate genes and related information tables4 genes and the corresponding cds length fig s1 flowchart ofthe construction of estimation model fig s2 the correlation of westmb and tmb as estimated by different gene panels fig s3 forestplots of hrs for os and dfs in the tcga and earlystage nsclcpatients study cohort fig s4 the performance of 23gene based tmbestimation model for the luad and lusc subtypes of nsclc tcgadata fig s5 forest plots of hrs for dfs in the earlystage nsclcpatients study cohort fig s6 forest plots of hrs for pfs of the nsclc patients in zs immunotherapy cohort fig s7 westmb is shownbased on muc16 a ttn b and pole c mutation status fig s8 thecorrelation of muc16 mutation status with overall survival a anddiseasefree survival b based on the earlystage nsclc patients figs9 the correlation of muc16 ttn and pold1 mutation status withprogressionfree survival pfs based on the rizvi cohort and ourimmunotherapy cohort fig s10 comparison of predictive performanceof response to icis by our 23gene panel with lyus 24gene paneladditional file the correlation of estimation models with genenumber to with os and dfs in the training setacknowledgementswe thank all patients that were involved in this study we also thankguoqiang wang jing zhao and shangli cai the medical department 3dmedicines inc shanghai peoples republic of china for their contribution tothe st | 0 |
sarcomatoid carcinomas scs are extremely rare aggressivemalignant tumors characterized by distinct cellular morphology the features of this tumor were ï¬rst described in by snover scs can occur in a wide variety of sitesincluding the respiratory tract digestive tract genitourinary tractbreast and thyroid glands however these tumors are rarein the digestive tract especially in the stomach as of april there are only six cases of gastric sarcomatoid carcinomagsc reported in the english medical literature these previousreports focused on the pathological and clinical manifestationsthem have not systematically described the radiologic appearanceof the tumor due to the more invasive nature and poorerprognosis of gsc than pure gastric adenocarcinoma gacand gastric lymphoma gl it is clinically beneï¬cial to narrowdown the diï¬erential diagnoses by understanding the computedtomography ct characteristics of gsc the present studyanalyzed our experience in diagnosing ï¬ve patients with gscin terms of the imaging ï¬ndings and clinical features to thebest of our knowledge our study represents the largest seriesof gscs to datein addition due to the rarity of gsc the diï¬erential diagnosisbetween gsc and other types of malignant gastric tumors hasnot received much attention so we also initially explored thediï¬erential diagnosis of gsc from gac and glmaterials and methodsthe protocol was approved by the medical ethics committeeof zhengzhou universityinformed consent was obtainedfrom all patientspatient selectionfrom august to january we searched the pathologyrecords and the picture archiving and communication systemspacs of our hospital the search terms included stomachand sarcomatoid carcinomas a total of ï¬ve patients wereidentiï¬ed as having sc and were enrolled in the present study weretrospectively reviewed all clinical data demographic featureslaboratory ï¬ndings clinical interventions and the histologicï¬ndings of the ï¬ve biopsy or operation specimensct evaluationfive gsc patients underwent ct examinations the ctscans were acquired with a 64row multidetector devicediscoveryct750hd ge healthcare waukesha wiunited states conventional axial scanning was performedbefore and after an intravenous iv injection of nonioniciohexol iopromide mgml ge medical systems mlkgand mls through a dualhead pump injector medradwarrendale pa united states the imaging parameters wereas follows tube voltage kv tube current ma ï¬eldof view fov mm matrix mm and sectionthickness mm finally a 20ml saline ï¬ush was performedat a rate of mlsgastric sarcomatoid carcinomacontrastenhanced ct scans were acquired with scanningdelays of s arterial phase ap and s portal venous phasepp after the iv injection of the contrast agent started the ctdose index volume for the three phases was msvimage analysistwo experienced radiologists and years of abdominal ctexperience performed a retrospective analysis of the ct imagesall analyses were performed with an aw47 workstation gehealthcare and the radiologists were blinded to the clinicalinformation of the patients the evaluated parameters includedthe tumor location gastric cardia gastric fundus gastric bodygastric angle and gastric antrum longaxis diameter shapegrowth pattern serosa condition attenuation and enhancementcharacteristics such as the enhancement pattern and degreeof enhancement the enhancement pattern of the tumor wasclassiï¬ed as homogeneous or heterogeneous based on the apimage the degree of enhancement of the tumor was based ondynamic ct imaging using hu attenuation where obviousenhancement was deï¬ned as hu moderate enhancementas hu and mildly enhancement as huthe gscs were staged with the union for internationalcancer control uicc tnm staging standard all imagingï¬ndings were compared with the postoperative pathologicalï¬ndings the accuracy rate the number of cts coincidentwith the pathological diagnosisthe number of actual pathologicaldiagnoses pathological evaluationthree patients underwent gastrectomy and two underwentendoscopic biopsy the three gastrectomy specimens measured cm cm cm cm cm cmcm cm cm respectively in two of theseand tumors the mucosal surface of the excised specimen showedulcerative masses of approximately cm cm cmand cm cm the remaining specimen was a soft massmeasuring cm cm cm for biopsy multiple sampleswere acquired and the diameter of each sample was cmaccording to the relevant literature the diagnostic criteria fsc were generally as follows the tumor originated fromthe stomach and the tumor consisted of both carcinomatousand sarcomatoid components and the sarcomatoid componentaccounted for more than of the tissue in addition if biopsywas performed the sarcomatoid component can be seen in everysample furthermore sarcomatoid regions express epithelialmarkers such as ck or emathe specimens were fully stretched ï¬xed and soaked in formaldehyde solution for h all biopsy specimenswere analyzed the specimens underwent routine dehydrationparaï¬n embedding sectioning into µm thick sectionsand hematoxylin eosin he staining immunohistochemicalstaining was performed using a roche benchmark xt automaticimmunohistochemical detector the antibodies used in thisstudy included ae1ae3 ckl ck818 epithelial membraneantigen ema vimentin p40 p63 and antigen ki67 ki67all antibodies listed above were purchased from dako dakoglostrup denmarkfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable comparison between gsc and gac glage median age rangemain symptomsepigastric discomfortpainintermittent vomitingacute hematemesisbloody stooldysphagialocationcardia and fundusbodyantrumthe longaxis diameter median size rangeshapefocal thickeningdiffuse thickeningmassserosal surfacebare areaclearunclearulcersyesnodensity characteristicsheterogeneoushomogeneousenhancement patterheterogeneoushomogeneouslymph node involvementyesnoliver involvementyesnotherapyresectionchemotherapyresection and chemotherapyneoadjuvant chemotherapyradiation therapygscgacgl years years years cm cm cm comprehensive comparative analysiseach patient with gsc was matched by age ± years year ofdiagnosis and sex to four patients with gac gl patients witheach disease were retrieved from pacs patients with gsc werecompared with those with gac gl in terms of demographicclinical and ct characteristics table resultspatient characteristicsthe patients included four men and one woman ranging inage from to years with a median age of years theclinical and ct features of these patients are summarized intables all patients had nonspeciï¬c symptoms includingabdominal discomfort epigastric discomfort nausea or vomitingthe other presenting symptoms included hematemesis or weightloss three patients underwent radical resectionin whichonly one patient was treated with adjuvant chemotherapyafter surgery and two patients chose to deny treatment inaddition we also reviewed the upper gastrointestinal radiographyresults figure the laboratory ï¬ndings revealed that patient was positivefor tumor abnormal protein tap and patient was positivefor carbohydrate antigen ca125 before treatmenthemoglobin and erythrocyte count decreased in three patientsfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and pathological factors of the ï¬ve gsc patientscasesexage yearscomplaintlocationmaximumdiametertumor markercmanemiatherapymetastasismmfmmsuddenhematemesisepigastricdiscomfortintermittentvomitingepigastric painepigastric painepigastric painlesser curvatureremnant stomachcardia andfunduscardia and fundusfunduscardia and fundusnormaltap ca125 normalnarrnnonenonerc yespresentpositive noabsentnegative f female m male age in years r radical gastrectomy rn remnant gastrectomy c chemotherapyna not availabletable computed tomography features of the ï¬ve gsc patientscasegross features of the tumorulcersgrowth modedensity characteristicsenhancement patterfocal thickeningfocal thickeningmassfocal thickeningfocal thickeningintracavityintracavityintracavityintracavityintracavityheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousheterogeneousheterogeneoushomogeneousheterogeneousheterogeneousmarginunclearunclearunclearclearunclear yespresentpositive noabsentnegativefigure characteristics of xray examinations of a 65yearold male patient with gsc ab reveals that there is a huge niche with irregular shapes at the smallcurvature of the stomach the niche is located inside the outline of the stomach the niche is surrounded by transparent bands with different widths that is ringembankments with irregular outlines the surrounding mucosa is thickened interrupted and the local gastric cavity is narrowedpatients and and platelet count was elevated in fourpatients patients and pathological featuresmicropathologically the gastric tumor cells showed inï¬ltrativegrowth the cytological characteristics ofthe tumor cellsshowed obvious malignant characteristics microscopicallythe spindle cell structure and the nucleus were obviouslyatypical pleomorphic and enlarged mitotic ï¬gures were visiblefigures 2ab on immunohistochemical examination thetumor cells showed positive staining for ae1ae3 cklck818 ema p40 vimentin the ki67 index was higher than figures 2ci all ï¬ve tumors were diagnosed as gscin addition the sarcomatoid component showed spindle cellsarcomatoid morphologyct findingsof the cases of gsc were in the gastric fundus and cardiafigure was in the gastric body and was in the gastricfundus of these tumors one was a recurrence in the remnantstomach the ct manifestations of this tumor included localthickening n mass formation n the longaxisfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure histological and immunohistochemical features of gsc ab hematoxylineosin he staining showing tumor cells demonstrated spindleshapedstructures signiï¬cant atypical nuclei pleomorphic nuclei and giant nuclei mitotic ï¬gures visible tumor cells showed inï¬ltrative growth cells were stained withhematoxylin and eosin stain magniï¬cation a b by immunohistochemistry the tumor cells were positive for ae1ae3 c ckl d ck818 e emaf p40 g and vimentin h moreover of them were positive for ki67 i the ï¬nal diagnosis was sc [magniï¬cation ci ]diameter of the lesions ranged from to cm mean size cm in addition ulcers with an irregular base and slightlyraised borders were observed in of cases among the threepatients who underwent surgery two lesions invaded the gastricserosa and the remaining lesion invaded the gastric bare areaamong the two patients with biopsyproven gsc one patientexhibited tumor invasion of the gastric bare area the majorchanges in the ct imaging characteristics were an irregularouter layer of the gastric wall and obscuration of the perigastricfat initially the ct ï¬ndings were interpreted as gac in fourcases and gl in the tumor showed predominantly inhomogeneous densityand the radiodensity values were hu in the noncontrastphase heterogeneous enhancement was seen in four casesdue to necrotic or cystic areas and the other tumor revealedhomogeneous enhancement the radiodensity values on the apimages ranged from to hu and to hu in thevenous phase after contrast medium injection two tumorsshowed obvious enhancement and moderate enhancementwas seen in the other three tumors the peak tumor valuewas observed in the portal phase one of the three patientswho underwentlymphsurgery demonstrated evidence ofin one patientthe boundary betweennode involvementthe lesion and the left lobe of the liver was unclear andthe area with low attenuation was conï¬rmed by pathologythe liver withas a metastatic lesion in the rightcircular enhancement the remaining patientshowed noevidence of metastasis among the two patients with biopsyspecimens one patient was identiï¬ed as having lymph nodemetastasis on ctlobe ofct staging versus pathological stagingof gscnone of the gscs were staged as t1t2 by ct or pathologythe accuracy of ct staging t3 and t4 gsc was and respectively the overall diagnostic accuracy of ctfor determining the t stage of gsc was none of the gscs were staged as n2n3 by ct or pathologythe accuracy of ct in staging n3 and n4 gsc was and respectively the overall diagnostic accuracy of ct fordetermining the n stage of gsc was the comparison of tn staging based on ct and pathology isshown in table frontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomafigure sarcomatoid carcinoma of the stomach in 62yearold women a unenhanced ct image of stomach reveals an intraluminal mass of homogeneousattenuation with an irregular surface at the gastric fundus and cardiac region bd contrastenhanced ct image shows obvious homogeneous enhancement ofmass with the peak value of the tumor on the portal phase in perigastric lymph nodes an enlarged and signiï¬cantly enhancement lymph node can be seenb arterial phase of contrast enhancement image c portal phase of contrast enhancement image d portal phase of contrast enhancement coronal imagediscussiontable ct and pathological tn staging for comparisonsarcomatoid carcinoma is an extremely rare and complicatedmalignant tumor composed of malignant epithelial componentsand atypical spindle cells however the spindle cells of scsappear to show evidence of epithelial diï¬erentiationforexample showing epithelial markers or epithelial ultrastructuralinstead of a speciï¬c line of mesenchymalcharacteristicsdiï¬erentiation moreoverliteratureemphasizes that the sarcomatous components occupy ofthe elements involved in the present study our patientstumor cells displayed atypical spindle shapes that expressed theepithelial phenotypethe currentsome ofsarcomatoid carcinomas can occur in almost any an wherecarcinoma can occur in the digestive system the incidencesof scs in the esophagus and liver are relatively high but scsare exceedingly rare in the stomach we could ï¬nd only sixprevious reports in the english literature table between and patients with sc conï¬rmed by pathologywere retrospectively analyzed with only ï¬ve tumors occurringin the stomach the average age of the reported patientswas years range and that in our series was years range a previous study reported that the sexcaseno no no no no ctt4an0t3n0t3n1t3n0t4an1na not available t tumor n nodepathological staget4an1t3n0nanat4an0distribution of male to female gsc patients was and thecorresponding proportion in our patients was ithas been noticed that scs are more common in male patientsand sex is a probable risk factor gsc patients may present withepigastric pain or discomfort dysphagia nausea and vomitinghematemesis and emaciation due to thickening of the gastricwall pain or discomfort in the upper abdomen is common thesymptoms can last from a few days to several years withoutobvious speciï¬cityin the present study of the cases of gsc were recognizedin the proximal stomach and the remaining tumor was founddistal to the stomach four cases of gsc in the present study hadfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatable clinical and imaging features of six previously reported cases of gcscasegenderageyearslocationsize cmshapeulcersenhanceappearancerecurrencemetastasistherapyprognosis mmffmmremnant stomachgreater curvaturelesser curvaturegastroesophageal junctionremnant stomachdistal stomachpolypoidpolypoidpolypoidulceratedpolypoidmassnenenenenehypernenonesurgerysurgerysurgeryendoscopysurgeryna mo d mo d mo d mo d mo dthe patient succumbed to heart failure before the surgical treatment an autopsy was performed yespresentpositive noabsentnegative hyper hyperdensene no evaluate mo month d dieareregarded asa longaxis diameter less than cm and the remaining tumorhad the largest longaxis diameter among our patients cmthe location distribution and longaxis diameters of the gscs inour patients were similar to those in previous reports in the actual diagnosis processthe diagnosis of sc has always been diï¬cult for cliniciansand pathologists especially the diï¬erential diagnosis fromcarcinosarcoma carcinosarcomastrulybiphasic neoplasms composed of distinct malignant epithelialcarcinomatous and mesenchymal sarcomatous componentsthe sarcoma components show typical specialized diï¬erentiation howeverthe termssarcomatoid carcinoma and carcinosarcoma have been usedinterchangeably in some cases therefore the understanding ofthese tumors has been hampered nevertheless we can try tofocus on whether there is a diï¬erence between these tumors froma new perspective the ct ï¬nding sc in the stomach have notbeen previously scientiï¬c reported or even detailed descriptionthere are only four simple descriptions chunchao reported that a patient with a giant sc presented a mass witha cm diameter in the antrum and body of stomach whichinï¬ltrated the gastric serosa the hepatic ï¬exure of the colon andgallbladder were also involved on ct contrastenhanced ctimages showed obvious enhancement of the two lesions sato reported a patient with sc of the remnant stomachand the radiographic examination showed an elevated lesionwith a large ulcer at the gastric cardiac lesser curvature thatmeasured cm in diameter the other two reports only describeda soft tissue mass or a large tumor in the dilated stomach on the other hand within in the upper gastrointestinal tractalthough there are fewer reports of carcinosarcoma localized inthe stomach this type of tumor is still more common than sc gastric carcinosarcoma showed an elevated lesion or thickenedgastric walls in of all reviewed cases tomoaki reported a 79yearold man with gastric carcinosarcomaand his veins showed severe invasion enhanced abdominalct showed irregular thickening and slight enhancement of thegastric wall on the side of the lesser curvature with suspiciousbulky lymph nodes yoshiyuki reported a 70yearoldjapanese woman who presented with a soft tissue mass adjacentto the lesser curvature of the stomach that was lobulated andct revealed an ulcer on the lesion the contrastenhanced ctimages showed heterogeneous enhancement of the mass theï¬nal pathological diagnosis was gastric carcinosarcoma inthe present study we found that gsc showed local thickeningof the gastric wall and mass formation often accompaniedby ulcers the site of the disease was mostly in the proximalpart of the stomach but these tumors can also occur in theremnant stomach the signal of the tumor was homogeneousor heterogeneous on plain ct scans after contrast mediuminjection of tumors demonstrated heterogeneousenhancement on ap images due to cystic areas or necrosis inthe lesions in this study the enhancement degree of all tumorsreached a peak in the pp after contrast enhancement for thesetumors the enhancement degree in the delayed phase wasnot signiï¬cantly reduced the overall enhancement mode wasdelayed enhancement in addition ct showed that four patientshad invasion into the gastric serosal region or gastric bare areatwo patients had the characteristics of enlarged perigastric orretroperitoneal lymph nodes and uneven enhancement and onepatient had invasion into the adjacent liver tissue these ï¬ndingsreï¬ect the metastatic and highly invasive characteristics of gscoverall ct and contrastenhanced ct can clearly show theprimary lesion inï¬ltration range lymph node metastasis anddistant metastasis of gsctomographic diagnosis of gsc has not been attemptedbecause of the rarity of this entity according to the ï¬ndingsof our study gsc needs to be diï¬erentiated from gac andgl on ct adenocarcinoma is the most common pathologicaltype of gastric tumor and is mainly distributed in the antrumseldomly in the body and fundus of the stomach the incidenceof gac is high in men and the median patient age is years the most common ct signs of gac are localor extensive thickening of the gastric wall mass formationincluding fungoidestype polypoidtype masses rough orsmooth serous surfaces and continuous interruption of themucosal layer tumors involving the mucosal surface can appearenhanced s after injecting a contrast agent the peakvalue for tumors invading the muscular layer usually appearsafter s and after the mucosal surface is strengthenedthe duration is longer primary gl accounts for ofmalignant gastric tumors and is predominantly situated in thegastric antrum gastric body and gastric fundus the incidenceof gl is high among males with a median patient age of yearsthe clinical symptoms included epigastric pain bleeding earlysatiety and fatigue the most common ct manifestations ofgl are diï¬use thickening of the gastric wall or a homogeneousfrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinomatissue mass with slight attenuation or an appearancesoftsimilar to that of the normal gastric wall for gl becauseof hemorrhage necrosis submucosal edema or infarction thegastric wall may be heterogenous on ct gl originates froma submucosal process and gastric mucosa is commonly intactin the early stage but shows interruptions or ulceration in thelater stage after contrast medium injection most gl showedhomogeneous and slight enhancement in the delayed phase lymphoma is considered when distant structures the mesenteryretroperitoneum or other parts of the abdomen have lymphnode metastasis the ct ï¬ndings may only reï¬ect features of gsc but cannotaccurately diagnose gsc l one explore the origin of thesarcomatous portion immunohistochemistry ihc also failedto conclusively establish the origin of gsc rodrigues usedï¬uorescence in situ hybridization fish to conï¬rm that sc andadenocarcinoma have a common origin that is the epithelium while primary gl originated from gastric submucosallymphoid tissuethe main treatment for localized lymphomas is eradicationof helicobacter pylori and surgical treatment whereas advanceddisease often requires radiation or chemotherapy alone surgery is the only treatment option for patients with gacadjuvant chemotherapy and chemoradiotherapy are also oftenused targeted therapy isin the exploration stage however there are currently no speciï¬c national comprehensivecancer network guidelines for the treatment of only gscbecause the tumor is relatively rare although complete surgicalresection is the most important treatment method for examplewhile chemotherapy is considered in clinical practice whetherchemotherapy can be applied for gsc and the eï¬cacy ofchemotherapy remain controversial domblides ï¬rstevaluated the eï¬cacy of immune checkpoint inhibitors icis forsc and found that lung sc patients exhibited high response ratesand prolonged overall survival os with icis this studyprovides a new idea for the treatment of gscbecause gl tends to be conï¬ned to the gastric wall forprolonged periods before tumor spread its prognosis is betterthan that of gac previous literature has found that scin the parotid gland lung hypopharynx liver and pancreashave poor prognoses due to metastasis or recurrence with asurvival period of a few months similarly gscpatients also died or developed metastasis or recurrence withina few months or it was already in the advanced stage at theï¬rst diagnosis all these clinical manifestations suggest that gschas a poorer prognosis than gac and gl in additiongsc can metastasize through the blood and lymph nodesand the most common sites of metastasis are the local lymphnodes and liver this conclusion is consistent with ourresearch resultsconclusionthe incidence rate of gsc is extremely low so clinicians andradiologists are not familiar with the features of this tumorbased on systematic research of this rare tumor and comparisonswith common gastric cancers we found that gsc is morecommon in men who are approximately years old and isoften accompanied by ulcers the disease is mostly located in theproximal part of the stomach and can also occur in the remnantstomach with delayed enhancement on contrastenhanced ctimages these characteristics can provide a reference for furtherresearch on gscs in the future however an accurate diagnosisof gsc depends on the combination of clinical imaging andhistopathological features due to the aggressive nature and poorprognosis of the tumor rapid clinical intervention and detailedfollowup with ct are essentialdata availability statementthe original contributions presented in the study are includedin the supplementary material further inquiries can bedirected to the corresponding authorethics statementthe studies involving human participants were reviewed andapproved by the medical ethical committee of the zhengzhouuniversity the patientsparticipants provided their writteninformed consent to participate in this study written informedconsent was obtained from the individuals for the publication ofany potentially identiï¬able images or data included in this author contributionsyl manuscript preparationliterature research and dataanalysis pl literature research and data analysis kf manuscriptreview and data collection kc guidance of pathologicalknowledge sy guidance of imaging knowledge jj imaging datacollection and analysis wl and xz manuscript editing jgstudy conception and design manuscript review and guarantor ofintegrity of the entire study all authors have read and approvedthe ï¬nal manuscriptfundingthis work was supported by the national natural and sciencefund of china no references zhu cc li mr lin tl zhao g sarcomatoid carcinoma of the stomach acase report and literature review oncol lett doi ol20153460 snover dc levine gd rosaij thymic carcinoma five distinctivehistological variants am j surg pathol zhou dk gao bq zhang w qian xh ying lx wang wl sarcomatoidcarcinoma of the pancreas a case report world j clin cases doi 1012998wjccv7i2236 xie y xiang y zhang d yao x sheng j yang y sarcomatoidthe doi 103892mmr2018and review ofthe pancreasreportcaseofcarcinomaliterature mol med repafrontiers in oncology wwwfrontiersinaugust volume 0cliu gastric sarcomatoid carcinoma sato a oki e kohso h endo y uchida h hiroshige s sarcomatoidcarcinoma of the remnant stomach report of a case surg today doi 101007s0059501204027 nakayama y murayama h iwasaki h iwanaga s kikuchi m ikeda s gastric carcinosarcoma sarcomatoid carcinoma with rhabdomyoblastic andosteoblastic diï¬erentiation pathol int doi 101111j144018271997tb04540x robeycaï¬erty ss grignon dj ro jy cleary kr ayala ag ordonezng sarcomatoid carcinoma of the stomach a report of three caseswith immunohistochemical and ultrastructural observations cancer doi 101002109701421990040165730co2n ruess da kayser c neubauer j fichtnerfeigl s hopt ut wittel uacarcinosarcoma of the pancreas case report with comprehensive literaturereview pancreas doi 101097mpa0000000000000904 fujiie m yamamoto m taguchi k iwanaga a ohgaki k egashira a gastric carcinosarcoma with rhabdomyosarcomatous diï¬erentiation a casereport and review surg case rep doi 101186s407920160176z tanimura h furuta m carcinosarcoma of the stomach am j surg doi 101016000296106790325x kitamura s study on carcinosarcoma of stomach gan kumagai k kawai k kusano h matsuo k irie j tsuchiyama h a caseof socalled carcinosarcoma of the stomach gan no rinsho bekki t fujikuni n tanabe k yonehara s amano h noriyuki t thegastric carcinosarcoma with severe venous invasion a case report surg caserep doi 101186s4079201804218 ikeda y kosugi s nishikura k ohashi m kanda t kobayashi t gastriccarcinosarcoma presenting as a huge epigastric mass gastric cancer doi 101007s1012000604054 cid³n eu cuenca ij gastric adenocarcinoma is computed tomography ctuseful in preoperative staging clin med oncol doi cmos2641 hallinan jt venkatesh sk gastric carcinoma imaging diagnosis stagingand assessment of treatment response cancer imaging doi gossios k katsimbri p tsianos e ct features of gastric lymphoma eurradiol doi 101007s003300050069 rodrigues dn hazell s miranda s crespo m fisher c de bono js sarcomatoid carcinoma of the prostate erg ï¬uorescence insituhybridization conï¬rms epithelial origin histopathology doi 101111his12493 levine ms rubesin se pantongragbrown l buck jl herlinger h nonhodgkins lymphoma of the gastrointestinal tract radiographic ï¬ndings ajram j roentgenol doi 102214ajr16818976941 russo ae strong ve gastric cancer etiology and management in asia and thewest annu rev med doi 101146annurevmed081117 domblides c leroy k monnet i mazi¨res j barlesi f gounant v eï¬cacy of immune checkpoint inhibitors in lung sarcomatoid carcinoma jthor oncol doi 101016jjtho202001014 niu x sarcomatoid carcinoma in the parotid gland a review of years ofexperience laryngoscope doi 101002lary27474 li s jiang l he q wei w wang y zhang x the prognostic signiï¬canceof jmjd3 in primary sarcomatoid carcinoma of the lung a rare subtypeof lung cancer onco targets ther doi 102147otts22 dai l fang q li p liu f zhang x oncologic outcomes of patients withsarcomatoid carcinoma of the hypopharynx front oncol doi103389fonc201900950 seo n kim mj rhee h hepatic sarcomatoid carcinoma magnetic resonanceimaging evaluation by using the liver imaging reporting and data system eurradiol doi 101007s00330019060528 shi y chen j chen h hong x sarcomatoid carcinoma of the gallbladdera case report j int med res doi conï¬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 liu liang feng chen yue ji li zhao and gao 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' | 0 |
specialty sectionthis was submitted tomolecular and cellular oncologya section of the frontiers in cell and developmentalbiologyreceived april accepted july published august citationchen w yang j fang h li l andsun j relevance functionof lincror in the pathogenesisof cancerfront cell dev biol 103389fcell202000696cancer is a serious disease that aï¬ects human health being one of the main causes of death all overthe worldwide according to research in of new tumor cases and of the cancerassociated deaths occurred in lowincome and developing countries kumar and sharawat noorolyai owing to a shortage in eï¬ective screening methods and lack of identiï¬cationof early symptoms most patients were already in advanced stages when they were diagnosedwith cancer bray koo additionally some clinical studies have shownthat polarity and adhesion of cancer cells was decreased leading to heir increased mobility andinvasion which is a key step in the development of cancer yan therefore studies haveshown that the high mobility of cancer cells is the main factor leading to high mortality rates inpatients with cancer currently there are many ways employed in the treatment of cancer includingsurgery radiotherapy chemotherapy biotherapy and targeted therapy nie howeverin the past years the survival rate of patients with cancers remains dismal nakashima abbreviations bc breast cancer cenas competing endogenous rna emt epithelialmesenchymal transition hcchepatocellular cancer ipscs induced pluripotent stem cells lincror long intergenic nonprotein coding rna regulatorof reprogramming lncrnas long noncoding rnas pc pancreatic cancerfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorin the process of developing human antitumorthereforestrategiesto ï¬nd new earlyitbiomarkers and thus identify potential regulatory mechanisms toimprove the survival rate of patients with cancersis particularly importantover the past decades ncrnas constitute more than of the rnas made from the human genome but mostof the known noncoding rnas ncrnas havebeen discovered and remain largely unstudied bhan slack and chinnaiyan transfer rna trna and ribosomal rna rrna constitute themajority of ncrnasfollowed in abundance by messengerrnas mrnas thus the remaining ncrnas includingcircular rna circrna small nuclear rna snrna smallnucleolar rna snorna microrna mirna and long nonfor ¼ ofcoding rna lncrna together accounttotalncrna despite their low abundancethese ncrnas havebeen reported to play critical roles in transcription posttranscriptional processing and translation such as epigeneticsposttranscriptional regulation chromatin modiï¬cation andregulation of the cell cycle huarte kondo peng 2017b additionally because ncrnas can be packagedinto extracellular vesicles ev including exosomes meldolesi they have been shown to provide a mechanism forintercellular communication through the transfer of mirnaand lncrna to recipient cells both locally and systemicallysun it is important to note that the expressionof ncrnas their posttranscriptional modiï¬cation particularlylncrnas and their subcellular distribution have been shownto be important to when assigning their potential functionpalazzo and lee recently nextgeneration sequencingand bioinformatics technology have revealed that circrnasplay crucial role in diagnosis and prognosis of various diseasespamudurti brieï¬y circrnas are singlestrandedtranscripts generated by backsplicing jeck and sharpless with covalently linked headtotail closed loop structures withneither 5cid483cid48 polarity nor a polyadenylated tail memczak that range in length from a few hundred to thousandsof nucleotides and are widely expressed in mammals therebyshowing higher stability compared to that in linear rnas chenj and exhibiting a celltype or developmentalstagespeciï¬c expression pattern barrett and salzman wang j j many functions of circrnas have alsobeen identiï¬ed including their role as mirna sponges bindingto rnabinding proteins and protein decoys and functioningas regulators of transcription hansen du yang y interestingly many circrnas havebeen shown to be dysregulated in pathophysiological processesand circrnas are known to regulate the expression of geneby acting as mirna sponges in a mechanism that is termedas competitive endogenous rna cerna mechanism zheng wang j j for example circmto1have been demonstrated to harbor conventional mirna bindingsites and has been identiï¬ed as an inhibitor of mirna9 inhepatocellular carcinoma hcc han additionallyour previous study has demonstrated that mirna plays arole in limiting the development of liver ï¬brosis by markedlyblocking the activation and proliferation of hepatic stellatecells hscs suggesting that mirnas might be involved inthe development and progression of several forms of cancersyang j yang of notelncrnaswhich are mainly transcribed by rna polymerase ii are anew kind of ncrna that are longer than nucleotidesma owing to the lack of open reading frameslncrnas have extremely limited or no protein coding capacityruan li j these new regulatorswere initially regarded as transcriptional noise with no speciï¬cbiologicalfunctions kim and sung recently ourlaboratory found that epigenetic silencing of lncrna anrilpromoted the progression of liver ï¬brosis thereby indicating thatlncrnas were associated with the progression of cancers yang interestingly increasing evidence have shown thatcellular events including diï¬erentiation proliferation invasionapoptosis and migration have all been associated with lncrnasguttman additionally there has been new evidencesuggesting that lncrnas may regulate a variety of biologicaland disease processes from gene transcription and translationto posttranslational modiï¬cations davalos and esteller pang more importantlylncrnas have beenreported to be used as tumor suppressor genes or oncogenesthus aï¬ecting the proliferation and metastasis of various typesof tumors during tumorigenesis chen q n lu subsequent studies have demonstrated thatlncrnas may serve as cernas for mirnas and in chromatinremodeling during the development of cancers huang wang c j figure illustrates the functionsof lncrnas at the molecular level regarding certain cancerassociated human lncrnasit was demonstrated that lincror was demonstrated to be predominantly upregulated intumors peng 2017a the abnormal expression of lincror in tumors has been suggested to be one of the mainleading factors driving the development the main ways torevert this eï¬ect would be to aï¬ect cell growth migrationand invasionthus leading to the inhibition of epithelialmesenchymal transition emt enhancement of the sensitivityto chemotherapy etc chen 2016a zhao forexample the expression level of lincror in hcc tissues wasinhibited compared with the adjacent tissues at the same timethe downregulation of lincror was linked to the aggressiveprocess of the disease in patients with hcc furthermore theability of migration and invasion of hcc cells may be delayedby the low expression level of lincror in this review weattempted to introduce the latest research on the biologicaleï¬ects potential clinical applications and molecular mechanismsof lincror in human tumors and discuss its prognostic andtherapeutic valuesoverview of lincrorlincror isamong lncrnasand importantcarcinogenic kb lncrna located in chromosome which was initially identiï¬ed as a highly expressed transcriptof pluripotent and embryonic stem cells chen 2016bstudies found that the octamerbinding transcription factor a novelfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure paradigms for the function of long ncrnas recent studies have identiï¬ed a variety of regulatory paradigms for the mechanism by which long ncrnasfunction many of which are highlighted here transcription from an upstream noncoding promoter pink can negatively or positively affect the expression ofthe downstream gene purple by inhibiting the recruitment of rna polymerase ii or inducing chromatin remodeling respectively an antisense transcript orangeis able to hybridize to the overlapping sense transcript purple and block the recognition of the splice sites by the spliceosome thus resulting in an alternativelyspliced transcript alternatively hybridization of the sense and antisense transcripts can allow dicer to generate endogenous sirnas by binding to speciï¬cprotein partners a noncoding transcript blue can modulate the activity of the protein serve as a structural component that allows the formation of a largerrnaprotein complex or alter where the protein localizes in the cell long ncrnas green can be processed to yield small rnas such as mirnaspirnas and other less wellcharacterized classes of small transcriptsoct4 srybox transcription factor sox2 and nanoghomeobox nanog key pluripotency factors could regulatelincror wang howeverlincror was alsofound to be expressed in several ans including lungliver breast and colon since its discovery research in thisï¬eld has been extensively expanded during the past yearsrevealing the important role of lincror in tumorigenesislincror has been suggestedadditionally upregulation ofto mediate the reexpression offetal and cardiomyocytehypertrophyrelated genes wang li inmany reportsrecent years have revealed thatlincror is positively correlated with the clinicopathologicalcharacteristics and poor prognosis of tumorsincluding thestages of advanced tumor node metastasis tnm positivelymph node metastasis lnm and lower survival rate buthigher recurrence rateregarding lincror and tumorigenesisthe upregulation ofcurrent evidence have strongly indicated thatlincrormay exert an impact on a variety of cancers pan furthermore both tumorigenesis and metastasis havebeen shown to be induced by lincror via activation of theemt in various cancers hou huang zhan for example lincror was demonstratedto be upregulated thereby promoting emt in hcc li j besides it was also reported that selfrenewal anddiï¬erentiation of glioma stem cells was signiï¬cantly aï¬ectedby lincror zhang feng moreimportantly the chemoresistance of pancreatic cancer pcand breast cancer bc li as well as radioresistance of colorectal cancer crc cells were observed to beelevated by lincror yang p moreover lincror has also been shown to exert a signiï¬cantly eï¬ect onthe stem celllike characteristics and tumorigenic potential ofpc recentlyit was also reported that lincror could beused as a biomarker in the ï¬eld of diagnosis and prognosisof bc and oral cancer arunkumar zhao notably increasing studies showed that lincror couldbe used as a cerna thus exerting its impact in the posttranscriptional network of tumor pathogenesis for examplein triplenegative bc lincror has been shown to serve asa cerna therefore promoting the migration and invasion ofbc cells signal overall lincror is a typicallncrna that plays important regulatory roles in interactionwith mirnas and maintenance of stem cell pluripotencytriggering the emt as well furthermore lincror has alsobeen involved in various key roles under hypoxia and in thepromotion of tumorigenesis figure therefore lincrormay be considered as an oncogene aï¬ecting the progressionof tumor and a promising predictor for the poor prognosis inpatients with cancer the transition of lincror from basicresearch to clinical application requires further investigations asearly as possiblefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure lincror is a typical lncrna that plays important regulatory roles in interacting with mirnas and maintaining stem cell pluripotency as well astriggering the emt as well lincror is also involved in various key roles under various stresses and in epigenetic regulationregulatory role of lincror invarious types of cancerincreasing evidence has shown that the lincror was abnormalexpression in many cancers and its dysregulation was associatedwith cellular functions fu spinelli additionally studies found that the expression level of lincror was substantially upregulated in samples of papillarythyroid carcinomas ptcs and ptcs cell lines as well as inmetastatic ptcs samples and ptcs cell lines zhang simultaneously cell migration and invasion could be regulatedby lincror via aï¬ecting emt pastushenko and blanpain more importantly studies demonstrated that lincrorwas abnormally expressed in several cancers and led to elevatedthe invasion and metastasis of cancer cells to promoting theprogression of tumors hashemian li 2020bcthis review summarizes the status of lincror research invarious human cancers and discusses its mechanism and clinicalsigniï¬cance in the development and progression of tumor theexpression pattern functional role and regulatory mechanism oflincror are summarized in table and depicted in figure breast cancerbc which accounts for a quarter of all female cancer casesis the most commonly diagnosed cancer and the leading causeof cancerassociated deaths among women worldwide li z in it was estimated that there would be million or so newly diagnosed cases of female bc bray hannafon the main risk factors forbc which is the diï¬cult to change due to prolonged exposureto endogenous hormones is diï¬cult to control rudel bray however comprehensive treatmentapproaches have resulted in relatively good clinical outcomesfor some patients with bc rudel goel kumler nevertheless it has been reported that aboutonethird of the patients with bc have the potential for cellmetastasis chemotherapy resistance and even recurrence goel kumler hence there is an urgent need todevelop new therapies targeting various molecular mechanismsof tumorigenesis for the treatment of bcthe level ofthe level ofhou the expression ofinvestigated the expression level of lincrorin patients hou their results revealedlincror was increased in bc tissuesthatmoreoverlincror in theperipheral blood ofthe patients with bc was shown tobe closely related to tnm phase and lnm in additionthe woundhealing assay showed that overexpression of lincror increased bc cells mcf10a mobility transwell assayrevealed that lincror overexpression remarkably increased themigration ability hou more importantly theyfound that ectopic expression of lincror induced an emtprogram in mcf10a cells fluorescence activated cell sorteranalysis demonstrated that the subpopulation of the stem cellphenotype cd44highcd24low was elevated in mcf10a cellstransfected with lincror plasmid mechanistically the resultsof bioinformatic analysis and rna immunoprecipitation analysisfrom hou demonstrated thatlincror functions asa cerna to regulate mir205 activity toward prevention ofthe degradation of transcripts of mir205 target genes suchas zeb1 and zeb2 from degradation additionallyit wasshown that the expression levels of mir205 members weredecreased upon lincror overexpression in mcf10a cellshou more importantly zhao recentlydemonstrated that crisprcas9generated brca1knockdownadiposederived stem cells stimulated a more aggressive behaviorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorlateillateoaglatenahzlatenehclatecillateihzlateilnusdnanaylatenapalategnahzlateuohsecnereferbezrmrorcniilmkppbtprmrorcniilgonanrmrorcniilnirehdacehzerorcnilsosfdmnlegatsmnttmeinosavnitmeinosavninoitargminoitarefilopicnegocnoldetaugerpuamoncraciraulllecotapehllseuceomyrotaugerllnoitaerroclacniilcleorlanoitcnufleorinosserpxesepytrecnacsrecnacnamuhnirorcnlielbatirmrorcnilevruccormnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnactsaerbtmeinosavnipbezrorcnilliavvrusroopmnlegatsmntnoitargminoitarefilorphtwicnegocnoldetaugerpurecnaccitaercnapmxofprmrorcniilncsfrmrorcniprmrorcniililirmrorcnilecnatsserigurdtmeinosavnitmelecycllecinosavniliavvrusroopmnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnacdoryhtisosfdmnlegatsmntinoitargmecyclllecissotpopaicnegocnoldetaugerpurecnacgnulecnatsseriyparehtodaritmeinosavnisosfdmnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnaclatcerooclin bc cells than wildtype adiposederived stem cells zhao therefore we believe that crisprcas9 may beused to in the treatment of bc by inhibiting the expressionof lincror during the progression of bc conclusively thelincrormirnas axis has been reported to closely aï¬ect theoccurrence and development of bcpancreatic cancerpc is the fourth most common cause of cancerrelated mortalityworldwide leading to approximately deaths annuallysiegel sabater the year relativesurvival of patients with pc remained at approximately for siegel hence pc has been proposedto be one of the top two cancers in terms of fatalities in thenext decade rahib surgical resection remainsthe exclusive potential curative treatment xiong however approximately half ofthe patients present withmetastasis at the time of diagnosis missing the opportunityfor an eï¬ective treatment vincent xiong a growing body of literature has demonstrated that bothmetastasis and limited eï¬ective biomarker for the diagnosis andtreatment are the main obstacles for the eï¬cient medical therapyof pc vincent boj basuroy thus it is an absolute necessity to identify potential biomarkersand therapeutic targets in pczhan have highlighted the oncogenic eï¬ectsof lincror in the initiation and progression of pc theirstudy demonstrated that the level of lincror was signiï¬cantlyelevated in pc tissues zhan moreoverthewoundhealing assay and boydens chamber assay results showedthat lincror silencing reduced the migratory capability andmetastasis of pc cells zhan another study bychen showed that the proliferation rates of shrorcellsin which the level of lincror was suppressed were evidentlylower than those of shnccells this result was conï¬rmed bycolony formation assay suggesting that lincror accelerated thegrowth of pc cells chen 2016a interestingly silencingof lincror was shown to result in increased levels of theepithelial markers ecadherin and αcatenin and decreased levelsof mesenchymal markers ncadherin and vimentin indicatingthat lincror plays an important role in the regulation ofemt in pc cells zhan chen moreimportantly microarray analysis identiï¬ed zeb1 as potentialtarget gene of lincror further the expression of lincrorand zeb1 were observed to be negatively correlated with thatof p53 suggesting that lincror might mediate migration andmetastasis in pc cells may partly via activation of zeb1 throughthe inhibition of the expression of p53 zhan interestingly the ï¬uorescence in situ hybridization and luciferasereporter assay results showed that the expression of lincrorwas demonstrated to be negatively correlated to that of mir145mir145 can induce posttranscriptional silencing of its targetedgenes by binding to the mrna utr or lincror speciï¬csites indicating that lincror can act as a cerna to decreasemir145 in pc cells thereby activating expression of nanogthus leading to the proliferation of pancreatic cancer stem cellspcscs gao additionally li furtherfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure underlying molecular mechanisms of lincror in multiple cancers a lincror binds to mir205 to upregulate zeb2 while it also regulated theexpression of emt markers b lincror could interact with mir145 to inhibit fscn1 and upregulated emtassociated proteins while it decreases g0g1 arrestand facilitated drug resistance c lincror facilitated emt through upregulate ezhz and regulated zeb2 by competitively binding to mir145 and zeb2overexpression leads to increased emt in addition lincror binds to mir8765p to upregulate foxm1 d lincror downregulated through emt production andrepress the expression of mir145 e lincror binds to mir68333p while also regulating the process of emt f lincror upregulated zeb1 to attenuate theexpression of p53 while also decreasing the expression of mir145 to increase the level of nanog and reduce that of mir124 to suppress pkm2 detailedmechanisms of lincror in other cancers are provided in the reviewproved that the impact of lincror can be partly reversed byoverexpression of mir124 consistently lincror was observedto exhibited a negative correlation with the expression of mir li hence a lincrormir124ptbp1pkm2axis was identiï¬ed in pc shedding new light on the lncrnabased diagnosis and therapeutic approaches in pc li 2020b notably recent studies showed that pc cellderivedevs could be used as eï¬ective carriers of paclitaxel to theirparental cells thereby bringing the drug into cells through anendocytic pathway and increasing its cytotoxicity saari additionally it was demonstrated that vesiclecontainingncrnas could serve as evassociated pc detection markersworst thus the presence of lincrors in evs frompatients with pc could serve as a potential diagnostic biomarkerand a novel target for the therapy of patients with pc this isworthy of further and wider research attentionhepatocellular carcinomaas the sixth most international commonly occurring cancer in hcc has become the fourth cause of cancerassociateddeaths worldwide it has been estimated that new casesand deaths will occur each year bray brieï¬yhcc has been reported to account for of all the livercancer cases half of which have been detected in china omata bray as such hcc poses a huge threatto the worldwide health especially that of the chinese peopleomata about of the patients is expected torecrudescent within years after hepatectomy and of thepatients will die from this tumor vigano thereforeon the basis of studying the pathogenesis of hcc it is apparent tolook for more eï¬ective molecular markers and therapeutic targetsfor the management of hccli c and chen reported that theexpression level of lincror was obviously elevated in hcctissues and four cell lines compared to the corresponding nontumor tissues and normal liver cell lines respectively suggestingthat lincror might be critical regulator in the progression ofhcc furthermore biological function assay demonstrated thatlincror could play promoting role in regulating migration andinvasion of hcc chen moreover downregulationof lincror could result in a signiï¬cant increase in g1g0phase and an obvious decrease in s phase li c more importantly silencing of lincror could lead to theincreased expression of ecadherin and decreased expressionlevel of ncadherin in hcc cell lines li c furtherconï¬rmed that lincror could bind to the zeste homolog ezh2 thereby aï¬ecting the expression of ecadherin furtherindicating that lincror could regulate the progression of emtmoreover lincror was further determined to be associatedwith dna repair currently mounting studies have identiï¬edreliable indicators of dna damage such as phosphorylatedhistone h2ax γh2ax chen uncover thatfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorlincror could obviously decrease theoverexpression ofexpression level of γh2ax illuminating the suppressive eï¬ectsof the overexpression of lincror on dna repair in hccfurther research demonstrated that lincror could interactwith mir and dramatically downregulate the expression ofmir in hcc cells li c the abovementionedresults revealed that lincror might play a promoting role inthe proliferation migration invasion and emt of the hcc cellwhich was contrary to the inï¬uence of mir enrichmentli c it suggested that overexpressed mircould eï¬ectively reverse the promotion of hcc tumorigenesisinduced by the overexpression of lincror li c liand his colleagues proposed a mechanistic model that lincrorpromotes hcc tumorigenesis and autophagy partly throughnegatively regulating the expression of mir aside fromthat it has been reported that mir145 represses emt tumormigration and invasion by directly targeting the utrs ofzeb2 in the tumor the decrease in mir and increase inzeb2 can obviously reversed the inhibition of cell migrationand invasion mediated by the lincror knockdown thereforeit was suggested that targeting the lincrormir145zeb2axis might represent a novel therapeutic application in hccli c similarly zhi similarly showedthat the migration and invasion of cells was reduced by theknockdown of lincror moreover they further conï¬rmedthat foxm1mediated activation of lincror contributes tothe poor sensitivity of hcc cells to sorafenib via partiallyregulating the mir8765pfoxm1 axis which forms a positivefeedback loop further evaluation of the regulatory mechanisminvolving this axis may provide new insights for exploringa potential therapeutic strategy for the management of hcczhi consequently these studies may oï¬er newinsights regarding the pathology of hcc and provide potentialstrategies for lncrnadirected treatment however both thein vivo inï¬uence and other underlying mechanisms of lincrorstill remain to be determined and clariï¬ed in the future researchthe prognosiscolorectal cancerthere are approximately million new crc cases and crcrelated deaths worldwide each year thus making crc thethird most common cancer in the world torre although the treatment of crc has signiï¬cantly improvedin recent decadesremains unsatisfactoryespecially in case of advanced tumors with distant metastasesbogousslavsky torre current studiesresults showed that approximately of cases with crchave synchronous liver metastases during the time of diagnosiskawaguchi these patients have inherently lowsurvival rates of less than within years with an even worseprognosis hu kawaguchi thus there isan urgent need to better understand the progression of crc andto identify novel and sensitive biomarkers for the diagnosis andtreatment of patients with crcyang detected the expression of lincror in crctissues compared to normal tissues by using qrtpcr theyfound that the expression of lincror was remarkably increasedin crc tissues compared with normaltissues similarlylincror was shown to be overexpressed in ï¬ve crc cell linesyan and sun li 2020a then they also performeda series of functional assays to clarify the biological eï¬ects ofthe aberrant expression of lincror on proliferation viabilityapoptosis migration and invasion of crc cells knockdownof lincror was shown to eï¬ectively inhibit the proliferationof crc cells whereas its overexpression obviously increasedthe proliferative capacity of crc cells accordingly silencing oflincror strongly inhibited the migratory and invasive abilitiesof crc cells compared with that in the control cells li 2020a in contrast the migratory and invasive ability of cells wasactivated following the overexpression of lincror the mts345dimethylthiazol2yl53carboxymethoxyphenyl24sulfophenyl2htetrazolium assay results showed thatthelincror could enhance the viability ofoverexpression ofcrc cells furthermore the ï¬ow cytometric analysis resultsrevealed that the percentage of apoptotic cells in lincroroverexpression group was reduced by ± indicatingthat the overexpression of lincror could inhibit apoptosisin the crc cell lines li 2020a more importantly arecent study revealed the role of lincror in the emt it wasrevealed that the upregulation of lincror could increase theexpression of ncadherin and vimentin as well as decrease thelevel of ecadherin leading to the promotion of the progressionof emt zhou yan and sun meanwhilethe high expression of lincror in crc was also conï¬rmedby hu mechanistically li 2020a provedthat that lincror could bind to mir68333p which wasdetermined to be signiï¬cantly downregulated in crc tissuesadditionally a negative correlation was exhibited between theexpression of lincror and mir68333p in bc tissues antiago2 rna immunoprecipitation assay further conï¬rmed theseresults li 2020a besides rescue assays demonstratedthat downregulation of mir68333p could partly reversed theinhibition of tumorigenesis induced by lincror knockdown inbc cells li and his colleagues uncovered that lincror exertedits oncogenic role through negatively regulating the expressionof mir68333p during the progression of crc which mightgive new insights into molecular diagnosis and treatment li 2020a in addition li 2020a further uncoveredthat lincror could mediate the expression level of smc bysponging mir68333p in crc cells thus promoting crcprogression as for the eï¬ects of lincror on radiotherapyresistance yan and sun showed that overexpression oflincror increased the ability of crc cells for radiotherapyresistance collectively these ï¬ndings indicated that lincrormight be engaged in the metastatic process of crc cells andcould promote the development of crc through a variety ofmolecular mechanismslung cancerlung cancer is the leading cause of cancerrelated deathsworldwide bray nonsmall celllung cancernsclc accounts for about of the lung cancer typesincluding squamous cell carcinomalung cancerand lung adenocarcinoma herbertz bray brainard and farver although there are variousapproaches for its diagnosis and treatments the 5year overallsurvival os rate for patients with advanced lung cancer is lesslarge cellfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorthan zhou therefore in order to carry outan early diagnosis and treatment of lung cancer the search forvaluable and eï¬cient tumor markers is very urgentin recent yearslincror has appeared as an importantregulator oflung cancer research from qu demonstrated that the expression of lincror was increasedin nsclc tissues compared to that in the normal tissuesthe overexpression of lincror was shown to be closely relatedto the poor prognosis of lnm histological grade and stageof tnm pan qu another study bypan demonstrated that the decreased expressionof lincror could obviously impair the proliferative capacityof lung adenocarcinoma lad cells cause a g0g1 phasearrest and increase the ratio of apoptotic lad cells moreoverdownregulation of lincror substantially inhibited the invasiveand metastatic ability of lad cells meanwhile forced expressionof lincror was observed to reduce the expression of ecadherinand βcatenin which are the characteristic biomarkers ofepithelial cells whereas it increased the expression of ncadherinand vimentinthus displaying a mesenchymal phenotypeconversely downregulation of lincror was demonstrated toresult in increased the expression of epithelial markers anddecreased the expression of mesenchymal markers this resultuncovered the fact that the prometastatic eï¬ects of lincrorwere induced by the regulation of the expression of a number ofgenes involved in cell metastasis and emt progress meanwhileoverexpression of lincror was shown to enhance the resistanceof lad to docetaxel dtx pan suggestingthat lincrorinduced resistance of lad cells to dtx andemt through regulation the expression of mir145 whichwas predicted to interact with lincror more importantlyfurther research conï¬rmed that mir145 could bind to lincror and its downregulation could partly inhibit the resistanceof lad cells to dtx and emt aside from that pan andhis colleagues discovered that a decrease in the expression ofmir145 and increase in the expression fscn1 could obviouslyreverse the inhibition of cell proliferation chemoresistanceand emt mediated by lincror knockdown they identiï¬edthat dysregulation of the lincrormir145fscn1 axis wasassociated with the therapeutic resistance and emt transition inlad cells thereby providing potential therapeutic strategies formanaging drug resistance in patients with lad pan taken together these studies collectively suggested that lincrorcould activate the malignant phenotype of nsclc cells with theguidance of a mechanism involving mirnas hence more eï¬orts | 0 |
"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 table1clinical 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 clinica 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 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 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 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 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 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 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 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 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 investigated 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 clinica 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 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 index has been used toevaluate histologic activity in patients with clinically quiescent ulcerative colitis as a result this study reported relatively low sensitivityand speciï¬city for fecal calprotectin in the prediction of geboesscore sensitivity speciï¬city and cut oï¬ µgg in another recent study the nancy index has been used toevaluate the histologic activity of ulcerative colitis and a high sensitivity and a low speciï¬city were ï¬nally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand speciï¬city for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a speciï¬city of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconï¬icts should be explored in the endoscopic and clinical activity ofthe disease the inclusion and exclusion criteria of these studies andpossibly in the diï¬erent indexes used for the evaluation of the histologicactivity of the disease another notable issue is that all of these studieshave been conducted on a relatively low number of patients with ulcerative colitis so the need for a study with a large sample size is stillstrongly felt moreover a large global study may be helpful in this regard prediction of relapse and response to treatmentas mentioned previously ibd has recurrence and relief periods sopredicting response to treatment and relapse is one of the significant 0cf khakikhatibi clinica chimica acta challenges in ibd management the ï¬rst evidence of the eï¬cacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the ï¬rststudies in this regard in this study ibd patients in clinical remissionwere followed up for one year for the assessment of recurrence afterpreparing a stool sample to measure calprotectin this study has alsoshown that fecal calprotectin levels were higher in ibd patients withrecurrent disease and it was found that fecal calprotectin had a sensitivity of and a speciï¬city of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and speciï¬city of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a speciï¬city of versus a sensitivity of and a speciï¬city at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and speciï¬city rates for fecal calprotectin in the prediction of relapse a sensitivity of and a speciï¬city at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and ï¬nally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas | 0 |
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 | 2 |
" exosomes are extracellular vesicles containing a variety of biological molecules including micrornasmirnas we have recently demonstrated that certain mirna species are selectively and highly enriched inpancreatic cancer exosomes with mir1246 being the most abundant exosome mirnas have been shown tomediate intercellular communication in the tumor microenvironment and promote cancer progression thereforeunderstanding how exosomes selectively enrich specific mirnas to initiate exosome mirna signaling in cancercells is critical to advancing cancer exosome biologyresults the aim of this study was to identify rna binding proteins responsible for selective enrichment ofexosome mirnas in cancer cells a biotinlabeled mir1246 probe was used to capture rna binding proteins rbpsfrom panc1 cells among the rbps identified through proteomic analysis srsf1 eif3b and tia1 were highlyassociated with the mir1246 probe rna immunoprecipitation rip and electrophoretic mobility shift assay emsaconfirmed the binding of srsf1 to mir1246 lentivirus shrna knockdown of srsf1 in pancreatic cancer cellsselectively reduced exosome mirna enrichment whereas gfpsrsf1 overexpression enhanced the enrichment asanalyzed by next generation small rna sequencing and qrtpcr mirna sequence motif analysis identified acommon motif shared by of srsf1associated exosome mirnas emsa confirmed that shared motif decoysinhibit the binding of srsf1 to the mir1246 sequences we conclude that srsf1 mediates selective exosome mirna enrichment in pancreatic cancer cells bybinding to a commonly shared mirna sequence motifkeywords srsf1 exosome mirna mir1246 pancreatic cancer exosomes are endosomederived extracellular vesiclesevs that can be transferred from cancer cells tostromal cells in the tumor microenvironment [ ]these membrane vesicles are nm in size andcontain proteinsincludinglipids and nucleic acids correspondence weiqundingouhscedu1department of pathology university of oklahoma health sciences centeroklahoma city stanton l young blvd bmsb 401a oklahoma city ok usa6stephenson cancer center university of oklahoma health sciences centeroklahoma city ok usafull list of author information is available at the end of the small rnas such as micrornas mirnas[ ]exosomemediated intercellular communication betweencancer cells endothelial cells [ ] fibroblasts [ ] orimmune cells [ ] can facilitate tumor progressionfurthermore cancer exosomes are released into the circulation and contribute to premetastatic niche formation in distant ans [ ]how cancer exosomes interact with stromal cells topromote tumor progression has been extensively investisignaling eventgated one criticalin the tumormicroenvironmentisthe exosome mirnamediatedintercellular communication [ ] studies haveshown that exosome mirna signaling promotes tumor 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 0cxu cell communication and signaling page of progression in various model systems [ ] notablyit has been reported that mirnas contained in exosomes are delivered to recipient cells in the tumormicroenvironment or distant ans where they canregulate target gene expression and promote tumorangiogenesis and metastasis [ ]in the context of exosome mirna signaling we andothers have reported that certain mirna species areselectively enriched in cancer exosomes as compared toexosomes derived from normal epithelial cells [ ] results from several studies have also indicated thatselective enrichment of exosome mirnas is relevant totumor progression for example exosome sortingof mir193a was found to promote colon cancer progression likewise mir122 a cancer exosomeenriched mirna [ ] was shown to reprogram glucose metabolism in a premetastatic niche to facilitatemetastasis in a breast cancer model system moreover the exosome enriched mir1246 was reportedto promote tumor invasion in both breast cancer and oral squamous cell carcinoma it seems clearthat selective enrichment of exosome mirnas drivescancer exosome mirna signaling in the tumor microenvironment which in turn reinforces tumor invasiveness and progression however how exosome mirnasare enriched or how exosome mirna signaling is initiated in cancer cells remains largely unknown elucidating the mechanisms ofselective exosome mirnaenrichment in cancer cells may help identify new cancertherapeutic opportunities that are urgently neededrecentreports have indicated that certain rnabinding proteinsrbps are involved in exosomemirna sorting in eukaryotic cells and the type ofrbps involved seems to differ among various modelsystems [ ] suggesting that exosome mirnasorting is a tissue or celltype specific processfurthermore there have been no reports on the identification of rbps that regulate exosome mirna sorting in pancreatic cancer cells we have recentlycharacterized the biogenesis of exosome mir1246 which is the most highly enriched mirna inpancreatic cancer cellderived exosomes the aimof this study was to utilize our established cell modelsystems to identify rbps that are involved in exosomemirna loading in pancreatic cancer cells using alabeled mir1246 probe as bait we fished out several rbpsincludingserine and arginine rich splicing factor srsf1eukaryotic translation initiation factor subunit beif3b and t cellrestricted intracellular antigen tia1 we found that srsf1 a recently claimedoncoproteininregulating exosome mirna enrichment in pancreaticcancer model systemsfrom pancreatic cancer cellspredominantlyinvolved ismethodscell culturelines panc1the human pancreatic cancer celllinemiapaca2 and bxpc3 and breast cancer cellmdamb231 were obtained from the american typeculture collection atcc manassas va usa cellswere cultured following atccs instructions except thatexosomedepleted fetal bovine serum fbs and horseserum wereapplied whenever needed exosomedepleted fbs and horse serum were prepared by pelleting the serum exosomes at g for h at °ccells were routinely incubated in a humidified environment at °c and co2exosome isolationexosomes were isolated from the culture medium utilizing a combination of centrifugation ultracentrifugationand filtration as we recently described [ ] withminor modifications in brief the culture medium ofpanc1 cells was precleared by g centrifugationfor min at °c and the resulting supernatant was filtered through a μm pvdf centrifuge filter thelarge size evs were trapped in the filter and recovered inpbs the filtered supernatant was then applied to a μm pvdf centrifuge filter the medium size evswere trapped in the second filter and resuspended inpbs the small size evs exosomes in the final supernatant were recovered by ultracentrifugation g min at °c the isolated exosomes were verified bywestern blot detecting positive and negative exosomemarker proteins and nanop analysis nanosightns300 system malvern instruments uk measuringboth sizes and concentrations of the isolated exosomesfig mirna binding protein pulldownpulldown experiment was performed using the pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific briefly pmol of biotinlabeled mir1246or polya rna oligonucleotides integrated dna technologies were hybridized to μl streptavidin magnetic beads prod1862766 thermo fisher scientificthe mir1246biotinstreptavidin beads were incubatedwith panc1 lysate for min at °c the lysatebeadmixture was washed three times with washing bufferfrom the abovementioned kit to elute bound proteins μl of elution buffer was applied and a magnetic separator was applied to separate the beads from the elutedprotein following the manufacturers protocol pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific proteins were separated by sdspage beforemass spectrometry ms analysis 0cxu cell communication and signaling page of fig verification of the exosomes derived from panc1 cells a representative western blot analysis of cd63 nonreducing condition cd81flotillin and calnexin in the evs isolated from panc1 cells positive exosome markers are only detected in small evs exosomes b representativenanop tracking analysis of exosomes small evs derived from control and srsf1 knockdown panc1 cells three individual experimentswere performed for both a and bliquid chromatographymass spectrometry lcmsmassspectrometry ms measurementthe experiment was performed by the laboratory formolecular biology and cytometry research core facilityat ouhsc proteins were digested with trypsin according to the fasp protocol briefly the eluate was buffer exchanged in m urea the proteins were reducedwith mm dithiothreitol and then alkylated with mm iodoacetamide the peptides were eluted dried andresuspended liquid chromatography tandem mass spectrometry was performed by coupling a nanaoacquityuplc waters corp manchester uk to a qtofsynapt g2s instrument waters corp manchesteruk each protein digest about ng of peptide wasdelivered to a trap column μm mm nanoacquity uplc nanoease column μm beh c18 waterscorp manchester uk at a flow rate of μlmin in solvent a mm ammonium formate ph inhplc grade water tandem mass spectra were generated in the trapping region of the ion mobility cell byusing a collisional energy ramp from v low massstartend to v high mass startend the pusherionmobility synchronization for the hdmse method wasperformed using masslynx v41 and driftscope v24lockspray of glufibrinopeptideb mz wasacquired every s and lock mass correction was appliedpost acquisitionprotein identificationraw ms data were processed by plgs proteinlynxglobal server waters corp manchester uk for peptide and protein identification msms spectra weresearched against the uniprot human database containing reviewed sequences with the followingsearch parametersfull tryptic specificity up to twomissed cleavage sites carbamidomethylation of cysteineresidues was set as a fixed modification and nterminalprotein acetylation and methionine oxidation were set asvariable modificationssmall rna library preparation and next generationsequencingtotal rna was extracted from cell and exosome pelletsusing the trizol reagent invitrogenlife technologiescarlsbad california the small rna libraries were constructed and run on the illumina miseq platform as werecently described [ ]rna immunoprecipitation assaypanc1 cells or mdamb231 celllysates were prepared using ip buffer mm trishcl ph mmnacl mm edta mm pmsf and triton x the lysate was sonicated for min on ice andinsoluble material was removed by centrifugation supernatants were collected and protein concentrations weremeasured the supernatant was precleared by proteing dynabeads¢thermo fisher scientific and thenmixed with antibody srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 in a ratio of at °c overnight withgentle rotation to capture the antibodyproteinrnacomplexes μl of protein g magnetic beads wereadded and the complexes were rotated for h at °cthe sample was separated by magnetic separation trizol reagent invitrogenlife technologies was appliedto isolated rna from the complex the mirna expression was analyzed by qrtpcr 0cxu cell communication and signaling page of coimmunoprecipitation coipcoimmunoprecipitation coip using panc1 cell lysate and antibody of srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 was performed as described previously and the protein complex was detected by westernblotwestern blot analysiswestern blot was performed as we recently described[ ] primary antibodies raised against srsf1 santacruz sc33652 eif3b santa cruz sc137214 tia1santa cruz sc166247 betaactin a5441 and glyceraldehyde 3phosphate dehydrogenase gapdh santacruz sc47724 were used for detection nuclear andcytoplasmic protein extraction was extracted followingrockland nuclear cytoplasmic extract protocol and verified by histoneh3 cst 4499s andgapdh santa cruz sc47724 detection antibodiesused for exosome marker detection include cd63cd81 santa cruz bio technology inc ca usaflotillin1 and calnexin cell signaling technology incma usaquantitative realtime reverse transcription polymerasechain reaction qrtpcrqrtpcr was performed as we described [ ] withspecific primers cel54 ²gcgcgcccgtaatcttcataatcc3² mir1246 ²gcgcgatggatttttggagcag3² mir320c ²gcaaaagcuggguugagagggu3² and mir320d ²gcgaaaagcuggguugagagga3²srsf1 shrna expression plasmid constructiontarget specific oligonucleotides were designed using online tool rnai codex cold spring harbor laboratoryand were synthesized integrated dna technologieswith the addition of overhangs according to the cuttingsite of bamh1 and ecori the shrna expression plasmid was constructed by annealing the oligonucleotidesto psihh1 vector following the user manual of psihh1 shrna system sbi system bioscience the oligonucleotide sequences for shrna of srsf1 eif3b ortia1 are provided in supplemental table 3rd generation packaging plasmidslentivirus transductionlentiviral ps were produced as previously described using the shrna expression plasmid andthepmd2gaddgene plasmid pmdlrregp addgeneplasmid and prsvrev addgene plasmid the packaging plasmids were cotransfectedwith the lentiviral expression vector into t cellsusing the polyethyleneimine polysciences inc to produce replication deficient lentivirus after transfectionthe supernatant was pooled and filtered with a μmmembrane and concentrated by ultracentrifugation toacquire lentivirus infection was performed by usinglentivirus in the presence of μgml polybrene sigmaaldrich approximately h postinfection cells wereselected by treating with μgml puromycin invivogen san diego cagfpsrsf1 expressionthe gfpsrsf1 expression plasmid was a gift from drmassimo caputi dna transfection was performedusing lipofectamine thermo fisher scientific topanc1 cells and the expression of gfpsrsf1 wasverified by western blotgstsrsf1 protein purificationbl21 thermofisher scientific c600003 competentcells transformed with pgex6psrsf1 dna addgeneplasmid were cultured at °c for hand after od600 reached to bacteria weretreated with mm isopropyl βd1thiogalactopyranoside for h at °c gsttaggedsrsf1 was purifiedwith glutathione sepharose beads ge health carethe purity of the recombinant proteins was determinedby sdspage with coomassie blue stainingelectrophoretic mobility shift assay emsaird800 labeled mir1246 μm integrated dnatechnologies was mixed with μl of gst slurry stsrsf1 in binding buffer tris ph mm kcl mm mgcl2 mm np40 dtt mm glycerol and incubated at room temperature for minavoiding light 5x loading buffer kcl mm tris ph mm glycerol xylene cyanol bromophenol blue was then added and the complexwas separated on a native gel polyacrylamide m tris ph m glycine m edta apstemed at voltage for min the signal was detected using the licor odyssey imaging systemlicor inc usadesign of decoy motif mimicsthe decoy motif mimics were designed by permutationand combination of the identified motif sequences in thelength of nucleotides the secondary structure of thedesigned sequences was analyzed in rnafold webserveruniversityselfcomplementary were selected decoy mimics ²uuggacuaggacuaggau3² decoy mimics ²aggaaggaaggaagga3²sequences withoutof vienna 0cxu cell communication and signaling page of bioinformatics analysisthe mirna motif analysis was performed using memesuite the protein profile analysis for the result ofmass spectrometry was performed using david bioinformatics abcc at saicfrederick inc the rnabinding protein and mirna sequence binding analysiswas performed using the database of rnabindingspecificities rbpdb srsf1 expression in cancertissues was examined using oncomine thecorrelation of gene expression with cancer patient survival was extracted from the human protein atlasscilifelab sweden statisticsstatistical analyses were performed using graphpadprism software graphpad software inc la jolla causa the heatmap was made in rstudio rstudio incwith the ggplot2 package students ttest was applied to determine significant differences among controland experimental groupsresultsidentification of mir1246 associated proteinsbecause rbps are involved in exosome mirna sortingwe first sought to identify proteins that bind to mirnashighly enriched in cancer exosomes mir1246 the mosthighly enriched mirna in pancreatic cancer exosomeswas biotinlabeled and incubated with a cellular lysatefrom panc1 cells the biotinmir1246 probe wascaptured with streptavidincoated magnetic beads biotinlabeled polya mimics were used as control themirnaprotein complexes were eluted and the proteinswere analyzed by liquid chromatographymass spectrometry in triplicate table there were total of proteins specifically pulled down by the mir1246 probeinterestingly about half of the proteins that associatewith mir1246 are vesicleassociated proteins supplement fig 1a based on the intensity of detection rnabinding property and cancer relevance we ranked therbps using the database for annotation visualizationand integrated discovery david this resulted in tencandidate rbps that complex with the mir1246 sequenceexosomestable among them srsf1 also called sfrs1 waspredictedsequencethe mir1246and arerelevantto eukaryotictobindtotable over view of the result of mass spectrometryexperiments conditionpoly a panc1number of proteins detectedpoly a mdamb231mir1246 panc1mir1246 mdamb231table mir1246 rna binding protein candidates obtainedfrom the mass spectrometric analysisprotein full nameprotein symbolsrsf1serineargininerich splicing factor park7eif3bthoc4acocddx5tia1if5a1eif2aimdh2parkinson disease protein eukaryotic translation initiation factor subunit btho complex subunit alyref export factorcytoplasmic aconitate hydrataseprobable atpdependent rna helicase ddx5tcellrestricted intracellular antigen1eukaryotic translation initiation factor 5a1eukaryotic translation initiation factor 2ainosine5²monophosphate dehydrogenase supplement fig by in silico analysis using the database of rnabinding specificities rbpdb levelsverification of srsf1 binding to mir1246rna immunoprecipitation rip was performed to verifythe association of several identified rbps with mir1246including srsf1 eif3b and tia1 igg and gapdhantibody was used as controls for immunoprecipitationas shown in fig 2a mir1246 expression is more than12fold higher in the srsf1precipitants as compared tothat of igg precipitants indicating a specific associationof srsf1 with mir1246 mir1246 expression wasmoderately increased in the tia1precipitants and nearigg controlin the eif3b precipitants coimmunoprecipitation coip experiments were performed to verify the immunoprecipitation proceduresdata not shown to directly determine the binding ofsrsf1 to the mir1246 sequence glutathionestransferase gst conjugated human srsf1 protein wasexpressed in bl21 competent e coli captured by glutathione sepharose beads and eluted by glutathione thepurity of eluted gstsrsf1 protein was shown by sdspage and coomassie blue staining supplement fig the binding of gstsrsf1 to a fluorescenttaggedmir1246 probe was determined by rna emsa asshown in fig 2b binding of the labeled probe was specific to gstsrsf1 but not gst and increased withgreater protein input the specific binding of gstsrsf1 to the mir1246 probe was evident as the unlabeled mir1246 probe effectively competed with thelabeled mir1246 probe in a concentrationdependentmanner fig 2c the detected bands were semi quantified and the kd was calculated from the detected signalsfig 2d these data confirmed the direct binding ofsrsf1 to the mir1246 sequence 0cxu cell communication and signaling page of fig srsf1 binds to mir1246 a qrtpcr detection of mir1246 in igg gapdh srsf1 eif3b and tia1 immunoprecipitants of panc1 lysaten p student ttest bc emsa detection of the srsf1mir1246 complex hot probe ird800 labeled mir1246 mimics cold probemir1246 mimics n direct binding of gstsrsf1 and mir1246 b and concentrationdependent competition between the cold and hotmir1246 probe for binding to gstsrsf1 c d semiquantification of srsf1 and mir1246 binding in c and calculated dissociationconstant n exosome mirna enrichment by srsf1 in cancer cellsbecause srsf1 is a key splicing factor that is essential toeukaryotic cells a knockout model could not beestablished thereforeto determine whether srsf1mirna binding activity is relevant to exosome mirnaenrichment we established a lentivirus srsf1 shrnaconstruct to knockdown srsf1 expression in panc1cells fig 3a interestingly though srsf1 protein wasdetected both in the nucleus and cytoplasm the knockdown was more pronounced in the cytoplasm fig 3bknockdown of srsf1 did not significantly alter the concentration and size distribution of the exosomes releasedby panc1 cells fig 1b cellular and exosome rnafrom control and srsf1shrna cells were isolated andsmall rna sequencing was performed among the highly enriched panc1 exosome mirnas expressionof mirnas was significantly downregulatedin exosomes derived from srsf1shrna panc1 cellsas compared to exosomes derived from control panc1cells fig 3c strongly indicating the involvement ofsrsf1 in exosome mirna enrichment a heatmapshowing the expression of the top mirnas enrichedin panc1 exosomes demonstrates the dramatic dropin expression levels of mirnasin srsf1shrnapanc1 exosomes compared to panc1 exosomesfig 3d notably mir1246 was the highest enrichedexosome mirna data not shown and its expressionin exosomes wassignificantly reduced by srsf1knockdown fig 3d on the other hand among of the mirnas less enriched in exosomes only were expressed at lower levels in exosomesderived from srsf1 knockdown cells as compared toexosomes derived from wild type panc1 cells fig3c suggesting that srsf1 knockdown mainly affectsexosome enriched mirnasto further confirm the effect of srsf1 knockdown onexosome mirna enrichment the expression levels ofseveral representative mirnas were quantified by qrtpcr srsf1 knockdown in panc1 cells significantlyreduced exosome levels of mir1246 mir320c andmir320d confirming the small rna sequencing resultsfig 3e in contrast knockdown of eif3b or tia1 didnot reduce exosome mir1246 expression suggestingthat these rbps may not promote exosome mirna 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig cellular and exosome mirna profiles after srsf1 knockdown in panc1 cells a detection of srsf1 knockdown by shrnas in panc1 cellsb panc1 srsf1 protein levels in nuclear and cytoplasmic fractions nc normal control c venn diagram of overlap of mirnas detected by nextgeneration small rna sequencing in srsf1 knockdown and control panc1 cells and exosomes d heatmap showing the expression of top exosome mirnas in cells and exosomes after srsf1 knockdown e qrtpcr analysis of mir1246 mir320c and mir320d in exosomes derivedfrom control and srsf1 knockdown panc1 cells p students ttest shown are representatives of three independent experiments aeenrichment supplement fig and our observationswere extended to two additional pancreatic cancer celllines miapaca2 and bxpc3 fig 4af in additionexpression of let7c which is less enriched in exosomeswas unchanged in exosomes after srsf1 knockdowndata not shownto verify the involvement of srsf1 in exosomemirna enrichment in cancer cells we also exogenouslyoverexpressed srsf1 in panc1 cells a gfpsrsf1 expression plasmid was introduced into panc1 cells andsrsf1 overexpression was confirmed by western blotfig 5a expression of mir1246 mir320c and mir320d in the exosomes derived from gfpsrsf1 panc1cells was analyzed by qrtpcr fig 5bc as shown infig 5b overexpression of gfpsrsf1 increased exosome expression of mir1246 and rescued mir1246levels in exosomes derived from srsf1shrna cellslevels of mir320c and mir320d were also increased inexosomes derived from the gfpsrsf1 cellsfurthersupporting the involvement of srsf1 in exosomemirna enrichment fig 5cdidentification of rna sequence motifs involved inexosome mirna enrichmentaccording to the rbpdb srsf1 binds specifically to amotif present in the mir1246 sequence supplementfig qrtpcr analysis of mir1246 mir320c mir320d expression in exosomes derived from srsf1 knockdown bxpc3 and miapaca2 cells ac qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown bxpc3 cells n p student ttest df qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown miapaca2 cells n p students ttest 0cxu cell communication and signaling page of fig qrtpcr analysis of exosome enriched mirnas derived from srsf1 overexpression panc1 cells a confirmation of gfpsrsf1overexpression in panc1 cells b qrtpcr detection of mir1246 in exosomes derived from wild type and srsf1 knockdown panc1 cells withgfpsrsf1 overexpression c qrtpcr detection of mir320c in exosomes derived from gfpsrsf1 overexpression panc1 cells d qrtpcrdetection of mir320d in exosomes derived from gfpsrsf1 overexpression panc1 cells p students ttest n for bdfig to understand the contribution of specific rnamotifs involved in exosome mirna enrichment we applied an unbiased approach to identify the rna motifsthat contribute to exosome mirna enrichment for thispurpose we analyzed the rna sequences of the mirnas highly enriched in cancer exosomes and regulatedby srsf1 using the bioinformatics tool meme suite a 6bp length motif was found to be shared in of the exosome enriched mirnasincluding mir fig ac to test whether the binding of srsf1to mir1246 depends on this motif two decoy mimicswere designed according to the shared motif sequencesand their secondary structure determined with thernafold webserver httprnatbiunivieacatcgibinrnawebsuiternafoldcgi the binding of the decoymimics to srsf1 protein was determined by rnaemsa analysis addition of decoy motif did not alterthe binding of srsf1 to the mir1246 probe fig 6dwhereas decoy motif competed with mir1246 binding to srsf1 in a concentrationdependent manner fig6df indicating that srsf1 directly interacts with thissequence motifdiscussionthe role of exosome mirna signaling in promotingcancer progression has been intensely investigated andwell recognized in recent years [ ] the higher enrichment of certain mirnas in cancer exosomes [] indicates that exosome mirna encapsulation isan active cellular process that initiates exosome mirnasignaling in the tumor microenvironment however thespecificselectivecellular processresponsiblefor 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig srsf1associated exosome mirna sequence motif analysis a the motif commonly shared among srsf1associated exosome mirnas bvenn diagram showing the number of srsf1associated exosome mirnas that share the motif c list of mirnas sharing the common motif demsa analysis demonstrating the inhibition of gstsrsf1 binding to mir1246 by rna decoys d1 decoy ²uuggacuaggacuaggau3² d2decoy ²aggaaggaaggaagga3² e concentrationdependent inhibition of gstsrsf1 binding to mir1246 by d2 f semiquantification ofthe detected bands in fig 5e and the calculated dissociation constantexosome mirna enrichment has not been well established in eukaryotic cells the most significant findingfrom the present study is that we have identified srsf1as a mediator of exosome mirna enrichment in pancreatic cancer cells a specific mirna sequence motif wasalso identified that may be involved in the exosomemirna enrichment process these findings provide newinsight into how mirnas are enriched in cancer cellexosomesexosomemediated mirnasignalinginitiatetowe recently reported that exosome mir1246themost highly enriched mirna in pancreatic cancer cellderived exosomes is derived from rnu2 a smallnuclear rna important for mrna splicing alongthis line of our research we sought to determine howthis mirna is enriched in cancer exosomes using ourestablished model systems in the present study we haveprovided severallines of evidence demonstrating thatsrsf1 a vital splicing factor and established oncoprotein is significantly involved in exosome mirnaenrichment in pancreatic cancer cells the first line ofevidence indicating srsf1 involvementin exosomemirna enrichment was obtained from the biotinlabeled mir1246 pulldown experimentfollowed byproteomic analysis among the rbps identified severalwere selected based on their detection intensity relevance to extracellular vesicles and reported connectionsto human cancer including srsf1 eif3b andtia1 of note srsf1 was the only rbp among themthat was also predicted by the rbpdb to bind to a motifin the mir1246 sequence furthermore the direct binding of srsf1 to the mir1246 sequence was verified byrip and rna emsa analysis strongly indicating thephysical interaction of srsf1 and not eif3b or tia1with the mir1246 sequence the most convincing evidence demonstrating the involvement of srsf1 in cancer exosome mirna enrichment was the observationthat knockdown of srsf1 significantly reduces exosomemirna enrichmentfor a majority of the selectivelyenriched exosome mirnas without altering the expression levels of less enriched exosome mirnas these results were based on small rna sequencing andconfirmed by rtpcr analysis the observations werealso extended to additional human pancreatic cancer celllines including miapaca2 and bxpc3srsf1 was initially identified as a splicing factor ineukaryotic cells but srsf1 was later revealed toindependent ofshuttle between the nucleus and cytoplasm to regulate rna metabolism mirna procession and othercellular eventsthe mrna splicingprocess importantly srsf1 is overexpressed indifferent cancer types and is considered a potent oncogene [ ] moreover srsf1 over expression in different types of cancer is associated with worse prognosissupplement fig while the full spectrum of srsf1function remains to be determined our results revealthat srsf1 binds to specific mirnas and is significantlyinvolved in exosome mirna enrichment in cancer cellsthis function is likely independent ofthe splicingprocess as the reduced expression of the detected exosome mirnas after srsf1 knockdown is greater thantheir expression change in the cells because exosomemirna signaling contributes to tumor developmentthrough intercellular communication in the tumormicroenvironmentthe involvement ofsrsf1 in exosome mirna signaling initiation likelyrepresents a part of its oncogenic action which maylead to new therapeutic strategies to intervene withexosome mirna signaling in cancer several rbpshave been previously identified as mediators of exosome mirna sorting in various model systemsincluding major vault protein in colon cancer cells hnrnpa2b1 in t cells and ybx1 in hek293tcells the identification of srsf1 involvement inexosome mirna enrichmentin pancreatic cancercells further supports the notion that the cellular exosome mirna sorting process in eukaryotic cells maydiffer among different cell types[ ]we have also identified a mirna motif commonlyshared by the srsf1associated exosome mirnasusing the meme suite program memesuitethis motif was specifically bound by srsf1 as evidenced by our rna emsa analysis a similar motifalbeit slightly shorter was identified in our recentreport that describes exosome mir1246 enrichmentin pancreatic cancer cells our results reinforcethe concept that specific mirn | 0 |
acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratiï¬cation in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple ï¬elds such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical signiï¬cance theseï¬ndings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression proï¬lesof aml specimens and the corresponding clinical followupdata were downloadedidentiï¬cation of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identiï¬ed between high and low score groups stratiï¬ed bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsigniï¬cance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identiï¬cation of gene coexpressionnetworksthrough highthroughput expression proï¬les ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbrieï¬y the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirnamrna and mirnalncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a proteinprotein interaction ppi networkof the degs identiï¬ed in the cerna network the interactingpairs with a conï¬dence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplanmeier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical signiï¬cance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskalwallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression proï¬les and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range years oldaccording to the fab classiï¬cation there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score was range from to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentiï¬cation of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identiï¬ed mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplanmeier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identiï¬ed and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identiï¬cation ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta ï¬nal lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aproteinprotein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been conï¬rmed tobe associated with immune microenvironment and leukemiaprogression association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and proteinprotein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi proteinprotein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayï¬nd new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplanmeier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors then we identiï¬ed diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identiï¬ed lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and speciï¬city ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of proammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic signiï¬cance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsigniï¬cance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identiï¬ed lncrnas with clinical signiï¬cance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identiï¬ed further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironmenthome of the leukemic blasts blood rev doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and 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American Joint Committee on Cancer AJCC Cancer Staging Manual 8th edition we explored the characteristics of central lymph node metastasis CLNM of papillary thyroid microcarcinoma PTMC in elderly patients ¥ years of age Our goal was to provide references for establishing a lymph node dissection scheme in such patientsMethods We retrospectively analyzed the clinical data of thyroid cancer patients admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Then we screened and analyzed eligible PTMC cases in strict accordance with our inclusion and exclusion criteriaResults The study included patients including men and women Median age was ± years The maximum diameter range of the cancer foci was mm and the median was ± mm Unilateral lobectomy had been performed in cases total thyroidectomy in cases and lateral cervical lymph node dissection in cases There were cases of CLNM and cases of lateral cervical lymph node metastasis The sensitivity of preoperative ultrasound in predicting CLNM was but its accuracy was only Multivariate logistic regression analysis showed that multiple cancer foci area under the curve [AUC] extrathyroidal expansion of cancer focus AUC and irregular nodules AUC were independent risk factors for CLNM of PTMC in elderly patients P Overall predictability for PTMCCLNM was Conclusion Preoperative color Doppler ultrasound is not recommended as the basis for cervical lymph node dissection in PTMC patients For multiple cancer foci irregular nodules and elderly patients with PTMC extrathyroidal expansion we recommend a prophylactic central lymph node dissecting Nonsurgical observation of PTMC in elderly patients with low risk should be carefully selectedKeywords elderly patients thyroid cancer papillary carcinoma microcarcinoma central lymph node metastasisIntroductionIn the World Health anization defined thyroid cancer TC with a maximum tumor diameter of mm as microcarcinoma Up to now doctors in different countries and regions of the world still differ significantly on how to treat such diseases including on whether to perform preventive lymph node dissection in the central region of the cancer As the AJCC raised the age factor in the clinical staging of TC from to in it can be seen that the medical community Cancer Management and Research Fu 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 0cFu Dovepresstends to be more conservative in general on surgical treatment of TC Based on clinical staging of TC in the AJCC Cancer Staging Manual 8th edition this study aimed to explore the characteristics of CLNM of PTMC in the elderly population ¥ years so as to provide some references for developing clinical treatment plans for such patients mm underwent concurrent total thyroidectomy and bilateral central lymph node dissection Centralregion lymph nodes were dissected in the following areas upper boundarylower hyoid margin lower boundarysuperior sternal fossa upper margin of the unknown artery and externallateral margin of the carotid sheath and lower boundaryanterior vertebral fasciaMethodsPatientsWe retrospectively analyzed the data of patients with PTMC admitted to the Head and Neck Surgery Center of Sichuan Cancer Hospital Chengdu China from January to September Eligible patients were screened in strict accordance with our inclusion and exclusion criteria for relevant data statistics and analysis Inclusion criteria were as follows Patient age was ¥ years All of the patients had been newly diagnosed and newly treated with no previous history of thyroid surgery Postoperative pathological diagnosis based on paraffinembedded sections was papillary carcinoma Benign nodules with or without pathology and mm in maximum diameter suggested by preoperative color ultrasound Patients complete medical records were available All of the surgeons had years experience in thyroid surgery Patients had undergone surgical resection of glandular lobes and isthmus on the affected side as well as lymph node dissection in the central region with or without lateral cervical lymph node dissection Exclusion criteria were as follows Postoperative pathology indicated mixed carcinoma with papillary and other types of carcinoma Patient had refused lymph node dissection in the central area on the affected side There were multiple cancer lesions with the sum of the maximum diameter mmSurgical TechniqueAll of the patients had been operated on according to the at least principle namely lymph node dissection at least in the central area on the cancerous side Total thyroidectomy bilateral centralarea lymph node dissection with or without lateral cervical lymph node dissection were performed on patients with preoperative cytological confirmation of bilateral multilobed carcinoma or lateral cervical lymph node metastasis Those with papillary microcarcinoma in one glandular lobe and benign nodules in the other glandular lobe ie multiple nodules with maximum diameter of Statistical AnalysisWe used SPSS software version SSPS Inc Chicago Illinois US to statistically analyze all of the data In our analysis of risk factors for lymph node metastasis in the central region we performed singlefactor analysis using Ï2 test We ran multivariate logisticregression analysis on statistically significant positive univariate influencing factors as well as univariate and multivariate receiver operating curve ROC analysis on the previously analyzed risk factors to predict lymph node metastasis in the central regionResultsWe screened a total of PTMC cases Of these met the inclusion criteria including men and women with a maletofemale ratio of Patients age range was years old with a median of ± years The maximum diameter range of the cancer lesion was mm median ± mm There were cases with singleleaf singlefocus with singleleaf multifocus and with multileaf multifocus Unilateral lobectomy was performed in cases and total thyroidectomy in cases and lateral cervical lymph node dissection in cases In terms of staging of cases were in stage T1 in stage T3 and in stage T4 Postoperative pathology showed CLNM in cases and lateral cervical lymph node metastasis in cases Evaluation of Central Lymph Nodes by Color Doppler UltrasoundPreoperative ultrasound examination indicated stage cN1a cases and stage cN0 cases Postoperative pathology confirmed stage pN1a cases and stage pN0 cases which were significantly different Predictive sensitivity specificity positive predictive value PPV and negative predictive value NPV were and respectively Table submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Comparison of Preoperative Ultrasound Predictions of CentralRegion Lymph Node MetastasisNTotalUltrasonic StagingcN1acN0Pathological stagepN1apN0TotalSensitivity Specificity PPV NPV Matching rate Abbreviations PPV positive predictive value NPV negative predictive valueUnivariate AnalysisIn this study singlefactor analysis of patients with PTMC age ¥ years showed that distribution nodule morphology calcification and extrathyroidal expansion of cancer focus significantly influenced centralregion lymph node metastasis P However patients gender thyroid stimulating hormone TSH levels thyroglobulin Tg levels nodular goiter Hashimotos thyroiditis maximum diameter of cancer focus nodular boundary and nodular blood flow had no statistical significance on such metastasis Table Multivariate LogisticRegression AnalysisFactors that had been statistically significant in univariate analysis results were further included in multivariate logisticregression analysis variables that might be clinically relevant but had been negative in univariate analysis were also included We found that distribution morphology and extrathyroidal expansion of cancer focus were independent risk factors for CLNM P while gender TSH Tg nodular goiters Hashimotos thyroiditis nodular boundary blood flow calcification and maximum diameter had no predictive significance Table ROC Curve AnalysisWe performed ROC curve analysis according to the independent risk factors obtained in our multivariate logistic regression analysis of CLNM as discussed above and we calculated areas under the curve AUCs Figures and DiscussionDisease Status of TCThe incidence of TC has been on the rise globally over the past years which has been confirmed by most current to International Agency studies1 According for Research on Cancer IARC data for a total of new cases malefemale and deaths malefemale were reported in countries around the world respectively accounting for and of all new cancer cases and deaths4 On the one hand due to the great influence of medical ultrasound and cytologicalpuncture diagnosis the proportion of PTMC in new cases of TC has increased significantly According to the data the overall incidence of PTMC in the United States has increased by over the past years with average annual new cases accounting for about On the other hand PTMC incidence in the elderly is significantly higher than that in the general adult population Some studies have shown that the average annual growth rate of PTMC in patients years old is times that in adults years old8 These results indicate that we should pay sufficient attention to elderly PTMC patients As the 8th edition of the AJCC Cancer Staging Manual raise PTC staging age from to years old it further confirms this viewTherapeutic ControversiesAs we all know diagnosis and treatment of PTMC have always been controversial especially in elderly patients In Japans Kuma hospital Ito defined the maximum diameter of thyroid cancer foci ¤10cm no cervical and distant lymph node metastasis and cytological biopsy of thyroid cancer foci as nonhighly malignant and no invasion of the trachea and recurrent laryngeal nerve as the judgment criteria for lowrisk PTMC After analyzing the data of patients they concluded that immediate surgical treatment of all PTMC patients was more harmful than beneficial so they suggested that lowrisk PTMC patients should choose active observation Among them elderly PTMC patients over years old were considered to be the most suitable group for observation1112 Conversely Megwalu UC of the National Cancer Center Plainview New York US reviewed cases in which senile PTMC patients age ¥ received nonoperative therapy His data analysis shows that the overall 5year survival rate was and the surgery was P which suggests that surgery for such patients has a survival advantage although more highquality investigative studies are necessary1 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer suggested that suspicious malignant thyroid nodules with a maximum diameter of cm be followed up to cm for cytological Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressTable SingleFactor Analysis of CentralRegion Lymph Node MetastasisFactorsGenderMaleFemaleTSH levelsNormalAbnormalTg levelsNormalAbnormalNodular goiterYesNoHashimotos thyroiditisYesNoDistribution of carcinomaUnilateral glandular lobes single fociUnilateral glandular lobes multiple fociBilateral glandular lobes multiple fociMaximum diameter¤ mm mm x ¤ mmBoundaryClearUnclearEchoLow or noStrong or mixedExtrathyroidal expansionYesNoCalcificationYesNoBlood flowRichNot richNumber n107Central Lymph Node Metastasis CLNMÏ2PYes n No n Abbreviations TSH thyroidstimulating hormone Tg thyroglobulinpuncture and other treatments but immediate surgical treatment is still recommended for highrisk patients In this study we performed surgical treatment on all PTMC patients with clear diagnoses including stage T1 T3 and T4 Although the study sample size needs to be further expanded we still believe that microcarcinoma is not equal to early cancer The percentage of differentiated tumor cells in elderly PTC patients is relatively higher than in youth and children leading to shorter life expectancy Choosing followup for middleaged and elderly submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu et alTable Multivariate LogisticRegression Analysis of Lymph Node Metastasis in the Central RegionFactorsGenderDistribution of carcinomaMaximum diameterTumor formation patternBoundaryExtrathyroidal expansionCalcificationBlood flowβSEWaldPOR CI ORAbbreviations SE Standard error OR odds ratio CI confidence intervalPTMC patients may be feasible but for those with longer life expectancy early surgery can significantly reduce future progress of tumors may not only but also reduced the forward of surgery as a result of basic diseases such as cardiovascular increase intolerance Therefore for PTMC patients age ¥ with good survival expectations we believe surgical intervention is still necessary which is also consistent with Shindo et al13Risk FactorsIn the past there have been many studies analyzing PTMCCLNM but few such reports address elderly patients with PTMC Due to air interference in the tracheal cavity it is relatively difficult to diagnose CLNM of the neck using ultrasound which has a high falsenegative rate In this study the accuracy of ultrasound in predicting CLNM was Therefore it is questionable whether dissection of such lymph nodes can be performed only by preoperative ultrasound Chung et al14 found that in young PTMC patients multiple cancer foci enlarged nodules extrathyroidal expansion of cancer focus and vascular invasion are independent risk factors for PTMCCLNM and lateral cervical lymph node metastasis but they did not clearly identify calcified nodules as an independent risk factor By analyzing the data of PTMC patients Oh et al15 showed that the rate of lymph node metastasis in patients with calcified nodules was higher than in patients whose nodules were not calcified they concluded that calcified nodules were an important risk factor for PTMC cervical lymph node metastasis Haugen et al16 have a similar view In this study the metastasis rates of the central cervical region and lateral cervical lymph nodes were and respectively The Ï2 test showed that distribution nodular morphology calcification and extrathyroidal expansion of cancer focus were risk factors for centralregion lymph nodes P Figure Areas under the curve AUC Distribution of carcinoma AUC extrathyroidal expansion AUC tumor formation pattern AUC Cancer Management and Research submit your manuscript wwwdovepresscom DovePress 0cFu DovepressAcknowledgmentsAll authors made substantial contributions to conception and design acquisition of data or analysis and interpretation of data took part in drafting the article or revising it critically for important intellectual content gave final approval of the version to be published and agree to be accountable for all aspects of the work We thank LetPub for its linguistic assistance during the preparation of this manuscriptDisclosureThe authors report no conflicts of interest for this workFigure Multiple independent risk factors predicted lymph node metastasis in the central region Areas under the curve AUC which was consistent with Liu et al17 However our multivariate logisticregression analysis found that only distribution of cancer lesions Ï2 P nodule morphology Ï2 P and extra thyroidal expansion of cancer focus Ï2 P were independent risk factors for such metastasis The AUCs of these factors were and respectively and overall predictability was In summary we believe that active followup and observation should be carefully selected for elderly patients with PTMC especially for those with multiple cancer foci extrathyroidal expansion of cancer focus and irregular morphology preventive centralarea lymph node dissection is also appropriate Although we did not find nodular calcification maximum tumor diameter Hashimotos thyroiditis or other variables to be independent risk factors we believe this result may have a certain relationship with the small sample size which we will further expand in the future for related studies and supplementsEthical ApprovalThis study was approved by the Institutional Review Board of Sichuan Cancer Hospital and Institutional Ethics Committee and performed according to the ICH GCP principleInformed ConsentWe obtained written informed consent from all of the individual participants included in the studyReferences Megwalu UC Observation versus thyroidectomy for papillary thyroid microcarcinoma in the elderly J Laryngol Otol doi101017S0022215116009762 Davies L Welch HG Current thyroid cancer trends in the United States JAMA Otolaryngol Head Neck Surg doi101001jamaoto20141 Kilfoy BA Zheng T Holford TR et al International patterns and trends in thyroid cancer incidence Cancer Causes Control doi101007s1055200892604 Bray F Ferlay J Soerjomataram I et al Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin doi103322caac21492 Hughes DT Haymart MR Miller BS et al The most commonly occurring papillary thyroid cancer in the United States is now a microcarcinoma in a patient older than years Thyroid doi101089thy20100137 Davies L Welch HG Increasing incidence of thyroid cancer in the JAMA United States doi101001jama295182164 Kuo EJ Goffredo P Sosa JA Aggressive variants of papillary thyroid microcarcinoma are associated with extrathyroidal spread and lymphnode metastases a populationlevel analysis Thyroid doi101089thy20120563 Hay ID Hutchinson ME GonzalezLosada T Papillary thyroid microcarcinoma a study of cases observed in a 60year period Surgery discussion doi101016j surg200808035 Simard EP Ward EM Siegel R et al Cancers with increasing incidence trends in the United States through CA Cancer J Clin doi103322caac20141 Cramer JD Fu P Harth KC Analysis of the rising incidence of thyroid cancer using the surveillance epidemiology and end results national cancer data registry Surgery doi101016jsurg201010016 Ito Y Miyauchi A Kudo T et al Trends in the implementation of active surveillance for lowrisk papillary thyroid microcarcinomas at Kuma Hospital gradual increase and heterogeneity in the acceptance of this new management option Thyroid doi101089thy20170448 Ito Y Miyauchi A Kihara M Patient age is significantly related to the progression of papillary microcarcinoma of the thyroid under observation Thyroid doi101089thy20130367 Shindo M Wu JC Park EE The importance of central compartment elective lymph node excision in the staging and treatment of thyroid cancer Arch Otolaryngol Head Neck Surg papillary doi101001archotol1326650submit your manuscript wwwdovepresscom DovePress Cancer Management and Research 0cDovepress Fu Liu LS Liang J Li JH et al The incidence and risk factors for central lymph node metastasis in cN0 papillary thyroid microcarcinoma a metaanalysis Eur Arch Otorhinolaryngol doi101007s0040501643020 Chung YS Kim JY Bae JS Lateral lymph node metastasis in papillary thyroid carcinoma results of therapeutic lymph node dissection Thyroid doi101089thy20080244 Oh EM Chung YS Song WJ The pattern and significance of the calcifications of papillary thyroid microcarcinoma presented in preoperative neck ultrasonography Ann Surg Treat Res doi104174astr2014863115 Haugen BR Alexander EK Bible KC American Thyroid Association Management Guidelines for adult patients with thyroid nodules and differentiated thyroid cancer the American Thyroid Association Guidelines task force on thyroid nodules and differenthyroid cancer Thyroid doi101089 tiated thy20150020Cancer Management and Research Publish your work in this journal Cancer Management and Research is an international peerreviewed access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient The manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use Visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors Dovepress Submit your manuscript here wwwdovepresscomcancermanagementandresearchjournalCancer Management and Research submit your manuscript wwwdovepresscom DovePress 0c' | 2 |
"Ectopic expression of BANCR was achieved through pCDNA-BANCR transfection with an empty pCDNA3.1 vector used as a control. The expression levels of BANCR were detected by qPCR. Cell transfection Plasmid vectors (pCDNA3.1-BANCR and pCDNA3.1) for transfection were prepared using DNA Midiprep or Midiprep kits (Qiagen Hilden Germany) and transfected into SPC-A1 or A549 cells. The siRNAs si-HDAC1 si-HDAC3 si-BANCR or si-NC were transfected into SPC-A1 or A549 cells (Additional file 3: Table S2). A549 and SPC-A1 cells were grown on six-well plates to confluency and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturers instructions. At 48 h post-transfection cells were harvested for qPCR or western blot analysis. Cell viability assays Cell viability was monitored using a Cell Proliferation Reagent Kit I (MTT) (Roche Applied Science). The A549 cells transfected with si-BANCR (3000 cells/well) and A549 or SPC-A1 cells transfected with pCDNA-BANCR were grown in 96-well plates. Cell viability was assessed every 24 h following the manufacturers protocol. All experiments were performed in quadruplicate. For colony formation assays pCDNA-BANCR-transfected SPC-A1 or A549 cells (n?=?500) were placed in a 6-well plates and maintained in media containing 10% FBS. The medium was replaced every 4 days; after 14 days cells were fixed with methanol and stained with 0.1% crystal violet (Sigma-Aldrich). Visible colonies were then counted. For each treatment group wells were assessed in triplicate. Flow cytometry analysis of apoptosis SPC-A1 and A549 cells were harvested at 48 h post-transfection by trypsinization. After staining with FITC-Annexin V and propidium iodide cells were analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (BD Biosciences). Cells were discriminated into viable cells dead cells early apoptotic cells and apoptotic cells. The ratio of early apoptotic cells was compared to that for controls from each experiment. All samples were assayed in triplicate. Wound-healing assay For the wound-healing assay 3??105 cells were seeded in 6-well plates cultured overnight and transfected with pCDNA-BANCR or the control vector. Once cultures reached 85% confluency the cell layer was scratched with a sterile plastic tip and washed with culture medium then cultured for 48 h with medium containing 1% FBS. At different time points images of the plates were acquired using a microscope. The distance between the two edges of the scratch was measured using Digimizer software system. Cell migration and invasion assays For the migration assays at 48 h post-transfection 5??104 cells in serum-free media were placed into the upper chamber of an insert (8-?m pore size; Millipore). For the invasion assays 1??105 cells in serum-free medium were placed into the upper chamber of an insert coated with Matrigel (Sigma-Aldrich). Medium containing 10% FBS was added to the lower chamber. After incubation for 24 h the cells remaining on the upper membrane were removed with cotton wool. Cells that had migrated or invaded through the membrane were stained with methanol and 0.1% crystal violet imaged and counted using an IX71 inverted microscope (Olympus Tokyo Japan). Experiments were independently repeated three times. Tail vein injections into athymic mice Athymic male mice (4-weeks-old) were purchased from the Animal Center of the Chinese Academy of Science (Shanghai China) and maintained in laminar flow cabinets under specific pathogen-free conditions. SPC-A1 cells transfected with pCDNA-BANCR or the empty vector were harvested from 6-well plates washed with phosphate-buffered saline (PBS) and resuspended at 2??107 cells/ml. Suspended cells (0.1 ml) were injected into the tail veins of 9 mice which were sacrificed 7 weeks after injection. The lungs were removed and photographed and visible tumors on the lung surface were counted. This study was carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Our protocol was approved by the Committee on the Ethics of Animal Experiments of Nanjing Medical University (Permit Number: 200933). All surgery was performed under sodium pentobarbital anesthesia and all efforts were made to minimize suffering [37]. Western blotting analysis Cells were lysed using RIPA protein extraction reagent (Beyotime Beijing China) supplemented with a protease inhibitor cocktail (Roche CA USA) and phenylmethylsulfonyl fluoride (Roche). The concentration of proteins was determined using the Bio-Rad protein assay kit. Protein extracts (50 ?g) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) then transferred to nitrocellulose membranes (Sigma) and incubated with specific antibodies. ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad) with GAPDH used as a control. Antibodies (1:1000 dilution) against E-cadherin and N-cadherin were purchased from BD. Antibodies against vimentin MMP-2 and MMP-9 were purchased from Cell Signaling Technology (MA USA). Fluorescence immunohistochemistry Cells were fixed in 4% paraformaldehyde following a standard protocol. Mouse anti-E-cadherin and -N-cadhherin polyclonal antibodies (1:100; BD) were used as primary antibodies with TRITC-labeled anti-Rabbit IgG (1:200; Sigma) used as a secondary antibody. Sections were mounted onto slides using Gel Mount Aqueous Mounting Medium (G0918 Sigma) and examined with an Olympus BX51 microscope (Olympus Optical Tokyo Japan). Statistical analysis Students t-test (2-tailed) one-way ANOVA and the MannWhitney U test were used to analyze data along with SPSS 16.0 (IBM IL USA). P-values of less than 0.05 were considered statistically significant. Competing interests The authors have no actual or potential conflicts of interest to declare. Authors contributions Conception and design: MS XHL WD. Development of the methodology: XHL EBZ FQN RK. Acquisition of data: KMW FYJ TPX. Analysis and interpretation of data: ZJL RX JFC. Writing revision of the manuscript: MS WD ZXW. Administrative technical and material support: XHL MS. Study supervision: WD ZXW. All authors read and approved the final manuscript. Supplementary Material Additional file 1: Table S1 Clinicopathological characteristics and BANCR expression of 113 patient samples of NSCLC. Click here for file Additional file 2: Figure S1 Effects of EZH2 SUZ12 and HDAC3 on BANCR expression. (A) Analysis of BANCR expression levels by qPCR following treatment of SPC-A1 and A549 cells with si-EZH2 and SUZ12.(B) Analysis of HDAC3 mRNA expression levels by qPCR in NSCLC cell lines. HDAC3 expression was upregulated more significantly in SPC-A1 H1650 and H1975 cells compared with 16HBE cells. (C) A549 and SPC-A1 cells were treated with selective HDAC3 inhibitor RGFP966 (dissolved in DMSO) at concentrations of 15?M for 24 hours. Analysis of BANCR expression levels by qPCR following treatment of SPC-A1 and A549 cells with RGFP966. *P?<?0.05 N.S. no significant. Click here for file Additional file 3: Table S2 Sequence of primers and siRNA. Click here for file Acknowledgements Xiang-Hua Liu was supported by the National Natural Scientific Foundation of China (No. 81301824). Zhao-Xia Wang was supported by the National Natural Scientific Foundation of China (No.81272601) the Medical Key Talented Person Foundation of the Jiangsu Provincial Developing Health Project (No.RC2011080) the Innovation Team Project of the Second Affiliated Hospital Nanjing Medical University the Jiangsu Provincial Personnel Department the Great of Six Talented Man Peak Project (No.09-B1-021) and 333 high class Talented Man Project (No. 2011-III-2630). Ming Sun was supported by Jiangsu province ordinary university graduate student research innovation project for 2013 (CXZZ13_0562). Jin-fei Chen was supported by the National 973 Basic Research Program of China (Grant No. 2013CB911300). Jin-song Yang was supported by the Medical Science Development Subject in Science and Technology Project of Nanjing (Grant No. ZKX13017). 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Meeting on the epithelial-mesenchymal transition EMBO Rep 2008 9 322 326 10.1038/embor.2008.30 18323854 Bremnes RM Veve R Gabrielson E Hirsch FR Baron A Bemis L Gemmill RM Drabkin HA Franklin WA High-throughput tissue microarray analysis used to evaluate biology and prognostic significance of the E-cadherin pathway in non-small-cell lung cancer J Clin Oncol 2002 20 2417 2428 10.1200/JCO.2002.08.159 12011119 Liu D Huang C Kameyama K Hayashi E Yamauchi A Kobayashi S Yokomise H E-cadherin expression associated with differentiation and prognosis in patients with non-small cell lung cancer Ann Thorac Surg 2001 71 949 954 discussion 954-945 10.1016/S0003-4975(00)02545-5 11269479 Cesana M Cacchiarelli D Legnini I Santini T Sthandier O Chinappi M Tramontano A Bozzoni I A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA Cell 2011 147 358 369 10.1016/j.cell.2011.09.028 22000014 Kilkenny C Browne W Cuthill IC Emerson M Altman DG Animal research: reporting in vivo experiments: the ARRIVE guidelines J Gene Med 2010 12 561 563 10.1002/jgm.1473 20607692 PLoS One PLoS ONE plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco USA 24586426 3930593 PONE-D-13-47163 10.1371/journal.pone.0088881 Research Article Mathematics Statistics Biostatistics Medicine Non-clinical medicine Health care policy Health statistics Health systems strengthening Health economics Cost effectiveness Socioeconomic aspects of health Public health Socioeconomic aspects of health Social and behavioral sciences Economics Health economics Health care sector Economic Outcomes of Maintenance Gefitinib for Locally Advanced/Metastatic Non-Small-Cell Lung Cancer with Unknown EGFR Mutations: A Semi-Markov Model Analysis Economic Outcomes of Maintenance Gefitinib Zeng Xiaohui 1 Li Jianhe 2 3 Peng Liubao 2 * Wang Yunhua 1 Tan Chongqing 2 3 Chen Gannong 4 Wan Xiaomin 2 3 Lu Qiong 2 Yi Lidan 2 1 PET-CT Center the Second Xiangya Hospital of Central South University Changsha Hunan Peoples Republic of China 2 Department of Pharmacy the Second Xiangya Hospital of Central South University Changsha Hunan Peoples Republic of China 3 School of Pharmaceutical Sciences Central South University Changsha Hunan Peoples Republic of China 4 Department of Surgery the Second Xiangya Hospital of Central South University Changsha Hunan Peoples Republic of China Hoheisel J¶rg D. Editor Deutsches Krebsforschungszentrum Germany * E-mail: [email protected] Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: JL LP. Performed the experiments: GC QL LY. Analyzed the data: XZ CT. Contributed reagents/materials/analysis tools: YW XW. Wrote the paper: XZ CT. 2014 20 2 2014 9 2 e88881 9 11 2013 12 1 2014 2014 Zeng et al This is an open-access article distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Background Maintenance gefitinib significantly prolonged progression-free survival (PFS) compared with placebo in patients from eastern Asian with locally advanced/metastatic non-small-cell lung cancer (NSCLC) after four chemotherapeutic cycles (21 days per cycle) of first-line platinum-based combination chemotherapy without disease progression. The objective of the current study was to evaluate the cost-effectiveness of maintenance gefitinib therapy after four chemotherapeutic cycles stand first-line platinum-based chemotherapy for patients with locally advanced or metastatic NSCLC with unknown EGFR mutations from a Chinese health care system perspective. Methods and Findings A semi-Markov model was designed to evaluate cost-effectiveness of the maintenance gefitinib treatment. Two-parametric Weibull and Log-logistic distribution were fitted to PFS and overall survival curves independently. One-way and probabilistic sensitivity analyses were conducted to assess the stability of the model designed. The model base-case analysis suggested that maintenance gefitinib would increase benefits in a 13 6 or 10-year time horizon with incremental $184829 $19214 $19328 and $21308 per quality-adjusted life-year (QALY) gained respectively. The most sensitive influential variable in the cost-effectiveness analysis was utility of PFS plus rash followed by utility of PFS plus diarrhoea utility of progressed disease price of gefitinib cost of follow-up treatment in progressed survival state and utility of PFS on oral therapy. " | 1 |
" child maltreatment leads to enormous adverse short and longterm health outcomes the aim ofthis study is to estimate the burden of disease and the cost of illness attributable to child maltreatment in japanmethods an incidencebased topdown cost of illness analysis was conducted to estimate medical costs andburden of disease attributable to child maltreatment based on a societal perspective the assessment includedshortterm and longterm medical costs and burden of disease measured by disabilityadjusted life years dalysthat generates mortality and morbidities based on several national surveys and systematic review we consideredthe main types of child maltreatment as exposure for which the incidence was obtained from literature reviewbased on population attributable fractions pafs burden of disease of physical and mental health consequencesattributable to child maltreatment were estimated then dalys were converted into monetary value the lifetimeeconomic burden was finally estimated by combining with medical costs and subject to sensitivity analysisresults the lifetime disease burden expressed in dalys was estimated at dalys ci dalys for the cohort victims in based on the incidence according to literature review the overall lifetimeeconomic burden was billion usd equivalent to million times of gross domestic product gdp per capitaamong the total economic burden costs of suffering and pain based on dalys were accounting for theseestimates were times of conservative estimates which used incidence data from official reported casess this study found that the national lifetime cost was huge and equivalent to million gdp percapita and its burden of disease was approximately equal to that of colon and rectum cancers or stomach cancerour findings particularly in terms of revealed the considerable burden of disease in long term and potential effectsof the strengthened maternal and child care as the preventive strategykeywords child maltreatment burden of disease study lifelong health consequences disabilityadjusted life yeardaly costofillness correspondence gairuoyanipssgojp1department of health policy national center for child health anddevelopment tokyo japan3department of empirical social security research national institute ofpopulation and social security research uchisaiwaicho chiyodakutokyo 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 0cmo bmc public health page of child maltreatment is a raising concern in public healthand social welfare in japan the reported number ofsuspected cases of child maltreatment is increasing from in to in according to theministry of health labour and welfare mhlw ofjapan child maltreatment is categorised into four essential types physical sexual or psychological includingwitnessing domestic violence wdv abuse and neglect exposure to multiple types and repeated episodes ofmaltreatment during childhood is associated with highrisks to enormous adverse health outcomes causing asignificant social and economic burden on individualsfamilies and societies those adverse outcomes duringchildhood include child death injuries and disabilitiesdevelopmental and behavioural problems moreover therelated physical and mental health conditions persistinto adulthood leading to the onset of chronic diseasesdepression drug alcohol misuse and risk sexual behaviour suicide ideation [ ]the number ofthe related analysis ofthe government has introduced a couple of protectivemeasures with increasing public budget [] assessment of costs and burden of disease helps developmentof resource allocation and priority setting in public sector paralleling with growing concerns on child maltreatmenttheprevalence health consequences and economic burdenis increasing so far for the economic burden there aretwo typical research frameworks one is a comprehensively costs evaluation from healthcare social educa[]tional areas and loss in productivity another one is to measure related economic and diseaseburden [ ] wada reported the socialcosts of child abuse in japan included direct costs ofdealing with abuse and the indirect costs related to longterm damage from abuse during the fiscal year onthe other hand the first framework is likely to underestimate longterm deleterious effects of child maltreatmenton which evidence derived from longitudinal studies isless available compared to that on the shortterm counterpart by integrating previous evidence our costofillness study aimed to assess lifetime economic anddisease burden of mortality and morbidities attributed tochild maltreatment based on the later frameworkinorder to address the evidence gap we extended cost calculations for monetary values converted from disabilityadjusted life years dalys covering related mortalityand morbidities methodsan incidencebased victims estimated by incidencetopdown approach or attributable risk approach measuring the proportion of a disease that is due to exposureto risk factor was applied in this study from a societalperspective we employed the following steps to estimate the total economic burden constituted by directand indirect costs population attributable fraction paf wasgenerated to estimate longterm impactscosts attributed to child maltreatment shortterm and longterm direct medical costs wereassessed by using national expenditure databasesindirect costs measured include productivity losscaused by abusive head trauma and economicburden deriving from dalys finally sensitivity analyses were performed for theplausible range of the discount rate and theincidence prevalenceestimating pafin the topdown approach paf for each disease i measured that how health outcomes and their associatedcosts may be attributed to child abuse using the following formula [ ]pafi ¼ p rri °p rri ¾¾ ¾ °p prevalence of child abuse rr the relative risk of theoutcome i in those who experienced child abuse compared with those who did notrisk ratio rr or odds ratio orseveral previous related systematic reviews and metaanalyses summarised the relevant health consequences[ ] as adverse childhood experiences acesoften intertwine with child maltreatment cluster in childrens lives and cumulatively lead to poor health outcomes we pooled the ors from a recent systematicreview and metaanalysis for the effect of multiple aceson health rather than that for each category of childmaltreatmentin japan thethe pooled prevalencea literature review was performed to synthesize the evidence on epidemiological characteristicsthe consequencesreview focused on thosepublished between december and march onmedline pubmed web of science scopus and ciniis japanese literature details of the search strategy search terms used and inclusion and exclusion criteria are provided in the additional file we combinedour review results with those studies in japan includedin an existing systematic review and calculated thesimple sizeweighted mean incidenceprevalenceinthe median value was also calculated toaddition 0cmo bmc public health page of examine the robustness supplementary table theannual incidence rate was obtained by the formula incidence rate ¼prevalenceaverage durationdue to the lack of local data on the average duration we adopted that published in australia theaverage years for physical abuse and years forsexual abuse based on this findingthe weightedaverage of years was used for other categories ofabusedirect medical costsshortterm medical costsfor abusive head trauma aht is the leading causeof death due to child abuse among children youngerthan years old we estimated its hospitalizationcosts as shortterm medical costs by multiplying theincidence of aht under years old the agespecific population in and admission medical fee per case there were two reported incidences one is the possibleincidence considering countable possibility ofaht cases at most and another one is the presumptiveincidence representing victims had intracranial injuriesor intentionalinjuries with certain icd10 code weused the possible incidence for the general calculationand the latter one in sensitivity analysis the total possible aht cases aged under years was about times ofthe presumptive counterparts longterm medical costsfor longterm medical costs we used national healthcare expenditures and patient survey tosimulate disease burden ofrelevant health consequences by sex and age group above and then multiplied with pafs to calculatethe attributable costs in the victim cohort of [ ] on the other hand we did not include selfharm and collective violence because of the limitationto distinguish the two in the reported overallinjurycasesindirect costsin this study we considered differential and loss of earning as a result of human capital depreciation is causedby mortality and morbidities it was presented as a monetary value of dalys and gdp per capita [ ]dalys and its monetary valuethe disease burden indicator daly aggregates yearsof life lost for premature death and years lost due todisability for morbidities related data wereobtained from the who global burden of diseasegbd using the pooled ors as described bykaren we matched each relatedhealth outcome with the cause of disease burdenin the who gbd categories though it was difficultto match some outcomes with the cause of gbdsupplementary table then monetary value was converted from daly attributable to child maltreatment by multiplying dalyand gdp per capita with adjustment of purchasingpower parity in productivity losses due to aht fatal casesproductivity loss due to fatal cases of child maltreatment was calculated based on the reported fatal caseswhich figure was obtained from official data andthe average lifetime income subject to discounting in there were abuserelated deaths reported injapan not including family suicide with the averageonset age of years the discounted lifetime income from to years old was calculated by assuming the longterm growth in labour productivityto be per year dalys losses of survival ahtfor disease burden due to survival aht we considered sequelae such as vision loss brain damage andreduced life span and longterm health consequences as developing diseases in adulthood we calculated the disease burden of aht in bymultiplying average cases and the estimated meanlifetime daly loss per case at different severity mildmoderate and severe longterm dalys losses of other diseasesthen the longterm health consequences were calculatedusing the following formuladaly losses ¼ Ï pafi 03original dalyi°i different child abuse related health outcome°¾¾sensitivity analysesa discount rate of is generally performed which wasrecommended in the domestic guideline for costeffectiveness analysis whereas especially in the usa discount rate of has been selected and applied inthe cost estimate reports of centers for disease controland a best practices for the social return on investmentanalysis recommended by experts and guidelines assuch the parameter potentially affects the finally resultswe adopted a plausible range of to for sensitivityanalysis 0cmo bmc public health page of in addition we also calculated costs and diseaseburden using the incidenceprevalence data based onofficially reported child abuse cases to calculate theconservative incidence of child abuse by categorieswe obtained the official data of victim cases reportedby child consultation facilities in and thendivided them by the total population number in corresponding age data by sex were not availablecocurrentinformation was not available and theoverlapped cases were not considered supplementarytable the initial victim age is assumed to be years old according to an ageweighted incidence calculation based on official reported cases we assumed the probable abuserelated death cases to be times of the reported cases based on the ratio of thepresumptive and the possible incidence of aht casesamong children aged under years resultsthe main results showed in tables were discounted at and conservative estimates were given for sensitivityanalysesthe pooled incidence prevalence and disease burdenthe estimations on different types of child maltreatment incidence draw from literature reviews variedregarding differences between sex except physicalabuse girls suffered more than boys in sexual abuseand witnessing domestic violence table the estimated lifetime disease burden associated with childmaltreatment onset in was considerable dalys with a ci of dalys to dalys table the top causes of totaldisease burden due to child abuse were suicide attempts cardiovascular disease and depression cancercostofillness analysis for child maltreatmenttable demonstrates lifetime costs attributed to childabuse onset in the total direct cost was estimatedtable estimated incidenceprevalence of child abuse in japanestimates aincidence bmalefemalephysical abuse sexual abuse psychological abusewdv c other d prevalencemalefemaleneglect a sample sized weighted mean valueb incidence rate prevalence average durationc wdv witnessing domestic violenced not specified as wdv often expressed as emotionalpsychological abuseto be usd million 95ci million11 million while the total indirect cost was estimatedto be usd million 95ci million52 million accounting for of the total lifetimecosts which were almost million times gdp percapita economic loss initiated from dalys in longterm costs of suffering and pain accounted for ofthe overall estimatessensitivity analysesconservative estimates based on the reported cases incidence showed a tendency similar to that observed in thedisease burden based on the literature review amongwhich psychological abuse including wdv accountedfor the majority of reported child abuse cases however the incidence estimated from the review weremuch higher than those reported by child protectionagencies the conservative estimation leading to about times difference gap on child maltreat burden bydifference discount rate table discussionour results indicated that disease and economic burdenattributable to child maltreatment is substantial in particular that originated from the longterm health consequences accounts for the majoritybased on literature review the pooled incidence ofchild maltreatment in japan is much higher than officially reported which is consistent with the findingsof other studies [ ] because of difficulty toidentify the actual cases and a public attitude to consider child abuse as a private affair in the society theofficially reported cases are likely to represent the tipof an icebergthe fourpsychological abuse including wdv representedthe majority of reported cases the results of the literature review also showed a gender difference in theprevalence oftypes of child abuse sizeweighted mean values girls were found to be morelikely to experience the harmful practices comparedto boys particularly sexual abuse this tendency wasalso observed in other countries in east asia and pacific region comparing those living in othercountries in the east asia and pacific region [ ]japanese children tended to less likely to experiencephysical abuse boys vs girls vsalthough it is difficult to directly compare the results across different study settings due to the different methodologies parameters and target populationsadopted the ingredients of the lifetime economic anddisease burden considered in our studyincludingmedical costs and monetary value of disease burdenare similar to that adopted in previous studies [ 0cmo bmc public health page of table longterm daly lost attributable to child abuse in japandiseases attributed to child abuse asuicide attemptdalys confidence intervalcancercardiovascular diseasedepressionrespiratory diseaseliver or digestive diseaseanxietyproblematic drug useabusive head traumaproblematic alcohol usediabetessexually transmitted infectionsviolence victimisationviolence perpetrationtotaldalys monetary value billion usa simple size weighted mean prevalence at discounted rate] still our results showed that the disease burdenwas about times of the conservative estimationdue to the huge gap of incidence generated from literature and that officially reported the number isconsistent with an australian research that showed awide distribution ofthe annual prevalence rangingfrom to in the conservative lifetimecourse simulation the initial victim age is assumed tobe years old according to an ageweighted incidencecalculation based on official reported cases whichwas also consistent with previous studies our study in particular highlighted dalys in longterm attributable to child maltreatment accountingin the overallfor a relevant proportion lifetime costs the estimation of disease burden attributed to child maltreatment dalys wascomparable to the total dalys due to colon and rectum cancers dalys in or stomachcancer dalys in to our knowledge this is the first study to estimatelifetime economic burden of child maltreatmentinjapan based on an epidemiological model the idea ofthis method is to convert diseaseinduced losses ofwellbeing into economic terms by multiplying theannual number oflost life years due to disease bysubreginal per capita income so far few studies hadever taken this part of costs into account potentiallyleading to an underestimation of health and economictable lifetime costs attributable to child abuse for the first time in ciitems of the costs usd milliondirect costs medical costsshortterm ahtlongterm other diseasesindirect costsabuse death a productivity lossessurvival aht dalys blongterm loss of other diseases btotal costsaht abusive head traumaa we used times of base line data for range costs of child abuseb costs of suffering and pain dalys converted into monetary value by multiplying a gross domestic product per capita million gdp per capita 0cmo bmc public health page of table sensitivity analyses on incidence resource and discounted ratesensitivity analysisliterature based estimation adisease burden in dalys 95cieconomic burden usd million 95cidr dr dr conservative estimation bdr dr dr dr discounted ratea estimated based on literature review simple size weighted average prevalenceb estimated based on the number of consultation cases disposed about child abuse at child guidance centres probable estimate of abuse death was assumedabout times confirmed aht casespossible cases of the costs of conservative estimatechild maltreatmentimpacts ofin addition weadopted conservative calculation methodology in thesensitivity analyses to estimate the burden of childmaltreatment for more reliable range estimationsthere are several limitations to this study first thecooccurrence of multiple types of child abuse isprevalent resulting in difficulties to identify theadverse effects separately in order to minimize possible consequent overestimation we used the pooledors of multiple adverse childhood health experiencesinstead of each types of child maltreatment and itsseverity second we focused on the economic burdendue to the mortality and morbidity of child maltreatment but did not consider nonhealth human capitalaspectslikeother economic burden estimation studies the availability of data on the related medical costs were limited wehealthconsequences and explored their unit costs for the estimates to address the knowledge gap thirdtargeted majorneverthelessthereproductiverecently in japan a continuum ofintensive supports to mothers and childrearing families encompassingcycle has been widelyimplemented in most local authorities such an integral approach serves as an essential preventive strategy against child maltreatment and other harmfulpractices by early detection and intervention of highrisk households in pregnancy postpartum and childrearing periods thisstudy can provide decisionmakers information on the economic burden of childmaltreatment as well as an important input in futureeconomic evaluations costeffectiveness analysis oncurrently ongoing intervention and policy in additionour results hint an emphasis on preventive interventions on suicide attempts and depression which aretop causes of the attributable disease burden due tochild maltreatmentour study demonstrated that lifetime disease and economic burden due to child maltreatment in japan is substantial its disease burden was approximately equal tothe burden of colon and rectum cancers or stomach cancer in particular it is important to include the longterm disease burden in future studies related to diseaseburden and cost of illness for both technical and policyperspectivessupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12889020093978additional file table a1 studies included in the quantitativesynthesis table a2 health outcomes and pooled ors used in this studyaht not included table a3 incidence rate by age and average onsetage based on the number of consultation cases disposed about childabuse at child guidance centersadditional file systematics review 2018520findpossible literature including japanese studies on risk of health outcomesattributable to child maltreatment figure a1 study selection prismaflow diagramabbreviationsdalys disabilityadjusted life years pafs population attributable fractionsgdp gross domestic product mhlw ministry of health labour and welfarewdv witnessing domestic violence aht abusive head traumaicd international classification of diseases gbd global burden of diseaserr risk ratio or odds ratio aces adverse childhood experiencesacknowledgementswe are grateful thank members of health informatics department kyotouniversity of public health school for their kind supportauthors contributionsmx and gr designed the study mx did the calculation and draft themanuscript gr and ty takahashi contributed to the revise ty tachibanatb and nt critically reviewed and provided important intellectual feedbackon the revise all authors have read and approved the manuscriptfundingthis study is granted by health labour sciences research grant japanagency for medical research and development and as part of an ipss 0cmo bmc public health page of project on the realization of japans plan for dynamic engagement of allcitizens the funders did not have any role in the study design datacollection and analysis interpretation of data or in writing the manuscriptavailability of data and materialsall the raw data is publicly accessible from respective official website asreference national healthcare expenditures and patient survey the datasets analysed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare no conflict of interestauthor details1department of health policy national center for child health anddevelopment tokyo japan 2department of health informatics kyotouniversity school of public health kyoto japan 3department of empiricalsocial security research national institute of population and social securityresearch uchisaiwaicho chiyodaku tokyo japan4maternalchild psychiatry department of psychosocial medicine nationalcenter for child health and development tokyo japan 5faculty ofeconomics saitama university sakuraku japanreceived march accepted august referencesgilbert r widom cs browne k fergusson d webb e sjtl j burden andconsequences of child maltreatment in highincome countries lancetnumber of consultation cases disposed about child abuse at child guidancecenters in japan in japanese [httpswwwestatgojpstatsearchfilespage1layoutdatalisttstat000001034573cycle8tclass1000001108815tclass2000001108820second21]definition and present condition of child abuse in japanese [httpswwwmhlwgojpseisakunitsuitebunyakodomokodomo_kosodatedvabouthtml] accessed july currie j spatz widom c longterm consequences of child abuse andneglect on adult economic wellbeing child maltreatment hughes k bellis ma hardcastle ka sethi d butchart a mikton c jones ldunne mp the effect of multiple adverse 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molhepatol metrics population attributable fraction paf [httpwwwwhointhealthinfoglobal_burden_diseasemetrics_pafen] accessed july norman re byambaa m de r butchart a scott j vos t the longtermhealth consequences of child physical abuse emotional abuse and neglecta systematic review and metaanalysis plos med 2012911e1001349kalmakis ka chandler ge health consequences of adverse childhoodexperiences a systematic review j am assoc nurse pract unicef child maltreatment prevalence incidence and consequences inthe east asia and pacific region new york unicef rothman kj epidemiology an introduction oxford university press joyce t huecker mr pediatric abusive head trauma shaken babysyndrome [updated aug ] in statpearls [internet] treasure islandfl statpearls publishing available from httpswwwncbinlmnihgovbooksnbk499836 yamaoka y fujiwara t fujino y matsuda s fushimi k incidence and agedistribution of hospitalized presumptive and possible abusive head traumaof children under months old in japan j epidemiol httpsdoi102188jeaje20180094japanese population projection [httpwwwstatgojpdatajinsui2016np] accessed july summary of patient survey [httpswwwmhlwgojpenglishdatabasedbhsssps_2014html] accessed july kirigia jm mburugu gn huka gs the indirect cost of disability adjusted lifeyears lost among the elderly in kenya int arch med httpsdoi1038232483 mortality and global health estimates [httpwwwwhointghomortality_burden_diseaseen] accessed july japan gdp gross domestic product [httpscountryeconomycomgdpjapanyear2016] accessed july the results of verification of death cases caused by child abuse threport [httpswwwmhlwgojpstfseisakunitsuitebunya0000173329_00001html] accessed july miller tr steinbeigle r wicks a lawrence ba barr m barr rg disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e154550 httpsdoi101542peds20141385shiroiwa t fukuda t ikeda s takura t moriwaki k development of anofficial guideline for the economic evaluation of drugsmedical devices injapan value health moore se scott jg ferrari aj mills r dunne mp erskine he devries kmdegenhardt l vos t whiteford ha burden attributable to childmaltreatment in australia child abuse negl publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
"conceptualization data curation investigation project administration supervision validation visualization writing review and editing Goneim I and Ibraheim A performed data curation Kamal NM was involved in literature review writingoriginal draft writing review and editing Alsofiani F and Alawur A performed literature review and writingoriginal draft All authors had read and approved the final manuscriptInformed consent statement Written informed consent in the patients native language was obtained from her fatherConflictofinterest statement The authors declare that they have no conflict of interestCARE Checklist statement The authors have read the CARE Checklist and the manuscript was prepared and revised according to the CARE Laila M Sherief Department of Pediatric Hematology and Oncology Faculty of Medicine Zagazig University Zagazig EgyptLaila M Sherief Amr Ibraheim Department of Pediatrics Faculty of Medicine Zagazig University Zagazig EgyptEsmael Goneim Department of Pediatric Oncology Tanta Cancer Institute Tanta EgyptNaglaa M Kamal Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Cairo EgyptNaglaa M Kamal Fuad A alsofiani Abdulraouf H Alawur Department of Pediatrics Alhada Armed Forces Hospital Taif Saudi ArabiaCorresponding author Naglaa M Kamal MD Full Professor Department of Pediatrics and Pediatric Hepatology Faculty of Medicine Cairo University Kasralainy Cairo Egypt naglakamalkasralainyeduegAbstractBACKGROUND thalassemia intermedia TI is one of the hemoglobinopathies It constitutes of thalassemia cases yet being associated with a better quality of life than thalassemia major TMCASE SUMMARY We recently reported the first case of acute lymphoblastic leukemia ALL from Egypt in a child with TM and we herein report the first case of ALL from Egypt in a child with TI In this report literature was reviewed for cases of malignancies associated with TI and the possible factors underling the relationship between the two entities We stress that physicians should have a high index of suspicion of malignancies in thalassemia patients if they present with any suggestive symptoms or signsKey words Acute lymphoblastic leukemia Thalassemia intermedia Children Malignancies Iron overload Hydroxyurea Case reportThe Authors Published by Baishideng Publishing Group Inc All rights reservedWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIChecklist Access This is an access that was selected by an inhouse editor and fully peerreviewed by external reviewers It is distributed in accordance with the Creative Commons Attribution NonCommercial CC BYNC license which permits others to distribute remix adapt build upon this work noncommercially and license their derivative works on different terms provided the original work is properly cited and the use is noncommercial See httpcreativecommonslicensesbync40Manuscript source Unsolicited manuscriptReceived January Peerreview started January First decision April Revised May Accepted June in press June Published online August PReviewer Fujioka K Moschovi MA SEditor Dou Y LEditor A EEditor Li JHCore tip Cases have been reported for malignancies in patients of thalassemia major However rare case reports have been reported for malignancies in patients of thalassemiaintermedia as it is a nontransfusion dependent anemia Physicians should have high index of suspicion to diagnose malignancies in patients with thalassemiaintermediaCitation Sherief LM Goneim E Kamal NM Ibraheim A Alsofiani F Alawur A Acute lymphoblastic leukemia in a thalassemia intermedia child A case report World J Clin Pediatr URL wwwwjgnetcom22192808fullv9i11htm dx105409wjcpv9i11INTRODUCTIONThalassemia represents the most common singlegene disorder worldwide The total annual incidence of symptomatic individuals with thalassemia is estimated at in throughout the world of whom nearly have thalassemia intermedia TI which is intermediate in severity between the milder thalassemiaminor and the more severe transfusiondependent thalassemiamajor TM[]We herein report the first case from Egypt with TI who developed acute lymphoblastic leukemia ALLCASE PRESENTATIONChief complaintsThe reported patient is a 15yearold girl with TI who presented at the age of years with pallor decreased growth rate and decreased activity She had severe microcytic hypochromic anemia with hemoglobin Hb of gdLHistory of present illnessPediatric hematologist workup proved the diagnosis of TI Her Hb electrophoresis showed HbA HbF and HbA2 Genetic molecular testing revealed compound heterozygosity for cd27 GT and cd39 CT mutations Hydroxyurea at a dose of mgkg per day was started in addition to folic acidShe was then followed at the pediatric hematology unit at regular intervals to monitor her tolerance to drug therapy with special attention to hematological toxicity There were no significant side effects during seven years of therapy and the patient showed good response with occasional need for blood transfusions She underwent splenectomy during her late teensHistory of past illnessAt the age of years she developed generalized bone aches abdominal pain persistent fever and dyspnea and so she was referred to our hospitalPhysical examinationOn physical examination there was severe pallor tachypnea tachycardia and hepatomegalyLaboratory examinationsInitial complete blood picture showed a Hb level gdL white blood cell count of 109L and platelets count of 109LSerum electrolytes cerebrospinal fluid analysis and kidney and liver function tests were normal expect for mild elevation of total serum bilirubin which was mgdLSerum ferritin was ngdL Serological studies including EpsteinBarr virus cytomegalovirus human immunodeficiency virus hepatitis C virus and hepatitis B virus were negative Lactate dehydrogenase was UL and serum uric acid was mgdLWJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIImaging examinationsHer chest Xray was normal Abdominal ultrasonography revealed hepatomegaly with calcular cholecystitis and bilateral diffuse renal enlargement Echocardiography showed mitral valve prolapse with trivial mitral regurgitationMULTIDISCIPLINARY EXPERT CONSULTATIONThe pediatric haematologistoncologist assessment requested bone marrow biopsy which was carried by the hematopathologistBone marrow examination revealed blast cells in a hypercellular marrow with depressed erythropoiesis and granulopoiesies and normal thrombopoiesis Immunophenotyping showed lymphoblasts that are CD10 positive CD19 positive CD34 positive TDT positive HLADR positive CD13 positive and CD33 positiveCytogenetic examination showed a normal karyotype with a DNA index of and negative t1221 t119 BCRABL or 11q23 translocationsmutationsFINAL DIAGNOSISA final diagnosis of Bacutelymphoblasticleukemia ALL with aberrant expression of CD13 and CD33 was achievedTREATMENTInduction chemotherapy of the total XV protocol with prednisone vincristine Lasparaginase doxorubicin cyclophosamide cytarabine 6mercaptopurine and intrathecal chemotherapy was commencedShe received multiple packed red cell transfusions which eventually led to elevation of serum ferritin to ngdL Thus she was started on oral chelation therapy with deferasirox with no complicationsThe patient eventually went into complete remission She then received consolidation chemotherapy of standard risk of the total XV protocol with times of high dose methotrexate HDMTX 6mercaptopurine and intrathecal chemotherapyShe received multiple packed red blood cell transfusions and other supportive measures during the periods of induction and consolidation The transfusions therapy was given according to the guidelines of pediatric oncologists who usually transfuse if Hb level is less than gdL and if associated with pulmonary or cardiac comorbidities or exposed to invasive procedure and hemorrhage and they transfuse with Hb less than gdL The transfusions were not associated with any complications Deferasirox was stopped in consolidation phase during infusion of high dose methotrexateThe main problem observed during the periods of induction and consolidation therapy was increased requirement of blood transfusions as well as repeated infections as during this period the child received intensive chemotherapy which caused bone marrow suppressionOUTCOME AND FOLLOWUPThe child is still in complete remission while being now in the continuation phase for standard risk week fortyDISCUSSIONWe have recently reported the first case from Egypt with thalassemia major TM who developed ALL[] herein we report similarly the first report from Egypt for a patient with TI who developed ALL to highlight that the coexistence of malignancy and beta thalassemia is not rareA thorough look in literature for previously reported cases of malignancies in WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TIpatients with TI revealed only one report in from Turkey on a years old boy with TI who developed ALL[] Other previous reports on malignancies associated with TI described nonHodjkinlymphoma[] chronic myeloid leukemia[] Hodjkin lymphoma[] hepatocellular carcinoma[] and thyroid malignancies[] Table To our knowledge our patient is the second worldwide and the first from EgyptAlthough reported cases of malignancies associated with TI are few but it raises the attention of physicians to have high index of suspicion of malignancies in this group of patients when they present with unexplained new symptoms or proposed symptoms and signs of malignancySpecial concern about management plans in these patients as they usually require more frequent blood transfusions as the chemotherapy causes suppression of the bone marrow which adds to the base line chronic hemolysisIn spite that reported cases of malignancies in TI are scarce which makes our trial to find causal relationship between TI and cancer development beyond the scope of our report but we tried to search literature foe possible contributing factors Those factors cant rise to the level of conclusions and definitely need to be proved and validated by larger prospective cohort with large control groups multicenter worldwide studies addressing all possible hypothesesIndeed the most practical logical thinking about that underlying factors for the development of malignancy in TI is being multifactorial[]In a large multicenter study on thalassemia patients from Iran the proportion of patients with cancer was higher in those with TI than those with TM and respectively[] They explained it by the fact that bone marrow in TM patients is suppressed by the regular transfusions while it is very active with high turnover in those with TI[] They suggested that this can lead to a higher rate of DNA repair faults and mutations with subsequent higher rate of hematological malignancies[]Another potential factor is the prolonged use of hydroxyurea Conflicting data are there regarding its carcinogenic potential Hydroxyurea as an antimetabolite interferes with both DNA synthesis and repair mechanisms with later accumulation of mutations and subsequent chromosomal damage Although no studies have yet investigated the relationship between hydroxyurea and the development of cancers in thalassemia but clinically concerns have been raised regarding its potential leukemogenic potential[] Other authors were against this assumption[] The BABYHUG clinical trial which compared hydroxyurea with placebo treated controls refuted this assumption and did not suggest any increased risk of genotoxicity[]Overall there is no evidence to suggest an increased risk of carcinogenesis in patients with thalassemia with hydroxyurea and further studies will need to be designed to establish any potential relationship[]One more probable factor is that patients with TI being having milder disease than those with TM with fewer blood transfusions might lead to delayed diagnosis and even if diagnosed usually there is underestimation of their iron overload problem and sometimes the deceiving relatively mildly elevated ferritin as compared to TM which has been shown to underestimate the true iron burden in TI patient with ultimate fate that these patients accumulate iron but it usually goes unnoticed unchelated and unmonitored Anemia hypoxia and ineffective erythropoiesis suppress the expression of hepcidin by increasing expression of growth differentiation factor and hypoxiainducible transcription factors with the resultant increased intestinal iron absorption and in turn adds to the problem of iron overload[]The longstanding iron overload with its deposition in different body ans with the wellknown association between excess iron and cancer development can be a predisposing factor for all types of malignancies through direct and indirect effects[]Iron can directly damage DNA by nontransferrinbound iron with the consequent inactivation of tumorsuppressor genes such as p53 or their products The indirect effects include the formation of reactive oxygen species ironinduced lipid peroxidation and altered immune system with decreased immune surveillance suppression of tumoricidal action of macrophages and alteration of cytokine activities TI patients usually survive longer than TM patients with enough time for iron overload to develop[]Some authors suggested that improved management protocols of thalassemia patients have led to increased survival with most of them reaching adult age with the consequent occurrence of diseases associated with long life span like malignancies[] This assumption can partially explain other reports in elder patients but it cant work in our patient and the Turkish one who are teenagersMany authors suggested that the occurrence of malignancies in thalassemia patients could be a pure coincidence or a combination of genetic and environmental factors[]WJCPwwwwjgnetcomAugust Volume Issue 0cTable Previously reported cases of thalassemia intermedia who developed malignanciesSherief LM ALL in a child with TINumber of patientsType of malignancyAcute lymphoblastic leukemiaNonHodgkin lymphoma NHLNHL Hodgkin lymphoma HLNHL HL chronic myeloid leukemia CMLCMLHLHepatocellular carcinoma HCCHCCHCCHCCHCCThyroid cancerSome patients have thalassemia major and others have thalassemia intermedia in references and Ref[][][][][][][][][][][][]We can sum up to a clear message that whatever the pathogenesis of malignancies in thalassemias the most important message is to alarm physicians to have high index of suspicion for malignancies if their thalassemia patients develop suggestive symptoms and signs Worsening anemia leukocytosis fever boneache lymphadenopathy and splenomegaly are alarming to look for leukemias and other hematological malignanciesREFERENCES Galanello R Origa R Betathalassemia Orphanet J Rare Dis [PMID ]Sherief LM Kamal NM Abdelrahman HM Hassan BA Zakaria MM First report of acute lymphoblastic leukemia in an Egyptian child with thalassemia major Hemoglobin [PMID ]TuÄcu D KarakaÅ Z G¶k§e M AÄaoÄlu L Un¼var A SarıbeyoÄlu E Ak§ay A DevecioÄlu O Thalassemia Intermedia and Acute Lymphoblastic Leukemia Is it a Coincidental Double Diagnosis Turk J Haematol [PMID 104274tjh20140068]Chehal A Loutfi R Taher A Betathalassemia intermedia and nonHodgkin's lymphoma Hemoglobin [PMID 101081hem120015025]Benetatos L Alymara V Vassou A Bourantas KL Malignancies in betathalassemia patients a singlecenter experience and a concise review of the literature Int J Lab Hematol [PMID 101111j1751553X200700929x]Karimi M Giti R Haghpanah S Azarkeivan A Hoofar H Eslami M Malignancies in patients with betathalassemia major and betathalassemia intermedia a multicenter study in Iran Pediatr Blood Cancer [PMID 101002pbc22144]Alavi S Safari A Sadeghi E Amiri S Hematological malignancies complicating thalassemia syndromes a single center experience Blood Res [PMID 105045br2013482149]Jabr FI Aoun E Yassine H Azar C Taher A Betathalassemia intermedia and Hodgkin lymphoma Am J Hematol [PMID 101002ajh20478]BnaPignatti C Vergine G Lombardo T Cappellini MD Cianciulli P Maggio A Renda D Lai ME Mandas A Forni G Piga A Bisconte MG Hepatocellular carcinoma in the thalassaemia syndromes Br J Haematol [PMID 101046j13652141200304732x]Mancuso A Sciarrino E Renda MC Maggio A A prospective study of hepatocellular carcinoma incidence in thalassemia Hemoglobin [PMID ]Restivo Pantalone G Renda D Valenza F D'Amato F Vitrano A Cassar F Rigano P Di Salvo V Giangreco A Bevacqua E Maggio A Hepatocellular carcinoma in patients with thalassaemia syndromes clinical characteristics and outcome in a long term single centre experience Br J Haematol [PMID 101111j13652141201008180x]Fragatou S Tsourveloudis I Manesis G Incidence of hepatocellular carcinoma in a thalassemia unit Hemoglobin [PMID ] WJCPwwwwjgnetcomAugust Volume Issue 0cSherief LM ALL in a child with TI Maakaron JE Cappellini MD Graziadei G Ayache JB Taher AT Hepatocellular carcinoma in hepatitisnegative patients with thalassemia intermedia a closer look at the role of siderosis Ann Hepatol [PMID ]Poggi M Sorrentino F Pascucci C Monti S Lauri C Bisogni V Toscano V Cianciulli P Malignancies in thalassemia patients first description of two cases of thyroid cancer and review of the literature Hemoglobin [PMID ]Halawi R Cappellini MD Taher A A higher prevalence of hematologic malignancies in patients with thalassemia Background and culprits Am J Hematol [PMID 101002ajh24682]McGann PT Flanagan JM Howard TA Dertinger SD He J Kulharya AS Thompson BW Ware RE BABY HUG Investigators Genotoxicity associated with hydroxyurea exposure in infants with sickle cell anemia results from the BABYHUG Phase III Clinical Trial Pediatr Blood Cancer [PMID 101002pbc23365] WJCPwwwwjgnetcomAugust Volume Issue 0cPublished by Baishideng Publishing Group Inc Koll Center Parkway Suite Pleasanton CA USA Telephone Email bpgofficewjgnetcom Help Desk wwwf6publishingcomhelpdesk wwwwjgnetcom Baishideng Publishing Group Inc All rights reserved\x0c" | 2 |
"progression-free survival (PFS) compared with placebo in patients from eastern Asian with locally advanced/metastatic non-small-cell lung cancer (NSCLC) after four chemotherapeutic cycles (21 days per cycle) of first-line platinum-based combination chemotherapy without disease progression. The objective of the current study was to evaluate the cost-effectiveness of maintenance gefitinib therapy after four chemotherapeutic cycles stand first-line platinum-based chemotherapy for patients with locally advanced or metastatic NSCLC with unknown EGFR mutations from a Chinese health care system perspective. Methods and Findings A semi-Markov model was designed to evaluate cost-effectiveness of the maintenance gefitinib treatment. Two-parametric Weibull and Log-logistic distribution were fitted to PFS and overall survival curves independently. One-way and probabilistic sensitivity analyses were conducted to assess the stability of the model designed. The model base-case analysis suggested that maintenance gefitinib would increase benefits in a 13 6 or 10-year time horizon with incremental $184829 $19214 $19328 and $21308 per quality-adjusted life-year (QALY) gained respectively. The most sensitive influential variable in the cost-effectiveness analysis was utility of PFS plus rash followed by utility of PFS plus diarrhoea utility of progressed disease price of gefitinib cost of follow-up treatment in progressed survival state and utility of PFS on oral therapy. The price of gefitinib is the most significant parameter that could reduce the incremental cost per QALY. Probabilistic sensitivity analysis indicated that the cost-effective probability of maintenance gefitinib was zero under the willingness-to-pay (WTP) threshold of $16349 (3per-capita gross domestic product of China). The sensitivity analyses all suggested that the model was robust. Conclusions Maintenance gefitinib following first-line platinum-based chemotherapy for patients with locally advanced/metastatic NSCLC with unknown EGFR mutations is not cost-effective. Decreasing the price of gefitinib may be a preferential choice for meeting widely treatment demands in China. This study was funded by National Natural Science Foundation of China (No.81173028) (URL: http://www.nsfc.gov.cn/Portal0/default152.htm). The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. Introduction Lung cancer the most commonly diagnosed form of cancer is also the leading mortality cause of cancer in males [1]. Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancer cases and the majority of patients with NSCLC have locally advanced/metastatic disease when they are diagnosed with carcinoma [2] [3]. Platinum-based combination therapies are recommended as first-line chemotherapy for unselected patients with locally advanced/metastatic NSCLC [4] [5]. However the duration of them (46 chemotherapeutic cycles 21 days per cycle) are limited by cumulative toxicities and response rates (20%35%) and median overall survival (712 months) are modest [6] [7]. On the basis of previous investigations efforts to improve treatment outcome have focused on the specific goal of prolonging tumour response progression-free survival (PFS) and overall survival (OS) with well tolerated maintenance treatment in patients who have attained tumor control during first-line treatment [8][12]. Because of these trials and other findings both erlotinib and pemetrexed (for patients with histologies other than squamous cell carcinoma) have been approved by clinical guidelines as a category 2A recommendation for switch maintenance therapy and also been approved by FDA in patients without disease progression after 46 chemotherapeutic cycles of first-line therapy [4] [13] [14]. In The Lancet Oncology recently Li Zhang et al based on a double-blind randomised phase 3 trial reported that maintenance gefitinib significantly prolonged PFS compared with placebo in patients from 27 centres across China with locally/metastatic NSCLC which indicates that gefitinib should be considered as a maintenance treatment choice in eastern Asian patients [15]. Several economic studies were conducted of maintenance therapy [16][23]. Two analyses concluded that maintenance erlotinib is cost-effective versus best supportive care for locally advanced/metastatic NSCLC [16] [17]. Except for the study by Greenhalgh et al [18] the 4 other studies of maintenance pemetrexed indicated that the new therapy was not cost-effective [19][22]. The evaluation from Zhu J et al on the basis of the clinical trial suggested that the maintenance gefitinib therapy was cost-effective for locally advanced/metastatic NSCLC patients with activating EGFR mutations [23]. However it is unclear whether the new therapy is cost-effective in patients with unknown EGFR mutations after first-line platinum-based combination chemotherapy without disease progression. The objective of the current study was to evaluate the long-term cost-effectiveness (10 year time horizon) of maintenance gefitinib therapy after four chemotherapeutic cycles of stand first-line platinum-based chemotherapy for locally advanced/metastatic NSCLC patients with unknown EGFR mutations from a Chinese health care system perspective. Materials and Methods A previously constructed semi-Markov model was used to compare the long-tern impact of maintenance gefitinib treatment versus placebo after 4 chemotherapeutic cycles of first-line platinum-based chemotherapy for patients with locally advanced/metastatic NSCLC [22] on the basis of the double-blind randomised phase III trial from China by Li Zhang et al [15]. The model along with two-parametric Weibull and Log-logistic distribution were used for calculating the direct medical costs life-years gained (LYGs) and quality-adjusted life-years (QALYs) gained of the practice presented in the trial [15]. Due to the perspective of the Chinese health care system only direct medical costs related to the practice were estimated including maintenance gefitinib therapy treatment of major adverse events routine follow-up treatment for patients without progression follow-up treatment for progressive disease and terminal-phase cost. Costs in this study were estimated in US dollars (USD) corresponding to the 2011 consumer price index and assuming an average exchange rate of 1 USD to 6.45 Chinese Yuan (RMB). Utilities for the model were derived from the literature. The future costs and outcomes were discounted at 3% annually in compliance with the request of China Guidelines for Pharmacoeconomic Evaluations (version 8) [24]. Effectiveness data were stemmed from the multicentre double-blind randomised clinical trial [15] which is the only phase III trial compared maintenance gefitinib treatment in patients with locally advanced/metastatic NSCLC according to our literature search. In brief 296 patients with histological or cytological NSCLC in stage IIIb or IV between September 28 2008 and August 112009 who were 18 years or older and had a WHO performance status of 02 and more than 12 weeks life expectancy after completion of four chemotherapeutic cycles first-line platinum-based chemotherapy without disease progression were eligible for the maintenance gefitinib or placebo treatment (1?1 randomization ratio). Eligible patients continued to take either gefitinib (250 mg per day) or placebo orally until disease progression intolerable toxicity withdrawal of consent serious non-compliance with protocol or dose delay or interruption >14 days. In this report there were 40 and 39 patients were deemed know EGFR mutation status in gefitinib group and placebo group respectively. Therefore there were 108 patients and 109 patients with unknown EGFR mutation received maintenance gefitinib and placebo treatment separately. The primary endpoint of the trial was progression-free survival and the survival analysis revealed that median PFS for patients with unknown EGFR mutation was 6.0 months in gefitinib group and 2.7 months in placebo group (HR 0.40 [95% CI 0.290.54]; p<0.0001). Median OS was not significantly different between the two groups (HR 0.84 [95% CI 0.621.14]; p?=?0.26; median OS 18.7 months vs 16.9 months). The incidence of adverse events in gefitinib group was more frequent than that in placebo group (80% vs. 53%). The cumulative probabilities of serious adverse events were 7% and 3% in the maintenance gefitinib and placebo groups respectively. The model outcomes were presented as costs LYGs and QALYs from the perspective of the Chinese health care system. Sensitivity analyses of input parameters with the high/low values and various distributions were conducted to assess the stability of the model at a value of recommended willingness-to-pay (WTP) threshold of $16349 (3per-capita gross domestic product GDP) based on the cost-effectiveness guidelines of Word Health Organization (WHO) [25]. Model Structure The simplified model structure was shown in Figure 1 which comprised 3 mutually exclusive health states: PFS (entry state); progressed survival (PS state) and death. Patients move from one state to another during each Markov cycle length of 3 weeks (short enough to detect all clinically relevant events) until time horizon termination of 10-year (>95% patients died). Two-parametric Weibull survival and Log-logistic distribution analyses using R for Statistical Computing version 2.15.2 (R Foundation Wien Austria) were fitted to the PFS and OS curves respectively on the basis of survival data extracted from the published Kaplan-Meier curves [15] by using GetData Graph Digitizer software (version 2.24). Table 1 shows the Weibull and Log-logistic distribution parameters of model estimated. The estimated Weibull parameters are used to measure the time-dependency transition probabilities from PFS to PS state according to the following formula:where the ? defines the scale of the distribution the ? gives the shape the u is the Markov cycle and tu indicates that t is calculated as integer multiples of the cycle length of the model. The transition probabilities of death at current t due to the following formula:where the ? and ? are the theta and kappa from the estimated Log-logistic parameters indications of the u and the tu are the same as above. 10.1371/journal.pone.0088881.g001 Figure 1 Markov model of locally advanced/metastatic non-small-cell lung cancer. 10.1371/journal.pone.0088881.t001 Table 1 Weibull and Log-logistic parameters of model estimated for progression-free and overall survival curves respectively. Progression-free survivala Scale Mean (Range) Shape Mean (Range) Adjusted R2 Correlation Coefficient Placebo arm 0.10443 (0.04509/0.16377) 1.29221 (0.99662/1.58780) 0.9729 ?0.995165 Gefitinib arm 0.10231 (0.06622/0.13840) 0.83852 (0.71474/0.96230) 0.9782 ?0.998386 Overall survival b Theta Mean (Range) Kappa Mean (Range) Adjusted R2 Correlation Coefficient Placebo arm ?6.54311 (?7.16112/?5.92510) 2.09373 (1.89823/2.28923) 0.9855 ?0.999986 Gefitinib arm ?5.04069 (?5.53622/?4.54516) 1.54139 (1.38359/1.69919) 0.9801 ?0.999852 a R output for Weibull regression fitted to progression-free curves of locally advanced/metastatic non-small-cell lung cancer patients derived from the Phase III trial [15]. b R output for Log-logistic regression fitted to overall curves of locally advanced/metastatic non-small-cell lung cancer patients derived from the Phase III trial [15]. Medical Costs and Utilities Medical costs for each strategy (Table 2) from the perspective of Chinese health care system were based on outlining current practice [15] which reflected the effectiveness of maintenance gefitinib treatment in Chinese patients with locally advanced/metastatic NSCLC. Direct medical costs related to the practice were estimated including maintenance gefitinib therapy treatment of major adverse events routine follow-up treatment for patients without progression follow-up treatment in PS state and terminal-phase cost. Prices of gefitinib follow-up treatment cost in PS state and terminal-phase cost were obtained from our previous study in which we have calculated healthcare costs associated with the time- and health status-related treatment resources that advanced NSCLC may anticipate based on health expenditure data for 253 cases of advanced NSCLC registered at the Second Xiangya Hospital of Central South University in China between 2006 and 2010 [26]. The aggregate annual medical costs for patients in either PFS or PS state and monthly healthcare costs accumulated during the terminal 3 months were estimated and evaluated using 95% confidence intervals through bootstrapping with the R software (version 2.14.0; R Foundation Vienna Austria) [26]. According to Gefitinib Patients Assistance Program of the pharmaceutical producer in China NSCLC patients receive donations of gefitinib after six months treatment [23]. Therefore six months was applied to calculate the total cost of the maintenance drug. Routine follow-up treatment cost for patients without progression including computed tomography scan physician visit and other examinations and drugs was derived from the literature by Wu B et al [27]. Based on expert opinion only diarrhoea and other grade 3/4 adverse events were considered to estimate the costs of treatment-associated toxicity. Therefore the unit costs of diarrhoea treated and liver protected were multiplied by published rates of corresponding events to populate the model analysis (we assumed patients with grade 3/4 alanine aminotransferase aspartate aminotransferase or aminotransferases increased should receive treatment of liver protected). The unit costs of diarrhoea and liver protected were estimated according to local charges in China." | 1 |
"At present the relationship between hypothyroidism and the risk of breast cancer is still inconclusive Thismetaanalysis was used to systematically assess the relationship between hypothyroidism and breast cancer riskand to assess whether thyroid hormone replacement therapy can increase breast cancer riskMethods The relevant s about hypothyroidism and the risk of breast cancer were obtained on the electronicdatabase platform Relevant data were extracted and odd ratios OR with corresponding confidence intervalsCI were merged using Stata SE softwareResults A total of related studies were included in the metaanalysis including cohort studies and casecontrol studies The results show that hypothyroidism was not related to the risk of breast cancer odd ratios CI In the European subgroup we observed that patients with hypothyroidism have a lower risk ofbreast cancerodd ratios CI Furthermore no significant correlation was observed betweenthyroid hormone replacement therapy and the risk of breast cancer odd ratios CI Conclusion Hypothyroidism may reduce the risk of breast cancer in the European population and no significantcorrelation was observed between hypothyroidism and breast cancer risk in nonEuropean populations Due to thelimited number of studies included more largescale highquality longterm prospective cohort studies areneededKeywords Hypothyroidism Thyroid hormone replacement therapy Breast cancer MetaanalysisBackgroundAs a global public health problem breast cancer has anincreasing incidence on a global scale [] According tothe US cancer statistics breast cancer has becomethe most common malignant tumour in women withabout new cases each year accounting for of new malignant tumours in women [] Therefore theidentification of risk factors for breast cancer and the Correspondence Yanhuangdr163com Ruobaolidr163com2Department of Oncology Affiliated Hospital of Weifang Medical UniversityWeifang China3School of Basic Medicine Weifang Medical University Weifang ChinaFull list of author information is available at the end of the adoption of effective early prevention and interventionmeasures are of great significance for patients withbreast cancerThe physiology and pathology of the breast are closelyrelated to the endocrine of the body [] As the largestendocrine an in the human body the thyroid glandhas specific regulation effects on various hormone levelsand cell growth and development in the body whichbrings new enlightenment to the research of breast cancer [] In Kapdi et alfirst proposed thathypothyroidism maybe increase the risk of breast cancer[] Since then many scholars have studied the relationship between hypothyroidism and the risk of breast 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 0cWang BMC Cancer Page of []cancer However the relationship between the two diseases remains controversial [] Some studies haveshown that hypothyroidism increases the risk of breastcancerthathypothyroidism reduces the risk of breast cancer []Besides some studies have found no correlation betweenthyroid disease and breast cancer risk [] Thereforewhether hypothyroidism can increase the risk of breastcancer is worthy of further studystudiesshownSomehaveTwo metaanalyses have previously been studied forhypothyroidism and breast cancer risk [ ] Based onprevious research we have included more prospectivestudies and Asian population studies to assess the relationship between hypothyroidism and breast cancer risksystematically Besides the impact of thyroid hormonereplacement therapy on breast cancer risk was exploredin this metaanalysisMethodsSearch strategyRelevant clinical literature was extracted by systematicretrieval of PubMed Medline EMBASE Springer Webof Science and Cochrane Library electronic databasesup to date to October Our search strategy includedorhypothyroidism or HT and thyroid diseases orbreast cancer or BC or breast neoplasms or mammarmy cancer and risk orincidence At the sametime we manually screened out the relevant potentialliterature in the references extracteddysfunctionthyroidtermsforInclusion and exclusion criteria The inclusion criteria Types of studies Published studies exploring therelationship between hypothyroidism and breastcancer risk Subject Female Exposure factors Primary hypothyroidism thediagnosis needs to be based on the detection ofthyroid function Outcome indicators the occurrence of primarybreast cancerThe exclusion criteria Nonprimary hypothyroidism due to other causes Non observational studiesInsufficient information was provided or no fulltext Unable to obtain full text or quality assessment ofthe literature Studies were repeated or publications overlappedData extraction and quality assessmentTwo researchers separately conducted literature screening data extraction and literature quality evaluationand any differences could be resolved through discussionor a third inspector Information secured from the enrolled literature included first authors surname year ofpublication country ofthe population sample sizefollowup time and data on the relationship betweenhypothyroidism and the risk of breast cancerThe NewcastleOttawa Scale NOS was used to assessthe quality of the study from three aspects cohort selection cohort comparability and outcome evaluation []NOS scores of at least six were considered highqualityliterature Higher NOS scores showed higher literaturequalityStatistical analysisAll data analysis was performed using Stata120 softwareMetaanalysis was performed according to the PRISMAguidelines The OR and 95CI from included studieswere treated with the combined effect size After thatthe heterogeneity test was conducted When P ¥ orI2 was performed it mean that there was no apparent heterogeneity and the fixedeffect model shouldbe applied for a merger When P or I2 ¥ indicated high heterogeneity the randomeffect model wasapplied Combined effect size if OR indicates thathypothyroidism is an unfavorable factor for breast cancer If OR is the opposite Publication bias Begg funnel plot and Egger test linear regression test were usedto research publication bias detection of the literatureincluded If P indicates obvious publication biasResultsProcess of study selection and description of qualifiedstudiesA total of studies were identified on our online databases After exclusion of duplicate references129 s were considered After screening the andtitle s were excluded After careful review ofthe full texts studies have been excluded because ofthem did not provide relevant data and s didnot have fulltext Nineteen s published between and met the inclusion criteria Fig A total of samples from studies involvingwere enrolled in this metaanalysis [ ] Sixcohort studies and casecontrol studies were includedin the study Twelve s were studied in the European population five in the North American populationand two in the Asian population All s are of highquality because of NOS score no less than The chiefcharacteristics of the enrolled materials are detailed inTable 0cWang BMC Cancer Page of Fig Flow chart of search strategy and study selectionRelationship between hypothyroidism and breast cancerriskThere were studies reported the relationship betweenhypothyroidism and breast cancer risk With obviousheterogeneity I p among these studies so a random effect model was used for assessmentThe pooled analysis suggested that was not related tothe risk of breast cancer OR CI P 0001Fig explorethefurtherrelationshipSubgroup analysis of hypothyroidism and risk of breastcancerTobetweenhypothyroidism and breast cancer risk subgroup analysis was conducted from three aspects study typepopulation distribution and followup time The resultsof subgroup analysis were shown in Table In theEuropean subgroup we observed that patients withhypothyroidism have a lower risk of breast cancer OR CI P In the subgroup witha followup date of more than four years patients withhypothyroidism can reduce the risk of breast cancerwith borderline significance OR CI In otherP found thathypothyroidism was not related to the risk of breastcancersubgroups weRelationship between thyroid hormone replacementtherapy and breast cancer riskA total of studies reported the relationship betweenthe use of thyroid hormone replacement therapy and therisk of breast cancer [ ] Asobvious heterogeneity observed the fixedeffect modelwas usedI p The result suggestedthat patients who received thyroid hormone replacementtherapy was not related to the risk of breast cancerOR CI 109P Fig Publication biasFigure 4a shows the results of publication bias for the relationship between hypothyroidism and breast cancerrisk which were evaluated by funnel plots and Eggerstest The Begg test Pr and the Egger testP were used to detecting publication bias showedthat there was no possibility of publication bias Asshown in Fig 4b there were no publication biases in the 0cWang BMC Cancer Table Main characteristics of the included studies in ouranalysisStudySampleYearRegionAdamiKalacheHoffmanBrintonMosesonSmythSheringTalaminiSimonTurkenKuijpensCristofanilliSandhuHellevikDitschGraniSøgaardWengKimSwedenUKSwedenUSACanadaIrelandIrelandItalyUSAPragueNetherlandsUSACanadaNorwegianGermanyItalyDanishUSAKoreaMedianMean ageyearsNANA ± NANA ± ± ¥ ± ¥Page of NOSFollowupyearsNANANAStudydesignCasecontrolCasecontrolCohortCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCohortCasecontrolCohortCohortCasecontrolCasecontrolCohortCasecontrolCohortStudyIDAdami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Overall Isquared p ES CIES CI WeightWeightNOTE Weights are from random effects analysisFig Relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Table Stratiedanalysis of the relationship between hypothyroidism and breast cancer riskVariableOR95CINoofstudiesPHeterogeneityI2RegionEur orth AmericaAsiaStudy designCasecontrolCohortFollowup date ¤ Page of ModelusedFixedRandomedFixedRandomedFixedFixedRandomedPhStudyIDHoffman Kuijpens Sandhu Ditsch Cristofanilli Simon Moseson Brinton Adami Weng ES CIES CI WeightWeightOverall Isquared p NOTE Weights are from random effects analysisFig Relationship between thyroid hormone replacement therapy and breast cancer risk 0cWang BMC Cancer Page of A ]rr[golB ]rh[golBegg's funnel plot with pseudo confidence limitsEgger's publication bias plotse of log[rr]Begg's funnel plot with pseudo confidence limitstceffe idezdradnatstceffi edezdradnatsprecisionEgger's publication bias plotse of log[hr]precisionFig Publication bias assessment a hypothyroidism b thyroid hormone replacement therapy Metaanalysis estimates given named study is omitted Lower CI Limit Estimate Upper CI Limit Adami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Fig Sensitivity analysis for relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Page of s on the study of thyroid hormone replacementtherapy The Egger test was P and the Begg testwas Pr Sensitivity analysisThe results of sensitivity analysis are generally stableand the primary source of heterogeneity is in the research of Cristofanilli []Fig So we excludedthe literature of Cristofanilli and analyzed the otherstudies The results revealed that the hypothyroidismcould reduce the risk of breast cancer was borderlineOR096 95CI092 P andsignificantthere was no heterogeneityI2 P cohortstudy ofDiscussionMore than years ago Beatson used thyroid extracts to treat patients with metastatic advanced breastcancer The condition was significantly alleviated sparkinginterest in exploring the relationship between thyroid andbreast cancer [] Subsequently a prospective study enrolled women and women with earlier diagnosisof hypothyroidism observed the occurrence of breast cancer during followup showed that low serum free thyroxine levels increased the risk of breast cancer [] In aprospective women withhypothyroidism and hyperthyroidism found thathypothyroidism slightly reduced the risk of breast cancer[] However a prospective cohort study of women with autoimmune hypothyroidism and women with normal thyroid function indicated that autoimmune hypothyroidism was not associated with breastcancer risk [] Besides some animal experiments alsoreflect the relationship between the two [ ] Animalexperiments by López Fontanafound thathypothyroidism mice inhibit the development of breastcancer and promote the apoptosis of breast cancer cellsdue to the low expression of βchain protein and activation of the apoptotic pathway on the tumour cell membrane [] Due to the inconsistency ofthe aboveconclusions we performed a metaanalysis to evaluate therelationship between hypothyroidism and breast cancerrisketalA total of studies were included in this metaanalysis and the results showed that patients withhypothyroidism not related to the risk of breast cancerHowever there was significant heterogeneity among theincluded studies After subgroup analysis and sensitivityanalysis we found that Cristofanillis research may causeheterogeneity [] Cristofanillis research is a retrospective study and the diagnosis of hypothyroidism patientswas based on the information recorded in the medicalrecords which may lead to the bias risk of misclassification and have a positive impact on the positive results ofthis study [] After excluding Cristofanillis researchwe found that patients with hypothyroidism had a lowerrisk of breast cancer with borderline significance [] Theresults of the metaanalysis are inconsistent with the findings of Hardefeldt and Angelousi [ ] Perhaps because our study included more prospective studiesand Asian population cohort study In addition we evaluated the risk of breast cancer in thyroid hormone replacement therapy and show that patients who received thyroidhormone replacement therapy was not related to the riskof breast cancerIn the analysis of the European population the resultsshow that hypothyroidism may reduce the risk of breastcancer We also found that patients with hypothyroidismcan reduce the risk of breast cancer was borderline significance in the subgroup with more longer followupdate However the relationship between the two was notobserved in North American and Asian populationsThe possible reasons for these disparities may be as follows First followup time may be the main contributorsto this difference A longer followup is required to demonstrate the relationship between hypothyroidism andbreast cancer risk In the metaanalysis five studies provided North American population data and two reported Asian population data However only one ofseven nonEuropean studies followup time for morethan years Second the differences may be attributedto different ethnicities sharing different genegene andgeneenvironmental backgrounds Third social and environmental factors are another critical cause for thisdifference With these in mind our findings suggest thathypothyroidism may reduce the risk of breast canceronly in the European population and more largescalehighqualitylongterm prospective cohort studies arestill needed to study on different human populationsThe following may explain the potential relationshipbetween hypothyroidism and the risk of breast cancerHealthy mammary epithelial cells can express a largenumber of T3 receptors and breast cancer cells have asimilar ability to bind to T3 [] T3 has an estrogenlike effect that promotes the growth of mammary glandlobes and stimulates normal breast tissue differentiation[ ] Therefore T3 can mimic the effect of estrogenon the proliferation of breast cancer cells When theconcentration of T3 is low in vivo it may inhibit theproliferation of breast cancer cells Hypothyroidism mayreduce the risk of breast cancer by affecting T3concentrationSome basic experiments support this theory In GonzalezSancho studied the relationship betweenT3 and breast cancer [] It was found that there is anoverexpressed T1 gene in human breast cancer cellsand T3 inhibits the proliferation of mammary epithelialcells by inhibiting the expression of cyclin D1 and T1thereby inhibiting the proliferation of breast cancer cells 0cWang BMC Cancer Page of Author details1School of Clinical Medicine Weifang Medical University Weifang China 2Department of Oncology Affiliated Hospital of Weifang MedicalUniversity Weifang China 3School of Basic Medicine WeifangMedical University Weifang ChinaReceived December Accepted July foundthat MartinezIglesias[] Afterthathypothyroidism can inhibit the growth of breast cancercells [] In Tosovic conducted a prospectivestudy of T3 levels associated with breast cancer risk andfound that T3 levels in postmenopausal women werepositively correlated with breast cancer risk in a doseresponse mannerthathypothyroidism through lower levels of T3 could reducethe incidence of breast cancer Our metaanalysis resultsalso confirm the above conjecture[] Therefore we suspectHowever this conclusion needs to be taken with caution as this study has several limitations First the studies that have been included do not adjust for importantrisk factors for breast cancer Second in subgroup analysis for example there are only two s in Asianstudies and we should be cautious about the results ofAsian analysis Third the results of this metaanalysis indicate that there is a large heterogeneity between studiesFourth followup time at different endpoints cannot beuniform Finally publication bias cannot be avoidedentirelyConclusionHypothyroidism may reduce the risk of breast cancer inthe European population and no significant correlationwas observed between hypothyroidism and breast cancerrisk in nonEuropean populations Furthermore therewas no obvious correlation between thyroid hormone replacement therapy and breast cancer risk It is necessaryto conduct a large sample size strictly controlled prospective study of hypothyroidism patients further todemonstrate the relationship between hypothyroidismand breast cancer riskAbbreviationsOR Odd ratios CI Confidence intervals NOS NewcastleOttawa ScaleAcknowledgementsNot applicableAuthors contributionsStudy design BW ZL RLYH and TL Data extraction BW ZL TL and YH Dataanalysis BW ZL RLand YH Manuscript writing BW and RL Manuscriptedition RL and YH All authors have read and approved the manuscriptFundingNo sources of funding were used to conduct this study or prepare this letterAvailability of data and materialsAll the published s and data were available onlineEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsNoneReferencesSiegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin httpsdoi103322caac21442Praestegaard C Kjaer SK Andersson M StedingJensen M Frederiksen KMellemkjaer L Risk of skin cancer following tamoxifen treatment in morethan breast cancer patients a cohort study Breast cancer httpsdoi101007s1228201506605 Mittra I Hayward JL Hypothalamicpituitarythyroid axis in breast cancerLancet httpsdoi101016s0140673674903444Adami HO Rimsten A Thoren L Vegelius J Wide L Thyroid disease andfunction in breast cancer patients and nonhospitalized controls evaluatedby determination of TSH T3 rT3 and T4 levels in serum Acta Chir ScandDargent M Berger M Lahneche B Thyroid function in patients with Cancerof the breast Acta Mustacchi P Greenspan F Thyroid supplementation for hypothyroidism Anlatrogenic cause of breast cancer JAMA Kapdi CC Wolfe JN Breast cancer Relationship to thyroid supplements forhypothyroidism JAMA httpsdoi101001jamaKuijpens JL Nyklictek I Louwman MW Weetman TA Pop VJ Coebergh JWHypothyroidism might be related to breast cancer in postmenopausalwomen Thyroid httpsdoi101089thy200515 Weng CH Chen YH Lin CH Luo X Lin TH Thyroid disorders and breastcancer risk in Asian population a nationwide populationbased casecontrolstudy in Taiwan BMJ 201883e020194 httpsdoi101136bmj 2017020194Sogaard M Farkas DK Ehrenstein V Jensen JO Dekkers OM SorensenHT Hypothyroidism and hyperthyroidism and breast cancer risk anationwide cohort study Eur J Endocrinol httpsdoi101530EJE150989 Angelousi AG Anagnostou VK Stamatakos MK Geiopoulos GAKontzoglou KC Mechanisms in endocrinology primary HT and risk forbreast cancer a systematic review and metaanalysis Eur J Endocrinol httpsdoi101530EJE110838 Hardefeldt PJ Eslick GD Edirimanne S Benign thyroid disease is associatedwith breast cancer a metaanalysis Breast Cancer Res Treat httpsdoi101007s1054901220193Stang A Critical evaluation of the NewcastleOttawa scale for theassessment of the quality of nonrandomized studies in metaanalyses Eur JEpidemiol httpsdoi101007s106540109491zKalache A Vessey MP McPherson K Thyroid disease and breast cancerfindings in a large casecontrol study Br J Surg httpsdoi101002bjs1800690731 Hoffman DA McConahey WM Brinton LA Fraumeni JF Jr Breast cancer inhypothyroid women using thyroid supplements JAMA Brinton LA Hoffman DA Hoover R Fraumeni JF Jr Relationship of thyroiddisease and use of thyroid supplements to breast cancer risk J Chronic Dis httpsdoi1010160021968184900626 Moseson M Koenig KL Shore RE Pasternack BS The influence of medicalconditions associated with hormones on the risk of breast cancer Int JEpidemiol httpsdoi101093ije2261000Shering SG Zbar AP Moriarty M McDermott EW O'Higgins NJ Smyth PPThyroid disorders and breast cancer Eur J Cancer Prevent Smyth PP Smith DF McDermott EW Murray MJ Geraghty JG O'Higgins NJA direct relationship between thyroid enlargement and breast cancer J ClinEndocrinol Metab httpsdoi101210jcem813Talamini R Franceschi S Favero A Negri E Parazzini F La Vecchia CSelected medical conditions and risk of breast cancer Br J Cancer httpsdoi101038bjc1997289 0cWang BMC Cancer Page of Simon MS Tang MT Bernstein L Norman SA Weiss L Burkman RT DalingJR Deapen D Folger SG Malone K Marchbanks PA McDonald JA Strom BLWilson HG Spirtas R Do thyroid disorders increase the risk of breast cancerCancer Epidemiol Biomarkers Prevent Turken O NarIn Y DemIrbas S Onde ME Sayan O KandemIr EG Yaylac IMOzturk A Breast cancer in association with thyroid disorders Breast CancerRes 200355R110 httpsdoi101186bcr609 Cristofanilli M Yamamura Y Kau SW Bevers T Strom S Patangan M Hsu LKrishnamurthy S Theriault RL Hortobagyi GN Thyroid hormone and breastcarcinoma Primary hypothyroidism is associated with a reduced incidenceof primary breast carcinoma Cancer httpsdoi101002cncr20881 Hellevik LR Vierendeels J Kiserud T Stergiopulos N Irgens F Dick ERiemslagh K Verdonck P An assessment of ductus venosus tapering andwave transmission from the fetal heart Biomech Model Mechanobiol httpsdoi101007s1023700901554Sandhu MK BrezdenMasley C Lipscombe LL Zagorski B Booth GLAutoimmune hypothyroidism and breast cancer in the elderly BreastCancer Res Treat httpsdoi101007s10549008 Ditsch N Liebhardt S Von Koch F Lenhard M Vogeser M Spitzweg CGallwas J Toth B Thyroid function in breast cancer patients Anticancer Res Grani G Dicorato P Dainelli M Coletta I Calvanese A Del Sordo M DeCesare A Di Matteo FM D'Andrea V Fumarola A Thyroid diseases inwomen with breast cancer La Clin Terapeut 20121636e401Kim EY Chang Y Lee KH Yun JS Park YL Park CH Ahn J Shin H Ryu SSerum concentration of thyroid hormones in abnormal and euthyroidranges and breast cancer risk a cohort study Int J Cancer httpsdoi101002ijc32283 Beatson GT On The Treatment Of Inoperable Cases Of Carcinoma Of TheMamma Suggestions For A New Method Of Treatment With IllustrativeCases1 Lancet Lopez Fontana CM Zyla LE Santiano FE Sasso CV CuelloCarrion FDPistone Creydt V Fanelli MA Caron RW Hypothyroidism reduces mammarytumor progression via Betacateninactivated intrinsic apoptotic pathway inrats Histochem Cell Biol httpsdoi101007s004180171544x MartinezIglesias O GarciaSilva S Regadera J Aranda A Hypothyroidismenhances tumor invasiveness and metastasis development PLoS One 47e6428 httpsdoi101371journalpone0006428 Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast cancer cell lines J Steroid Biochem Mol Biol httpsdoi101016s0960076096001173 Alyusuf RH Matouq JA Taha S Wazir JF The pattern of expression and roleof triiodothyronine T3 receptors and type I ²deiodinase in breastcarcinomas benign breast diseases lactational change and normal breastepithelium Appl Immunohistochem Mol Morphol httpsdoi101097PAI0b013e3182a20917 Pereira B Rosa LF Safi DA Bechara EJ Curi R Control of superoxidedismutase catalase and glutathione peroxidase activities in rat lymphoidans by thyroid hormones J Endocrinol httpsdoi101677joe01400073 GonzalezSancho JM Figueroa A LopezBarahona M Lopez E Beug HMunoz A Inhibition of proliferation and expression of T1 and cyclin D1genes by thyroid hormone in mammary epithelial cells Mol Carcinog httpsdoi101002mc10046Tosovic A Bondeson AG Bondeson L Ericsson UB Malm J Manjer JProspectively measured triiodothyronine 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acute myeloid leukemia aml is the most common type ofacute leukemia in adults caused by the clonal expansion ofundiï¬erentiated myeloid progenitor cells although mostpatients with aml can achieve complete remission by inductionchemotherapy the recurrence rate remains high and thus is themain factor aï¬ecting the outcomes of aml patients relapseoften develops from minimal residual disease in the protectivebone marrow bm microenvironment a comprehensiveunderstanding of the bm microenvironment is conducive to thediagnosis and personalized treatment of aml leukemic cellscytogenetics and molecular aberrations are the main factorsfor risk stratiï¬cation in patients with aml in additionthe bm microenvironment also plays a very important role thein the homing and survival ofbm microenvironment contains various componentssuchas immune cells stromal cells endothelial progenitor cellsextracellular matrix growth factors and cytokines theinteraction between leukemic cells and bm microenvironmentaï¬ects resistance to chemotherapy in aml the modulationof bm microenvironment in aml is currently undergoingpreclinical research and early clinical trials molecular inhibitorssuch as cxcr4 inhibitors vla4 inhibitors and eselectininhibitors are currently undergoing clinical trials immunecells and stromal cells are important components of the bmmicroenvironment and are also the main uencing factorsof leukemia development the estimate program isa common method to explore the microenvironment of manytumors recently it has also been used to explore theprognostic genes in the microenvironment of aml patients most studies have focused on diï¬erentially expressedgenes degs however the interaction and relationship betweengenes are open to investigate moreover the coding genes wereextensively explored but regions that encoded lncrnas andmirnas were less wellstudiedweighted gene coexpression network analysis wgcnais an algorithm commonly used in systems biology toexplore the correlation between gene sets and the clinic functionalization is achieved by constructing a freescalecoexpression gene network wgcna can identify highlyrelated genes and aggregate them into the same genetic modulecurrently wgcna is used in multiple ï¬elds such as cancer andnervous system or to identify potential biomarker candidates ornew therapeutic targets from genetic data the competition endogenous rna cerna hypothesis was anew regulatory mechanism between noncoding rna ncrnaand messenger rna mrna proposed by salmena in in this theory crosstalk between cernas is achieved byabbreviations aml acute myeloid leukemia auc area under curve bmbone marrow cam cell adhesion molecules cerna competing endogenousrnas deg diï¬erentially expressed gene david the database for annotationvisualization and integrated discovery go gene ontology icam1 intercellularadhesion molecule il10ra interleukin receptor subunit alpha keggkyoto encyclopedia of genes and genomes ppi proteinprotein interactiontcga the cancer genome atlas tlr6 toll like receptor tlr8 toll likereceptor tom topological overlap matrix wgcna weighted correlationnetwork analysisimmunerelated cerna network of amlcompetitively combining shared mirnas in recent yearsthe cernas hypothesis has attracted widespread attention in thestudy of molecular and biological mechanisms of tumorigenesisand development for example previous studies have foundthat lncrnarelated cernas were involved in the biologicalprocesses of glioblastoma and breast cancer theresearch on cernas of leukemia was generally based on thediï¬erential genes screened by leukemia and normal controls but no known module based on cernas network related tomicroenvironment in leukemia has been set upinformation ofthe aml cohortin this study mrnas mirnas and lncrnas data andclinicalfrom the cancergenome atlas tcga database were used to calculate theimmune and stromal scores of these aml cases using theestimate algorithm diï¬erentially expressed mrnas andlncrnas were applied to wgcna to identify the modulesmost relevant to the aml immune microenvironment thenthe immunerelated lncrnamirnamrna cerna networkwas established to screen genes with clinical signiï¬cance theseï¬ndings will help to better understand the role oftumormicroenvironment in aml and shed light on the developmentand progression of amlmaterials and methodsdata acquisitionall data sets of aml patients were downloaded from tcgadatabase1 the data used in this study met the following criteria excluding samples combined with other malignancies samples with lncrnas and mirnas and mrnas detection datafinally all lncrnas mrnas and mirnas expression proï¬lesof aml specimens and the corresponding clinical followupdata were downloadedidentiï¬cation of differentially expressedgenesthe estimate algorithm2 was used to calculate the immunescores and stromal scores of aml samples diï¬erentiallyexpressed genes degs such as lncrnas mirnas and mrnaswere identiï¬ed between high and low score groups stratiï¬ed bythe median value of immune scores and stromal scores usinglimma package all q values use fdr to correct the statisticalsigniï¬cance of the multiple test lncrnas and mrnas withlog fc and fdr were regarded as diï¬erentiallyexpressed while mirnas with log fc and fdr were regarded as diï¬erentially expressed then all the degs wereentered into r version auckland nz united states forcluster analysis based on the expression value of each sample inits respective data set the results were expressed in a clustergrameach column represents a sample and each row represents theexpression level of a given gene1portalgdccancergov2sourcefenetprojectsestimateprojectfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amldavid3 wasgo and pathway enrichment analysesthe database for annotation visualization and integrateddiscoveryenrichedbiological themes of degs functions particularly go geneontology terms and kegg kyoto encyclopedia of genes andgenomes pathway enrichment p was set as thecutoï¬ criterionapplied to analyzeweighted gene coexpression networkanalysiswgcna is an algorithm for identiï¬cation of gene coexpressionnetworksthrough highthroughput expression proï¬les ofmrnas or lncrnas with diï¬erent characteristics pairwisepearson correlation analysis was used to evaluate the weightedcoexpression relationship between all datasettopics in theadjacency matrix in this study wgcna was used to analyzemrnas and lncrnas to obtain the mrnas or lncrnas mostrelevant to aml immune microenvironmentcerna network construction andanalysisaccording to the results of wgcna we selected all mrnasand lncrnas in the most relevant module turquoise anddiï¬erentially expressed mirnas to construct a cerna networkbrieï¬y the associated cerna network in aml was constructedfollowing three stages prediction of lncrnamirna inorder to make lncrnas and mirnas map into the interactionssuccessfully we used the miranda4 and pita5 to get targetedlncrnas that mirnas may regulate prediction of mirnamrna three online databases miranda4 targetscan6 andmirwalk7 were used simultaneously for target mrna prediction construction of lncrnamirnamrna cerna networkthe cerna network was constructed based on the negativelyregulating target relationships of mirnamrna and mirnalncrna correlation pairsppi network constructionthe retrieval of interacting genes string database8 wasutilized to construct a proteinprotein interaction ppi networkof the degs identiï¬ed in the cerna network the interactingpairs with a conï¬dence score greater than were considered assignificant and were retained the degree represents the numberof interaction partnerssurvival analysiskaplanmeier plots weretherelationship between the overall survival of aml patientsand the expression level of mrnas lncrnas and mirnasthe statistical signiï¬cance of the correlation was tested by theconstructed to illuminatelogrank test and p was considered significant theanalysis was conducted using the r package of survivalstatistical analysisgraphpad prismtm san diego ca united states or rversion auckland nz united states software was usedfor all data analyses diï¬erences across groups were comparedusing kruskalwallis test for continuous variables diï¬erenceswere considered significant when p resultsimmune and stromal scores areassociated with aml clinicalparameterswe obtained gene expression proï¬les and clinical informationof aml patients from tcga database supplementarymaterial among them were male and were female with a median age range years oldaccording to the fab classiï¬cation there were cases of m0 m1 cases m2 cases m3 cases m4 cases m5 cases m6 cases and m7 case the estimate algorithm was used tocalculate the immune scores and stromal scores of aml patientsthe median immune score was range from to and the median stromal score was range from to we analyzed the relationship between immune scores andstromal scores and clinical parameters of aml patients caseswith m4 subtype aml had the highest immune scores while caseswith m3 subtype had the lowest immune scores p figure 1a similarly m4 cases had the highest stromal scoreswhereas m0 subtypes had the lowest p figure 1baccording to cytogenetics aml patients were divided intothree groups favorable intermediatenormal and poor therewas an obvious correlation between the cytogenetic risk andthe immune scores p figure 1cfavorable vsintermediate p favorable vs poor p intermediate vs poor p but no significant correlationbetween the cytogenetic risk and the stromal scores was observedp figure 1dscoreand high immunestromalusing the median immune or stromal score as a thresholdaml patients were divided into two groups with lowimmunestromalscoresurvival analysis showed that the survival rate of aml patientswith low immune scores was significantly higher than that ofpatients with high immune scores p figure 1ehowever there was no significant diï¬erence in survival betweenpatients with low stromal scores and those with high stromalscores p figure 1f3httpdavidncifcrfgov4httpwwwmicrorna5genieweizmannacilpubsmir07mir07_exehtml6httpwwwtargetscan7http12920671508tringdbidentiï¬cation of differentially expressedgenes based on immune scoresand stromal scoressetting the cutoï¬ criteria as log fc and fdr we identiï¬ed mrnas figure 2a and lncrnasfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure immune and stromal scores are associated with aml clinical parameters ab distribution of immune scores a and stromal scores b for aml fabsubtypes cd the correlation between immune scores c and stromal scores d and aml cytogenetic risk ef kaplanmeier survival curve based on immunese and stromal scores f aml acute myeloid leukemiafigure heatmap of differentially expressed genes in the high and low immunestromal score groups a mrnas b lncrnas and e mirnas based onimmune scores c mrnas d lncrnas and f mirnas based on stromal scoresfigure 2b based on immune scores and mrnasfigure 2c and lncrnasfigure 2d based onstromal scores setting the cutoï¬ criteria as log fc and fdr we identiï¬ed and mirnas based onimmune scores figure 2e and stromal scores figure 2frespectively the degs oflow vs high immunethefrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top go terms in each of biological process were performed for functional enrichment clustering analysis a top25 significant go terms based onupregulated genes in immune scores b top25 significant go terms based on upregulated genes in stromal scores c top25 significant go terms based ondownregulated genes in immune scores d top25 significant go terms based on downregulated genes in stromal scores go gene ontologyscore or stromal score groups were illustrated in the heatmap figure aml and thus require further research to determine theirbiological contributionfunctional enrichment analysis of degsbased on the david the databasefor annotationvisualization and integrated discovery gene annotation tool weperformed go analyses of both upregulated and downregulateddegs the top go biological process indicated that theupregulated degs based on immune or stromal scores wereprimarily enriched in neutrophil degranulation regulationof immune response signal transduction and ammatoryresponse figures 3ab while the downregulated degs basedon immunestromal scores were primarily enriched in rrnaprocessing regulation of translation regulation of transcriptionand cell diï¬erentiation figures 3cd subsequently weperformed kegg kyoto encyclopedia of genes and genomespathway enrichment and interrelation analysis kegg analysisrevealed that the upregulated degs were mainly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingpathway hematopoietic cell lineage and cell adhesion moleculescams pathways figures 4ab while the downregulateddegs were mainly enriched in ribosome metabolism pi3kaktsignaling pathway transcriptional dysregulation in cancer andmirnas in cancer figures 4cd above analyses revealedthatthese degs play a vital role in the development ofconstruction of weighted correlationnetwork analysis and identiï¬cation ofkey modulesbased on the results of survival analysis degs based on immunescores were selected for subsequent analysis the best β valuein the lncrnamrna coexpression network was which wascalculated using the diï¬erential lncrnas diï¬erentialmrnas and their expression data in leukemia samples nextthe method of dynamic tree cutting was used to producecoexpression modules finally modules of lncrnamrnacoexpression networks were generated and the heat map plot oftopological overlap matrix tom was shown figure 5a eachmodule was calculated and plotted with its corresponding clinicalcharacteristics correlation analysis showed that turquoisemodule displayed the highest relationship with aml immunescores r which included mrnas and lncrnasfigure 5b these mrnas were further used to performthe gene enrichment analysis the genes were most relatedto neutrophil degranulation immune response ammatoryresponse signal transduction and tolllike receptor signalingpathway figure 5c in addition genes were highly enriched ininfection osteoclast diï¬erentiation nodlike receptor signalingfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure top kegg pathway analysis were performed for functional enrichment clustering analysis a top25 significant kegg pathways based onupregulated genes in immune scores b top25 significant kegg pathways based on upregulated genes in stromal scores c top25 significant kegg pathwaysbased on downregulated genes in immune scores d top25 significant kegg pathways based on downregulated genes in stromal scores kegg kyotoencyclopedia of genes and genomespathway metabolic pathways and hematopoietic cell lineage bykegg analysis figure 5dcerna network constructionsince the turquoise module showed the highest relationshipwith aml immune scores we selected lncrnas and mrnas inthe turquoise module and diï¬erentially expressed mirnasbased on immune scores to construct a cerna network firstlybased on the pita and mircode online database that matchespotential mirnas with lncrnas a total of lncrnamirnapairs contained lncrnas and mirnas then we searchedfor the mrnas targeted by the diï¬erentially expressed mirnasusing three target gene prediction websites miranda mirwalkand targetscan using these websites we detected and target mrnas respectively based on the venn intersectionanalysis target mrnas were selected subsequently wematched the predicted target genes with the mrnas in theturquoise module then we constructed the cerna networkby integrating the mirnalncrnamrna interactions at lasta ï¬nal lncrnamirnamrna cerna network was constructedwith lncrnas mirnas and mrnas figure 6aproteinprotein interaction ppinetwork analysisto further explore the interplay among the mrnas in cernawe constructed a ppi network based on the string theretrieval of interacting genes online database figure 6b inthe network tlr8 toll like receptor icam1 intercellularadhesion molecule tlr6 toll like receptor and il10rainterleukin receptor subunit alpha had higher degrees and respectively supplementary table the genes encoding these proteins have been conï¬rmed tobe associated with immune microenvironment and leukemiaprogression association between mrnas mirnasand lncrnas in cerna and overall amlsurvivalwe further analyzed the prognostic values of mrnas mirnasand lncrnas in the cerna network subjects were dividedinto highexpression and lowexpression cohorts accordingto the median value ofthese genes for overall survivalfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure wgcna was used to analyze genetic modules a cluster diagram of coexpression network modules based on topological overlap in mrna andlncrna b study the relationship between each module with their corresponding clinical characteristics c go analysis showed the gene symbols and geneinteractions in the turquoise module d kegg analysis was used to study the pathway enrichment in the turquoise module wgcna weighted correlation networkanalysis go gene ontology kegg kyoto encyclopedia of genes and genomesthrough the package survivalthe highexpression and lowexpression cohorts were splitfor the logrank testin rsoftware out of lncrnas ac0099485 cmahp cta331p31 fcgr2c grk6p1 linc00539 linc01272 mipepp3psmb8as1 rp11266l95 rp11320g101 rp11421f163rp11439e1910 rp11792a83 and stag3l2 out of mirnas hsamir125b5p and hsamir3383p and out of mrnas agpat3 ankrd27 cbx2 ccnd2cd300lb cyth1 erg gdf11 igf1r kiaa0513 kiaa0930larp1 lfng lpcat1 nrep nudt16 pou2f2 ppm1hptafr qsox2 rab3d ralgps2 siglec7 slc43a2 srsf6tnfaip2 tns3 trib1 zbtb5 znf70 and znrf1 wereassociated with overall survival according to the logrank testp representative genes are shown in figure age ¥ years vs years and risk group favorablevsintermediatenormal vs poor were also associated withoverall survival according to the logrank test p and respectively the above mentioned lncrnas mrnas and mirnas were brought into further multivariatecox proportional hazard regression analysis with age and riskgroup finally mrnas ccnd2 erg lpcat1 nudt16ralgps2 tnfaip2 and znf70 lncrnas cmahp fcgr2cpsmb8as1 rp11266l95 rp11320g101 rp11792a83 andstag3l2 and mirnas hsamir125b5p and hsamir3383p were independently associated with overall survival table discussionin recent years studies about the roles of gene mutationsand chromosomalin the occurrence anddevelopment of aml and their prognostic values have madesignificant progress however the bm microenvironmentwhich also plays an important role in the pathophysiologytranslocationsfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure cerna network construction and proteinprotein interaction network a a lncrnamirnamrna cerna network was constructed by lncrnas mirnas and mrnas b a ppi network was constructed based on the string database the rectangle represents micrornas the circle represents mrnasand the triangle represents lncrnas the red represents upregulation in ishigh and the green represents downregulation in ishigh the size of the dot representsthe regulatory capacity of the mrna and larger points indicate stronger regulatory capability cerna competing endogenous rnas ppi proteinprotein interactionprocess in aml are poorly understood therefore mosttreatments previously targeted only tumor cells butfewtargeted the tumor microenvironmentindepth researchon the microenvironment of leukemia will help to furtherunderstand the mechanism of leukemia development and mayï¬nd new targets for microenvironment treatment this studyscreened microenvironmentrelated genes based on the tcgadatabase and further established microenvironmentrelatedlncrnamirnamrna cerna networks through wgcnafirst we calculated the immune scores and stromal scores ofaml patients based on the estimate algorithm and found thatthese scores were related to the fab typing of aml in additionimmune scores were significantly correlated with cytogenetic riskand overall survival estimate is a new algorithm to inferthe level of stromal and immune cells in tumor tissues andtumor purity using gene expression data high immunescores in the bm samples from patients with poor prognosisindicated that more immune cells were recruited in their bmfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlfigure correlation between mrnas mirnas and lncrnas in cerna and overall aml survival in tcga kaplanmeier survival curves with the logrank testwere performed for the representative mrnas mirnas and lncrnas cerna competing endogenous rnas aml acute myeloid leukemia tcga the cancergenome atlasmicroenvironment this may be due to that aml cells activelyshape the bm environment and immune cells to promote diseaseprogression through cellular structural and functional changes however there was no significant correlation betweenstromal scores and cytogenetic risk or survival of aml patientssuggesting that the proportion of stromal cells were comparablein diï¬erent group possible explanation may be that stromal cellsplay an important role in solid tumors while its role inleukemia is not as strong as in solid tumors then we identiï¬ed diï¬erentially expressed mrnas mirnasstromaland lncrnas based on the immune scores orscores functional enrichment analysis indicated thatthesedegs were mainly involved in immune and ammatoryresponses consistent with these results previous studies haveshown that the biology of the immune system is essentialfor the formation of a complex bm microenvironment in recent yearsthe immunologicalcharacteristics of aml has increased and the developmentof eï¬ective aml immunotherapy strategies has attractedwidespread attention the understanding ofin recent years important advances in cerna coexpressionnetwork research have developed rapidly the disruption offrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amltable multivariate cox proportional hazard regression analysis of lncrnas mrnas and mirnasgenesccnd2erglpcat1nudt16ralgps2tnfaip2znf70cmahpfcgr2cpsmb8as1rp11266l95rp11320g101rp11792a83stag3l2hsamir125b5phsamir3383phr 95ci pthe cerna network balance is a major cause for tumorigenesis therefore understanding the complex interactions betweendiï¬erent cerna networks will lead to an indepth understandingof gene regulatory networks and has implications for cancertreatment in addition the lncrnamirnamrna cernanetwork can predict the prognosis of the disease for examplea lncrnamirnamrna cerna network was established basedon rnaseq data of breast cancer from tcga which consistsof mirnas lncrnas and mrnas multiplexcox regression analyses showed that four of these lncrnasadamts9as1 linc00536 al3914211 and linc00491 havesignificant prognostic value wang identiï¬ed lncrnas mirnas and mrnas from a database toconstruct a lncrnamirnamrna cerna network inthe network a univariate and multivariate cox proportionalhazard regression analysis was used to establish a survivalmodel with target mrnas hoxa9 insr krit1 mybspry2 ube2v1 wee1 and znf711 where auc area undercurve is indicating the sensitivity and speciï¬city ofprognostic prediction however the screening of diï¬erentialgenes in this study was based on leukemia patients andnormal people and did not focus on tumor microenvironmentso far there is no cerna research based on the leukemiamicroenvironment in our research the screening of diï¬erentialgenes was based on the immune score then wgcna wasused to identify the modules most relevantto the amlimmune microenvironment then using wgcna and mirnaprediction websites a lncrnamirnamrna cerna networkconsisting of lncrnas mirnas and mrnaswas constructedsubsequently we built a ppi network predicting theinteraction among the proteins encoded by the degs inthe cerna network tlr8 icam1 tlr6 and il10ra hadhigher degrees tlr8 and tlr6 are members of the tolllike receptor family which is upstream to the transcriptionittransportationthe innate immune system andfactor nfκb and part ofplays an importantin progression of aml roleicam1 is one of the cams a large class of transmembraneproteinsinvolved in the binding of cells to another cellor extracellular matrix and involved in cell proliferationdiï¬erentiation movementandtissue structure the protein encoded by il10ra is areceptor for interleukin which has been shown to mediatethe immunosuppressive signal ofinterleukin and thusinhibits the synthesis of proammatory cytokines and isreported to promote survival of progenitor myeloid cellsthrough the insulin receptor substrate2pi3kakt pathway these results indicated that this novel cerna networkwere closely associated with immune microenvironment andprogression of amlapoptosisfurthermore lncrnas mirnas and mrnas withprognostic signiï¬cance were screened out which could be usedas biomarkers for prognosis among the genes with prognosticsigniï¬cance in our module of immunerelated cerna networkthere were aml related reports about cbx2 ccnd2 ergigf1r larp1 lfng nudt16 pou2f2 ptafr rab3dsiglec7 srsf6 tnfaip2 trib1 zbtb5 and znrf1 themost reported of which were erg ccnd2 and ifg1r ergtranslocation was involved in the occurrence and developmentof aml and high expression of erg was a poor prognosticfactor for patients with normal karyotype aml ccnd2mutations were more common in cbfaml and it was also afrequent mutation event in t aml ccnd2 leadedto increased phosphorylation of retinoblastoma proteins leadingto significant cell cycle changes and increased proliferation ofaml cell lines nicolas chapuis found that igf1spontaneous lesions played a key role in pi3kakt activation ofaml cells providing strong evidence for targeting igf1r as apotential new therapy for aml the functions of agpat3ankrd27 cd300lb cyth1 gdf11 kiaa0513 kiaa0930lpcat1 nrep ppm1h qsox2 ralgps2 slc43a2 tns3and znf70 in aml have not been reported we identiï¬ed lncrnas with clinical signiï¬cance among them only cmahpwas reported to be related to mllpositive aml andother lncrnas have not been reported in leukemia twomirnas including hsamir125b5p and hsamir3383p havebeen reported to be associated with a variety of cancers but no studies have been reported related to aml all theseunreported mrnas mirnas and lncrnas may be potentialnovel biomarkers or therapeutic targets for amlis importantto note that our current research haslimitations we selected the target data from the tcga publicdatabase only through the biological algorithm method weshould further verify the results of this in clinical patientsin further studyconclusionin summary a comprehensive bioinformatics analysis wasperformed on the aml dataset in tcga with an emphasison the immune microenvironment using wgcna andfrontiers in oncology wwwfrontiersinaugust volume 0cwang immunerelated cerna network of amlmirna prediction programs an immunerelated lncrnamirnamrna cerna network was established and degs withprognostic value were further identiï¬ed further studies of thesegenes are needed in the clinic and may provide new insights intothe pathogenesis of aml this study increases our understandingof the complex interactions between aml tumor cells and the bmmicroenvironment and may provide novel prognostic factors andtherapeutic targetsdata availability statementall data sets of aml patients were downloaded from the cancergenome atlas tcga database portalgdccancergovauthor contributionsyfl cw and zj conceptualization and design sw dataacquisition and writing original draft sw ly yx and dzmethodology sw and cw data analysis and interpretationyjl and yfl writing review and editing yfl cw and zjproject administration all authors contributed to the andapproved the submitted versionfundingthis work was supported by the national natural sciencefoundation of china grant numbers and u1804191and the henan medical science and technology research projectgrant number acknowledgmentswe thank the tcga database for the availability of the datasupplementary materialthe supplementary materialonline202001579fullsupplementarymaterialfor this can be foundwwwfrontiersins103389foncatreferences ferrara f schiï¬er ca acute myeloid leukaemia in adults lancet doi 101016s0140673612617279 zeng z shi yx samudio ij wang ry ling x frolova o targetingthe leukemia microenvironment by cxcr4 inhibition overcomes resistanceto kinase inhibitors and chemotherapy in aml blood doi 101182blood200805158311 shafat ms gnaneswaran b bowles km rushworth sa the bone marrowmicroenvironmenthome of the leukemic blasts blood rev doi 101016jblre201703004 bullinger l döhner k döhner h genomics of acute myeloid leukemiadiagnosis and pathways j clin oncol doi 101200jco ayala f dewar r kieran m kalluri r contribution of bonemicroenvironment to leukemogenesis and leukemia progression leukemia doi 101038leu2009175 uy gl rettig mp motabi ih mcfarland k trinkaus km hladnik lm a phase study of chemosensitization with the cxcr4 antagonist plerixaforin relapsed or refractory acute myeloid leukemia blood doi 101182blood201110383406 rashidi a uy gl targeting the microenvironmentin acute myeloidleukemia curr hematol malig rep doi 101007s11899 austin r smyth mj lane sw harnessing the immune system in acutemyeloid leukaemia crit rev oncol hematol doi 101016jcritrevonc201604020 yehudairesheï¬ s attiasturgeman s sabbah r gabay t musallam rfridmandror a abnormal morphological and functional nature ofbone marrow stromal cells provides preferential support for survival of acutemyeloid leukemia cells int j cancer doi 101002ijc yoshihara k shahmoradgoli m martÃnez e vegesna r kim h torresgarciaw inferring tumour purity and stromal and immune cell admixture fromexpression data nat commun doi 101038ncomms3612 yan h qu j cao w liu y zheng g zhang e identiï¬cation 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chrysoperla nipponensis okamoto which has the unique diapause phenotype distinguishable from nondiapause adult is an ideal model anism for studying the mechanism of reproductive diapause however there is no reliable and effective reference genes used for the reproductive diapause study of c a0nipponensis therefore in this study we evaluated the expression stability of candidate reference genes tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin αtub in adults under diapause and nondiapause induction conditions using four statistical algorithms including genorm normfinder bestkeeper and ct method results showed that arp3 and tub1 were the most stable reference genes in all samples and in the adult tissues group arp3 and rps5 were the most stable reference genes in the development degree group αtub and ef1a were unstable reference genes under the conditions of this study meanwhile to verify the reliability of the reference genes we evaluated the relative expression levels of vg and vgr in different treatments significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was respectively observed when using arp3 as the reference gene but not when using an unstable reference gene the reference genes identified in this work provided not only the basis for future functional genomics research in diapause of c a0nipponensis and will also identify reliable normalization factors for realtime quantitative realtime polymerase chain reaction data for other related insectskey words chrysoperla nipponensis okamoto reference genes qrtpcr reproduction diapausedue to the advantages of high sensitivity rapidity specificity and accuracy bustin et a0al valasek et a0al vanguilder et a0al shakeel et a0al quantitative realtime polymerase chain reaction qrtpcr has been widely used in the study of animals plants and microanisms roy et a0al jia et a0al zhang et a0 al ding et a0 al sun et a0 al qrtpcr is the most commonly used method for the expression analysis of target genes however the reliability of qrtpcr results in different samples is determined by a variety of factors among which the use of stably expressed reference genes is an important link for accurate detection of gene expression changes by qrtpcr bustin et a0 al at present several commonly used reference genes for data normalization include tubulin actin ribosomal protein elongation factor 1α glyceraldehyde3phosphate dehydrogenase 18s ribosomal rna and other genes bustin vanguilder et a0al however more and more studies have found that these reference genes do not show consistent expression patterns under different experimental conditions and even affect the reliability of experimental results therefore in order to obtain stable and reliable normalization factors reference genes with stable expression are usually used for correction and standardization to reduce errors between samples selection and evaluation of reference genes have become a necessary step before quantifying the expression of target genes accuratelynowadays there have been many studies on the selection of reference genes for insects such as sesamia inferens helicoverpa armigera aphis gossypii myzus persicae etc lu et a0 al shakeel et a0al zhang et a0al ma et a0al kang et a0al in these studies four statistical algorithms including genorm normfinder bestkeeper and ct method were mainly used to analyze the ct values obtained by qrtpcr of reference genes under various experimental conditions shakeel et a0 al finally the stability of the candidate reference genes was determined according to the geometric mean value of each gene ranked using different the authors published by oxford university press on behalf of entomological society of americathis 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 cited for commercial reuse please contact spermissionsoupcom 0c of insect science vol no algorithms and the most suitable reference gene was selected for the target gene expression analysis xiao et a0al kang et a0al chrysoperla nipponensis okamoto as one of the important predatory natural enemies of agricultural and forestry pests prefers to eat aphids thrips and other pests okamoto nie et a0al because of its characteristically wide geographical distribution and broad range of host prey niijima syed et a0al it has good prospects for widespread application in biological control mcewen et a0 al memon et a0 al reproductive diapause is an important way for c a0nipponensis adults to escape from adverse environments xu et a0al at present there have been many reports on the diapause of c a0nipponensis xu et a0al found that the body color of c a0nipponensis was green during the reproductive period but turned brown and yellow during the diapause period chrysoperla nipponensis belongs to the photoperiodic sensitive insect the adult diapause was induced by short photoperiods xu et a0al chen et a0al found that different photoperiods affected the material content eg protein and glycogen of c a0nipponensis diapause induced by the short photoperiod was beneficial to the storage of c a0nipponensis chen et a0al we expect an exponential increase of diapause research on c a0nipponensis at the molecular level in the near future thus stable and reliable reference genes are important for accurately quantifying gene expression of c a0nipponensisribosomal proteins and ribosomal rna have been used as reference genes in previous diapause studies for example williams et a0al used ribosomal protein rp49 as a reference gene to study the natural variation of drosophila melanogaster diapause williams et a0 al and sim and denlinger used ribosomal protein large subunit rpl19 as a reference gene in a study of ovarian development of culex pipiens during overwintering diapause sim and denlinger this indicates that under the same experimental conditions the selected reference genes in different species research are also differentin this research candidate reference genes were selected including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub whose expression profiles were measured by the qrtpcr the stability was analyzed by four statistical algorithms genorm normfinder bestkeeper and ct method in different developmental stages reproductive and diapause of adults and among different tissues the optimal reference genes under different conditions were determined which contributed to the accurate expression of target genes for future researchmaterials and a0methodsinsecta stable population of c a0 nipponensis was maintained in an artificial climate chamber rsz intelligent artificial climate chamber changzhou guohua jiangsu province under the following conditions a0± °c temperature a0± relative humidity rh and long photoperiod of l d h in our laboratory the eggs were collected by cutting the stalk and incubated in fingertip tubes a0cm in diameter and a0cm in height the primary hatching larvae were fed megoura japonica matsumura whose host plant was vicia faba l a0the adults were paired immediately after emergence in a bottle a0cm in diameter and a0cm in height fed a dry brewers yeast feed mixed with sucrose in a ratio of and then minced in a mortar and sifted through mesh and honey water the diapause adults used in this study were kept under the conditions of short photoperiod of ld h in all processes from eggs larvae pupae to adults whereas the nondiapause adults were kept under the conditions of long photoperiod of ld a0hsample collection development degree samples from individuals from varying developmental stages included female adults in the diapause induction period d under the short photoperiod the diapause maintenance period d under the short photoperiod the diapause termination period d under the short photoperiod and the reproduction period d under the long photoperiod each sample which included three to four females was independently replicated three times as three biological replicates adult tissues reproduction seven different tissues were collected from reproductive adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of reproductive adults under long photoperiod were collected on the 10th day after emergence each tissue required about a0mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues diapause seven different tissues were collected from diapause adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of diapause adults under short photoperiod were collected on the 20th day after emergence each tissue required about a0 mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues samples from reproductive and diapause adult tissues all samples samples from group and all treatments were immediately frozen with liquid nitrogen and stored in an ultralow temperature refrigerator at °c prior to rna extractiontotal rna extraction and cdna synthesisin this study total rna was extracted using minibest universal rna extraction kit takara japan and dnase i a0 was used for digestion of the membrane rna integrity was estimated by agarose gel electrophoresis rna concentration and purity were measured with a nanodrop one spectrophotometer thermo scientific then 1μg rna was reversetranscribed into the firststrand complementary dna cdna according to the hiscript ii q rt supermix for qpcr gdna wipers vazyme nanjing china instructions and stored at °c all cdna was diluted 10fold with dnasernasefree sterile water before usecandidate reference gene selection and primer a0designaccording to several commonly used reference genes candidate reference gene sequences were obtained by screening from the existing transcriptome of c a0 nipponensis in the laboratory namely tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub in order to ensure the predictive accuracy of the selected sequences we conducted blast alignment all primers were designed using primer premier based on the following criteria gc content annealing temperature °c and primers length a0 bp and the specificity of each pair of amplicons was determined by qrtpcr followed by agarose gel electrophoresis and melting curve analysis the amplification efficiency of the pcr was calculated by using the formula e a0 a0 slope the slope was obtained by the standard curve which was generated by qrtpcr of a series of continuously diluted cdna samples 0c of insect science vol no realtime qrtpcr analysisthe 20µl total reaction volume were configured according to the protocol of chamq sybr qpcr master mix vazyme contained 10µl a0à chamq sybr qpcr master mix a0µl a0μm of each gene specific primer a0µl of cdna and a0µl of ddh2o the amplification reaction program was set as follows predenaturation at °c for a0s followed by cycles of denaturation at °c for a0s annealing at °c for a0 s the melting curves were analyzed in the °c temperature range after amplification step the reaction was performed on a roche lightcycler96 instrument to obtain ct values amplification curves melting curves and standard curves all samples were carried out in four technical and three biological replicates and the negative control no template was performed in paralleldata analysisct values for all samples were exported into an excel spreadsheet and were used to analyze the stability of candidate reference gene expression by genorm normfinder bestkeeper and ct method the comprehensive ranking were performed following methods adapted from xiao et a0 al the optimal number of genes was determined by the pairwise variation vnn1 between the normalization factors calculated by genorm among the four algorithms genorm and normfinder need to convert the original ct value according to the corresponding requirements before analysis in genorm the stability ranking of genes was determined by the expression stability value m value in bestkeeper the stability ranking of genes was determined by the coefficient of variation cv and sd in normfinder the stability ranking of genes was determined by the gene expression stability value sv in ct method the stability of genes was ranked according to the sd of genes ct values in four statistical algorithms genes with the lowest value were the most stably expressedvalidation of reference a0genesin most insects vitellogenin vg and vitellogenin receptor vgr play important roles in the reproductive process of female insects vg is taken up by developing oocytes through receptormediated endocytosis rme thereby promoting the development of oocytes and the formation of eggs in this process vgr is the main receptor mediating endocytosis previous studies have shown that reproductive diapause arrests development of oogenesis and vitellogenesis tatar and yin and the expression levels of the vg and the vgr in nondiapause female were significantly higher than those in reproductive diapause female jiang et a0al in order to evaluate the effectiveness of the selected reference genes the expression levels of the target genes vg and vgr were respectively detected by qrtpcr in the different development degree and tissues of adults and the most unstable reference gene was used for comparison in parallel the reaction system and program were the same as for qrtpcr of reference genes and four technical and three biological replicates were performed for each treatment the relative expression levels of vg and vgr were respectively calculated in excel using the ct method the differences of target genes expression levels were analyzed by tukeys test using spss software spss inc under different experimental conditionsresultsselection and primer performance of candidate reference a0genesthe candidate reference genes including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub were selected to identify the normalization factors for qrtpcr analysis and the sequence information has been submitted to genbank and the accession numbers are shown in supp table online only to determine the amplification specificity of the primers agarose gel electrophoresis and melting curve analysis were performed all primer pairs showed a single band and a single peak fig a0 to obtain correlation coefficient r2 and amplification efficiency e of pcr a standard curve was generated with the 10fold dilution series of cdna the results showed that the amplification efficiency ranged from to and the correlation coefficient varied from to supp table [online a0only]expression profiling of candidate reference a0genesto evaluate expression levels of the candidate reference genes the cycle threshold ct values under different groups were obtained by qrtpcr and represented by boxplot fig a0 the results indicated that ct values of all candidate reference genes were different under different conditions and also varied under the same condition overall ct values ranged from to among them the genes with higher expression abundance were tub1 rps26e and actin followed by 128up arp3 arpc5 rps5 and ef1a the genes with lower expression abundance were αtub and gapdh according to the ct value range of each candidate reference gene the genes with relatively stable expression were tub1 and arp3 whereas the most unstable genes were ef1a actin and αtubexpression stability of candidate reference genes under different conditionsin this study four statistical algorithms were used to analyze the expression stability of the candidate reference genes under different conditions including genorm normfinder bestkeeper and ct method as different statistical algorithms would generate different ranking patterns the comprehensive ranking of genes was finally determined through the geometric mean of sequencing bestkeeper the original ct values were used for analysis evaluated the stabilities of the candidate reference genes according to the cv and sd of the ct values ct method the difference values of original ct values were used for analysis performed stability ordering according to the mean sd of ct value genorm and normfinder the original ct values were converted for analysis sequenced the candidate reference genes according to the stability values the lower the stability value the more stable the gene expressiondevelopment degree of a0adultsthe expression stability of the candidate reference genes at different periods of reproductive and diapause female showed that the top four ranked genes identified by the genorm bestkeeper normfinder and δct method were similar but the rank order was slightly different arp3 and rps5 were the first and second stably expressed genes in the four statistical algorithms supp table [online only] and comprehensive ranking analysis fig a0 as for the third and fourth ranked gene tub1 and actin identified by comprehensive ranking analysis were the same as those generated by genorm and normfinder while bestkeeper selected arpc5 and tub1 ct method selected actin and rps26e supp table [online only] αtub was ranked by genorm bestkeeper normfinder and δct method as the least stable gene among the candidate reference 0c of insect science vol no fig specificity a and product length b of qrtpcr amplification for ten candidate reference genesgenes during different development degrees of adults supp table [online a0only]adult a0tissuesthe expression stability ranking of candidate reference genes in different tissues of reproductive and diapause females was varied according to the four statistical algorithms in different tissues of reproductive females the top four genes were rps5 arp3 arpc5 and tub1 respectively fig a0 but the rank order of the four genes was significantly different among different statistical algorithms rps5 was ranked first by genorm and ct method and was ranked third and fourth by normfinder and bestkeeper respectively arp3 was ranked first by bestkeeper and was ranked second third and fifth by normfinder δct method and genorm respectively arpc5 was ranked first and second by genorm and δct method and was ranked fourth and fifth by normfinder and bestkeeper respectively tub1 was ranked first and second by normfinder and bestkeeper and was ranked fifth and sixth by δct method and genorm respectively supp table [online only] however the four statistical algorithms found that ef1a was ranked as the least stable gene in the tissues from reproductive females supp table [online only]in different tissues of diapause females the top four genes were different from those of different tissues of reproductive females tub1 was ranked first by the comprehensive ranking followed by rps26e arp3 and 128up fig a0 through analysis it was found that ct method and normfinder displayed the same rankings for expression stability of candidate reference genes under this condition supp table [online only] tub1 was identified as the most stably expressed gene by genorm ct method and normfinder although it was ranked fifth by bestkeeper rps26e ranked steadily among the four statistical algorithms was ranked second by ct method and normfinder whereas it was ranked first and third by genorm and bestkeeper respectively arp3 was ranked first by bestkeeper with the smallest coefficient of variation whereas it was ranked third by ct method and normfinder in addition to being ranked third by genorm 128up was ranked fourth by bestkeeper ct method and normfinder supp table [online only] however in tissue from diapause females actin was consistently identified as the gene with the most unstable expression by the four statistical algorithms supp table [online only]in the reproductive and diapause female tissues the expression stability of the candidate reference genes was different in order to accurately determine the expression of the target genes the expression stability of candidate reference genes under the two conditions was analyzed according to the comprehensive ranking 0c of insect science vol no fig expression profiles of candidate reference genes in c a0nipponensis expression data are displayed as ct values for each reference gene using a box and whisker plot in different experimental conditions the line across the box is the median the box indicates the 25th and 75th percentiles the whiskers represent the 10th and 90th percentilesfig expression stability and comprehensive ranking of reference gene measured by the geomean method a a0lower geomean value indicates more stable expressiontub1 and ef1a were the most stable and unstable genes respectively whereas arp3 128up and arpc5 were ranked second third and fourth respectively fig a0 tub1 was the best candidate reference gene identified by normfinder and ct method and was ranked third and sixth by bestkeeper and genorm respectively arp3 was the most suitable candidate reference gene selected by bestkeeper and was ranked second by normfinder and ct method and fifth by genorm 128up and arpc5 were the most stable candidate reference genes identified by genorm whereas they were ranked separately fifth and sixth by normfinder and ct method and seventh and fifth by bestkeeper respectively supp table [online only] ef1a was identified as the least stable gene by genorm normfinder and δct method although bestkeeper selected actin as the least stable gene supp table [online only]all a0samplesin order to determine the best reference gene suitable for the different conditions of adults the stability of the ten candidate reference genes was ranked for all samples arp3 was identified as the most stable gene by the comprehensive ranking followed by tub1 arpc5 and rps5 whereas ef1a was identified as the least stable gene fig a0 0c of insect science vol no but the most and least stable genes identified by different statistical algorithms were slightly different arp3 was selected as the most stable reference gene by bestkeeper and ct method although tub1 and arpc5 were selected as the most stable reference gene by normfinder and genorm respectively ef1a was selected as the least stable reference gene by genorm and ct method despite it being ranked ninth by bestkeeper and normfinder supp table [online only]the best combination of candidate reference genes under different conditionsaccording to the pairwise variation vnn1 between the normalization factors and cutoff value calculated by genorm the number of reference genes required for optimum normalization in each experimental condition was determined the cutoff value of vnn1 a0 suggested that n reference genes were enough to make gene expression normalization otherwise n reference genes were needed the analysis results showed that all v23 were indicated that the optimal number of reference genes under each condition was two fig a0 more specifically arp3 and rps5 were the most stable gene combinations under adult developmental stage and reproductive adult tissues conditions tub1 and rps26e were the most stable gene combinations under adult diapause tissues conditions and arp3 and tub1 were the most stable gene combinations under adult tissues and all samples groups table a0relative expression levels of target genes vg and vgr genbank mt308983 mt522179 in the whole adult and from tissues of reproductive and diapause adults respectively when the most stable reference genes arp3 andor rps5 were used as normalization factors at different periods of reproduction and diapause the expression patterns of the target genes vg and vgr were consistent with low expression in the diapause period and rich expression in the late diapause and reproduction period however when the most unstable reference gene αtub was used as a normalization factor neither the target gene vg nor vgr showed a consistent expression pattern fig a0 under different tissue conditions when the most stable reference genes tub1 andor arp3 were used as the normalization factors the expression level of the target gene vg in the fat body of the reproductive female was significantly higher than that in the ovary of the reproductive female the expression level of the target gene vgr in the fat body of the reproductive female was lower than the ovary of the diapause female while when the most unstable reference gene ef1a was used as the normalization factor the expression pattern differed with normalization by tub1 and tub1arp3 fig a0 in general when the most stable reference genes were used as the normalization factors the accurate expression pattern of the target gene could be obtainedvalidation of reference a0genesthe stability of reference gene is very important for the analysis of expression level of target gene vg and vgr which are important for insect reproduction were selected to verify the applicability of the selected reference gene we examined the discussionqrtpcr has become an important means to explore gene expression level due to its high sensitivity rapidity specificity and accuracy and was widely used in physiology studies that investigated insect diapause such as drosophila melanogaster williams fig pairwise variations vnn1 was calculated by genorm to determine the optimal number of reference genes for accurate normalization in different conditions the cut off values under indicate that no additional genes are required for the normalization 0c of insect science vol no et a0al culex pipiens sim and denlinger leptinotarsa decemlineata lehmann et a0 al chrysopa septempunctata liu et a0al and pieris melete wu et a0al however the selection of appropriate reference genes was the key to accurately analyze the gene expression level for example under conditions of injury heatstressed and experimentally varied diets table recommendation for the best combination of reference genes based on the genorm and comprehensive rankings under various experimental conditionsgroupreference genemostdevelopment degreeadult tissues reproductionadult tissues diapauseadult tissuesall samplesarp3rps5tub1tub1 arp3 rps5arp3rps26earp3tub1leastαtubef1aactinef1aef1athe best reference gene was different in drosophila melanogaster ponton et a0al under biotic factors and abiotic stress inappropriate selection of the reference genes in locusta migratoria resulted in significant differences in the expression level of the target gene chitin synthase chs1 yang et a0al diapause of most insects was mainly affected by photoperiod and temperature in past studies the screening of reference genes of drosophila melanogaster ponton et a0al leptinotarsa decemlineata shi et a0al helicoverpa armigera zhang et a0al bombyx mori guo et a0al and harmonia axyridis qu et a0al under main environmental factors was completed by genorm normfinder bestkeeper and ct method in this study the expression profiles of candidate reference genes of c a0nipponensis were analyzed under different conditions by the same four statistical algorithms genorm vandesompele et a0 al normfinder andersen et a0al bestkeeper pfaffl and ct method silver et a0al different algorithms produced different stability rankings in order to obtain statistically consistent and accurate results we finally ranked the gene based on their stabilities determined by fig validation of selected reference genes under different periods a and tissues b of reproductive and diapause female in c a0 nipponensis relative expression levels of the vg and vgr in different samples using different normalization factors the most and least stable genes asterisks indicate significant differences in the expression levels of the vg and vgr r reproduction period d1 the diapause induction period d2 the diapause maintenance period d3 the diapause termination period 0c of insect science vol no comprehensive analysis method xiao et a0 al and selected the most stable reference genes under each condition as far as we know actin which played an important role in cell contraction and cytoskeletal maintenance was found in virtually all eukaryotic cells and was considered as an ideal reference gene for many anisms sürencastillo et a0al shakeel et a0al for example actin was used as a reference gene for normalization in the determination of genes related to reproductive and nutritional signaling such as vitellogenin of chrysopa septempunctata liu et a0al however in this study three genes related to actin were selected for analysis among which actin was similar to actin of c a0septempunctata which was also a member of the neuroptera in our study actin was the most unstable gene in the diapause female tissues but the actinrelated protein arp3 which was structurally homologous with actin showed better stability arp3 was selected as the most stable reference gene in adults of different developmental levels and all samples while it was the second most stable gene in the reproductive adult tissues and all adult tissues although arp3 was ranked as the third most stable gene in the diapause adult tissues it showed relatively stable expression in the expression profile tubulin which played an essential role in maintaining cell shape movement and intracellular material transport was also often used as a reference gene but different types of tubulin have different stability for example in the study of helicoverpa armigera the expression of βtub was relatively stable compared with that of αtub under almost all conditions zhang et a0 al similarly in this study αtub showed unstable expression and was the least stably expressed gene in adults of different developmental stages whereas tub1 was considered to be the most stably expressed reference gene in diapausing adult tissues and all adult tissues and was the second most stable gene in all samples ribosomal protein rp widely distributed in various tissues played an important role in protein biosynthesis and was widely used as a reference gene in many insects lu et a0al koyama et a0 al sun et a0 al in this study rps5 was considered to be stable in the tissues of reproductive females and ranked second among different developmental stages of adults elongation factors ef was a protein factor which promoted polypeptide chain to extension during the translation of mrna and was recommended as the ideal reference gene under different conditions of a variety of insects chapuis et a0 al ponton et a0 al however some studies showed that ef1α was one of the most unstable genes under certain conditions fu et a0 al in our study ef1a was found to be the most unstable gene in the reproductive adult tissues all tissues and all samples and the second least stable gene in the adults of different developmental stages and diapause adult tissues therefore it was not suitable for the study of c a0nipponensisrecently an increasing number of studies have demonstrated the importance of using multiple stably expressed reference genes for the accuracy of qrtpcr analysis ling and salvaterra yuan et a0 al kang et a0 al however this does not mean that the more reference genes increase the reliability of the results the study has indicated that either too few o | 0 |
" according to the who most chronic diseases including cancer can be prevented by identifyingtheir risk factors such as unhealthy diet smoking and physical inactivity this research examined the effectiveness ofa theorybased educational intervention on colorectal cancerrelated preventive nutritional behaviors among asample of anizational staffmethods in this interventional study employees of shahid beheshti university of medical sciences wererandomly divided into two groups intervention and control with cluster sampling the data gathering tool was aresearchermade questionnaire containing two parts of 10dimensional information and health belief modelconstructs the educational intervention was conducted for month and in four sessions in the form of classroomlecture pamphlet educational text messages via mobile phones and educational pamphlets through the officeautomation system two groups were evaluated in two stages pretest and posttest data were analyzed usingspss18 software analysis of covariance ancova and independent ttest intergroup comparisonsresults two groups were evaluated for variables such as age sex education level and family history of colorectalcancer and there was no significant difference between the two groups p after the months sinceintervention except for the mean score of perceived barriers which was not significant after intervention the meanscores of knowledge perceived susceptibility perceived severity perceived benefits perceived selfefficacybehavioral intention and preventive behaviors were significantly increased after the intervention in the interventiongroup compared to the control group p implementation of educational intervention based on health belief model was effective for thepersonnel and can enhance the preventative nutritional behaviors related to colorectal cancerkeywords educational intervention health belief model nutritional behavior colorectal cancer correspondence mohtashamghaffarisbmuacir1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iranfull 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 0crakhshanderou bmc medical education page of nearly million new cases of colorectal cancer arediagnosed every year worldwide with nearly half of theaffected patients losing their lives due to the disease approximately of men in and of women in are diagnosed with crc during their life time the incidence of colorectal cancer in iran ranges from to per annually with a death rate of about per hundred thousand and it accounts for approximately of all gastrointestinal cancerrelated deaths according to the latest cancer record in iran colonand rectum cancer ranked third in female cancers andfifth in male cancers the global incidence of crc is predicted to increase by to more than million newcases leading to million cancer deaths by therisk of colon cancer increases with age and is higher inmen than in women various factors are involved inthe development of various types of cancerincludingcolorectal cancer which can be attributed to geneticenvironmental and dietary factors among the riskfactors of colorectal cancer nutritionalfactors areconsidered to be the most important and preventableones so that to of cases can be prevented byproper nutrition [ ] colorectal cancer is also morecommon in iran than in other asian countries [ ]therefore the need to educate people about the nutritionalbehaviors associated with colorectal cancer is becomingmore and more evident theories and models identifyfactors that influence health and behavior which meansthat they can be used to develop programs the most effective training programs are based on the theorydrivenapproaches which are rooted in behaviorchanging modelsalso selecting appropriate model or theory is the first stepin the process of planning a training program [ ] asone of the most widely applied theories of health behaviorthe health belief model hbm posits that six constructspredict health behavior perceived susceptibility perceivedseverity perceived benefits perceived barriers perceivedselfefficacy and cues to action fig the hbmposits that when an individual perceives a serious threatalong with a way to reduce the threat they will be morelikely to take action to reduce the threat the hbmhas been applied to predict a wide variety of healthrelatedbehaviors such as being screened for the early detection ofasymptomatic diseases the model has been applied tounderstand patients responses to symptoms of disease lifestyle behaviors and behaviors related to chronicillnesses which may require longterm behaviormaintenance in addition to initial behavior change the research hypotheses are an intervention based onthe hbm can significantly promote colorectal cancer preventive behaviors the score for each and every constructof the hbm eg perceived awareness and susceptibilityperceived severity perceived benefitsbarriers and perceivedselfefficacy is increased significantly after the interventionin the experimental group as compared to the controlmethodsstudy design and samplingthis interventional study was conducted at shahidbeheshti university of medical sciences tehran iranfrom october to june fig health belief models components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbachs alphafor face validity a survey was done on employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tools questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbachs alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartletts research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedont know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring rangecorrect response dont knowresponse incorrect response strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individuals perception of his orher competence to successfully perform a behavior behavioral intention refers to a persons perceived probability orsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items Ï2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartletts test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of educationfemalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that onesintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the researcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med gimenogarca az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarca r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran anz j surg yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis becker mh the health belief model and sick role behavior health educmonogr janz n champion v strecher vj the health belief model k glanz bk rimerjanz nk becker mh the health belief model a decade later health educ qlp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c" | 0 |
" according to the who most chronic diseases including cancer can be prevented by identifyingtheir risk factors such as unhealthy diet smoking and physical inactivity this research examined the effectiveness ofa theorybased educational intervention on colorectal cancerrelated preventive nutritional behaviors among asample of anizational staffmethods in this interventional study employees of shahid beheshti university of medical sciences wererandomly divided into two groups intervention and control with cluster sampling the data gathering tool was aresearchermade questionnaire containing two parts of 10dimensional information and health belief modelconstructs the educational intervention was conducted for month and in four sessions in the form of classroomlecture pamphlet educational text messages via mobile phones and educational pamphlets through the officeautomation system two groups were evaluated in two stages pretest and posttest data were analyzed usingspss18 software analysis of covariance ancova and independent ttest intergroup comparisonsresults two groups were evaluated for variables such as age sex education level and family history of colorectalcancer and there was no significant difference between the two groups p after the months sinceintervention except for the mean score of perceived barriers which was not significant after intervention the meanscores of knowledge perceived susceptibility perceived severity perceived benefits perceived selfefficacybehavioral intention and preventive behaviors were significantly increased after the intervention in the interventiongroup compared to the control group p implementation of educational intervention based on health belief model was effective for thepersonnel and can enhance the preventative nutritional behaviors related to colorectal cancerkeywords educational intervention health belief model nutritional behavior colorectal cancer correspondence mohtashamghaffarisbmuacir1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iranfull 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 0crakhshanderou bmc medical education page of nearly million new cases of colorectal cancer arediagnosed every year worldwide with nearly half of theaffected patients losing their lives due to the disease approximately of men in and of women in are diagnosed with crc during their life time the incidence of colorectal cancer in iran ranges from to per annually with a death rate of about per hundred thousand and it accounts for approximately of all gastrointestinal cancerrelated deaths according to the latest cancer record in iran colonand rectum cancer ranked third in female cancers andfifth in male cancers the global incidence of crc is predicted to increase by to more than million newcases leading to million cancer deaths by therisk of colon cancer increases with age and is higher inmen than in women various factors are involved inthe development of various types of cancerincludingcolorectal cancer which can be attributed to geneticenvironmental and dietary factors among the riskfactors of colorectal cancer nutritionalfactors areconsidered to be the most important and preventableones so that to of cases can be prevented byproper nutrition [ ] colorectal cancer is also morecommon in iran than in other asian countries [ ]therefore the need to educate people about the nutritionalbehaviors associated with colorectal cancer is becomingmore and more evident theories and models identifyfactors that influence health and behavior which meansthat they can be used to develop programs the most effective training programs are based on the theorydrivenapproaches which are rooted in behaviorchanging modelsalso selecting appropriate model or theory is the first stepin the process of planning a training program [ ] asone of the most widely applied theories of health behaviorthe health belief model hbm posits that six constructspredict health behavior perceived susceptibility perceivedseverity perceived benefits perceived barriers perceivedselfefficacy and cues to action fig the hbmposits that when an individual perceives a serious threatalong with a way to reduce the threat they will be morelikely to take action to reduce the threat the hbmhas been applied to predict a wide variety of healthrelatedbehaviors such as being screened for the early detection ofasymptomatic diseases the model has been applied tounderstand patients responses to symptoms of disease lifestyle behaviors and behaviors related to chronicillnesses which may require longterm behaviormaintenance in addition to initial behavior change the research hypotheses are an intervention based onthe hbm can significantly promote colorectal cancer preventive behaviors the score for each and every constructof the hbm eg perceived awareness and susceptibilityperceived severity perceived benefitsbarriers and perceivedselfefficacy is increased significantly after the interventionin the experimental group as compared to the controlmethodsstudy design and samplingthis interventional study was conducted at shahidbeheshti university of medical sciences tehran iranfrom october to june fig health belief models components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zd and with an attrition rate of finally women subjects in the experimental and in thecontrol group were considered the random samplingmethod clustering and simple random sampling wasused in this study in order to choose from four facultiesfaculties of shahid beheshti university of medicalsciences four faculties were randomly selected and fromthese four faculties two faculties were assigned as intervention group and were considered as control grouprandom sampling method was used to select samplesfrom each clusterinclusion exclusion criteriabeing under years of age having satisfaction to participate in the study and not having serious diseases including gastrointestinal diseases were the inclusion criteriaalso not willing to continue with the study not completing the questionnaire in full and not attending in morethan two educational sessions were the exclusion criteriameasuresthe researchermade questionnaire was used for datacollection in this study three sources of existed toolsliterature review and expert view were used for itemgeneration this instrument consisted of two main partsas followpart one demographic questions about age gendereducational level and economic statuspart two constructs of the health belief model whichincludes knowledge perceived susceptibility perceivedseverity perceived benefits perceived barriersperceived selfefficacy behavioral intention andbehavior table validity and reliabilityface and content validities were applied for validationphase reliability was confirmed based on methods oftestretest and internal consistency cronbachs alphafor face validity a survey was done on employeesabout the difficulty in understanding the words andphrases the probability of misunderstanding the phrasesand lack of clarity in the meaning of the words somemodifications were made to the tools questions todetermine the content validity of the questionnaire twogastroenterologistsfive health education and healthpromotion specialists and one related expert were askedto complete the questionnaire the initial questionnairehad questions theconstructs of knowledgeperceived susceptibility perceived severity perceivedbenefits perceived barriers perceived selfefficacyintention and behavior had and questions respectively internal consistency was used todetermine the reliability of hbm structures the cronbachs alpha coefficient was for all structures andwas statistically acceptable the retest was used to ensure the reliability of the awareness variable in this way employees completed the questionnaire twice and theicc was obtained also construct validity wasperformed by exploratory analysis method the kmovalue was and bartletts research showed thetable description of study instrumentconstruct knowledge refers to a theoretical or practical understanding of asubject perceived susceptibility refers to subjective assessment of risk ofdeveloping a health problem perceived severity perceived severity refers to the subjectiveassessment of severity of a health problem and its potentialconsequences perceived benefits healthrelated behaviors are also influenced bythe perceived benefits of taking an actionno of items format items truefalsedont know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring rangecorrect response dont knowresponse incorrect response strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree perceived barriers healthrelated behaviors are also a function ofperceived barriers to taking action perceived selfefficacy refers to an individuals perception of his orher competence to successfully perform a behavior behavioral intention refers to a persons perceived probability orsubjective probability that he or she will engage in a given behavior items5 point likert scalestrongly disagree strongly agree items5 point likert scalestrongly disagree strongly agree items5point likert scalestrongly disagree to stronglyagreestrongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree strongly disagree disagree noidea agree strongly agree behavior refers preventative behaviors associated with colorectalcancer items5point likert scalealways to neveralways often sometimes rarely never 0crakhshanderou bmc medical education page of significant correlations among the items Ï2 df p therefore the data were suitable forconducting factor analysisinterventionboth intervention and control groups were pretestedusing the questionnaire the analysis of educational needsdetermined the educational methods educational package and the number of educational sessions was obtainedby the pretestreadabilitycomprehensibility and not complexity of educational contents for participants was obtained by pretesting materialssuch as pamphlets messages etc in a sample of employees who were not included in main researchresults assurance abouteducational intervention based on educational textmassagesover the course of days ten text messages were sentto the employees in the intervention group at am mostof which had been prepared according to the educationalobjectives ofthe constructs of knowledge perceivedsusceptibility perceived benefits perceived barriers andperceived selfefficacycounseling there waseducational pamphletstwo pamphlets were given to the employees during twoseparate sessions along with simultaneous provision ofindividuala possibility ofquestioning and answering any ambiguity regarding thecontent of pamphlets the first pamphlet containedsections on the signs and symptoms of colorectal cancerand the risk factors of this cancer and the secondpamphlet contained sections on methods of preventingthis cancereducational packages in the office automation systemeducational packages were uploaded on the staff automation system for days and the employees were askedto study it during the working hoursthe intervention was conducted month and followup months after the intervention the educationalcontents were taken from the trusted sources of theministry of health complemented by what the staffneeded to know about promoting nutritional behaviorsrelated to the prevention of colorectal cancer the education varied in form across the model constructs forperceived susceptibility the facts and figures of the incident rate of colorectal cancer were presented in theclass and for perceived severityimages of colorectalcancer problems were used also for perceived barrierseducational materials were used to somehow incite theindividuals to analyze the cost of optimal behavioragainst the costs of risks time etc involved in unhealthybehavior the educational content used for perceivedbenefits intended to raise awareness on the usefulness ofhealth promoting behaviors to reduce the risk of illnessor to understand the benefits of healthy behaviors infig the research process is presented in generalethical considerationsat first a permission was obtained from the universityto conduct the study and attend the healthcare centerthe samples were assured about the confidentiality oftheir specifications and information they were also toldthat their information will only be used for the purposeof this study and the data collection the participantswere allowed to enter and leave the study at any timesuitable conditions were provided for a proper understanding of questions and responses for the subjectsafter the end of the intervention period the controlgroup was also trained using the slides that were used totrain the intervention group an informed consent wasobtained from the participants the study on whichthese data analyses are based was approved by theethical board committee of shahid beheshti universityof medical sciencesdata analysisdata were analyzed by spss software kolmogorov smirnovtest was used to check the normality of the data to assessthe effectiveness of intervention on variables of knowledgeperceived susceptibility perceived severity perceived benefits perceived barriers perceived selfefficacy behavioralintention and behavior in the intervention and controlgroups two groups were evaluated in two stages pretestand posttest data were analyzed using spss18 softwareanalysis of covariance ancova and independent ttestintergroup comparisonsthe confidence level of and the significance level of were consideredin this studyresultsthe findings of this study showed no drop out until theend of study the questionnaire was completed in bothgroups in a complete and precise manner homogenizationwas done in the two groups by controlling variables such asage sex level of education and related family history theresults showed no significant relationship within thesevariables p table effectiveness of the educationalintervention in improving knowledge perceived susceptibility perceivedseverity perceived benefits perceived selfefficacybehavioral intention and behavior once age gender andlevel of education factors were adjusted was checkedthrough ancova the results revealed that the intervention was successful in improving constructs of thehealth belief model significantly in participants table the mean score ofintention and behavior in the 0crakhshanderou bmc medical education page of fig schematic diagram of designed interventions for colorectal cancer preventionexperimental and control groups before and after theintervention is presented in fig discussionthe purpose of this study was to investigate the effectsof educationalinterventions on the promotion ofcolorectal cancer prevention nutritional behaviors thekmo and bartletts test p results confirmed the suitability of the model for conducting factoranalysis the kmo is in the range if the value ofthe inedex is near to one the data are suitable for factoranalysis kaiser at least kmo to determinestable demographic and variables in intervention and control groups before the interventionvariablegroupintervention group n n control group n n agegenderlevel of educationfemalemalediplomaassociate degreeundergraduate degreeand higherhistory of specialdiet compliancefamily history of cancerchisquareyesnoyesnop value 0crakhshanderou bmc medical education page of table comparison of intervention and control groups in terms of health belief model constructs before and after the interventionp valueconstructsgroupsbefore interventionmean ± sd ± after interventionmean ± sd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± meandifference ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± knowledgeinterventioncontrolperceived susceptibilityinterventionperceived severityperceived benefitsperceived barriersperceived self efficacybehavioral intentionbehavioranalysis of covariance ancovacontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrolinterventioncontrol also bartlett test was used to confirm adequacy ofthe samples in the present study the mean score of behavioralconstruct increased after the intervention in the intervention group and there was significant differencebetween the two groups after the intervention in thisregard the results of this study are consistent with thefindings of abood hart roozitalabi alidoosti and davoodi studies behavioral intention is the thought of doing abehavior and is considered as the immediate determinant of that behavior the mean score in this construct aswell increased in the intervention group after the intervention and there was significant difference between thetwo groups after the intervention in the study of braun and gimeno the results were similar tothe results of present study selfefficacy is a keyprerequisite for behavior change there was significantdifference between mean score of perceived selfefficacyconstruct in the two groups after the intervention in thisfig mean scores of intention and behavior in the experimental and control groups before and after the intervention 0crakhshanderou bmc medical education page of regard the results of the study by braun alidoosti and hart are consistent with thisfinding perceived selfefficacy is considered as a strongmotivational source and in fact is an indicator of theability of individuals to anize themselves in pursuit ofcertain goals studies show that individuals with ahigh level of perceived selfefficacy have a greatercommitmentto engage in activities at a time ofchallenges and difficulties and spent more time andeffort on such activities such individuals are morelikely to contribute to maintaining healthy behaviors andretrieve them even after failure and they have strongerintention and motivation this not only improves thetarget adjustment but also ensures achievement andsustainability in pursuit of the goals another important factor is knowledge that can be pointed to itsrole in healthy behaviors this study showed a significant difference in the two group in terms of the meanscore of knowledge after the educationalinterventionthese results are consistent with the findings of roozitalab ho and gimeno studiesalso there was no significant difference in the controlgroup before and afterthe intervention althoughincreasing knowledge is an important step in changingattitudes and behaviors it is not a major contributor tocrc prevention achieving the intention to behave isinfluenced by individual and environmental factors so inaddition to enhancing individual aspects overcomingthe structural and environmental barriers of the healthsystem regarding the use of cancer prevention nutritional behaviors is also vital in the present study themean score of perceived susceptibility and perceived severity constructs showed a significant difference betweenthe intervention and control group after the educationalintervention studies by kolutek wang cengiz and donadiki reportedthe role of beliefs regarding public health threats perceived susceptibility and perceived severity in the healthpromotion behaviors becker believed that onesintention to selfcare is influenced by his or her perception of vulnerability and the severity of disease outcomes therefore the need for interventions to increasethe perception of society about the irreparable complications of diseases caused by unhealthy behaviors malnutrition habits seems necessary in this study there was asignificant difference between the two groups in terms ofthe constructs of perceived benefits after the educationalintervention this result is consistent with the findings ofgrace alidoosti and abood studies also in the present study the mean score of perceived barrier construct decreased after the interventionthis was a good result but it was not statistically significant in the present study the mean score of perceivedbarrier construct decreased after the intervention which isnot consistent with the results of studies by moatari grace and gimeno the study ofrajabi identified some of the most important causes of barriers to nutrition in preventionof cancer such as the difficulty of preventativemeasuresinappropriate economic status and fear ofcancer information therefore strategies that overcome the individual and environmental barriers thataffect nutritional behaviors should be addressed byplanners and policymakerslimitationsthe limitations of this study which could have had a relative effect on its findings include the short duration ofintervention the sample size the inability to follow thelong term effect of the intervention and the selfreportingof the subjects in responding to questions however theuse of this method in such studies is inevitable and maylead to a bias of the researcherdesired report in thisstudy anonymous questionnaire was used to minimizethis biasthe findings of this study confirmed the effectiveness ofhealth belief modelbased education in improvement ofcolorectal cancerrelated preventive behaviors on theother hands interventions based on hbm concepts couldpromote nutritional behaviors related to colorectal cancerprevention consequently offering educational programsincluding public information campaigns workshopsvideos websites exhibitions etc should be used to informpeople about crc symptoms and risk factors alsomodelbased education will have a greater effect on nutritional behaviors improvement by focusing on perceptionsand enhancing beliefs aboutthe applicability oftheprogram and understanding the benefits and barriersabbreviationscrc colorectal cancer hbm health belief modelacknowledgementsthis is a part of an msc dissertation in health education approved by theshahid beheshti university of medical sciences the authors of this paperwould like to express their gratitude and appreciation to all the contributorswho have somehow collaborated on the design guidance andimplementation of this projectauthors contributionsmgh sr as and mm designed the study mm and mgh wrote the firstdraft sr and asm conducted the analyses all authors contributed towriting revising and approved the final manuscriptfundingthis study is sponsored by shahid beheshti university of medical sciences intehran the funding agencies had no role in the design of study datacollection and analysis or presentation of the resultsavailability of data and materialsthe datasets used and analyzed during the current study are available fromthe corresponding author on reasonable request 0crakhshanderou bmc medical education page of ethics approval and consent to participatethe study on which these data analyses are based was approved by theethical board committee of shahid beheshti university of medical sciencesparticipants were provided information about the study and verbalconsented by proceeding to take the survey this implied verbal consent wasapproved by the ethical board committee of shahid beheshti university ofmedical sciencesconsent for publicationnot applicablecompeting intereststhe authors have no conflict of interestsauthor details1environmental and occupational hazards control research center schoolof public health and safety shahid beheshti university of medical sciencestehran iran 2school of public health and safety shahid beheshti universityof medical sciences tehran iranreceived december accepted august screening in general practice in central england j epidemiol communityhealth roozitalab m moatari m gholamzadeh s saberifiroozi m zare n the effectof health belief on participation of the official administrative personnel incolorectal cancer screening programs in shiraz university of medicalsciences govaresh alidosti m sharifirad g hemate z delaram m najimi a tavassoli e theeffect of education based on health belief model of nutritional behaviorsassociated with gastric cancer in housewives of isfahan city daneshvarmed davodi a anoosheh m memarian r the effect of selfcare education onquality of life in patients with esophageal cancer following esophagectomyzums j braun kl fong m kaanoi me kamaka ml gotay cc testing a culturallyappropriate theorybased intervention to improve colorectal cancerscreening among native hawaiians prev med gimenogarca az quintero e nicol¡sp©rez d parrablanco a jim©nezsosa a impact of an educational videobased strategy on the behaviorprocess associated with colorectal cancer screening a randomizedcontrolled study cancer epidemiol bandura a social cognitive theory handbook of social psychologicaltheories london sage bandura a social cognitive theory an agentic perspective annu revpsychol luszczynska a guti©rrezdo±a b schwarzer r general selfefficacy invarious domains of human functioning evidence from five countries int jpsychol ho tv effects of an educational intervention on breast cancer screeningand early detection in vietnamese american women oncol nurs forumkolutek r avci ia sevig u the effects of scheduled observation at homeon health beliefs related to breast and cervical cancer screening andattitudes of married women eur j oncol nurs 201418s25 wang wl hsu sd wang jh huang lc hsu wl survey of breast cancermammography screening behaviors in eastern taiwan based on a healthbelief model kaohsiung j med sci cengiz b bahar z use of the health belief model in screening methodsfor colorectal cancer eur j oncol nurs 201418s27 donadiki e jim©nezgarca r hern¡ndezbarrera v sourtzi p carrascogarrido p de andr©s al jimeneztrujillo i velonakis e health belief modelapplied to noncompliance with hpv vaccine among female universitystudents public health becker mh drachman rh kirscht jp a new approach to explaining sickrole behavior in lowincome populations am j public health ma gx shive s tan y gao w rhee j park m kim j toubbeh jicommunitybased colorectal cancer intervention in underserved koreanamericans cancer epidemiol moatari m roozitalab m saber f zare m gholamzadeh s effect ofeducation on health beliefs on knowledge and participation j res med rajabi r sharifi a shamsi m almasi a dejam s investigating the effectof package theorybased training in the prevention of gastrointestinal cancers publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsreferencesarnold m sierra ms laversanne m soerjomataram i jemal a bray f globalpatterns and trends in colorectal cancer incidence and mortality gut american cancer society colorectal cancer facts and figures available at httpswwwcancercontentdamcancerresearchcancerfactsandstatisticscolorectalcancerfactsandfigures 20172019pdf[accessed ]ansari r amjadi h norozbeigi n zamani f mirnasseri s khaleghnejad amalekzadeh r survival analysis of colorectal cancer in patients underwentsurgical operation in shariati and mehr hospitaltehran in a retrospectivestudy govaresh centers for disease control and prevention cdc colorectal cancer risk byage available at httpwwwcdcgovcancercolorectalstatisticsagehtm[accessed apr ] malekzadeh r bishehsari f mahdavinia m ansari r epidemiology andmolecular genetics of colorectal cancer in iran a review kz aa saadat a jalalian hr esmaeili m epidemiology and survival analysisof colorectal cancer and its related factors trauma monthly winter239ghaffari m mehrabi y rakhshanderou s safarimoradabadi a jafarian szeffectiveness of a health intervention based on who food safety manual iniran bmc public health hosseini sv izadpanah a yarmohammadi h epidemiological changes incolorectal cancer in shiraz iran anz j surg yazdizadeh b jarrahi a mortazavi h mohagheghi ma tahmasebi s nahvijoa time trends in the occurrence of major gi cancers in iran asian pac jcancer prev glanz k rimer bk viswanath k health behavior and health educationtheory research and practice john wiley sons ghaffari m rakhshanderou s safarimoradabadi a torabi s oral and dentalhealth care during pregnancy evaluating a theorydriven intervention oraldis becker mh the health belief model and sick role behavior health educmonogr janz n champion v strecher vj the health belief model k glanz bk rimerjanz nk becker mh the health belief model a decade later health educ qlp o review of translation and cultural adaptation process ofquestionnaires kellar sp kelvin ea munro's statistical methods for health care researchwolters kluwer healthlippincott williams wilkins abood da black dr feral d nutrition education worksite intervention foruniversity staff application of the health belief model j nutr educ behav hart ar barone tl gay sp inglis a griffin l tallon ca mayberry jf theeffect on compliance of a health education leaflet in colorectal cancer 0c" | 0 |
objective cannabinoids are able to reduce tumor growth in xenograft models but their therapeutic potential as anticancer drugs in humans is unclear yet in vitro studies of the effect of cannabinoids on cancer cells are often carried out in absence of serum or in low serum concentration ie serum conditions that limit cellular growth and therefore can increase the response of cells to additional challenges such as the presence of cannabinoids however the tumor microenvironment can be teaming with growth factors in this study we assessed the viability and proliferation of cancer cells treated with cannabidiol in presence of a serum concentration that commonly sustains cell growth serumresults the results show that cannabidiol exerts a markedly different effect on the viability of the human ht cancer cell line when cultured in presence of serum in comparison to serum displaying a cytotoxic effect only in the former situation in presence of serum no inhibitory effect of cannabidiol on dna replication of ht cells was detected and a weak inhibition was observed for other cancer cell lines these results indicate that the effect of cannabidiol is cell contextdependent being modulated by the presence of growth factorskeywords paclitaxel colon cancer cannabidiol serumintroductionthe cannabis plant has a therapeutic potential to treat a wide range of diseases including cancer phytocannabinoids are being tested in a0vitro and in a0vivo for the potential to fight different types of cancer cannabis extracts have recently been described to exert a cytotoxic effect on human cancer cell lines however in a0 vitro cancer models present limitations which reduce their predictive validity one of these limitations is to reproduce the nutritional environment of the cells using cell culture media and growth factors many in a0 vitro cancer studies use historical culture media with fetal calf serum fcs however it is usual correspondence albertosainzcgmailcom gh medical barcelona spainfull list of author information is available at the end of the to eliminate or reduce fcs concentrations ie fcs from the media at the moment of drug exposure to avoid confounding effects of growth factors present in serum as in many studies testing the cytotoxic properties of cannabinoids in cancer cells [ ]the deprivation of survival factors from the media can sensitize cells to a subsequent challenge pirkmajer and chibalin showed that the effects of serum starvation in cell cultures are unpredictable according to eastman serum should be kept in cell cultures to avoid both false positive and negative results due to its effects on cell proliferation stipulating the importance of replicating anic conditions to obtain clinically valid resultsin the present study we analyzed the viability response of different cancer cell lines to cannabidiol cbd in presence of a standard concentration of serum in comparison to a low serum concentration 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 0csainzcort a0et a0al bmc res notes page of main textmaterials and a0methodsmaterialscbd was supplied by schibano pharma ag waldsch¶nengrund switzerland mccoys 5a medium alamarblue® ab invitrogen were bought leibovitzs l15 medium l15 and rpmi and from thermofisher scientific barcelona spain paclitaxel ²6diamidino2phenylindole dapi dimethyl sulfoxide lglutamine penicillinstreptomycin and fcs were bought from sigmaaldrich madrid spain cell proliferation reagent wst1 and 5bromo2²deoxyuridine brdu cell proliferation elisa kit were bought from roche sigmaaldrich madrid spain paclitaxel was dissolved in dimethyl sulfoxide and cbd was dissolved in methanol at a0mm and kept at a0°c for a maximum of a0 months when needed paclitaxel and cbd were diluted conveniently in the cell media at the indicated final concentrations cellular controls without cbd or paclitaxel contained cell media without additivescell cultureht29 cells ref htb38 and sw480 cells ref ccl were obtained from american type culture collection ags cells were kindly provided by miguel a pujana catalan institute of oncology idibell barcelona spain and were originally obtained from nuria sala catalan institute of oncology idibell barcelona spain human colon cancer ht29 cells and sw480 cells were maintained in mccoys 5a and l15 media respectively human gastric cancer ags cells kindly provided by francesca mateo catalan institute of oncology bellvitge institute for biomedical research lhospitalet del llobregat spain were maintained in rpmi medium all of the media was supplemented with penicillinstreptomycin and a0nm lglutamine a0h before treatment cells were plated in 96well plates at cellswell a0 h later wells in triplicates received cbd and paclitaxel all assays with sw480 and ags cells included fcs while the assays using ht29 cells included either or fcscell viability and a0proliferation assaysfor the viability and proliferation assay based on resazurin and its redoxmediated reduction we used ab and measured the fluorescence of the wells using a plate readerfor the viability and proliferation assay based on cleavage of tetrazolium salts by mitochondrial dehydrogenase we used wst1for the proliferation based on the measurement of dna synthesis we added brdu to cells and detected its incorporation into dna following manufacturer instructionsto assess cell viability dapi was added to the cell suspension a0 min before the analysis by flow cytometry dapi emits higher fluorescence when bound to dna dapi enters rapidly through altered cell membranes allowing the detection of damaged cells the cell population was selected by gating in a forward scatter vs side scatter dot plot excluding aggregates and cell debris samples were analyzed using a gallios flow cytometerstatistical analysisdata was analysed using ibm spss statistics and real statistics using excelwe used shapirowilk test to assess data normality and nonparametrical independent samples kruskalwallis test to identify significant differences between each experimental condition we used dunn test as a posthoc analysis to identify which groups show statistically significant differencesresultsviability and a0proliferation of a0ht cells with a0serum deprivation fcswhen human colon cancer ht29 cells were incubated in media with serum adding cbd at a0µm reduced cell viability as assessed via the resazurin method which is based on evaluating mitochondrial reductive capacity fig a0 1a interestingly when cbd concentrations were a0 µm cell viability increased during the first a0 h differences between or and a0 µm were statistically significant p and p at a0h the increasing viability with cbd a0 µm disappeared while the blocking effect of a0µm cbd was more pronounced fig a0 1a this suggests that cbd can induce mitochondrial stress as reported by others looking at the morphology of cells the treatment with a0µm cbd led to changes in cell form such as massive cellular detachment cell rounding and presence of wrinkled cells characteristic of dead cells fig a0 1b in fact analyzing the presence of dead cells using dapi dye we found an increased percentage in samples incubated with a0 µm cbd when compared to control cells fig a01c thus the loss of mitochondrial activity observed at cbd a0 µm correlated with cell death of note at longer incubation times ie a0days massive cellular death was also observable at a0µm cbd data not shown in summary a0µm cbd shows cytotoxic activity on ht29 cells cultured in fcs 0csainzcort a0et a0al bmc res notes page of fig a ht cells were incubated with fcs and different concentrations of cbd for and h cell viability was assessed by incubation with ab the mean sd of three assays are shown b morphology of ht cells incubated with or without μm cbd for h representative images are shown bar µm c ht cell viability according to dapi staining see the materials and methods section ht cells were incubated without top or with μm cbd bottom for h stained with dapi and immediately analyzed by flow cytometry the cursor identifies dapipositive cells dead cells showing a higher percentage in cbdtreated cells a representative experiment c is shown p viability and a0proliferation of a0ht cells in a0 fcscontrary to the drop in viability of cells in fcs cbd did not inhibit the viability of ht29 cells even after a0days in media containing fcs fig a02a b an apparent increase in ht29 cell viability was observed at a0µm cbd as assessed by ab or wst1 fig a0 suggesting mitochondrial stress we sought to find whether in these conditions cbd could show additive or synergistic antiproliferative effects with the therapeutic drug paclitaxel paclitaxel partially decreased the viability of ht29 cells according to ab measurement but not wst1 thus cbd at a0µm does not grossly affect the viability of ht29 cells after a0days culture in presence of serumto ascertain whether cbd had any effect on proliferation of ht29 cells we measured the incorporation of brdu into dna no changes in dna synthesis were observed after a0days of incubation of ht29 cells with any concentration of cbd fig a02c although paclitaxel in itself did inhibit dna synthesis cbd did not increase the effect of paclitaxel fig a02c in summary cbd up to a0µm do not decrease the viability nor the proliferation of ht29 cells cultured in fcs none of these results showed statistically significant differencesviability and a0proliferation of a0sw480 and a0ags cellsto know whether other cancer cell lines behaved similarly to ht29 showing little or no response to cbd when cultured in fcs we used sw480 another colon cancer cell line and ags a gastric cancer cell lineags cells did not show changes of viability by incubation with cbd up to a0µm though a0nm paclitaxel did decrease their viability fig a0 3a higher paclitaxel concentrations resulted in a severe decrease of ags cells viability data not shown so we used a0nm paclitaxel to observe potential effects of cbd the viability of sw480 cells with cbd and fcs showed a trend to decline fig a03c surprisingly and contrary to ht29 cells a0µm cbd did actually impair dna replication in ags and sw480 cells fig a03b d in fact the inhibition of dna replication was additive to that produced by paclitaxel the assessment of dna replication in sw480 cells 0csainzcort a0et a0al bmc res notes page of showed significant differences between the control sample and a0µm cbd without paclitaxel p any other statistic analysis did not show significant resultsin summary in presence of fcs and during a0days of culture cbd does not affect the viability of ht29 sw480 and ags cells though cbd at a0µm does impair the proliferation of ags and sw480 cellsdiscussionin this study we investigated the effects of cbd and its combination with paclitaxel on the viability of three different cancer cells ht29 sw480 and ags under two different concentrations of serum a standard appropriate for cell growth for ht29 sw480 and ags and a restrictive one of for ht29 only for ht29 cells cbd only reduces cell viability under low fcs with no effects on viability or dna replication when cells were in fcs however for sw480 and ags dna replication was impaired under a0µm cbd with serum moreover the inhibition of dna replication in sw480 and ags cells by cbd and paclitaxel had an additive effectat low cbd concentrations ht29 cells showed a trend towards increased cell viability though the differences were not significant different concentrations of cbd have previously been shown to have opposing effects on cells thus a0µm cbd induces proliferation of t leukemia cells but at higher concentration kills the cells a low concentration cbd increases mitochondrial ca2 augmenting mitochondrial metabolism and cell growth but at high concentration it leads to fig ht cells were incubated for days with fcs and different concentrations of cbd in absence or presence of nm paclitaxel a the viability was assessed by incubation with ab the mean sd are shown n b the viability was assessed by incubation with wst the mean sd are shown n c before harvesting cells were incubated with brdu for h which incorporated into dna and dna synthesis was quantified the mean sd are indicated n fig ags cells and sw480 cells were incubated for days with different concentrations of cbd in absence or presence of nm paclitaxel ags or nm paclitaxel sw480 a c cell viability was assessed by incubation with ab the mean sd of three ags and six sw480 assays are shown b d before harvesting cells were incubated for h with brdu which incorporated into dna and dna synthesis was quantitated the mean sd of three assays ags and assays sw480 are shown p 0csainzcort a0et a0al bmc res notes page of excessive mitochondrial ca2 mitochondrial dysfunction and cell death appropriate culturing conditions are essential for the survival and growth of cells in many studies cell culture conditions are not sufficiently detailed which is essential for study replication one possible solution to address the potential effect of serum could be using culture media without fcs so the media does not need to be altered during drug exposition in any case neither higher serum concentrations nor lower serum concentrations represent the proper microenvironment of a cancer cell in the human body and both approaches could be valid to test the effects of a drug on cell lines the tumor microenvironment is enriched with metabolites including lactate and adenosine [ ] which increases tumor growth and may modulate the therapeutic effect of a drug in tumors that are highly glycolytic increasing mitochondrial activity as exerted by cbd may add metabolic stress to cells forcing them to decreased growth the effect of a drug on cells can be assessed effectively if the experimental conditions of the treatment are the same as the growing conditions before the treatment once growing conditions and treatment conditions differ from more than one variable drug treatment then the resulting effects cannot be associated only to the treatment but to the combination of variableslimitationsour results did not show statistically significant differences with the exception of the assessment of viability of ht29 cells under cbd treatment and the assessment of dna replication of sw480 under a0µm cbd the lack of statistically significant results could be due to the small sample size n for most of the assays our study was also not able to replicate the strongly inhibitory effect of cbd shown in other studies where cannabinoids were tested against cancer cells cultured with fcs fcs contains many growth factors and nutrients and differences in the fcs source could substantially modify the viability proliferation and differentiation of cultured cells there are also other studies where cancer cells were cultured with fcs and treated with cbd or other synthetic cbdlike molecules the results of these studies showed that cbd a0μgml reduced the viability of cancer cells and also had effects on other survival variables [ ] the cell lines used in these studies being different to the ones used in our study could account for the different results observedabbreviationsab alamarblue brdu bromo²deoxyuridine cbd cannabidiol dapi ²diamidinophenylindole fcs fetal calf serumacknowledgementswe would like to thank manuel reina for his expert adviceauthors contributionsschibano pharma ag participated in the idea of the study as and ee designed the study as and ee acquired analyzed and interpreted the data cm provided technical assistance and carried out some experiments as and ee drafted the work all authors read and approved the final manuscriptfundingthis study was partially funded by schibano pharma ag waldsch¶nengrund switzerland and gh medical barcelona spain the design of the study was prepared by as ee and cm and approved by schibano pharma ag and gh medicalavailability of data and materialsthe datasets used andor analyzed during the current study are available from the corresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting interestsas was employee at gh medical while performing this projectauthor details gh medical barcelona spain celltecub department of cell biology physiology and immunology faculty of biology university of barcelona av diagonal barcelona spain received may accepted august references ackermann t tardito s cell culture medium formulation and its implications in cancer metabolism trends cancer https doi101016jtreca n201905004 brand a singer k koehl ge kolitzus m schoenhammer g thiel a matos c bruss c klobuch s peter k kastenberger m bogdan c schleicher u mackensen a ullrich e fichtnerfeigl s kesselring r mack m ritter u schmid m blank c dettmer k oefner pj hoffmann p walenta s geissler ek pouyssegur j villunger a steven a seliger b schreml s haferkamp s kohl e karrer s berneburg m herr w muellerklieser w renner k kreutz m ldhaassociated lactic acid production blunts tumor immunosurveillance by t and nk cells cell metab https 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Treatment Strategy for Metastatic Spinal Tumors A Narrative ReviewSam Yeol Chang Sujung Mok Sung Cheol Park Hyoungmin Kim BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Seoul Korea Metastatic spinal tumors are common and their rising incidence can be attributed to the expanding aging population and increased survival rates among cancer patients The decisionmaking process in the treatment of spinal metastasis requires a multidisciplinary approach that includes medical and radiation oncology surgery and rehabilitation Various decisionmaking systems have been proposed in the literature in order to estimate survival and suggest appropriate treatment options for patients experiencing spinal metastasis However recent advances in treatment modalities for spinal metastasis such as stereotactic radiosurgery and minimally invasive surgical techniques have reshaped clinical practices concerning patients with spinal metastasis making a demand for further improvements on current decisionmaking systems In this review recent improvements in treatment modalities and the evolution of decisionmaking systems for metastatic spinal tumors are discussedKeywords Spinal metastasis Decisionmaking system Radiosurgery Minimally invasive surgical procedures Separation surgeryIntroductionThe spine has been identified as the most common site for malignant metastasis in the musculoskeletal system and vice versa spinal metastasis is considered the most common malignant lesion in the spine [] The incidence of metastatic spinal tumors has seen an increasing trend due to the growing aging population worldwide and continued improvements in survival rates among cancer patients [] Symptomatic spinal metastasis is often the first clinical manifestation for of cancer patients [] whereas up to of cancer patients may experience spinal metastasis at some point during the course of their disease [] The objectives of surgical treatment in patients with metastatic spine tumors are mostly palliative Spine surgeons make an effort to maintain or improve the patients quality of life during the remainder of their survival by reducing pain and preserving ambulatory function via surgical treatment []Because clinical manifestations and treatment responses vary widely among cancer patients a multidisciplinary decisionmaking process that integrates medical and radiation oncology together with surgery along with assistance from pathology and diagnostic radiology deemed is essential when deciding to conduct surgical treatment for spinal metastasis [] In the literature various decisionmaking systems have been developed and introduced to date in an effort to aid in this decisionmaking process Received Jul Revised Jul Accepted Jul Corresponding author BongSoon ChangDepartment of Orthopedic Surgery Seoul National University Hospital Daehakro Jongnogu Seoul KoreaTel Fax Email bschangsnuackrCopyright ¸ by Korean Society of Spine SurgeryThis is an Access distributed under the terms of the Creative Commons Attribution NonCommercial License httpcreativecommonslicensesbync40which permits unrestricted noncommercial use distribution and reproduction in any medium provided the original work is properly citedAsian Spine Journal ¢ pISSN eISSN ¢ wwwasianspinejournalAsian Spine JournalASJAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J [] However recent advancements in the treatment of metastatic spine tumors have injected more complexity into this decisionmaking process and demanded the evolution of the decisionmaking system itself these recent advances include the development of stereotactic spine radiosurgery SRS the introduction of minimally invasive surgical techniques and the evolution of various target therapies for individual primary cancers [] In this review we discussed the development and evolution of various decisionmaking systems in spinal metastasis treatment Current concepts and recent trends in radiotherapy and surgery for spinal metastasis are also includedDecisionMaking Systems for Managing Metastatic Spinal TumorsPrognostic factors for metastatic spinal tumorsWhen attempting to choose an appropriate treatment for a patient with spinal metastasis establishing an accurate estimation of the individuals life expectancy is the most crucial To do this one must first identify prognostic factors associated with the survival of patients with spinal metastasis As such many authors have conducted studies to try and identify prognostic factors associated with survival among spinal metastasis patients and have developed various decisionmaking systems in order to estimate survival based on these prognostic factors []In a recently published metaanalysis Luksanapruksa [] have identified independent prognostic factors associated with the survival of patients with spinal metastasis Among these factors nine factors can be classified as relating to the preoperative performance or neurological status of a patientfor example the Karnofsky Performance Score or Eastern Cooperative Oncology Group grade [] Meanwhile four factors involve the presence or the number of metastases spine bone or visceral and two factors were found to be related to primary tumors in the Tomita classification scheme [] Finally male sex and the time interval from cancer diagnosis to the start of radiotherapy are the two remaining prognostic factors independently associated with survival in spinal metastasis patients Among these primary tumor histology the presence and number of metastases and performance status are proposed as the three most important prognostic factors associated with survival in spinal metastasis patients not only in this study but also in most previous investigations []In other studies the patients age and comorbidities assessed using the American Society of Anesthesiologists physical status [] and Charlson Comorbidity Index [] have also been identified to be prognostic factors in spinal metastasis [] Other authors have identified laboratory abnormalities such as leukocytosis and low hemoglobulin and albumin levels as prognostic factors and included these into their decisionmaking systems [] In addition previous systemic treatment or chemotherapy has also been suggested as an independent prognostic factor by multiple authors [] Further not only preoperative chemotherapy but also the presence of available systemic treatments in the postoperative period has been hypothetically regarded as a potential prognostic factor in the literature [] In a recently published study by Chang [] the authors verified the presence of the remaining systemic treatment options to be independently associated with improved postoperative performance status and survivalClassificationbased decisionmaking systemsBased on these prognostic factors numerous decisionmaking systems or scoring systems have been developed and introduced to estimate the life expectancy among spinal metastasis patients [] In these classificationbased decisionmaking systems scores for each prognostic factor identified by multivariate logistic or proportional hazards regression analyses are integrated in order to obtain a total prognostic score that reflects the estimated survival of the patient Surgeons can adopt these prognostic scores to identify patients with an estimated survival profile that is sufficient to warrant surgical treatment Although the prognostic factors included in each system vary primary tumor histology and visceral metastases are included in most systems Table In the New England Spinal Metastasis Score NESMS was introduced by Ghori [] The NESMS was developed using multicenter data and retrospectively validated in the following investigations [] The NESMS consists of modified Bauer score components and score serum albumin level and ambulatory status of the patient More recently the NESMS was validated prospectively in the Prospective Observational Study of Spinal Metastasis Treatment trial which aimed to verify the 0cTreatment of Spinal Metastasis Table Prognostic factors in decisionmaking systemsReferencesBauer [] modifiedTomita [] To kuhashi [] revisedKa tagiri [] revisedGhori [] Paulino [] Primary tumorPerformance statusNo of vertebral metastasesBone metastasisVisceral metastasisPrevious systemic treatmentOther factorsOOOOOOOOOOOOOOOOOOOOOOOOOOBr ain metastasis WBC Hb platelet albumin bilirubin Creactive protein lactate dehydrogenaseSerum albuminAge WBC Hb brain metastasisWBC white blood cell Hb hemoglobin NESMS as a reliable predictive tool in spinal metastasis patients []There have been efforts to develop novel decisionmaking systems using evolving methodologies In the Skeletal Oncology Research Group S compared multiple prognostic survival algorithms including classic nomogram and boosting algorithms using the same retrospective dataset obtained from patients [] In their study the nomogram was found intuitive and demonstrated a comparable level of performance Then in the S used machinelearning algorithms to develop a novel prognostic model for metastatic spinal disease [] which was externally validated in subsequent studies []Although these various classificationbased decisionmaking systems are helpful and widely used for predicting the survival of spinal metastasis patients recent studies have reported that the degree of accuracy of these classic systems eg Tomita Tokuhashi decreases over time especially in cancers with poor prognoses such as lung cancer [] This pitfall of classificationbased decisionmaking systems reportedly stems from the inability of these systems to reflect survival improvement due to recent evolutions in systemic treatment for primary cancers [] Another existing limitation is that these systems cannot directly guide the selection of specific treatments appropriate for patients with spinal metastasisPrinciplebased decisionmaking systemsAs an alternative to these classificationbased decisionmaking systems that are incapable of reflecting recent advances in oncology and guiding specific treatments several authors have proposed principlebased decisionmaking systems These principlebased systems do not score the patient and estimate survival but instead provide advice regarding which treatment is more appropriate in individual cases based on the integration of rapidly evolving treatment modalities including target therapies radiosurgery and minimally invasive surgical techniquesThe neurologic oncologic mechanical and systemic NOMS decision framework was first introduced in [] The NOMS decision framework consists of neurologic N oncologic O mechanical M and systemic S considerations integrating novel multimodal therapies including SRS and minimally invasive surgical techniques [] As a neurological N assessment approach the grading system developed by Bilsky and Smith [] was used while surgical decompression is recommended for highgrade spinal cord compressions During oncological O assessment the responsiveness of spinal metastasis to currently available treatments especially the level of tumor sensitivity to radiotherapy is evaluated Mechanically M instability of the spinal column as determined by the Spinal Instability Neoplastic Score SINS indicates the need for surgical stabilization regardless of the neurologic or oncologic status [] Finally systemic S assessment focuses on the patients ability in tolerating the suggested treatment Meanwhile if the general condition performance status and medical comorbidities of a patient do not allow surgery to be performed radiotherapy is instead recommendedA modification of the NOMS decision framework has also been presented in the literature Paton introAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J duced the LMNOP system as an improvement to the NOMS approach [] With this development these authors added two additional key considerations to the NOMS as follows the location and levels of metastasis L and the patients response to previous therapy P The P in LMNOP stands for not only the response to prior therapy but also includes patient fitness and prognosis which was considered previously as part of the systemic S assessment in the NOMS decision framework The authors emphasized that the response of primary cancer to previous treatments including chemotherapy and radiotherapy is considered a significant factor when determining the appropriate treatment for spinal metastasis patients For instance it is anticipated that a patient diagnosed with symptomatic spinal metastasis at the initial presentation of primary cancer synchronous metastasis who would have multiple potential treatment options is more likely to experience a better prognosis than a patient diagnosed with spinal metastasis despite previous treatment metachronous metastasis Differences in the survival rates between the patients with synchronous and metachronous spinal metastasis have been confirmed in a previous research [] Therefore a more aggressive approach including surgical treatment can be considered for patients with a synchronous spinal metastatic lesion In summary principlebased decisionmaking systems relative to classificationbased systems are able to better incorporate evolving treatment modalities and guide the selection of appropriate treatments for patients in a timely fashion Table Current trends and future directions in the development of decisionmaking systemsAdvances in cancer biology and treatment modalities are necessitating the evolution of decisionmaking systems for spinal metastasis Possible future directions to take to improve decisionmaking systems include the following the use of multicenter or multinational databases the integration of histologyspecific data the application of computational methodologies such as machinelearning algorithms and the combination of classificationbased and principlebased systems Some recent studies are already covering these trendsThe size of the study sample under assessment determines the performance and accuracy of prognostic models Although spinal metastasis is found to be relatively common data from a larger sample population beyond that of just a single institution is usually required to develop a powerful enough prognostic model For this reason recently introduced prognostic models or decisionmaking systems are generated from multicenter databases [] Future prognostic models should also have the freedom to rely on even larger databases such as multinational tumor registriesBiologic therapy including molecular target therapy and immunotherapy is believed to be an emerging gamechanger in modern cancer treatment Genetic subtype analysis of the primary cancer histology which guides selection among t hese therapies has become more essential [] In a revised prognostic system proposed by Katagiri Table The NOMS decision frameworkNeurologic NOncologic OMechanical MSystemic SDecisionLowgrade ESCCno myelopathyHighgrade ESCC±myelopathyRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadiosensitiveRadiosensitiveRadioresistantRadioresistantRadioresistantRadioresistantStableUnstableStableUnstableStableUnstableStableStableUnstableUnstablecEBRTStabilization followed by cEBRTSRSStabilization followed by SRScEBRTStabilization followed by cEBRTAble to tolerate surgeryD ecompressionstabilization followed by SRSUnable to tolerate surgerycEBRTAble to tolerate surgeryDecompressionstabilization followed by SRSUnable to tolerate surgeryStabilization followed by cEBRTNOMS neurologic oncologic mechanical and systemic ESCC epidural spinal cord compression cEBRT conventional external beam radiation SRS stereotactic radiosurgery 0c [] the authors considered the availability of molecular target therapy when classifying the primary tumor For example lung cancer treated with targeted drugs was designated as an example of a moderategrowth tumor while lung cancer without targeted drugs is regarded as an example of a rapidgrowth tumor [] This application of genetic profiles to decisionmaking systems is likely to grow more specific and tailored corresponding to the evolution of molecular genetics in the futureAs previously described machinelearning algorithms have been applied in the development of prognostic models Classically prognostic models for spinal metastasis have been developed using logistic or proportional hazards regression analyses As part of its research efforts the S was able to develop prognostic models using machinelearning algorithms such as gradient boosting decision trees random forests and neural networks [] and these algorithms were externally validated elsewhere [] Like in other fields of medicine evolving computational methodologies including machinelearning algorithms should be assessed extensively in terms of their potential in the management of spinal metastasisFinally the combination of classificationbased and principlebased decisionmaking systems should be considered Classificationbased systems or prognostic models seek to estimate the patients remaining survival Based on this survival estimation principlebased systems then may suggest the most appropriate treatment option Until now surgeons and physicians have been employing these two separate systems in the same decisionmaking process A novel decisionmaking system could integrate these two systems together and provide survival estimations and appropriate treatment options simultaneouslyRadiotherapy for Metastatic Spinal TumorsTreatment of Spinal Metastasis single session of SRS achieved durable longterm control of spinal metastasis regardless of histology and tumor size Of note the only significant factor associated with tumor control was the radiation dose These results suggest that SRS can be effective even in cases of metastasis previously considered to be radioresistantSRS can also be a definitive treatment for the management of solitary metastasis without spinal cord compression [] Excellent local control rates of have allowed SRS to replace curative surgeries with high morbidities such as total en bloc spondylectomy for addressing these solitary metastases [] In patients with highgrade spinal cord compression SRS can be applied after separation surgery which will be discussed further in the following section Overall the effectiveness of SRS has been changing the role and extent of surgical treatment and in turn shifting the focus of the treatment of spinal metastasisVertebral compression fracture following stereotactic spine radiosurgeryA pitfall of SRS is an increase in vertebral compression fracture VCF following radiotherapy Risk estimates for VCF after SRS are reported to be up to as compared with just in relation to conventional radiotherapy [] The occurrence of VCF is dosedependent and caution is required if the radiation dosage exceeds Gy per fraction in highrisk patients [] Highrisk patients of older ages with lytic lesions andor with spinal malalignment can reportedly benefit from undergoing preventive stabilization surgery before SRS When determining the necessity of stabilization surgery before SRS SINS can be used to identify potentially unstable lesions [] SINS will be further covered in the following sectionStereotactic spine radiosurgery is triggering a paradigm shiftTiming of radiotherapy after surgeryEvidence in the literature supports that radiosurgery is a safe and effective modality for local tumor control with low associated complication rates in patients with spinal metastasis [] Technical improvements including intensitymodulated and imageguided radiation delivery and processing software have allowed SRS to be a gamechanger in the treatment of spinal metastasis [] A recent study by Yamada [] reported that a highdose Adequate timing of radiotherapy following surgery whose determination is related to the risk of wound complications continues to be debated among spine surgeons and radiation oncologists It is also controversial whether the interval can be shortened in patients receiving SRS Lee [] sent questionnaires to radiation oncologists and spine surgeons to gather opinions on the optimal timing of surgery and radiotherapy in spinal metastasis Based on the procured comments the auAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J thors recommended that the interval be at minimum weeks regardless of radiation modalities Interestingly as compared with radiation oncologists surgeons tended to favor a shorter interval of time between surgery and radiotherapy when SRS is performed although there was no statistically significant difference in this regard []A recently published systemic review also advocated for weeks with a minimum of days between surgery and radiotherapy [] When the rates of wound complications were compared between SRS and conventional radiotherapy many studies reported reduced wound complications in SRS patients [] However due to limited highlevel evidence no definite conclusion was made regarding whether the interval could be reduced in patients undergoing SRSSurgery for Metastatic Spinal TumorsSurgical indicationsThe objective of surgical treatment in spinal metastasis was to provide pain relief support neurological improvement and in turn enhance the quality of life during the remaining survival period Clinical benefits of direct surgical decompression in patients with metastatic spinal cord compression MSCC have been well described in the literature [] The most important prerequisite for surgical treatment in spinal metastasis has been identified as the sufficient enough estimated survival time to make surgery a reasonable approach Researchers have largely recommended that a minimum of to months of remaining survival should exist when considering whether to perform surgery [] At this point a number of prognostic scoring systems previously described are being used to estimate a patients survival The patient should also have a good enough general condition or performance status to in order to endure surgery If these conditions are satisfied then surgery can be performed in patients with symptomatic MSCC or mechanical instabilityIn spinal metastasis the instability is assessed by the SINS [] Table The SINS is an independent and unique tool that integrates clinical and radiological components to help surgeons decide whether to conduct surgery for stabilization A score of to points suggests impending instability while that of points or more indicates existing spinal instability which requires stabilization As previously mentioned the SINS has also been incorporated into principlebased decisionmaking systems such as the NOMS and LMNOP systems [] Recent studies have reported the reliability and accuracy of the SINS system in predicting spinal adverse events including VCF especially in those patients who received radiotherapy [] although some components may still require revision []Separation surgery and minimally invasive surgeryWhen considering the surgical techniques used for spinal metastasis the literature shows a trend toward the adoption of less invasive techniques which is thought to have Table Spinal Instability Neoplastic Score systemComponentLocationJunctional occiputC2 C7T2 T11L1 L5S1Mobile spine C3C6 L2L4Semi rigid T3T10Rigid S2S5PainaYesOccasional pain but not mechanicalPainfree lesionBone lesionLyticMixed lyticblasticBlasticRadiographic spinal alignmentSubluxationtranslation presentDe novo deformity kyphosisscoliosisNormal alignmentVertebral body collapse collapse collapseNo collapse with body involvedNone of the abovePosterolateral involvement of spinal elementsbBilateralUnilateralNone of the aboveScoreaPain improvement with recumbency andor pain with movementloading of spine bFacet pedicle or costovertebral joint fracture or replacement with tumor 0cTreatment of Spinal Metastasis primarily resulted from recent advances in radiotherapy as previously described [] With the use of advanced radiation techniques surgeons can minimize surgical morbidities by avoiding extensive debulking surgery [] Fig During the separation surgery circumferential spinal cord decompression is performed only to the extent required to facilitate safe radiosurgery In a study by Laufer [] separation surgery followed by postoperative SRS resulted in a low local progression rate Other studies have also reported that this hybrid surgeryradiosurgery approach is a safe and effective treatment option for MSCC []BCADFig A 68yearold male with spinal metastasis of renal cell carcinoma at T9 The patient also had lung metastasis A Preoperative MRI shows spinal cord compression and involvement of posterior elements and left rib head at the T9 level B Following a separation surgery without spinal instrumentation MRI at postoperative 2weeks shows a decompressed spinal canal but residual metastatic tumor around the left 9th rib head C In the postoperative 1year MRI the patient showed a nearcomplete response following a single session stereotactic radiosurgery 18Gy1 fraction which was performed weeks after the separation surgery MRI magnetic resonance imaging D Planning images for the postoperative stereotatic radiosurgery following a separation surgery Asian Spine Journal 0cSam Yeol Chang et alAsian Spine J Minimalaccess surgery is also used in treating spinal metastasis while reducing surgical morbidities In the anterior approach retractor systems and thoracoscopic assistance have been described [] Minimally invasive posterior approaches for decompression and corpectomy have also been introduced and reviewed in the literature [] Other minimally invasive techniques for spine surgery such as the intraoperative stereotactic navigation system and percutaneous pedicle screw instrumentation are being incorporated into spinal metastasis surgery as well []Studies comparing minimally invasive surgery and surgery showed that minimally invasive surgery provided equivalent or superior outcomes with reduced surgical morbidity and complications in spinal metastasis patients [] However because the quality of evidence is deemed low in the current literature no definite conclusion regarding the superiority of minimally invasive surgery over surgery can be derived and no strong recommendations have been made at this point []Role of curative surgery en bloc resectionMost surgeries for spinal metastasis are found palliative and the role of en bloc resection in spinal metastasis is decreasing even further due to improvements in radiotherapy Generally curative surgical resection of spinal metastasis has been considered in the context of a single metastasis of a slowgrowing tumor such as in renal cell thyroid and breast cancers Fig Favorable outcomes in this regard have been reported in the literature [] However some authors recently reported that curative ABECDFig A 63yearold male with spinal metastasis of thyroid carcinoma at T8 A Preoperative MRI shows pathologic fracture and spinal cord compression at the T8 level B Postoperative Xray at month shows removal T8 vertebra and reconstruction with an expandable cage following total en bloc spondylectomy C MRI at postoperative 3years shows a widely decompressed spinal canal with no tumor recurrence D Postoperative Xray at postoperative 5years shows wellmaintained instrumentation E Bone scan at postoperative 5years shows no evidence of bone metastasis MRI magnetic resonance imaging 0csurgical resection en bloc spondylectomy did not impact the oncologic outcomes of spinal metastasis patients [] Therefore a more careful and thorough decisionmaking process is required before performing curative resection surgery for spinal metastasis especially when the extended role of radiosurgery is consideredPostoperative complications and preventive measuresThe overall complication rate following surgery for spinal metastasis ranges from to in the literature [] Because surgery for spinal metastasis is performed to improve the quality of life of a patient surgeons should try to minimize all possible surgical and medical complications by implementing multidisciplinary interventions Among diverse complications those that require additional attention when found in spinal metastasis patients eg wound infection instrumentation failure and intraoperative bleeding are briefly discussed hereThe incidence of surgical site infection SSI is determined to be higher in spinal metastasis surgery reaching up to as compared with during other spine surgeries [] SSI has also been identified as the most common cause for reoperation in of reoperations following surgery for spinal metastasis [] Poor nutrition and exposure to adjuvant therapies chemotherapy and radiotherapy put spinal metastasis patients at risk for SSI [] Surgeons should provide adequate nutritional support perioperatively and secure a sufficient time interval between radiation and surgery as previously discussed to minimize SSIs Additional risk factors such as smoking obesity and medical comorbidities should also be considered []The second cause for reoperation in spinal metastasis surgery is the failure of instrumentation [] Risk factors associated with instrumentation failure include the number of operated levels prior chest wall resection and history of radiotherapy [] The necessity of additional fusion procedures while performing surgery for spinal metastasis is debated but highlevel evidence remains to be lacking at this time [] In future studies we suggest that instances of early and late failure be distinguished when assessing instrumentation failure because the mechanisms of failure between the two types seem to differ ie insufficient stability of the construct in the context of early failure versus the progression of deformity or lack of fusion in the context of late failureTreatment of Spinal Metastasis Intraoperative bleeding during spinal metastasis surgeries can be massive and can further lead to serious complications such as cardiovascular or cerebral events [] For the spinal metastasis of hypervascular tumors such as renal cell hepatocellular and thyroid cancers preoperative embolization is recommended Previous studies have verified the effectiveness of preoperative embolization in reducing intraoperative bleeding in spinal metastasis surgeries [] Surgery should be performed within hours following embolization to avoid the diminished effect of preoperative embolization []ConclusionsThe determination of appropriate treatment for a patient with spinal metastasis is a challenging task that requires multidisciplinary considerations Recent advances in radiotherapy and surgery for spinal metastasis have brought about improvements in the management of these patients Evolving decisionmaking systems are also crucial contributors to stateoftheart care of patients with metastatic spinal tumorsConflict of InterestNo potential conflict of interest relevant to this was reportedReferences White AP Kwon BK Lindskog DM Friedlaender GE Grauer JN Metastatic disease of the spine J Am Acad Orthop Surg Laufer I Sciubba DM Madera M Surgical management of metastatic spinal tumors Cancer Control Schiff D ONeill BP Suman VJ Spinal epidural metastasis as the initial manifestation of malignancy clinical features and diagnostic approach Neurology Klimo P Jr Schmidt MH Surgical management of spinal metastases Oncologist Nathan SS Healey JH Mellano D Survival in patients operated on for pathologic fracture implications for endoflife orthopedic care J Clin Oncol Curtin M Piggott RP Murphy EP Spinal metaAsian Spine Journal 0cSam Yeol Chang et alAsian Spine J static disease a review of the role of the multidisciplinary team Orthop Surg Ahmed AK Goodwin CR Heravi A Predicting survival for metastatic spine disease a comparison of nine scoring systems Spine J Barzilai O Fisher CG Bilsky MH State of the art treatment of spinal metastatic disease Neurosurgery Tomita K Kawahara N Kobayashi T Yoshida A Murakami H Akamaru T Surgical strategy for spinal metastases Spine Phila Pa Tokuhashi Y Matsuzaki H Oda H Oshima M Ryu J A revised scoring system for preoperative evaluation of metastatic spine tumor prognosis Spine Phila Pa Bauer HC Wedin R Survival after surgery for spinal and extremity metastases prognostication in patients Acta Orthop Scand Van der Linden YM Dijkstra SP Vonk EJ Marijnen CA Leer JW Dutch Bone Metastasis Study Group Prediction of survival in patients with metastases in the spinal column results based | 2 |
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 | 2 |
"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: 23824319101304 33 ChaoYK ChangHC WuYC LiuYH HsiehMJ et al (2012) Management of lung metastases from colorectal cancer: video-assisted thoracoscopic surgery versus thoracotomya case-matched study. Thorac Cardiovasc Surg60: 39840422228090 101150042 30118 Mol Cancer Res Mol. Cancer Res. Molecular cancer research : MCR 1541-7786 1557-3125 24202705 3946989 10.1158/1541-7786.MCR-13-0300 NIHMS538386 NEDD9 Depletion Leads to MMP14 Inactivation by TIMP2 and Prevents Invasion and Metastasis. McLaughlin Sarah L. 5 * Ice Ryan J. 5 * Rajulapati Anuradha 5 Kozyulina Polina Y. 1 Livengood Ryan H. 4 Kozyreva Varvara K. 5 Loskutov Yuriy V. 5 Culp Mark V. 3 Weed Scott A. 2 5 Ivanov Alexey V. 1 5 Pugacheva Elena N. 1 5 # 1Department of Biochemistry West Virginia University School of Medicine Mantown WV 26506 2Department of Neurobiology and Anatomy West Virginia University School of Medicine Mantown WV 26506 3Department of Statistics West Virginia University School of Medicine Mantown WV 26506 4Department of Pathology West Virginia University School of Medicine Mantown WV 26506 5Mary Babb Randolph Cancer Center West Virginia University School of Medicine Mantown WV 26506 #Corresponding author: Elena N. Pugacheva Mailing address: Department of Biochemistry and Mary Babb Randolph Cancer Center PO Box 9142 1 Medical Center Drive West Virginia University School of Medicine Mantown WV 26506. Phone: (304) 293-5295; Fax: (304) 293-4667; epugachevahsc.wvu.edu S.L. McLaughlin* and R. J. Ice* contributed equally to this work. 17 12 2013 07 11 2013 1 2014 01 1 2015 12 1 69 81 The scaffolding protein NEDD9 is an established pro-metastatic marker in several cancers. Nevertheless the molecular mechanisms of NEDD9 driven metastasis in cancers remain ill defined. Here using a comprehensive breast cancer (BCa) tissue microarray it was show that increased levels of NEDD9 protein significantly correlated with the transition from carcinoma in situ to invasive carcinoma. Similarly it was shown that NEDD9 overexpression is a hallmark of highly invasive BCa cells. Moreover NEDD9 expression is crucial for the protease-dependent mesenchymal invasion of cancer cells at the primary site but not at the metastatic site. Depletion of NEDD9 is sufficient to suppress invasion of tumor cells in vitro and in vivo leading to decreased circulating tumor cells (CTCs) and lung metastases" | 1 |
since leung reported a single case of lowdose aspirin lda induced multiple smallintestinal ulcers in many investigators have described ldainducedsmall intestinal mucosal injuries watanabe reportedthat all of their patients who used lda had small intestinallesions that were detected using capsule endoscopy 0cgastroenterology research and practicece iwamoto investigated patients whounderwent ce for occult bleeding and erosions wereobserved in cases of whom were taking lda ornonlda nonsteroidal antiammatory drugs endo described small intestinal lesions in of subjects who took aspirin mg for weeks shiotani performed ce on young healthy individuals beforeand after medium doses of enteric aspirin were administered for days and rabeprazole mg was administered for week and found large erosions that included small intestinal ulcers in of the subjects recently direct oral anticoagulants doacs have beenadministered as alternatives to warfarin dabigatran is adirect thrombin inhibitor and rivaroxaban apixaban andedoxaban are factor xa inhibitors doacs have significantlyfewer side eï¬ects than warfarin including intracranial hemorrhage hence the number of patients taking doacs isgradually increasing [] howeverthe ï¬ndings frommetaanalyses have shown that compared with warfarinthe incidence of gastrointestinal gi bleeding is higher inassociation with doacs [ ] the causes of gi bleedingin association with doacs were bleeding from colon astric cancers and diverticular hemorrhages comparedwith warfarin dabigatran and rivaroxaban are associatedwith higher risks of gi bleeding depending on their dosesand the risk of gi bleeding associated with apixaban iscomparable edoxaban is associated with significantlyless gi bleeding at low doses mg once daily comparedwith warfarin but the risk is significantly greater at highdoses mg once daily but unlike western countries the rate of gi bleeding for both dabigatran and rivaroxaban is equivalent to warfarin in asian countries furthermore edoxaban tends to cause less digestive tractbleeding than warfarin esophageal ulcers caused bydabigatran have been described by toya kasai and okada and okada the tartaric acidcoating on dabigatran causes esophageal mucosal disorders because dabigatran persists in the midesophagus ifit is consumed without water however there havebeen no reports of smalllesions in patientswho receive doacs here we aimed to evaluate smallintestinal mucosal injuries in patients taking doacs usingvideo capsule endoscopy vceintestinal methodsthis study was a prospective openlabel nonblinded multicenter and observational study from september tomarch pat5ents taking doacs namely dabigatranrivaroxaban and apixaban for atrial ï¬brillation at saitamamedical university hospital keio university hospital saitama medical center and yokohama municipal citizenshospital were enrolled patients with severe comorbiditiesincluding severe anemia and exacerbations of heart failurethat required blood transfusion crohns disease and ileuswere excludedthe hemoglobin hb and serum ferritin levels the esophagogastroduodenoscopy egd ï¬ndings and colonoscopicï¬ndings were examined vce pillcam sb2 given imagingfigure rednessfigure erosionltd yoqneam israel was performed to examine small intestinal lesions according to the doac used redness erosionulcer and angioectasia were checked redness was a red spotfigure and erosions were deï¬ned as small and superï¬cialmucosal disruptions denuded of villi figure ulcers weredeï¬ned as large submucosal disruptions with a central areacovered with exudate and angioectasia was a patchy erythematous lesion figure the images were analyzed using theproprietary rapid software by an expert n h whohad performed more than vce examinations blindlythe type and location of smallbowel lesions were registeredalso the proportion of lesions detected between types ofdoac was evaluated and the hemoglobin hb and serumferritin levels were compared between patients with and without smallbowel lesionsthe study protocol accorded with the tenets of therevised declaration of helsinkiand it wasapproved by the institutional review boards at our institutions written informed consent was obtained from all ofthe patients this study was registered with the universityhospital medicalinformation network clinical trialsregistry umin000011527 october 0cgastroenterology research and practicetable patients characteristicsparametermean age years rangesex n malefemalecomorbid disease natrial ï¬brillationparoxysmal atrial ï¬brillationhypertensionhyperlipidemiacerebral infarctiondoac n years dabigatranrivaroxabanapixabanbayaspirin ncelecoxib nppi nh2 blocker n mean hb gdl rangedoac direct oral anticoagulant ppi proton pump inhibitor hbhemoglobinlower portion in patients table erosions wereobserved in patients and they were present in theupper portion in in the middle portion in and in the lower portion in patientstable erosions tended to occur less frequently in themiddle portion however the diï¬erence was not significantp compared with the upper portion p compared with the lower portion angioectasia was observedin patients and was present in the upper portionin patients and in the middle portion in patient and was absent from the lower portion table there were no ulcers in any patients erosions tended to bemore frequent in patients taking dabigatran or apixaban thanin patients taking rivaroxaban this diï¬erence was not significant p table no significant diï¬erences wereobserved regarding angioectasia among the patients takingthe diï¬erent doacs none of these patients had activebleeding from small intestinal lesionsthe mean hb concentrations in the patients with andlesions were gdl and gdlwithout smallbowelrespectively a diï¬erence which was not significant p the mean ferritin levels in the patients with and withoutsmallbowel lesions were mgdl and mgdl respectively a diï¬erence which was not significant p discussionthis studys ï¬ndings showed that of the patients who tookdoacs had redness had erosionsor small ulcers had angioectasia and had no abnormalities in their small bowel smallbowellesions were observed in of patients thereforethere was a high incidence of smallbowel lesions in patientsfigure angioectasiaibm®spss® statistical software version ibm corporation armonk ny usa was used for the statisticalanalyses the data were analyzed using ttests and fishersexact test resultsthirtythree patients were enrolled to participate in thisstudy but patients withdrew their consent and vce wasperformed on patients the patients mean age was years range years and males and females participated in this study twenty patients had atrial ï¬brillation had paroxysmal atrial ï¬brillation had hypertension hadhyperlipidemia and had cerebral infarction eight patientstook dabigatran took rivaroxaban and took apixabanthe mean duration of doac use was months months additionally patient took bayaspirin patienttook celecoxib patients took proton pump inhibitorsppis and patients took h2 receptor antagonists theaverage hb concentration was gdl range gdl table twentytwo patients underwent egdand atrophic gastritis was present in patients hiatal hernias in patients gastric polyps in patients erosive gastritisin patients gastric ulcer or ulcer scar in patients reï¬uxesophagitis in patients endoscopic submucosal dissectionscar for early gastric cancer in patients and an esophagealulcer in patient nineteen patients underwent colonoscopyand colonic polyps were present in patients colonic diverticulum were present in patients and a rectal ulcer waspresent in patient none of these lesions detected by egdand colonoscopy had active bleedingthe patients evaluated with vce was redness in thelower esophagus was present in patient gastric erosionswere present in patients and gastric redness was presentin patient table the patient who had redness in thelower esophagus was taking apixaban smallbowel transitwas complete in of patients smallbowellesions were observed in of patients redness was observed throughout the small intestine in patients and it was present in the upper portionin in the middle portion in and in the 0cgastroenterology research and practicetable capsule endoscopy ï¬ndingsentire small intestine observation rate n detection rate n esophagusstomachupper small intestinemiddle small intestinelower small intestineentire small intestineredness lower n erosions n redness n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions n angioectasia n no abnormalities n redness n erosions nangioectasia nno abnormalities n table findings at each small intestinal site according to the directoral anticoagulant useddabigatranrivaroxabanapixabansiteï¬ndingupper small intestineredness nerosions nangioectasia nmiddle small intestineredness nerosions nangioectasialower small intestineredness nerosions nangioectasia nentire small intestineredness nerosions nangioectasia ntaking doacs however none of these patients had activebleeding and most of the lesions were mild patients withsevere anemia or active bleeding were excluded from thisstudy hence only patients with mild symptoms wereincluded ldainduced lesions cause redness erosions andulcers [] previous studies ï¬ndings that describe the characteristics of smallbowel injuries associated with chroniclda use suggest that ulcers are observed mainly in the distalpart of the small bowel [] in this study erosionstended to be observed less frequently in the middle portionof the small bowel in the patients taking doacs howeverthere were no significant diï¬erences regarding the distributions of the lesions there were no ulcers in any patientstherefore the intake of doac might not be related withsevere ulcers in the small intestinein this study angioectasia was observed in the upper andmiddle portions but not in the lower portion of the smallbowel kaufman used a transit timebased quartilemethod to evaluate patients with angioectasia whounderwent ce and found that most lesions were inthe ï¬rst quartile igawa reported that while therewere no diï¬erences regarding the location of type 1a angioectasia among patients with occult gastrointestinal bleedingtype 1b angioectasia was relatively less frequent in the lowerportion compared with that in the upper and middle portionsof the small bowel the data reported before thereforeangioectasia might not be aï¬ected by the intake of doacscomparison of vce ï¬ndings before and after the administration of d719acs is neededno significant relationships were determined in relationto the presence of the hb level or the serum ferritin levelbetween the patients with and without smallbowel lesionsin this study despite detecting abnormal ï¬ndings in thesmall bowel no active bleeding was seen by vce and therewas no severe anemia in any patients in this study furthermore compared with warfarin the incidence of gi bleedingis higher in association with doacs the causes of gi bleeding in association with doacs are bleeding from colon astric cancers and diverticular hemorrhages however noneof these lesions detected by egd and colonoscopy had activebleeding doacs did not aï¬ect bleeding from the upper gitract and the colon in this studyno significant diï¬erences were observed among thedoacs in relation to smallbowel lesions the ï¬ndings fromthe randomized evaluation of longterm anticoagulationtherapy trial of dabigatran showed that in the warfaringroup patients with gi tract bleeding had gastric canceror colonic cancer and that in the dabigatran group patients with gi tract bleeding had colonic cancer and patient had gastric cancer hence dabigatran mightinduce gi tract bleeding from colon cancer rivaroxaban apixaban and edoxaban compete directly with the s1pocket of factor xa and inhibit factor xa activity whereasdabigatran is a prodrug that is activated in the presence ofesterase in the gi tract plasma and liver the causes of themucosal damage by dabigatran were thought to be due todirect acting at the local area where it is absorbed in addition tartaric acid coats dabigatran tablets and the tabletscan cause mucosal damage if they are retained within theesophagus while we expected an increase in the frequencyof intestinal mucosal injury among the patients who tookdabigatran as a consequence of the tartaric acid coating this 0cgastroenterology research and practicestudys ï¬ndings did not demonstrate a higher rate of smallbowel lesions associated with the use of this doac themechanisms underlying mucosal injuries caused by doacsother than dabigatran remain unclear smallbowel lesionsincluding redness erosions and angioectasia might be moreeasily detected by performing ce on patients who takedoacs because the doacs might cause bleeding thatcould facilitate the detection of the lesionsthis study has several limitations while this was a multicenter study the sample size was small hence more patientsshould be accrued and investigated in the near future moreover this study only included data that described the patientsï¬ndings after the administration of the doacs and datadescribing the ï¬ndings before their administration wereabsent therefore it remains unclear whether small intestinallesions are directly induced by doacs studies of patientsï¬ndings before and after the administration of doacs areneeded in the near future furthermore patients with severeanemia and overt bleeding were excluded from this studyand most of the enrolled patients did not have bleeding orhad minor bleeding patients taking edoxaban were notincluded in this study because edoxaban was not available injapan when this study began conclusionlesionssmallbowelincluding redness erosions smallulcers and angioectasia were detected in of patientswho took doacs more patients using doacs should beinvestigated using ce in the near futureabbreviationsdoac direct oral anticoagulantvideo capsule endoscopyvceldalowdose aspirincapsule endoscopycegastrointestinalgihemoglobinhbegdesophagogastroduodenoscopyproton pump inhibitorppidata availabilitythis manuscript describes a study that was aimed at evaluating direct oral anticoagulant doac related to smallbowellesions using video capsule endoscopy we believe that ourstudy makes a significant contribution to the literaturebecause its ï¬ndings showed that many patients takingdoacs had smallbowel lesions however most lesions wererelatively mild and they did not cause bleedingconflicts of interestthe authors have no conï¬icts of interest to disclose that arerelevant to this studyauthors contributionshiroyuki imaeda was responsible for the conception anddesign and ï¬nal approval of the article minoru yamaokahiroyuki imaeda naoki hosoe kazuaki yoneno ryukanno hisashi mitsufuji takahiro sasaki and keijiyamamoto were responsible for enrollment of patientshiroyuki imaeda and naoki hosoe were responsible foranalysis and interpretation of the data minoru yamaokaand hiroyuki imaeda were responsible for drafting of thearticle naoki hosoe was responsible for the critical revision of the article for important intellectual content takanori kanai toshimasa yamamoto toshihide mimuraharuhiko ogata nobuo araki and hidetomo nakamotowere the supervisorsreferences w k leung i bjarnason v w s wong j j y sung andf k l chan small bowel enteropathy associated withchronic lowdose aspirin therapy lancet vol no t watanabe s sugimori n kameda small bowelinjury by lowdose entericcoated aspirin and treatment withmisoprostol a pilot study clinical gastroenterology andhepatology vol no pp j iwamoto y mizokami y saito smallbowel mucosalinjuries in lowdose aspirin users with obscure gastrointestinalbleeding world journal of gastroenterology vol no pp h endo k hosono m inamori incidence of smallbowel injury induced by lowdose aspirin a crossover studyusing capsule endoscopy in healthy volunteers digestionvol no pp a shiotani k haruma r nishi randomized doubleblind pilot study of geranylgeranylacetone versus placebo inpatients taking lowdose entericcoated aspirin lowdoseaspirininduced small bowel damage scandinavian journalof gastroenterology vol no pp s j connolly m d ezekowitz s yusuf dabigatranversus warfarin in patients with atrial ï¬brillation the newengland journal of medicine vol no pp m r patel k w mahaï¬ey j garg rivaroxaban versuswarfarin in nonvalvular atrial ï¬brillation new england journal of medicine vol pp c b granger j h alexander j j v mcmurray apixaban versus warfarin in patients with atrial ï¬brillation thenew england journal of medicine vol no pp c t ruï¬ r p giugliano e braunwald comparison ofthe eï¬cacy and safety of new oral anticoagulants with warfarinin patients with atrial ï¬brillation a metaanalysis of randomised trials lancet vol no pp c s miller a dorreen m martel t huynh and a n barkun risk of gastrointestinal bleeding in patients taking nonvitamin k antagonist oral anticoagulants a systematic reviewand metaanalysis clinical gastroenterology and hepatologyvol no pp 1683e3 d caldeira m barra a ferreira systematic reviewwith metaanalysis the risk of major gastrointestinal bleeding 0cgastroenterology research and practicewith nonvitamin k antagonist oral anticoagulants alimentary pharmacology therapeutics vol no pp t yamashita y koretsune y yang edoxaban vs warfarin in east asian patients with atrial ï¬brillationan engageaftimi subanalysis circulation journal vol no pp m hori m matsumoto n tanahashi rivaroxaban vswarfarin in japanese patients with atrial ï¬brillation circulation journal vol no pp y toya s nakamura k tomita dabigatraninducedesophagitis the prevalence and endoscopic characteristicsjournal of gastroenterology and hepatology vol no pp k kasai e ishida and y kobayashi two cases of esophagealulcer caused by dabigatran gastroenterological endoscopyvol pp m okada and k okada exfoliative esophagitis and esophageal ulcer induced by dabigatran endoscopy vol supplement pp e23e24 t vanassche j hirsh j ginsberg and j eikelboom anspeciï¬c bleeding patterns of anticoagulant therapy lessonsfrom clinical trials thrombosis and haemostasis vol pp h endo k hosono m inamori characteristics ofsmall bowel injury in symptomatic chronic lowdose aspirinusers the experience of two medical centers in capsule endoscopy journal of gastroenterology vol no pp i watari s oka s tanaka comparison of smallbowelmucosalinjury between lowdose aspirin and nonaspirinnonsteroidal antiammatory drugs a capsule endoscopystudy digestion vol no pp h endo k hosono t higurashi quantitative analysisof lowdose aspirinassociated small bowel injury using a capsule endoscopy scoring index digestive endoscopy vol no pp d kaufman g leslie n marya smallintestinalangioectasia characterization risk factors and rebleedingjournal of clinical gastroenterology vol no pp a igawa s oka s tanaka major predictors and management of smallbowel angioectasia bmc gastroenterologyvol no 0c' | 0 |
Acute myeloid leukemia AML is a complex hematological disease characterized by genetic and clinical heterogeneity The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients Compared with recurrent chromosomal translocations in AML t816p112p133 can be found in any age group but is very rare and typically associated with poor prognosisMethods Conventional cytogenetic studies were performed among AML patients recorded in our oncology database over the last years Fluorescence in situ hybridization FISH was carried out to detect the translocation fusion Array comparative genome hybridization aCGH was carried out to further characterize the duplication of chromosomesResults We identified three AML patients with t816p112p133 by chromosome analysis Two of the three patients who harbored an additional 1q duplication were detected by FISH and aCGH aCGH characterized a Mb and Mb gain in chromosome at band q321q44 separately in these two patients One patient achieved complete remission CR but relapsed months later The other patient never experienced CR and died years after diagnosisConclusion A 1q duplication was detected in two of three AML patients with t816p112p133 suggesting that 1q duplication can be a recurrent event in AML patients with t816 In concert with the findings of previous studies on similar patients our work suggests that 1q duplication may also be an unfavorable prognostic factor of the diseaseKeywords 1q duplication Acute myeloid leukemia t816p112p133 Prognostic factorBackgroundAcute myeloid leukemia AML is a common disease characterized by immature myeloid cell proliferation and bone marrow failure which can be subdivided into pathogenetically different subtypes [] Over the past two decades the incidence has increased by [ ] Furthermore AML has poor longterm survival with a Correspondence lzhang202003163com Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of ChinaFull list of author information is available at the end of the high relapse rate [] Therefore AML represents a substantial health problem that requires strict monitoring and innovative treatment strategies The development of newer effective treatment strategies is necessary for AML patientsTo date the detection of cytogenetic abnormalities has been regarded as a critical prognostic tool for AML treatment [] Hence it is urgently necessary to identify chromosomal rearrangements in AML patients and provide the whole spectrum of cytogenetic abnormalities for AML [] According to the World Health anization classification system updated in AML with recurrent genetic abnormalities including t821q22q22 The Authors Access 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 0cLiu a0et a0al Mol Cytogenet Page of t1517q24q21 t1517PMLRARA t11q23MLL inv16p131q22 and t1616p131q22 has been identified [ ] Nonrandom chromosomal abnormalities such as deletions and translocations have been detected in approximately of all adult AML patients Moreover chromosomal abnormalities have been recognized as genetic events that can cause and promote this disease [] Certain cytogenetic abnormalities including t821q22q22 t1517q24q21 and inv16p131q22 are associated with longer remission and survival while alterations of chromosomes 11q23 and complex karyotypes are associated with poor response to therapy and shorter overall survival [] Chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBMYH11 and t11q23MLL are usually found in AML patients [ ] However AML with t816p112p133KAT6ACREBBP is a very rare AML subtype and can be found in any age group from infancy to the eighth decade of life with a female predominance [] A majority of adult patients with t816p112p133 are therapy related [] and pediatric patients tend to be de novo [] There are approximately cases reported in the literature [] and the first t816p112p133 in an infant was described in [] Some AML patients with t816 p112p133 have a bleeding tendency and disseminated intravascular coagulopathy which are overlapping clinical features that mimic acute promyelocytic leukemia APL [] Unlike APL AML with t816p112p133 has an unfavorable treatment response and outcome [ ] As a sole chromosomal anomaly t816p112p133 is found in more than of reported cases and one or more additional chromosomal anomalies can be seen in the remaining cases [] The most common secondary chromosomal anomalies are total or partial trisomy and monosomy or deletion of the long or short arm of chromosome [ ] Comparatively the gain of 1q in variable sizes has also been frequently noticed in patients with t816p112p133 in these large studies [ ]Recurrent cytogenetic abnormality t816p112p133 is seldom associated with AML and the 1q duplication in AML patients with t816p112p133 has never been discussed In the present study a total of de novo or treatmentrelated AML patients were collected from our laboratory oncology database Among them three patients were detected with t816p112p133KAT6ACREBBP and two of these three showed an additional copy of partial chromosome 1qMethodsPatientsThis study was approved by the Institutional Review Board IRB of Oklahoma University IRB Number A total of AML patient samples were studied cytogenetically from to at the Genetics Laboratory of Oklahoma University Health Sciences Center Bone marrow samples were obtained from three of the patients who had t816p112p133Conventional cytogenetic analysisShortterm cultures of unstimulated bone marrow samples were established and harvested according to standard laboratory protocols Karyotype analysis was performed using Giemsa and trypsin techniques for Gbanding The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature ISCN Fluorescence in a0situ hybridization analysisFluorescence in a0 situ hybridization FISH assays were performed according to the manufacturers instructions in combination with our established laboratory protocols A PMLRARA dualcolor dualfusion translocation probe Abbott Molecular Inc Des Plaines IL USA subtelomerespecific probes for chromosome parm and qarm and whole chromosome painting WCP probes for chromosomes and were purchased from Cytocell Ltd NY USA A spectrum greenlabeled probe mapping to the 8p1121 region and a spectrum orangelabeled probe mapping to the 16p133 region were created in house with the following BACPAC clones RP11642I24[chr8 4167633641856494hg19] and RP11589C21[chr8 4187370242036222hg19] RP11619A23[chr16 37200763914571hg19] and RP1195J11[chr16 38603744025510hg19] Childrens Hospital Oakland Research Institute Oakland CA USA The KAT6A gene located on 8p1121 and the CREBBP gene located on 16p133 were covered by the greenlabeled and redlabeled homebrewed probes respectively All probes were validated before use Chromosome spreads were counterstained with 46diamidino2phenylindole DAPI4 in antifade medium Vector Laboratories Inc CA USA Digital images carrying specific hybridization signals were captured and processed on CytoVision version Applied Spectral Imaging Carlsbad CA USAaCGH analysisGenomic DNA was extracted from each of the three patients bone marrow pellets according to the standard operating procedure using the phenol and chloroform method with a commercially available DNA extraction kit Puregene blood kit Qiagen Valencia CA or Nucleic Acid Isolation System QuickGene610L FUJIFILM Corporation Tokyo Japan Two aCGH platforms NimbleGen and Agilent were used in this study For the 0cLiu a0et a0al Mol Cytogenet Page of NimbleGen aCGH platform human reference genomic DNA was purchased from Promega Corporation Promega Corporation Madison WI USA The patients DNA and the reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming and then equal quantities of both labeled products were mixed and loaded onto a a0K oligonucleotide chip Roche NimbleGen Inc Madison WI USA to hybridize at a0 °C for a0 h in a MAUI hybridization system BioMicro Systems Salt Lake City UT according to the manufacturers protocols with minor modifications The slides were washed with washing buffers Roche NimbleGen Inc after hybridization and scanned using a Roche Scanner MS Microarray Scanner Roche NimbleGen Inc Images were analyzed using NimbleScan software version and SignalMap software version Roche NimbleGen Inc The genomic positions were determined using GRCh36hg18 UCSC Genome Browser For the Agilent aCGH platform human reference genomic DNA was purchased from Agilent Corporation Agilent Corporation Santa Clara CA USA The patients DNA and the purchased reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming Agilent Corporation Patient DNA labeled with Cy3 was combined with a normal control DNA sample labeled with Cy5 of the same sex and hybridized to an Agilent à a0K oligo microarray chip Agilent Technologies by incubating in an Agilent Microarray Hybridization Oven Agilent Technologies After a0h of hybridization at a0°C the slides were washed and scanned using the NimbleGen MS Microarray Scanner Roche NimbleGen Inc Agilents CytoGenomics software Agilent Technologies was applied for data analysis The genomic positions were determined using GRCh37hg19 UCSC Genome BrowserCase presentationCase An 82yearold male presented with anemia was referred to us for AML evaluation His subsequent lab results and hospital records were not available in our clinical databaseCase A 28yearold female presented with disseminated intravascular coagulopathy was referred to rule out APL Her complete blood examination and bone marrow aspirate smears were not available Flow cytometry revealed monocytic cells positive for CD4 CD11b partial CD13 bright CD14 partial CD15 CD33 bright and HLADR partial but negative for CD3 CD7 CD34 CD117 MPO and TdT consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient achieved hematological CR on day and cytogenetic CR on day after induction chemotherapy and then relapsed a0months laterCase A 69yearold female with a medical history of breast cancer after lumpectomy chemotherapy and radiation presenting with generalized weakness pancyt ia and fever was referred to us for disease progression evaluation A complete blood examination showed a white blood cell count of à 109L with blasts a hemoglobin count of a0 gL and a platelet count of à 109L Her bone marrow aspirate smear demonstrated over myeloblasts Flow cytometry revealed that of the blast cells expressed CD45 moderate CD34 dim CD38 HLADR CD13 CD15 and CD33 and were negative for CD117 consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient started consolidation chemotherapy but had spontaneous regression and died a0years after AML diagnosisResultsIn case routine chromosome analysis detected an abnormal karyotype with a translocation between the short arms of chromosomes and Fig a01a in of cells consistent with a diagnosis of AML with t816p112p133 The nomenclature of the cytogenetic findings in patient was t816p112p133[]46XY[] No other consistent karyotypic aberrations were detected Thus this male patient was excluded from subsequent FISH and aCGH analysesIn case chromosome analysis demonstrated the same chromosome rearrangement between and in all cells Besides of these cells showed an extra chromosome segment attached to chromosome Fig a01b The karyotypes in patient were described as 46XXt816p112p133 add14p112[]46XY[] Negative FISH t1517q24q21PMLRARA further ruled out a diagnosis of APL data not shown Metaphase FISH analysis confirmed the t816p112p133KAT6ACREBBP fusion and demonstrated a part of chromosome on chromosome Fig a02a and b In addition to characterizing the extrachromosomal material aCGH was carried out aCGH confirmed the FISH findings and detected a a0Mb gain from chromosome at bands q321q44 a0bp GRCh36hg18 USCS Genome Browser Fig a03aIn case t816p112p133 with a gain of a similar chromosome segment on the long arm of chromosome was detected in of cells by karyotyping analysis Fig a0 1c FISH confirmed the KAT6ACREBBP fusion and revealed additional chromosome material Fig a02c and d Loss of the end portion of the chromosome long arm was not found by FISH Fig a03e aCGH further detected a gain from chromosome at bands 1q321q44 results for 0cLiu a0et a0al Mol Cytogenet Page of a Patient b Patient Fig Representative abnormal karyotypes of three patients with t816p112p133 a Karyotype of patient showing 46XYt816p112p133 as the sole abnormality b and c Karyotypes of patients and showing 46XXt816p112p133 and an additional chromosome segment attached to the short arm of chromosome and the long arm of chromosome respectively Translocated derivatives and are indicated by black arrows and derivatives and are indicated by red arrows 0cLiu a0et a0al Mol Cytogenet Page of c Patient Fig continued a0 bp GRCh37hg19 UCSC Genome Browser Fig a0 3b The molecular size was a0MbDiscussionAML is one of the most common diseases characterized by the proliferation of blast cells in bone marrow or peripheral blood which accounts for approximately of adult leukemia cases As reported previously common chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBfrequently observed and numerous MYH11 are uncommon chromosomal aberrations also exist in AML [] The detection of these fusion transcripts is important for the diagnosis and progression monitoring of AML patients []t1517PMLRARA and In previous large studies approximately AML cases with t816p112p133 have been reported [] Among them cases showed a gain by 1q of variable sizes [ ] As an uncommon entity t816 accounts for of all cases of AML [] In our study three patients with t816p112p133 were identified one man and two women The two women were both diagnosed with AML subtype M5 and showed an extra copy of 1q at the same bands q321q44 which were different from the nine reported cases above The clinical features and cytogenetic data of the cases of AML with t816p112p133 and 1q duplications are summarized in Table a0 To the best of our knowledge this is the first study of the delineation of 1q duplication by aCGH in AML patients with t816p112p133AML patients with this abnormality often show unique clinical and biological characteristics [] Compared with the current categories t1517 t821 inv16 and t11q23 in AML t816 is clustered closer to t11q23 and shares commonly expressed genes [] Xie et a0 al reported adult AML cases with t816p112p133 indicating that t816p112p133 commonly exhibits monoblastic or myelomonocytic differentiation and arises in patients with a history of cytotoxictreated cancer Patients with de novo AML with t816 or treatmentrelated AML with t816 without adverse prognostic factors have a good outcome [] Identifying adverse prognostic factors is of importance to the choice of therapy and evaluation of survival in AML patients with t816 0cLiu a0et a0al Mol Cytogenet Page of CREBBPKAT6A fusionKAT6A8p1121CREBBPKAT6A fusionKAT6ACREBBP fusionCREBBP16p133CREBBP16p133KAT6ACREBBP fusiona KAT6A8p1121c WCP14WCP1WCP14b WCP1WCP1TelVysion 3q WCP1WCP1WCP3WCP1d WCP3TelVysion 3p TelVysion 3p TelVysion 3q e Fig Metaphase FISH of patient a and c showing KAT6ACREBBP fusion signals WCP FISH indicating the extra chromosomal materials on chromosome and chromosome were both from chromosome b and d No loss of the end portion of the chromosome long arm was indicated eOver the past a0 years cytogenetic and molecular technologies have largely promoted the efficiency of the identification and characterization of this disease [] Compared with conventional cytogenetic analysis and FISH methods aCGH is an attractive method for the investigation of cancer genomes [] aCGH has higher resolution simplicity high reproducibility shorter turnaround time and precise mapping of aberrations Most importantly it avoids the need for cell culture and dividing cells [] Furthermore aCGH chromosomal analysis facilitates rapid detection and duplication of cytogenetic abnormalities previously undetectable by conventional cytogenetics [] In our investigation we applied aCGH to characterize the additional chromosome materials in patients and and interestingly found that the two patients 0cLiu a0et a0al Mol Cytogenet Page of revealed the same extra copy of 1q at bands q321q44 Patients with 1q duplication have also demonstrated a wide range of multiple malformations such as intellectual disability macrocephaly large fontanels prominent foreheads broad flat nasal bridges higharched palates retrognathia lowset ears and cardiac defects [ ] More recent studies have shown that a 1q gain is also related to a portion of solid tumors For instance the gain of 1q is well known as a poor prognostic biomarker of Wilms tumor [] and it plays an important role in predicting poor clinical outcome in patients with thyroid carcinoma as well [] In addition patients with a 1q duplication showed worse survival and high risk in acute leukemia Burkitt lymphoma and myeloproliferative neoplasms [] The outcomes of 1q duplication in the nine reported AML patients with t816p112p133 are summarized in Table a0 Seven patients data were available These seven patients two adult and five pediatric all received induction chemotherapy and six achieved CR At the time of last followup two adult patients and three of five pediatric patients had died Only two pediatric patients were alive We reported two adult patients here patient achieved CR but relapsed a0 months later and patient had spontaneous regression and died a0 years after diagnosis Taken together the findings suggest that 1q duplication might be associated with adverse outcomes in AML patients with t816p112p133 However the significance of the 1q duplication in AML with t816 needs to be further investigated Since such changes have been seldom reported the pathogenic effects of 1q duplication in AML patients with t816p112p133 require more studies to be delineatedConclusionThree patients were detected with t816p112p133 from an AML patient database Two female patients were identified with a 1q duplication by FISH and aCGH analyses Combining our investigation with the findings of published studies we conclude that 1q duplication is a recurrent finding in AML patients with t816 Our data also suggest that 1q duplication might be associated with unfavorable prognosis in these cases The understanding of cytogenetic data would contribute to the diagnosis and treatment evaluation of AMLFig aCGH results of patient and patient showing partial 1q gain duplicated 1q regions are indicated by red frames 0cLiu a0et a0al Mol Cytogenet Page of Table The previously reported AML cases with a0t816p112p133 and a01q duplicationSex Age years FAB type Karyotype1q BandsOutcome yearsLast stateCase Case FFHaferlach et al FM5M5M5aDiab et alM M4Diab et alDiab et alDiab et alDiab et alFFFFXie et alM Brown et alM Brown et alFM45M4M4M5M4M4M446XXt816p112p133 add14p112[]46XX[]46XXt816p112p133[]46idemadd3q27[]45XXt816p11p13der1013q10q10[]46XXder7t17q21q35t816p11p13[]46XX[]46XY1del1p22t816p11p1310der14t1014q112p112[]47XYdel1q11der1t18p11q112x2i5p10810der14t1014q112p112der16t8165XXt816p11p1318der21t121q12p13[]46XX[]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110q11p11[]46idemadd7p21[]46XX[46XYt816p11p13der14t114q31p11[]46XderXtX1q26q23t816p11p13der11t1111p11q1346XYder3t38q27q13del6p22t816p112p133del10q21q25add13p112del16p12del20p112del20q112q133[]46idemdel1p35p363del15q23add19p131[]46XYt816q27q13del12q21q241del13q21q3116der19t119q32p133mar[]46XYdel6p22t816p112p133[cp2]46XY[]47XderYtY1q12q21 6t816 p11p13[]47idemdel13q3q3 [checked with CAD data]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110 q11p11 []46idemadd7p21 []46XX []1q321q441q321q44CR after inductionRelapsed months laterspontaneous regression1q21NAPartial 1q gain CR for 1q121q111q311q23CR for CR for CR for NA1q32CR for monthsAliveDiedNADeadAliveDiedAliveNADead1q21No CRDied month after treatment1q11Early remission after course Relapsed at months and months after diagnosisDiedAML acute myeloid leukemia FAB FrenchAmericanBritishh M male F female NA not available CR complete remissionAbbreviationsAML Acute myeloid leukemia aCGH Array comparative genomic hybridization FISH Fluorescence in situ hybridization APL Acute promyelocytic leukemia WCP Whole chromosome painting CR Complete remissionAcknowledgementsNot applicableAuthors contributionsM Liu and YR gathered clinical information and drafted the manuscript YR YK and M Liu performed routine cytogenetic analysis and participated in the interpretation of the results M Li performed FISH analysis and participated in the interpretation of the results XL supervised the FISH analysis and helped draft the manuscript XW performed CGH array analysis and helped draft the manuscript LZ and SL conceived the study participated in its design and 0cLiu a0et a0al Mol Cytogenet Page of extensively reviewed and revised the manuscript All authors have read and approved the final manuscriptFundingThis study has received no funding supportAvailability of data and materialsAll data generated or analyzed during this study are included in this published Ethics approval and consent to participateThis study was approved by University of Oklahoma Institutional Review Board for the Protection of Human SubjectsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of China Department of Pediatrics University of Oklahoma Health Sciences Center Oklahoma City OK USA Department of Neurology The Second Hospital of Jilin University Jilin Peoples Republic of China Received April Accepted August References Braess J Acute myeloid leukemia Dtsch Med Wochenschr Luppi M 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" the development of a safe effective reversible nonhormonal contraceptive method for men hasbeen an ongoing effort for the past few decades however despite significant progress on elucidating the functionof key proteins involved in reproduction understanding male reproductive physiology is limited by incompleteinformation on the genes expressed in reproductive tissues and no contraceptive targets have so far reachedclinical trials to advance product development further identification of novel reproductive tractspecific genesleading to potentially druggable protein targets is imperativeresults in this study we expand on previous single tissue single species studies by integrating analysis of publiclyavailable human and mouse rnaseq datasets whose initial published purpose was not focused on identifyingmale reproductive tractspecific targets we also incorporate analysis of additional newly acquired human andmouse testis and epididymis samples to increase the number of targets identified we detected a combined totalof genes for which no previous evidence of male reproductive tractspecific expression was annotated manyof which are potentially druggable targets through rtpcr we confirmed the reproductive tractspecific expressionof novel orthologous human and mouse genes without a reported mouse model of these we ablated fourepididymisspecific genes spint3 spint4 spint5 and ces5a and two testisspecific genes pp2d1 and saxo1 inindividual or double knockout mice generated through the crisprcas9 system our results validate a functionalrequirement for spint45 and ces5a in male mouse fertility while demonstrating that spint3 pp2d1 and saxo1 areeach individually dispensable for male mouse fertilitys our work provides a plethora of novel testis and epididymisspecific genes and elucidates thefunctional requirement of several of these genes which is essential towards understanding the etiology of maleinfertility and the development of male contraceptiveskeywords contraception drug target male reproductive tract paralog sperm maturation spermatidspermatozoa correspondence coarfabcmedu thomasgarciabcmedu1dan l duncan comprehensive cancer center baylor college of medicine baylor plaza houston tx usa3department of pathology and immunology baylor college of medicine baylor plaza houston tx usafull 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 0crobertson bmc biology page of the world human population reached nearly eight billion people in august this number continues torise and is predicted to reach nearly ten billion by theyear the increasing need to promote familyplanning through the development of reliable contraceptive options available to both men and women is widelyrecognized currently there are numerous contraceptiveoptions available to women however identification of asafe nonhormonal contraceptive option for men is stillan ongoing challenge although several different fertilitycontrol alternatives for men have been investigatednone are currently clinically approved for use our understanding of the mechanisms underlying male reproductive physiology is still at an early stage as theidentification and elucidation of the function of key reproductive proteins is still an ongoing effort identifyingdruggable protein targets expressed in the male reproductive tract has been the focus of numerous studiesdedicated to the development of male contraceptionessentialroletheitsthe mammalian epididymis is a segmented ancomprised of a single highly coiled tubule with functionally and morphologically distinct regions that can besubdivided most simplistically into a proximal centraland distal region conventionally named the caput corpus and cauda regions respectively as mammalianspermatozoa transit through the epididymis they acquire the ability to recognize and fertilize an egg properties that they did not possess upon exiting the testis epididymisinconsideringaddition to maturing germ cells of the testis and spermatozoais a prime target for the development of a malecontraceptive to advance progress towards the development of a nonhormonal male contraceptive several previous highthroughput studies have been published thatidentified a number of human mouse and rat genes astestisspecific or epididymisspecific [ ] in schultz conducted the first study to identify malereproductive tractspecific genes using microarraysthroughgeneexpression analysis of meiotic and postmeiotic spermatogenic cells together with parallel analysis of available data from the ncbi unigene database the authorsidentified mouse genes as testisspecific which included genes with both known and unknown function atthe time in the following years through two additional microarraybased studies of rat testes and purifiedrat testicular cells johnston identified and additional or overlapping genes astestisspecific in as part of the continued effort to identify novel contraceptive targets the newer rnaseqbased transcriptomics methodology was utilized identifying human genes as testisspecific together withantibodybased protein profiling many of these genesaffymetrixbasedgenomewidewere characterized in terms of the spermatogenic cellpopulations showing expression the first highthroughput transcriptomics study to identify epididymisspecific genes was a mouse epididymal transcriptome studyin which rna isolated fromeach of the epididymal segments was analyzed bymicroarray analysisidentifying epididymisspecificgenes with distinct patterns of segmental gene regulation later in additional transcriptome profiling utilizing whole genome microarrays resulted in identification of previously unreported epididymisspecific transcripts inthe mouse and epididymisspecific transcripts inthe rat a significant number of the identified mouseand rat genes in these studies were not known at the timeand only the probe identification numbers were presentedwhen evaluating potential druggability in a targetbased drug discovery process one must consider theprotein properties that are required for safe and effectiveinhibition among the most significant is tissue expression specificity to minimize potential adverse effectsprotein function and whether protein activity or interaction with other proteins is potentially druggable sequence similarity to closely related paralogs that may beubiquitously expressed and whether genetically manipulated animal models demonstrate a functional requirement for the target of interest several noteworthyreview publications have mentioned numerous geneswhose critical functions high expression and specificityto the testes or epididymides make them viable nonhormonal male contraceptive targets [] howeveramong the identified genes a significant number either are required for fertility but are expressed in nonreproductive tissues or are reproductive tractspecific but when disrupted lead to subfertility ineither case both are ineffective and highly undesirableoutcomes for a potential male contraceptive targettherefore the identification of additional novel male reproductive tractspecific genes would allow for furtheradvances to be made in the quest to develop an effectiveand safe nonhormonal male contraceptivein this study newly acquired and previously published human and mouse rnaseq datasets [ ]were processed in parallel through a custom bioinformatics pipeline designed to identify novel reproductive tractspecific and reproductive tractenriched transcripts additional databases obtained from illuminating the druggable genome mouse genome informatics andensembl biomart were utilized to stratify the resultsinto subgroups based on protein druggability and on theavailability of a mouse model numerous reproductivetractspecific and reproductive tractenriched potentiallydruggable targets for which no published mouse modelexists congruent in expression across both mouse and human datasets were identified through our analysis and 0crobertson bmc biology page of verified through conventional polymerase chain reactionpcr we present the data in a manner that should bemost relevant and of substantialinterest to the malecontraceptive development field since identification ofnew targets worthy of consideration for further functionalanalysis in a knockout animal model and potential drugtargeting continues to be of vast importanceresults wethrough ouridentified four novelepididymisspecific genes spint3 spint4 spint5 andces5a and two novel testisspecific genes pp2d1 andsaxo1 worthy offunctional validation in an animalmodel through the crisprcas9 system we generatedfour individual gene knockouts spint3 ces5a pp2d1and saxo1 and one double knockout mouse modelspint45 revealing an essential requirement for spint4and spint5 in male mouse fertility and the potential utility of pursuing spint4 in humans as a nonhormonalcontraceptive targetresultsstudy approaches and datadespite significant advances in our understanding of thehuman and rodent testis and epididymis transcriptomemostly through microarraybased studies no prior studies have utilized purified human testis cells for the identification of human testisspecific transcripts no priorstudies have utilized the more stateoftheart rnaseqbased transcriptomics methodology for analysis of human epididymisspecific transcripts and no prior studieshave utilized rnaseq analysis of rodent reproductivetissues or cells to identify rodent reproductive tractspecific transcripts to address these gaps in knowledgeand to increase the number of identified reproductivetractspecific genes in both species using the most relevant highthroughput transcriptomics methodology weanalyzed in parallel on a custom bioinformatics pipelinea large number of published and newly acquired humanand mouse rnaseq datasets one hundred and sixtytwo previously published human and previously published mouse rnaseq datasets were retrieved from thesequence read archive sra the sra value for eachsample is listed in additional file table s1 and additional file table s2 we also generated new humanand new mouse reproductive tissue rnaseq samplesgeo accession gse150854 the final dataset is comprised of new and previously published human testisdatasets previously published purified humangerm cell datasets [ ] previously published purified human sertoli cell datasets [ ] new and previously published human epididymis segment datasets previously published mouse testis datasets new mouse epididymis datasets previouslypublished purified mouse germ cell datasets [ ]and previously published purified mouse sertoli celldatasets an additional previously publisheddatasets contributed to the nonreproductive humantissues and previously published datasets contributed to the nonreproductive mouse tissues figure 1a b summarizes all the samples acquired for thestudywe performed a principal component analysis tovisualize the variation in the samples after correcting forbatch effects human reproductive and nonreproductivetissues grouped according to sample type the reproductive tissue samples clustered by tissue type whetheror not they were newly generated or acquired from thesra fig 1c mouse data showed a similar variation inthe samples based on the tissue type fig 1d for bothhuman and mouse reproductive tissues samples separated by whether or not the rnaseq was performed onisolated cells or the whole tissue epididymal tissue wasdistinct from testis tissue in both human and mousefig 1c didentified in the mouseto identify potential male reproductive tractspecificdrug candidates we analyzed the aggregated rnaseqdata to find genes that were statistically significant in expression when compared to the nonreproductive tissuethat had the maximum expression for that gene thisgene list was then further refined by filtering for genesthat were lowly expressed in the nonreproductive tissuethat had the maximum expression for that gene tpmless than or equal to for human tpm less than orequal to for mouse finally this tpm filtered listwas then filtered for the genes that had a reproductivetissue or cell expression value greater than or equal to tpm for human or tpm for mouse fig 2aacross all the reproductive tissues candidate geneswere identified in the human and candidate geneswerefig 2badditional file fig s1 additional file table s3 andadditional file table s4 summarize the differentialfold change identity of the nonreproductive tissue withmaximal gene expression based on the differential geneanalysis fdr average and standard deviation tpm expression values and log2 cpm gene expression value forthe human and mouse samples respectively the resultsfrom the fdr and tpm expression value filtering forthe human and mouse samples are summarized inadditional file table s5 and additional file tables6file table s5 andadditional file table s6 report the log2 fold changefor the reproductive tissue or cell of interest comparedto the tissue with maximal gene expression the genesidentifiedandadditional file table s6 pass the filters in at least oneofinadditional file table s5 and additional file tables6 a value of zero for a given gene and fold expressionthe reproductive tissues or cells ofrespectively additionalinterestsamplesin additionalfile tables5 0crobertson bmc biology page of fig see legend on next page 0crobertson bmc biology page of see figure on previous pagefig summary of the human and mouse rnaseq samples used in the identification of novel male reproductive tractspecific drug targets thernaseq samples used in the human a and mouse b analyses are schematically shown principal component analysis was performed on thehuman c and mouse d nonreproductive and reproductive samples separately the colors of the circles next to the tissues listed in a and bcorrespond to the colors used in the circles for the pca in c and d sample size n values in red andor black denote the number of new redand previously published black samples included in our analysiscomparison indicates that for that comparison the genedid not pass the filters the majority of genes weredownregulated in the reproductive tissue ofinterestcompared to the maximal geneexpressing nonreproductive tissue additional file fig s2 from theanalysis the majority of the candidate genes that passedthe fdr and tpm filters were identified in the testis orspermrelated cells in both human and mouse samplesadditional file fig s2the majority of candidate genes identified in ourscreen that were testisspecific were already identified bythe human protein atlas andor our reanalysis offig identification of candidate drug male reproductive gene targets a diagrammatic representation of overall methodology used to identifyreproductive tractspecific candidate genes in humans genes and in mice genes the maximum gene expression was determinedacross all the nonreproductive tissue samples for each gene for a reproductive tissue or cell sample of interest genes were then filtered forsignificance using a false discovery rate fdr of less than or equal to based on the differential gene expression analysis for the nonreproductive tissue with maximum gene expression and reproductive tissue or cell sample of interest genes that passed the fdr filter werefiltered such that the average tpm expression value of the maximum expressing nonreproductive tissue was less than or equal to tpm andthe average tpm expression value of the reproductive tissue or cell of interest was greater than or equal to tpm b diagrammaticrepresentation of the number of human and mouse candidate genes in terms of the number of orthologs in the opposite species thenumber of genes previously or not previously identified in a prior transcriptomicsbased drug target report the availability and phenotypicoutcome of any reported mouse models and the number of novel genes without a reported mouse model congruent across both speciesthe main value in each bubble represents the total number of candidate genes identified regardless of tissue or cell identified in the numbers inparentheses comprise the total number of candidate genes that are either epididymisspecific or specific to testis and epididymis but not testisandor testis cellspecific only 0crobertson bmc biology page of the hpa testis datasets additional file fig s3 andadditional file table s7 thirtysix out of the genes that were identified across all the human epididymis tissue were also identified by the human combinednewly acquired and previously published datasets testiscandidate gene list finally the majority of the candidategenes identified from the combined newly generated and previously published human testis datasetswere shared with genes identified from the various testiscell datasets we identified more candidate genes in thenewly generated human epididymis tissues compared topreviously published data out of genes wereunique to the newly generated caput samples comparedto only out of genes which was unique to the previously published samples out of genes were uniquein the newly generated corpus samples compared to out of genes which were unique to the previously published corpus samples and genes were unique to thenewly generated cauda samples compared to genes inthe previously published cauda data with no overlap between the two cauda gene lists additional file fig s3and additional file table s7 there were candidategenes that overlapped between the newly generated human testis samples and mouse testis sample gene listswhile there were candidate genes that overlapped between the previously published human testis sample andmouse testis sample gene lists additional file fig s3and additional file table s7 across all human epididymis tissue samplesincluding the newly generated andpreviously published samples there were genes in common with the combined list of candidate genes across allthe mouse epididymis tissue samples there was a smalloverlap between the human and mouse samples when thenewly generated human caput corpus and cauda tissueswere individually compared to the mouse caput corpusand cauda tissues there was an overlap of and forrespectivelyadditional file fig s3 and additional file table s7this trend was continued for the candidate gene lists derived from the previously published human caput corpusand cauda samples when compared to the candidate genelist from the mouse caput corpus and cauda with and genes in common for the caput corpus and caudacomparisons respectively additional file fig s3 andadditional file table s7 additional file table s7 details the genes that are unique and in common for each ofthe comparisonscorpuscaputtheandcaudato assess the potential usefulness of the candidategenes identified in each human reproductive tissue asdrug targets we assigned the genes to a protein familyie gpcr or ion channel the majority of identifiedgenes were not from a traditional drug target family likekinases or enzymes the testis and germ cell datasetsprovided the most potential targets while the epididymisdatasets provided the fewest additionalfile figs4a the protein family classification for each candidate gene identified in each reproductive tissue is detailed in additional file table s8 the majority of thecandidate genes do not have a reported mouse modeladditional file fig s4b additional file table s9summarizes mouse model availability for each candidategene identified from human reproductive tissues or cellsfigure shows the complete list of novel human geneswithout a reported mouse model as identified in each ofthe respective cell andor tissue datasets digital pcrsheatmap and conventional pcrs demonstrating expression of a subset of the novel human reproductivetractspecific genes without a reported mouse modelthat we identified are shown in figs and respectively additional file fig s5 shows the complete listof previously identified human genes that remain without a reported mouse model as identified in each of therespective cell andor tissue datasets additional file fig s6 shows the complete list of male reproductivetractspecific human genes for which a previously generated mouse model shows male infertility phenotype asidentified in each of the respective cell andor tissuedatasetsreproductive tractspecific genes identified throughhuman datasetsthrough our bioinformatics analysis of previously published and newly acquired rnaseq datasets we identified a total of genes as reproductive tractspecific inhumans fig of these genes genes do not have amouse gene ortholog while genes have a mousegene ortholog fig of those with a mouse geneortholog have a single gene ortholog have thesame symbolin mouse while have a differentsymbol in mouse while have two or more orthologous mouse genes seventysix human genes had orthologous mouse symbols genes had orthologous mouse symbols and genes fam205a krtap106 magea10 or2ag1 pramef11 pramef2ssx2 ssx3 and ssx4b had greater than orthologous mouse symbols symbols additional file table s5 of the human genes that we identified asmale reproductive tractspecific have not been previously identified in a transcriptomicsbased male reproductive tractspecific study [ ] the sum of ourhuman data confirms the findings of out of genes from djureinovic after reidentificationof gene symbols from reported affymetrix ids and consideration of orthologous genes mouse to human andrat to human our human data confirm the findings of out of genes from johnston out of genes from schultz out of genes fromjohnston out of genes from johnston 0crobertson bmc biology page of fig two hundred and thirtythree novel human reproductive tractspecific genes that each have mouse orthologous genes but with noreported knockout mouse models the listed genes were identified in one or more datasets as indicated in the venn diagram underlined geneswere also identified in our studies as reproductive tractspecific in mouse genes genes written in blue encode either enzymes kinasesgpcrs ogpcrs transporters transcription factors or proteins involved in epigenetic regulation genes genes written in dark red wereidentified in both testis testis andor testis cell and epididymis genes out of genes from johnston and out of genes from jelinsky of the genes that have a mouse gene ortholog have notbeen previously identified as male reproductive tractspecific and of these human genes currently lackmouse phenotype information based on data obtainedfrom ensembl biomart mgi impc and ncbihuman testisspecificthree hundred and eightysix genes were identified astestisspecific through either the reanalysis of djureinovic testis datasets genes identified analysis ofour de novo testis datasets genes identified orboth additional file table s5 three hundred andthirteen genes were congruent across both datasetswhile genes were uniquely identified through our reanalysis of djureinovic s datasets and only genes[ac1363524 ankrd20a1 ankrd62fam230aggtlc2iqcm potec prnt and utf1] wereuniquely identified through our de novo datasetsadditional file table s5 interestingly of the genes we identified through djureinovic s reanalyzed datasets were not previously identified in theirreport or any of the other previous reports [ ]of these genes we randomly verified of thesegenes as testisspecific in humans through conventionalpcr fig we also verified through rtpcr an additional genessuch as allc cdkl3 cox7b2or2h1 and sppl2cthat had been identified throughprevious studies additional file fig s7 of the genes identified through either testis datasets havenot been previously identified of these genes haveone or more mouse orthologs and of these genes arelacking reported phenotype information of the novelgenes lacking a reported mouse model genes encodeenzymes adam20 cpa5 dusp21 naa11 plscr2prss38 and triml1 encode transcription factorsbhmg1znf560znf729 encode transporters slc22a14 slc25a52and encode proteins of unknown drug target typetgif2lyfoxr2prdm9 0crobertson bmc biology page of fig representative novel reproductive tractspecific human a and mouse b genes without a reported mouse model the listed genes wereidentified through our studies as reproductive tractspecific in both humans and mice the digital pcr heatmap depicts the average transcriptsper million tpm value per tissue per gene from the indicated human and mouse rnaseq datasets as processed in parallel through ourbioinformatics pipeline the data was obtained from published and newly acquired datasets white tpm black ¥ tpm the expressionprofile of the human and mouse housekeeping genes gapdh and eif3l is included as reference for data obtained from published datasetssuperscript values succeeding the sample names reference the publications as follows djureinovic fagerberg guo zhu kumar browne consortium helsel da cruz and zimmermann such as etdb smim36 bend2 btg4 cnbd1dppa2 efcab5 erich6 fthl17 iqcm mroh2bms4a5 oosp2 pnma6e ppp4r3c rbmxl3 rtl9spdye4 spem2 all of these genes are listed in fig and many of these genes are listed in figs andor to the best of our knowledge no prior studies haveutilized purified human testis cells for the identificationof human testisspecific transcripts through our analysis we identified genes as human testisspecificthrough one or more of the human germ cell datasetsbut not through either of the human testis datasetsadditionalfile table s5 seventysix genes wereidentified exclusively through one or more of the fivehuman spermatogonia datasets genes such as anp32dc13orf42 dscr4 or13g1 or2d2 or52e4 ssx2tle7 while genes were identified exclusivelythrough the human spermatocyte datasets genes suchas h2bfm mageb17 mageb18 or2b6 tcp10 andznf709 and genes were identified exclusivelythrough the human spermatid datasets genes suchas ac0132691 clec20a or7e24 pramef2 spata31a3 tmem191c znf679 thirtyfour geneswere identified through all three cell types datasetsgenes such as ccdc166 eloa2 fam47a heatr9 and spata31a1 many of these genes are listedin figs andor of the genes identified as human testisspecificthrough one or more of the human germ cell datasets genes have not been previously identified ofwhich have one or more equivalent mouse orthologswith of these genes having not been knocked out inmouse of these novel genes with no mouse model encode enzymes glt6d1 prss48 satl1 sult6b1tmprss7 tpte2 triml2 and ttll8 encodes anepigenetic protein taf1l encode gpcrs gpr156tas2r13 tas2r30 tas2r46 tas2r50 vn1r2 encodes a kinase cdkl4 encode ogpcrs such asor2d3 or3a2 or52e5 or8g5 or10j1and 0crobertson bmc biology page of adrenal glandadiposecolonbrainskeletal musclesmooth musclesmall intestinesalivary glandleukocytespancreasstomachprostatethyroidkidneyspleentestisheartlungliverskincorpusuteruscaudaovarycaputaac0221675ac1876531adam20al6720431bhmg1bsph1bx2760929c1orf105c2orf92c4orf51c16orf95ccdc196ces5acpa5dcaf8l1efcab5erich6etdbfer1l5glt6d1iqca1llcn6magea11mageb6mroh2bnaa11pp2d1ppp4r3cprr23aprr23bprr23cprss38saxo1slc22a14spag11aspag11bspata31a1spata31d1spata31d3spata31d4spint3spint4tcp10l2tex13dtex44tex48tex51tle7tpte2triml1trpc5oswfdc8wfdc9wfdc10awfdc10bwfdc13gapdhskeletal musclesmooth musclesmall intestinestomachprostatethyroidspleentestislungskinkidneyheartlivercorpusuteruscaudaovarycaputadiposebladderbraincoloneyebgm5767gm5294adam201700042g07rikgm4969bsph14933412e24rik4930558k02rik4933424g06rik1700011l22rik1700018b08rikgm6657ces5acpa5pet2efcab5erich6etdfer1l5glt6d14931409k22riklcn6magea4mageb3mroh2bnaa11pp2d14932429p05rikprr23a3prr23a3prr23a3prss38saxo1slc22a14spag11bspag11bspata31spata31d1bspata31d1bspata31d1bspint3spint4spint5tcp10ctex13c1tex44tex48gm35060tle7tptetriml1trpc5oswfdc8wfdc9wfdc10wfdc10wfdc13hprtfig rtpcr confirmation of reproductive tract specificity in both humans a and mice b the genes listed in this figure are novel as identifiedthrough our studies and without a reported mouse model humans do not have an equivalent proteincoding equivalent to mouse spint5 gapdh and hprt are included as housekeeping genes 0crobertson bmc biology page of fig three hundred and two novel mouse genes with human orthologs without a reported mouse model the listed genes were identified inone or more mouse datasets as indicated in the venn diagram underlined genes were also identified through our studies as reproductive tractspecific in human genes genes written in blue encode either enzymes kinases gpcrs ogpcrs transporters transcription factors orproteins involved in epigenetic regulation genes genes written in dark red were identified in both testis testis andor testis cell andepididymis genesnkx24or14a2 encode transcription factorsznf99 znf648 znf705b znf705g and znf709 encode transporters abcc12 slc35g3 slc35g5 and encode proteins of unknown drug target type suchas ac0080733 ac1135541 aknad1 al0496342dnlz ervw1 etda fbxw10 fer1l5 lmntd1lrrc72 nms prr23a prr23b prr23c pxt1rfpl4b and ssx4b many of these genes are listed infigs andor fam236a figs and and obp2b met candidatethreshold through our analysis of human testes datasetsbut did not meet candidate threshold from any of thegerm cell or sertoli cell datasets indicating potential expression in peritubular myoid cells leydig cells or othercell outside of the seminiferous epithelium fam36a hasnot been previously identified and neither mouse orthologs 1700011m02rik gm9112 have been knocked outobp2b was previously identified through djureinovic and johnston however of the equivalent mouse orthologs lcn4 obp2a obp2b only obp2ahas been knocked out revealing abnormal coathair pigmentation ism2 and magec2 were identified through both human sertoli cell datasets while also identified throughtestis andor germ cell datasets both genes have beenpreviously identified ism2 magec2 ism2knockout mice display nonreproductive phenotypes consistent with this finding our mouse data do notidentify ism2 as reproductive tractspecific in micemagec2 lacks a mouse ortholog for functional analysis in micehuman sertoli cellspecifickrtap23 krtap412 lhx9 and psg5 were identified through one or both human sertoli cell datasets butwere not identified through any of the testis or germ cell 0crobertson bmc biology page of datasets indicating sertoli cellspecific expression in thetestes additional file table s5 none of these geneshave been previously identified as reproductive tractspecific in humans although lhx9 and psg5 havemouse orthologs that have been knocked out []human krtap23 has mouse orthologs krtap52gm4559 gm40460 and gm45618 and human krtap412 has mouse orthologs krtap47 and gm11555none of these mouse orthologs have been knocked outfig and additional file table s5knockin mousepsg5 knockout mice display nonreproductive phenotypes [] however lhx9 knockout mice display absent testes and sterility due to an essential requirementfor lhx9 during mouse gonad formation a lhx9gfpcreerbyknockingin gfpcreer at the endogenous lhx9 locuscrossed with the rosa26tdtomato reporter mouse linerevealed cre recombinase activity in retinal amacrinecells developing limbstestis hippocampal neuronsthalamic neurons and cerebellar neurons thuslhx9 is not reproductive tractspecific in mice ourmouse data confirm this findinglinegeneratedhuman epididymisspecificto the | 0 |
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 | 2 |
" exosomes are extracellular vesicles containing a variety of biological molecules including micrornasmirnas we have recently demonstrated that certain mirna species are selectively and highly enriched inpancreatic cancer exosomes with mir1246 being the most abundant exosome mirnas have been shown tomediate intercellular communication in the tumor microenvironment and promote cancer progression thereforeunderstanding how exosomes selectively enrich specific mirnas to initiate exosome mirna signaling in cancercells is critical to advancing cancer exosome biologyresults the aim of this study was to identify rna binding proteins responsible for selective enrichment ofexosome mirnas in cancer cells a biotinlabeled mir1246 probe was used to capture rna binding proteins rbpsfrom panc1 cells among the rbps identified through proteomic analysis srsf1 eif3b and tia1 were highlyassociated with the mir1246 probe rna immunoprecipitation rip and electrophoretic mobility shift assay emsaconfirmed the binding of srsf1 to mir1246 lentivirus shrna knockdown of srsf1 in pancreatic cancer cellsselectively reduced exosome mirna enrichment whereas gfpsrsf1 overexpression enhanced the enrichment asanalyzed by next generation small rna sequencing and qrtpcr mirna sequence motif analysis identified acommon motif shared by of srsf1associated exosome mirnas emsa confirmed that shared motif decoysinhibit the binding of srsf1 to the mir1246 sequences we conclude that srsf1 mediates selective exosome mirna enrichment in pancreatic cancer cells bybinding to a commonly shared mirna sequence motifkeywords srsf1 exosome mirna mir1246 pancreatic cancer exosomes are endosomederived extracellular vesiclesevs that can be transferred from cancer cells tostromal cells in the tumor microenvironment [ ]these membrane vesicles are nm in size andcontain proteinsincludinglipids and nucleic acids correspondence weiqundingouhscedu1department of pathology university of oklahoma health sciences centeroklahoma city stanton l young blvd bmsb 401a oklahoma city ok usa6stephenson cancer center university of oklahoma health sciences centeroklahoma city ok usafull list of author information is available at the end of the small rnas such as micrornas mirnas[ ]exosomemediated intercellular communication betweencancer cells endothelial cells [ ] fibroblasts [ ] orimmune cells [ ] can facilitate tumor progressionfurthermore cancer exosomes are released into the circulation and contribute to premetastatic niche formation in distant ans [ ]how cancer exosomes interact with stromal cells topromote tumor progression has been extensively investisignaling eventgated one criticalin the tumormicroenvironmentisthe exosome mirnamediatedintercellular communication [ ] studies haveshown that exosome mirna signaling promotes tumor 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 0cxu cell communication and signaling page of progression in various model systems [ ] notablyit has been reported that mirnas contained in exosomes are delivered to recipient cells in the tumormicroenvironment or distant ans where they canregulate target gene expression and promote tumorangiogenesis and metastasis [ ]in the context of exosome mirna signaling we andothers have reported that certain mirna species areselectively enriched in cancer exosomes as compared toexosomes derived from normal epithelial cells [ ] results from several studies have also indicated thatselective enrichment of exosome mirnas is relevant totumor progression for example exosome sortingof mir193a was found to promote colon cancer progression likewise mir122 a cancer exosomeenriched mirna [ ] was shown to reprogram glucose metabolism in a premetastatic niche to facilitatemetastasis in a breast cancer model system moreover the exosome enriched mir1246 was reportedto promote tumor invasion in both breast cancer and oral squamous cell carcinoma it seems clearthat selective enrichment of exosome mirnas drivescancer exosome mirna signaling in the tumor microenvironment which in turn reinforces tumor invasiveness and progression however how exosome mirnasare enriched or how exosome mirna signaling is initiated in cancer cells remains largely unknown elucidating the mechanisms ofselective exosome mirnaenrichment in cancer cells may help identify new cancertherapeutic opportunities that are urgently neededrecentreports have indicated that certain rnabinding proteinsrbps are involved in exosomemirna sorting in eukaryotic cells and the type ofrbps involved seems to differ among various modelsystems [ ] suggesting that exosome mirnasorting is a tissue or celltype specific processfurthermore there have been no reports on the identification of rbps that regulate exosome mirna sorting in pancreatic cancer cells we have recentlycharacterized the biogenesis of exosome mir1246 which is the most highly enriched mirna inpancreatic cancer cellderived exosomes the aimof this study was to utilize our established cell modelsystems to identify rbps that are involved in exosomemirna loading in pancreatic cancer cells using alabeled mir1246 probe as bait we fished out several rbpsincludingserine and arginine rich splicing factor srsf1eukaryotic translation initiation factor subunit beif3b and t cellrestricted intracellular antigen tia1 we found that srsf1 a recently claimedoncoproteininregulating exosome mirna enrichment in pancreaticcancer model systemsfrom pancreatic cancer cellspredominantlyinvolved ismethodscell culturelines panc1the human pancreatic cancer celllinemiapaca2 and bxpc3 and breast cancer cellmdamb231 were obtained from the american typeculture collection atcc manassas va usa cellswere cultured following atccs instructions except thatexosomedepleted fetal bovine serum fbs and horseserum wereapplied whenever needed exosomedepleted fbs and horse serum were prepared by pelleting the serum exosomes at g for h at °ccells were routinely incubated in a humidified environment at °c and co2exosome isolationexosomes were isolated from the culture medium utilizing a combination of centrifugation ultracentrifugationand filtration as we recently described [ ] withminor modifications in brief the culture medium ofpanc1 cells was precleared by g centrifugationfor min at °c and the resulting supernatant was filtered through a μm pvdf centrifuge filter thelarge size evs were trapped in the filter and recovered inpbs the filtered supernatant was then applied to a μm pvdf centrifuge filter the medium size evswere trapped in the second filter and resuspended inpbs the small size evs exosomes in the final supernatant were recovered by ultracentrifugation g min at °c the isolated exosomes were verified bywestern blot detecting positive and negative exosomemarker proteins and nanop analysis nanosightns300 system malvern instruments uk measuringboth sizes and concentrations of the isolated exosomesfig mirna binding protein pulldownpulldown experiment was performed using the pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific briefly pmol of biotinlabeled mir1246or polya rna oligonucleotides integrated dna technologies were hybridized to μl streptavidin magnetic beads prod1862766 thermo fisher scientificthe mir1246biotinstreptavidin beads were incubatedwith panc1 lysate for min at °c the lysatebeadmixture was washed three times with washing bufferfrom the abovementioned kit to elute bound proteins μl of elution buffer was applied and a magnetic separator was applied to separate the beads from the elutedprotein following the manufacturers protocol pierce¢magnetic rnaprotein pulldown kit thermo fisherscientific proteins were separated by sdspage beforemass spectrometry ms analysis 0cxu cell communication and signaling page of fig verification of the exosomes derived from panc1 cells a representative western blot analysis of cd63 nonreducing condition cd81flotillin and calnexin in the evs isolated from panc1 cells positive exosome markers are only detected in small evs exosomes b representativenanop tracking analysis of exosomes small evs derived from control and srsf1 knockdown panc1 cells three individual experimentswere performed for both a and bliquid chromatographymass spectrometry lcmsmassspectrometry ms measurementthe experiment was performed by the laboratory formolecular biology and cytometry research core facilityat ouhsc proteins were digested with trypsin according to the fasp protocol briefly the eluate was buffer exchanged in m urea the proteins were reducedwith mm dithiothreitol and then alkylated with mm iodoacetamide the peptides were eluted dried andresuspended liquid chromatography tandem mass spectrometry was performed by coupling a nanaoacquityuplc waters corp manchester uk to a qtofsynapt g2s instrument waters corp manchesteruk each protein digest about ng of peptide wasdelivered to a trap column μm mm nanoacquity uplc nanoease column μm beh c18 waterscorp manchester uk at a flow rate of μlmin in solvent a mm ammonium formate ph inhplc grade water tandem mass spectra were generated in the trapping region of the ion mobility cell byusing a collisional energy ramp from v low massstartend to v high mass startend the pusherionmobility synchronization for the hdmse method wasperformed using masslynx v41 and driftscope v24lockspray of glufibrinopeptideb mz wasacquired every s and lock mass correction was appliedpost acquisitionprotein identificationraw ms data were processed by plgs proteinlynxglobal server waters corp manchester uk for peptide and protein identification msms spectra weresearched against the uniprot human database containing reviewed sequences with the followingsearch parametersfull tryptic specificity up to twomissed cleavage sites carbamidomethylation of cysteineresidues was set as a fixed modification and nterminalprotein acetylation and methionine oxidation were set asvariable modificationssmall rna library preparation and next generationsequencingtotal rna was extracted from cell and exosome pelletsusing the trizol reagent invitrogenlife technologiescarlsbad california the small rna libraries were constructed and run on the illumina miseq platform as werecently described [ ]rna immunoprecipitation assaypanc1 cells or mdamb231 celllysates were prepared using ip buffer mm trishcl ph mmnacl mm edta mm pmsf and triton x the lysate was sonicated for min on ice andinsoluble material was removed by centrifugation supernatants were collected and protein concentrations weremeasured the supernatant was precleared by proteing dynabeads¢thermo fisher scientific and thenmixed with antibody srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 in a ratio of at °c overnight withgentle rotation to capture the antibodyproteinrnacomplexes μl of protein g magnetic beads wereadded and the complexes were rotated for h at °cthe sample was separated by magnetic separation trizol reagent invitrogenlife technologies was appliedto isolated rna from the complex the mirna expression was analyzed by qrtpcr 0cxu cell communication and signaling page of coimmunoprecipitation coipcoimmunoprecipitation coip using panc1 cell lysate and antibody of srsf1 santa cruz sc33652eif3b santa cruz sc137214 tia1 santa cruz sc gapdh promab and igg santacruz sc2025 was performed as described previously and the protein complex was detected by westernblotwestern blot analysiswestern blot was performed as we recently described[ ] primary antibodies raised against srsf1 santacruz sc33652 eif3b santa cruz sc137214 tia1santa cruz sc166247 betaactin a5441 and glyceraldehyde 3phosphate dehydrogenase gapdh santacruz sc47724 were used for detection nuclear andcytoplasmic protein extraction was extracted followingrockland nuclear cytoplasmic extract protocol and verified by histoneh3 cst 4499s andgapdh santa cruz sc47724 detection antibodiesused for exosome marker detection include cd63cd81 santa cruz bio technology inc ca usaflotillin1 and calnexin cell signaling technology incma usaquantitative realtime reverse transcription polymerasechain reaction qrtpcrqrtpcr was performed as we described [ ] withspecific primers cel54 ²gcgcgcccgtaatcttcataatcc3² mir1246 ²gcgcgatggatttttggagcag3² mir320c ²gcaaaagcuggguugagagggu3² and mir320d ²gcgaaaagcuggguugagagga3²srsf1 shrna expression plasmid constructiontarget specific oligonucleotides were designed using online tool rnai codex cold spring harbor laboratoryand were synthesized integrated dna technologieswith the addition of overhangs according to the cuttingsite of bamh1 and ecori the shrna expression plasmid was constructed by annealing the oligonucleotidesto psihh1 vector following the user manual of psihh1 shrna system sbi system bioscience the oligonucleotide sequences for shrna of srsf1 eif3b ortia1 are provided in supplemental table 3rd generation packaging plasmidslentivirus transductionlentiviral ps were produced as previously described using the shrna expression plasmid andthepmd2gaddgene plasmid pmdlrregp addgeneplasmid and prsvrev addgene plasmid the packaging plasmids were cotransfectedwith the lentiviral expression vector into t cellsusing the polyethyleneimine polysciences inc to produce replication deficient lentivirus after transfectionthe supernatant was pooled and filtered with a μmmembrane and concentrated by ultracentrifugation toacquire lentivirus infection was performed by usinglentivirus in the presence of μgml polybrene sigmaaldrich approximately h postinfection cells wereselected by treating with μgml puromycin invivogen san diego cagfpsrsf1 expressionthe gfpsrsf1 expression plasmid was a gift from drmassimo caputi dna transfection was performedusing lipofectamine thermo fisher scientific topanc1 cells and the expression of gfpsrsf1 wasverified by western blotgstsrsf1 protein purificationbl21 thermofisher scientific c600003 competentcells transformed with pgex6psrsf1 dna addgeneplasmid were cultured at °c for hand after od600 reached to bacteria weretreated with mm isopropyl βd1thiogalactopyranoside for h at °c gsttaggedsrsf1 was purifiedwith glutathione sepharose beads ge health carethe purity of the recombinant proteins was determinedby sdspage with coomassie blue stainingelectrophoretic mobility shift assay emsaird800 labeled mir1246 μm integrated dnatechnologies was mixed with μl of gst slurry stsrsf1 in binding buffer tris ph mm kcl mm mgcl2 mm np40 dtt mm glycerol and incubated at room temperature for minavoiding light 5x loading buffer kcl mm tris ph mm glycerol xylene cyanol bromophenol blue was then added and the complexwas separated on a native gel polyacrylamide m tris ph m glycine m edta apstemed at voltage for min the signal was detected using the licor odyssey imaging systemlicor inc usadesign of decoy motif mimicsthe decoy motif mimics were designed by permutationand combination of the identified motif sequences in thelength of nucleotides the secondary structure of thedesigned sequences was analyzed in rnafold webserveruniversityselfcomplementary were selected decoy mimics ²uuggacuaggacuaggau3² decoy mimics ²aggaaggaaggaagga3²sequences withoutof vienna 0cxu cell communication and signaling page of bioinformatics analysisthe mirna motif analysis was performed using memesuite the protein profile analysis for the result ofmass spectrometry was performed using david bioinformatics abcc at saicfrederick inc the rnabinding protein and mirna sequence binding analysiswas performed using the database of rnabindingspecificities rbpdb srsf1 expression in cancertissues was examined using oncomine thecorrelation of gene expression with cancer patient survival was extracted from the human protein atlasscilifelab sweden statisticsstatistical analyses were performed using graphpadprism software graphpad software inc la jolla causa the heatmap was made in rstudio rstudio incwith the ggplot2 package students ttest was applied to determine significant differences among controland experimental groupsresultsidentification of mir1246 associated proteinsbecause rbps are involved in exosome mirna sortingwe first sought to identify proteins that bind to mirnashighly enriched in cancer exosomes mir1246 the mosthighly enriched mirna in pancreatic cancer exosomeswas biotinlabeled and incubated with a cellular lysatefrom panc1 cells the biotinmir1246 probe wascaptured with streptavidincoated magnetic beads biotinlabeled polya mimics were used as control themirnaprotein complexes were eluted and the proteinswere analyzed by liquid chromatographymass spectrometry in triplicate table there were total of proteins specifically pulled down by the mir1246 probeinterestingly about half of the proteins that associatewith mir1246 are vesicleassociated proteins supplement fig 1a based on the intensity of detection rnabinding property and cancer relevance we ranked therbps using the database for annotation visualizationand integrated discovery david this resulted in tencandidate rbps that complex with the mir1246 sequenceexosomestable among them srsf1 also called sfrs1 waspredictedsequencethe mir1246and arerelevantto eukaryotictobindtotable over view of the result of mass spectrometryexperiments conditionpoly a panc1number of proteins detectedpoly a mdamb231mir1246 panc1mir1246 mdamb231table mir1246 rna binding protein candidates obtainedfrom the mass spectrometric analysisprotein full nameprotein symbolsrsf1serineargininerich splicing factor park7eif3bthoc4acocddx5tia1if5a1eif2aimdh2parkinson disease protein eukaryotic translation initiation factor subunit btho complex subunit alyref export factorcytoplasmic aconitate hydrataseprobable atpdependent rna helicase ddx5tcellrestricted intracellular antigen1eukaryotic translation initiation factor 5a1eukaryotic translation initiation factor 2ainosine5²monophosphate dehydrogenase supplement fig by in silico analysis using the database of rnabinding specificities rbpdb levelsverification of srsf1 binding to mir1246rna immunoprecipitation rip was performed to verifythe association of several identified rbps with mir1246including srsf1 eif3b and tia1 igg and gapdhantibody was used as controls for immunoprecipitationas shown in fig 2a mir1246 expression is more than12fold higher in the srsf1precipitants as compared tothat of igg precipitants indicating a specific associationof srsf1 with mir1246 mir1246 expression wasmoderately increased in the tia1precipitants and nearigg controlin the eif3b precipitants coimmunoprecipitation coip experiments were performed to verify the immunoprecipitation proceduresdata not shown to directly determine the binding ofsrsf1 to the mir1246 sequence glutathionestransferase gst conjugated human srsf1 protein wasexpressed in bl21 competent e coli captured by glutathione sepharose beads and eluted by glutathione thepurity of eluted gstsrsf1 protein was shown by sdspage and coomassie blue staining supplement fig the binding of gstsrsf1 to a fluorescenttaggedmir1246 probe was determined by rna emsa asshown in fig 2b binding of the labeled probe was specific to gstsrsf1 but not gst and increased withgreater protein input the specific binding of gstsrsf1 to the mir1246 probe was evident as the unlabeled mir1246 probe effectively competed with thelabeled mir1246 probe in a concentrationdependentmanner fig 2c the detected bands were semi quantified and the kd was calculated from the detected signalsfig 2d these data confirmed the direct binding ofsrsf1 to the mir1246 sequence 0cxu cell communication and signaling page of fig srsf1 binds to mir1246 a qrtpcr detection of mir1246 in igg gapdh srsf1 eif3b and tia1 immunoprecipitants of panc1 lysaten p student ttest bc emsa detection of the srsf1mir1246 complex hot probe ird800 labeled mir1246 mimics cold probemir1246 mimics n direct binding of gstsrsf1 and mir1246 b and concentrationdependent competition between the cold and hotmir1246 probe for binding to gstsrsf1 c d semiquantification of srsf1 and mir1246 binding in c and calculated dissociationconstant n exosome mirna enrichment by srsf1 in cancer cellsbecause srsf1 is a key splicing factor that is essential toeukaryotic cells a knockout model could not beestablished thereforeto determine whether srsf1mirna binding activity is relevant to exosome mirnaenrichment we established a lentivirus srsf1 shrnaconstruct to knockdown srsf1 expression in panc1cells fig 3a interestingly though srsf1 protein wasdetected both in the nucleus and cytoplasm the knockdown was more pronounced in the cytoplasm fig 3bknockdown of srsf1 did not significantly alter the concentration and size distribution of the exosomes releasedby panc1 cells fig 1b cellular and exosome rnafrom control and srsf1shrna cells were isolated andsmall rna sequencing was performed among the highly enriched panc1 exosome mirnas expressionof mirnas was significantly downregulatedin exosomes derived from srsf1shrna panc1 cellsas compared to exosomes derived from control panc1cells fig 3c strongly indicating the involvement ofsrsf1 in exosome mirna enrichment a heatmapshowing the expression of the top mirnas enrichedin panc1 exosomes demonstrates the dramatic dropin expression levels of mirnasin srsf1shrnapanc1 exosomes compared to panc1 exosomesfig 3d notably mir1246 was the highest enrichedexosome mirna data not shown and its expressionin exosomes wassignificantly reduced by srsf1knockdown fig 3d on the other hand among of the mirnas less enriched in exosomes only were expressed at lower levels in exosomesderived from srsf1 knockdown cells as compared toexosomes derived from wild type panc1 cells fig3c suggesting that srsf1 knockdown mainly affectsexosome enriched mirnasto further confirm the effect of srsf1 knockdown onexosome mirna enrichment the expression levels ofseveral representative mirnas were quantified by qrtpcr srsf1 knockdown in panc1 cells significantlyreduced exosome levels of mir1246 mir320c andmir320d confirming the small rna sequencing resultsfig 3e in contrast knockdown of eif3b or tia1 didnot reduce exosome mir1246 expression suggestingthat these rbps may not promote exosome mirna 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig cellular and exosome mirna profiles after srsf1 knockdown in panc1 cells a detection of srsf1 knockdown by shrnas in panc1 cellsb panc1 srsf1 protein levels in nuclear and cytoplasmic fractions nc normal control c venn diagram of overlap of mirnas detected by nextgeneration small rna sequencing in srsf1 knockdown and control panc1 cells and exosomes d heatmap showing the expression of top exosome mirnas in cells and exosomes after srsf1 knockdown e qrtpcr analysis of mir1246 mir320c and mir320d in exosomes derivedfrom control and srsf1 knockdown panc1 cells p students ttest shown are representatives of three independent experiments aeenrichment supplement fig and our observationswere extended to two additional pancreatic cancer celllines miapaca2 and bxpc3 fig 4af in additionexpression of let7c which is less enriched in exosomeswas unchanged in exosomes after srsf1 knockdowndata not shownto verify the involvement of srsf1 in exosomemirna enrichment in cancer cells we also exogenouslyoverexpressed srsf1 in panc1 cells a gfpsrsf1 expression plasmid was introduced into panc1 cells andsrsf1 overexpression was confirmed by western blotfig 5a expression of mir1246 mir320c and mir320d in the exosomes derived from gfpsrsf1 panc1cells was analyzed by qrtpcr fig 5bc as shown infig 5b overexpression of gfpsrsf1 increased exosome expression of mir1246 and rescued mir1246levels in exosomes derived from srsf1shrna cellslevels of mir320c and mir320d were also increased inexosomes derived from the gfpsrsf1 cellsfurthersupporting the involvement of srsf1 in exosomemirna enrichment fig 5cdidentification of rna sequence motifs involved inexosome mirna enrichmentaccording to the rbpdb srsf1 binds specifically to amotif present in the mir1246 sequence supplementfig qrtpcr analysis of mir1246 mir320c mir320d expression in exosomes derived from srsf1 knockdown bxpc3 and miapaca2 cells ac qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown bxpc3 cells n p student ttest df qrtpcr detection of mir1246 mir320c and mir320d in exosomes derived from srsf1 knockdown miapaca2 cells n p students ttest 0cxu cell communication and signaling page of fig qrtpcr analysis of exosome enriched mirnas derived from srsf1 overexpression panc1 cells a confirmation of gfpsrsf1overexpression in panc1 cells b qrtpcr detection of mir1246 in exosomes derived from wild type and srsf1 knockdown panc1 cells withgfpsrsf1 overexpression c qrtpcr detection of mir320c in exosomes derived from gfpsrsf1 overexpression panc1 cells d qrtpcrdetection of mir320d in exosomes derived from gfpsrsf1 overexpression panc1 cells p students ttest n for bdfig to understand the contribution of specific rnamotifs involved in exosome mirna enrichment we applied an unbiased approach to identify the rna motifsthat contribute to exosome mirna enrichment for thispurpose we analyzed the rna sequences of the mirnas highly enriched in cancer exosomes and regulatedby srsf1 using the bioinformatics tool meme suite a 6bp length motif was found to be shared in of the exosome enriched mirnasincluding mir fig ac to test whether the binding of srsf1to mir1246 depends on this motif two decoy mimicswere designed according to the shared motif sequencesand their secondary structure determined with thernafold webserver httprnatbiunivieacatcgibinrnawebsuiternafoldcgi the binding of the decoymimics to srsf1 protein was determined by rnaemsa analysis addition of decoy motif did not alterthe binding of srsf1 to the mir1246 probe fig 6dwhereas decoy motif competed with mir1246 binding to srsf1 in a concentrationdependent manner fig6df indicating that srsf1 directly interacts with thissequence motifdiscussionthe role of exosome mirna signaling in promotingcancer progression has been intensely investigated andwell recognized in recent years [ ] the higher enrichment of certain mirnas in cancer exosomes [] indicates that exosome mirna encapsulation isan active cellular process that initiates exosome mirnasignaling in the tumor microenvironment however thespecificselectivecellular processresponsiblefor 0cxu cell communication and signaling page of fig see legend on next page 0cxu cell communication and signaling page of see figure on previous pagefig srsf1associated exosome mirna sequence motif analysis a the motif commonly shared among srsf1associated exosome mirnas bvenn diagram showing the number of srsf1associated exosome mirnas that share the motif c list of mirnas sharing the common motif demsa analysis demonstrating the inhibition of gstsrsf1 binding to mir1246 by rna decoys d1 decoy ²uuggacuaggacuaggau3² d2decoy ²aggaaggaaggaagga3² e concentrationdependent inhibition of gstsrsf1 binding to mir1246 by d2 f semiquantification ofthe detected bands in fig 5e and the calculated dissociation constantexosome mirna enrichment has not been well established in eukaryotic cells the most significant findingfrom the present study is that we have identified srsf1as a mediator of exosome mirna enrichment in pancreatic cancer cells a specific mirna sequence motif wasalso identified that may be involved in the exosomemirna enrichment process these findings provide newinsight into how mirnas are enriched in cancer cellexosomesexosomemediated mirnasignalinginitiatetowe recently reported that exosome mir1246themost highly enriched mirna in pancreatic cancer cellderived exosomes is derived from rnu2 a smallnuclear rna important for mrna splicing alongthis line of our research we sought to determine howthis mirna is enriched in cancer exosomes using ourestablished model systems in the present study we haveprovided severallines of evidence demonstrating thatsrsf1 a vital splicing factor and established oncoprotein is significantly involved in exosome mirnaenrichment in pancreatic cancer cells the first line ofevidence indicating srsf1 involvementin exosomemirna enrichment was obtained from the biotinlabeled mir1246 pulldown experimentfollowed byproteomic analysis among the rbps identified severalwere selected based on their detection intensity relevance to extracellular vesicles and reported connectionsto human cancer including srsf1 eif3b andtia1 of note srsf1 was the only rbp among themthat was also predicted by the rbpdb to bind to a motifin the mir1246 sequence furthermore the direct binding of srsf1 to the mir1246 sequence was verified byrip and rna emsa analysis strongly indicating thephysical interaction of srsf1 and not eif3b or tia1with the mir1246 sequence the most convincing evidence demonstrating the involvement of srsf1 in cancer exosome mirna enrichment was the observationthat knockdown of srsf1 significantly reduces exosomemirna enrichmentfor a majority of the selectivelyenriched exosome mirnas without altering the expression levels of less enriched exosome mirnas these results were based on small rna sequencing andconfirmed by rtpcr analysis the observations werealso extended to additional human pancreatic cancer celllines including miapaca2 and bxpc3srsf1 was initially identified as a splicing factor ineukaryotic cells but srsf1 was later revealed toindependent ofshuttle between the nucleus and cytoplasm to regulate rna metabolism mirna procession and othercellular eventsthe mrna splicingprocess importantly srsf1 is overexpressed indifferent cancer types and is considered a potent oncogene [ ] moreover srsf1 over expression in different types of cancer is associated with worse prognosissupplement fig while the full spectrum of srsf1function remains to be determined our results revealthat srsf1 binds to specific mirnas and is significantlyinvolved in exosome mirna enrichment in cancer cellsthis function is likely independent ofthe splicingprocess as the reduced expression of the detected exosome mirnas after srsf1 knockdown is greater thantheir expression change in the cells because exosomemirna signaling contributes to tumor developmentthrough intercellular communication in the tumormicroenvironmentthe involvement ofsrsf1 in exosome mirna signaling initiation likelyrepresents a part of its oncogenic action which maylead to new therapeutic strategies to intervene withexosome mirna signaling in cancer several rbpshave been previously identified as mediators of exosome mirna sorting in various model systemsincluding major vault protein in colon cancer cells hnrnpa2b1 in t cells and ybx1 in hek293tcells the identification of srsf1 involvement inexosome mirna enrichmentin pancreatic cancercells further supports the notion that the cellular exosome mirna sorting process in eukaryotic cells maydiffer among different cell types[ ]we have also identified a mirna motif commonlyshared by the srsf1associated exosome mirnasusing the meme suite program memesuitethis motif was specifically bound by srsf1 as evidenced by our rna emsa analysis a similar motifalbeit slightly shorter was identified in our recentreport that describes exosome mir1246 enrichmentin pancreatic cancer cells our results reinforcethe concept that specific mirn | 0 |
"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 | 0 |
"Accordingly the protease inhibitor E-64d partially suppressed TFP-induced cell death. Moreover we demonstrate that lysosomal targeting of TFP was critical for its cytotoxic activity because inhibition of vacuolar adenosine triphosphatase by BafA1 which dissipates the lysosomal pH gradient and prevents intra-lumenal entrapment of lysosomotropic compounds (e.g. phenothiazines) in their protonated forms18 largely abolished TFP-induced cell death. Overall these data demonstrate that phenothiazines can modulate lysosomal functions in human LC cells as the amount of endogenous LC3-II is correlated with autophagic vesicle formation and conversion of LC3-I into LC3-II is a hallmark of autophagic activity and changes in these processes were observed upon phenothiazine treatment especially in SCLC. The lysosome has recently emerged as a promising target for anticancer therapy because lysosome-initiated cell death can operate independently of p53 and downstream caspases19 20 pathways that are often functionally inactivated in a variety of human tumors.2122 23 Also in our studies lysosomal perturbation occurred despite mutation in p53 and was not influenced by intrinsic sensitivity/resistance toward conventional chemotherapeutic agents. From our data we cannot rule out that SCLC cells or tumors with wt p53 or p53 null status could potentially respond differently yet our opinion is that in this context p53 status is not the main factor that regulates phenothiazine monotherapy response of SCLC. However the disparate sensitivities that exist between but also within SCLC and NSCLC indicate intrinsic differences in lysosomal activity suggesting that not all SCLC cells will be equally susceptible to phenothiazine-based treatment. Hence we sought to reveal whether lysosome-associated parameters can be used to predict the sensitivity of human LC cells to phenothiazines. Indeed using a limited number of SCLC and NSCLC cell lines we found that sensitivity to phenothiazines inversely correlated with baseline LysoTracker Green retention which was generally higher in NSCLC. Meanwhile sensitivity to phenothiazines correlated positively with the magnitude by which LysoTracker retention was lost upon treatment which tends to be more pronounced in SCLC. This suggests that lysosomes in the tested panel of SCLC cells may be less abundant and/or have higher intra-lumenal pH and are therefore more prone to functional perturbations. These findings require however further validation in a wider panel of SCLC cell lines to make a final . Also as LysoTracker Green only stain lysosomes in live cells alternative methodologies need to be developed to assess definitively whether lysosomal mass and/or pH is altered in clinical SCLC specimens such as for example tissue microarray staining with LC3-II upon treatment. Nevertheless consistent with this idea a number of different SCLC cell lines (H69 H82 H592 and U-1285) exhibited increased sensitivity toward several well-known lysosome-disrupting agents such as TMX CQ quinacrine and ammonium chloride (NH4Cl) all of which elicited larger changes in LysoTracker retention in SCLC cells as compared with NSCLC counterparts. By contrast we found that the CaM antagonist W7 neither induced LC3-II accumulation nor was selectively cytotoxic in SCLC cells. In addition the expression of D2R was comparable in our cell lines panel. Therefore the enhanced cytotoxicity of phenothiazines in SCLC that we report here most likely derives from lysosomal dysregulation rather than preferential inhibition of CaM or neurotransmitter signaling two established activities of phenothiazines.5 In addition it is worth noting that NSCLC cell lines in general exhibited a higher basal autophagy than SCLC counterparts which may render them less sensitive to chemically induced lysosomal perturbation. Similar observations have been reported by other investigators implicating autophagy as a pro-survival mechanism in NSCLC.24 25 Multiple studies on human and mouse cell lines have demonstrated the cytotoxic potential of phenothiazines given as monotherapy.4 In addition a few case reports exist that described anecdotal evidence for the antitumor activity of phenothiazines in vivo.2627 28 While the clinical utility of phenothiazines for anticancer treatment needs to be further analyzed through in vivo using SCLC xenograft transgenic murine SCLC models and possibly also patient-derived xenografts we believe our data in SCLC cell lines provide an important rationale for such analysis. Indeed it has been shown that at advanced stages several types of human cancers rely on lysosomes/autophagy for survival and metastasis.29 This is not surprising given that tumors frequently experience metabolic and replication stress.30 31 Therefore it is conceivable that tumors with high stress load and relatively low lysosomal activity could be particularly sensitive to phenothiazines. Given that SCLC initially are typically sensitive to conventional CT but rapidly develop resistance our data support clinical trials where phenothiazines are administered as second-line treatment for tumors that become refractory to standard treatment. " | 1 |
"A pulmonary function test showed mild restrictive patterns. The patient underwent bronchoscopy and no endobronchial lesion was found. Bronchoalveolar lavage results were negative for malignant cells. Thick varicose septal veins and intraalveolar macrophages were identified on transbronchial lung biopsy. After seven months a follow-up chest CT scan revealed increased interstitial septal thickening (Fig. 1E F) new peribronchovascular cuffing and small amounts of bilateral pleural effusion (Fig. 1G). Finally the patient underwent video-assisted thoracoscopic surgery wedge resection of the right middle and lower lobes. In the surgical field abnormal hypervascularity was noted on the lung surface (Fig. 1H). Microscopic examination showed proliferation of thinwalled anastomosing vascular spaces lined by a single layer of endothelial cells lacking cytological atypia (Fig. 1I). Theses lesions were located along the lymphatic distribution and were highlighted by D2-40 (1:100 Dako Glostrup Denmark) immunohistochemical stain (Fig. 1J) characteristic of DPL. Presently the patient is alive without any symptoms and being observed without specific treatment such as low fat medium chain fat. DISCUSSION Diffuse pulmonary lymphangiomatosis is a rare condition in which diffuse proliferation of anastomosing lymphatic channels is observed. It manifests equally in both sexes mostly in children and young adults (3). Symptoms include dyspnea nonproductive cough bronchial casts milky sputum fever and recurrent pneumonia. However patients can present with the disease later in adulthood and often have symptoms dating back to their childhood (5). To the best of our knowledge only five cases of DPL in middle-aged patients have been reported in the English literature () (1-5) with the current report being the sixth. Ours is a unique one in the following aspects; although the disease progression revealed on serial follow-up CT required differentiation from lymphangitic metastasis the patient remained asymptomatic and did not need any treatment; also the extent of the disease markedly increased within one year and a neoplastic but possibly benign component was presumed. Making a preoperative diagnosis of DPL in adults is difficult. The most common radiologic finding is increased interstitial markings on chest radiography. CT demonstrates smooth or nodular interlobular septal and peribronchovascular thickenings. Patchy ground glass opacities are also seen. Pleural thickening pleural effusion and diffuse mediastinal soft tissue infiltration can also occur (5). Pulmonary edema pulmonary veno-occlusive disease Erdheim-Chester disease and lymphangiectasis can be considered possible diagnoses when smooth interlobular septal thickening is found. In this patient pulmonary edema was less likely since there was no evidence of congestive heart failure or pleural effusion. Prominent central pulmonary arteries were also not identified making pulmonary veno-occlusive disease less likely (6). The possibility of Erdheim-Chester disease characterized by proliferation of lipid-containing foamy histiocytes in the skeleton and other ans was ruled out by the absence of sclerotic changes in the diaphyses and metaphyses of long bones in the current case (7). The CT appearance of DPL is virtually identical to that of pulmonary lymphangiectasia (8). Pulmonary lymphangiectasia is a rare condition characterized by the diffuse dilatation of pulmonary lymphatics and classified as congenital or secondary (9). In this case there was no evidence of pulmonary hypertension or venous obstruction factors that can cause secondary lymphangiectasia. Congenital lymphangiectasia typically presents shortly after birth and is associated with high neonatal morbidity and mortality. Lymphangiomatosis typically presents in older children and is rarely seen in adults (10). Histopathologically lymphangiomatosis is characterized by an increased number of variable-sized lymphatic spaces. This should be distinguished from the findings of lymphangiectasia in which nonproliferative lymphatic channels are dilated (4). Other diseases with lymphatic distributions such as lymphangitic carcinomatosis sarcoidosis and pulmonary lymphoma can also be considered. In these diseases interlobular septal thickening tends to be nodular. There is no established treatment for DPL. " | 1 |
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' | 2 |
"variability around prevalence estimates of multimorbidity due to poorconsensus regarding its definition and measurement Medicationbased measures of morbidity may be valuableresources in the primarycare setting where access to medical data can be limited We compare the agreementbetween patient selfreported and medicationbased morbidity and examine potential patientlevel predictors ofdiscordance between these two measures of morbidity in an older ¥ years communitybased populationMethods A retrospective cohort study was performed using national pharmacy claims data linked to The IrishLongituDinal study on Ageing TILDA Morbidity was measured by patient selfreport TILDA and two medicationbased measures the RxRisk years and RxRiskV ¥ years which classify drug claims into chronic diseaseclasses The kappa statistic measured agreement between selfreported and medicationbased morbidity at theindividual patientlevel Multivariate logistic regression was used to examine patientlevel characteristics associatedwith discordance between measures of morbidityResults Two thousand nine hundred twentyfive patients were included years N and ¥ years N Hypertension and high cholesterol were the most prevalent selfreported morbidities inboth age cohorts Agreement was good or very good κ for diabetes osteoporosis and glaucoma andmoderate for high cholesterol asthma Parkinsons and angina κ All other conditions had fair or pooragreement Age gender marital status education poordelayed recall depression and polypharmacy weresignificantly associated with discordance between morbidity measuresConclusions Most conditions achieved only moderate or fair agreement between selfreported and medicationbased morbidity In order to improve the accuracy in prevalence estimates of multimorbidity multiple measures ofmultimorbidity may be necessary Future research should update the current RxRisk algorithms inline with currenttreatment guidelines and reassess the feasibility of using these indices alone or in combination with othermethods to yield more accurate estimates of multimorbidityKeywords Agreement Selfreport Rxrisk RxriskV Morbidity Polypharmacy Older people Correspondence caitrionacahirrcsiie Clionadh Mannion and John Hughes are joint first authors2Division of Population Health Sciences Royal College of Surgeons in IrelandDublin IrelandFull 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 0cMannion BMC Geriatrics Page of Key pointsKey findings and implications Agreement between patient selfreported morbidityand medicationbased measures of morbidity RxRisk and RxRiskV was mainly moderate or fairDiabetes was the only condition for which the levelof agreement was found to be very good The results of our study indicate that neithermeasure of morbidity is completely reliable and wesuggest that researchers may require multiplemeasures selfreport and medicationbased measures of morbidity to fully capture accurate prevalence estimates of multimorbidity Our study identified several limitations of thecurrent versions of the RxRisk indices which require updating if medicationbased measures ofmorbidity are to be used to assess the epidemiologyof chronic conditions and multimorbiditytheofIndeedattentionBackgroundMultimorbidity is commonly defined as the presence oftwo or more chronic medical conditions and its prevalence has been shown to increase with age [] As theworlds older population continues to grow multimorbidity has become an important public health issue caphealthcareturingresearchersprofessionals as well as policy makersforhealthcare systems to effectively adapt and manage thedelivery of healthcare to our growing older populationan accurate description of the epidemiology of chronicconditions is required However to date studies in theliterature reveal wide disparities in prevalence estimatesof multimorbidity ranging from to [ ] Thislarge variability is thought to be due to the lack of standards defining multimorbidity and validated methods forhow it should be measured [] A recent systematic review reported definitions of multimorbidity involving differenttheappropriateness of different measures of multimorbidityis also variable depending on both the outcome of interest as well as the type of data that is available []In additioncriteria[]Measures of multimorbidity include diagnosisbasedmeasures eg Charlson Index based on hospital diagnosis codes ICD codes [] medicationbased measureseg RxRisk and RxRiskV for those aged ¥ yearsbased on pharmacy data [] and patient selfreportDiagnosisbased measures of multimorbidity are themost common measures and are generally based on hospital or physician records [] Medicationbased measures of multimorbidity include the RxRisk and RxRiskV two algorithms which determine an individualscurrent comorbidities based on their dispensed medication The RxRisk indexes only include morbidities forwhich a medicine could be prescribed and include categories of morbidities based on the World Health anisation WHO Anatomical Therapeutic Classification ATC system [] The RxRisk and RxRiskVhave good reliability and criterion validity against ICD9diagnoses and have been shown to predict costs of caremortality and health care utilisation [] Previous studies have reported medicationbased measures of morbidity such as the Medicines Disease Burden Index MDBIand RxRiskV to be useful in epidemiological studieswhen adjusting for comorbidity [] However there arefew studies describing the use of these indices to directlymeasure chronic conditions Patient selfreport is also avalid method of identifying disease categories A study ofolder patients with multimorbidity reported good agreement between patient selfreport and general practitioner GP report for a wide range of diseases []A number of studies have compared the differentmeasures of multimorbidity with differing results [ ] A study of older primary care patients inIreland found that medicationbased measures ofmultimorbidity such as RxRiskV performed betterthan diagnosisbased measures of multimorbidity inpredicting emergency and ambulatory care sensitiveACS admissions [] Studies comparing patientselfreport and diagnosisbased measures of multimorbidity have reported a stronger association between selfreport measures of multimorbidity andqualitythandiagnosisbased measures [ ] However no previous research has compared selfreported morbidityin the primary care or community setting with theRxRisk measures of morbidity Comparison betweenselfreported morbidity data and pharmacy records isimportant in order to understand the relative meritsof each measure of morbidity and the potential formisclassification particularly in the community setting where access to medical or clinical data can belimitedfunctionaloutcomesandlifeofStudies have also indicated that agreement betweenselfreport measures and other measures of morbiditymight be influenced by patient recall bias [] Patientrecall has been reported to be influenced by age maritalstatus and education [] There is also some evidencethat cognition and memory influence patient recall []The impact of these factors needs to be explored furtherwhen assessing and comparing measures of morbidityThe aim of this study was to compare the agreementbetween patient selfreported morbidity and medicationbased morbidity RxRisk and RxRiskV and examine potential patientlevel predictors of discordance between theincludingdemographic cognitive and mental health factors in anolder community based populationtwo measures of morbidity 0cMannion BMC Geriatrics Page of MethodsThe STrengthening the Reporting of ObservationalStudies in Epidemiology STROBE guidelines were usedin the reporting of this study []Study populationThis was a retrospective cohort study using data froma national pharmacy claims database the Health Service ExecutivePrimary Care Reimbursement ServiceHSEPCRS General Medical Services GMS schemelinked to the first wave of The Irish LongituDinalstudy on Ageing TILDA TILDA is a nationally representative sample of community dwelling individualsaged ¥ years in Ireland The sampling framework isbased on the Irish Geodirectory a comprehensive anduptodate listing and mapping ofresidential addresses in Ireland compiled by the Ordinance SurveyOffice and participants aged ¥ years were randomlyselected using the RANSAM sampling procedureThis meant that each residential address in Irelandhad an equal probability of selection and thus ensured that the TILDA sample was representative ofthe Irish population aged ¥ years The first wave ofdata collection began in October through toFebruary N participants aged ¥ yearswhere participants completed a computeraided personal interview CAPI and a health assessment measuring their health economic and social circumstancesFurther information on TILDAs study design andsampling framework is described in detail elsewhere[]The HSEPCRS GMS scheme is the largest pharmacy claims dataset in Ireland covering more than of the general Irish population [] It is meanstested and provides free health servicesincludingmedications to eligible persons in Ireland Qualification for the GMS scheme is on the basis of incomerelated meanstesting Automaticforthose aged ¥ years occurred between July andDecembercurrent study period meanstesting was introducedbut with a higher income threshold than the generalpopulation As of of men and ofwomen in the general population aged ¥ yearswere eligible [] The HSEPCRS GMS pharmacyclaims data were available for consenting TILDAparticipants aged ¥ years with GMS eligibility N entitlementhoweversinceJanuaryWithin the HSEPCRSGMS pharmacy claims dataprescriptions are coded using the WHO ATC classification system and prescriber information defineddaily doses strength quantity method and unit ofadministration of each drug dispensed are all available Pharmacy claims data was extracted for yearprior to each participants TILDA interview GMSpatientstypically receive their medications on amonthly basis []ifthey had any ofSelfreported morbidityAs part of the TILDA interview participants wereasked to reportthe followingdoctordiagnosed chronic diseases high blood pressure or hypertension high cholesterol angina congestive heart failure heart attack diabetes stroke orministroke abnormal heart rhythm arthritis osteoporosis cancer Parkinsons disease emotional nervous or psychiatric problems alcohol or substanceabuse dementia serious memory impairment stomach ulcers glaucoma incontinence or chronic painParticipants were also asked to selfreport urinaryincontinence in the past months as well as painmoderate or severe and if they were taking medication for pain management If participants reportedthat they had arthritisthey were asked to clarifythe type of arthritis eg osteoarthritis rheumatoidarthritis some other kind of arthritis Similarlyifparticipants reported emotional nervous or psychiatric problems they were asked to clarify from a listof conditions eg anxiety depression emotionalproblems psychosis manic depressionfillsthatclassify prescription drugMedicationbased measures of morbidity Rxrisk andRxriskVThe RxRisk and RxRiskV indices were applied tothe HSEPCRS pharmacy claims data The RxRiskindex was applied to the population aged yearswhile the RxRiskV was applied to the populationaged ¥ years The RxRisk and RxRiskV are algorithmsintochronic disease classes for older populations basedon the WHO ATC classification system []Within the RxRiskV cardiac disease is separatedinto a number of categories anticoagulation antiplatelet agents arrhythmias congestive heart failureCHFhypertension hypertensionischaemic heartdisease IHDangina and ischaemic heart diseaseIHDhypertension [] For a medication to be eligible as a measure of morbidity per RxRisk and RxRiskV chronic disease classes a patient was required to have been dispensed two or more consecutive prescriptions of the medication in question egdonepezil was required to be dispensed on ¥ consecutive prescriptions to link this medication withthe RxRiskV condition dementia This definitionhas previously been used by other pharmacoepidemiological studies [] 0cMannion BMC Geriatrics Page of Comparison of selfreported morbidity with Rxrisk andRxriskVEach selfreported condition in TILDA was matched tothe equivalent RxRisk and RxRiskV condition at theindividual patient level for those aged years and ¥ years respectively This was performed by consensusbetween two pharmacists FM CM For some selfreported conditions the ATC classes of medicationsspecific to these conditions eg antiwere notthrombotic agents B01AC04 B01AC30 were matchedto the selfreported condition of a heart attack and alsoto stroke There were four selfreported TILDA conditions which could not be matched to an RxRisk or RxRiskV condition but the prevalence was low Appendix in Tables and The RxRisk and RxRiskV alsoreported conditions which patients had not been askedabout during their TILDA interview Appendix in Tables and Patientlevel characteristics associated with discordancebetween the two measures of morbidityPatient characteristics were assessed to determine discordance patient recall bias between selfreported morbidity TILDA and the RxRisk years and RxRiskV ¥ years medicationbased measures of morbidity These characteristics were age gender maritalstatus education poor delayed recall depression andpolypharmacy Marital status was subcategorised intomarried never married separated or divorced Educationwas categorised into primarynone secondary or thirdhigher level education Delayed recall based on participants being presented with words during the interview and being later asked to recall as many as possiblewas defined as poor where or fewer words wererecalled Depression was defined as scoring or greateron the Centre for Epidemiologic Studies DepressionScale CESD [] Polypharmacy was defined as reporting regular use of five or more prescription medications[]Statistical methodsAgreement between selfreported morbidity TILDAand the RxRisk and RxRiskV measures of morbiditypharmacy claims was assessed using Cohens Kappastatistic as neither source was considered to be a goldstandard for reporting morbidity Interpretation of thevalue of Kappa was as follows poor fair moderate good and verygood []Multivariate logistic regression was used to examinethe association between the patientlevel characteristicsand discordance between the two measures of morbidityAdjusted odds ratios OR and confidence intervalsCIare presented Discordance was defined asparticipants reporting to have the condition in the absence of any dispensed medication for the condition perRxRisk years or per RxRiskV ¥ years andparticipants reporting to not have the condition butmedication was found to be dispensed for the conditionper RxRisk years or RxRiskV ¥ years Allsignificance tests were twotailed Statistical significancewas set at P after adjustment for a false discoveryrate of [] Analyses were performed using Stata SEVersion statistical package StataCorp College Station TXResultsStudy populationIn total patients were included in this cohortstudy patients were aged years and were aged ¥ years Characteristics ofthe study participants are presented in Table On average patients aged years had SD conditionsper the RxRisk and patients aged ¥ years had SD conditions per the RxRiskV The proportion ofpatients with thirdhigher level education was relatively years N low across both age ¥ years N Poor delayed recall years N ¥ years N years N ¥ years N were significantlymore prevalent in the older cohort compared to theyounger cohort p polypharmacygroupsandAgreement between selfreported morbidity andmedicationbased measures of morbidity Rxrisk and RxriskVTables and present a comparison between the number and percentage of patients selfreported morbiditiescompared to the RxRisk Table aged years andRxRiskV Table aged ¥ years measures of morbidity High blood pressure or hypertension yearsN ¥ years N and highcholesterol years N ¥ years N were the most prevalent selfreportedmorbidities in both age cohorts in the TILDA datasetHigh cholesterol was also found to be highly prevalentin the RxRisk N and RxRiskV N measures of morbidity Other prevalentRxRisk and RxRiskV conditions included arthritisRxRisk N stomach ulcers RxRiskN RxRiskV N strokeRxRiskV N heart attack RxRiskVN and other heart trouble RxRiskVN There was very good agreement between the selfreported TILDA measure of diabetes and the RxRiskand RxRiskV measures κ There was also good 0cMannion BMC Geriatrics Page of Table Characteristics of study participants by age years and ¥ years years N Age¥ years N GenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimarynoneSecondaryThirdHigher LevelPoor delayed recall YesDepression YesPolypharmacy Yes Data presented as N or mean CI unless otherwise statedagreement between selfreported measures of osteoporosis κ and glaucoma κ and the RxRiskV measure of these morbidities in the older cohort Despite the high prevalence of high cholesterolin both measures of morbidity there was only moderate agreement κ RxRisk κ RxRiskVbetween the two measures There was moderateagreement also for asthma κ RxRisk Parkinsons κ RxRiskV and angina κ RxRisk V Agreement was fair for selfreported highblood pressure or hypertension RxRisk and RxRiskV heart attack RxRisk stroke RxRisk abnormalheart rhythm RxRiskV cancer RxRisk depression RxRisk and RxRiskV and pain RxRiskVand RxRisk measures of these conditions κ All other conditions had poor agreement κ including arthritis RxRisk chronic lungdisease and incontinence RxRiskV and emotionalnervous psychiatric problems anxiety and stomach ulcers RxRisk and RxRiskV Tables Patientlevel characteristics associated with discordancebetween the two measures of morbidityAge gender marital status education poor delayedrecall depression and polypharmacy were all associated with discordance between the two measures ofmorbidity Table Females were five times morelikely to have discordance in reporting osteoporosisOR Confidence Intervals CI P Females were also more likely to have discordance in reporting anxiety OR CI emotional problems OR CI and depression OR CI as well as use of pain medication OR CI and incontinence OR CI They were less likely to have discordance in reporting stroke and high cholesterol TablePatients who were never married were less likely tohave discordance in reporting a heart attack OR CI and stroke OR CI Patients with third level educationwere lesslikely to have discordance in reportinghypertension OR CI comparedto those with primary level education Table Patients with poor delayed recall and depression weremore likely to have discordance in reporting anxietyand depression In general discordance was higher inpatients with polypharmacy Table found thatagreement between patientDiscussionWithin a population based study of ageing in Irelandweselfreported morbidity and medicationbased measures ofmorbidity RxRisk and RxRiskV was generally notgood with most conditions achieving only moderateor fair agreement There was very good agreementκ between selfreported diabetes and pharmacy dispensing records across both age cohortsThis was the only morbidity common to both age cohorts for which the level of agreement was found tobe very good Many research studies confirm this 0cGlaucomaHigh CholesterolAsthmaHigh blood pressure orHypertensionCancer or a malignant tumourDepressionStroke cerebral vascular diseaseParkinsonHeart attack including myocardialinfarction or coronary thrombosisManic depressionEmotional nervous or psychiatricproblem such as depression oranxietyCirrhosis or serious liver damageStomach ulcersArthritis including osteoarthritis orrheumatismN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX Hyperlipidaemia C10AA01C10BX17 Asthma R03AAR03AL R03BAR03BX R03CAR03CC R03DAR03DX Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02C09BA09 C09DA01C09DA07 C02AB01C02AC05 C02DB02C02KX01 Malignancies L01AA01L01XX31 Depression N06AA01N06AG02 N06AXAntiplatelet therapy B01AC04B01AC30Parkinsons disease N04AA01N04BX02Antiplatelet therapy B01AC04B01AC30Bipolar disorder N05AN01 Anxiety N05BA01N05BA12Anxiety N05BA01N05BA12Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11 GORD Peptic ulcer A02B A02BB A02BC Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01L04AB01L04AB05 L04AD01 L04AX03Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Anxiety Mannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRisk algorithm yearsTILDAStandardErrorSelfreported morbidityDiabetes or high blood sugarRxRisk Pharmacy ClaimsMedicationbased Morbidity ATCKappaκNAny other heart troubleRheumatoid arthritis only Rheumatoid Arthritis M01AAM01CX M02AAM02AX L01BA01Ministroke or TIAL04AB01L04AB05 L04AD01 L04AX03Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06ATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient coprescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm []same level of agreement for diabetes [ ] Thiswas expected given that previous research has demonstrated the reliability of reporting to be better inmorbidities for which there are clear diagnostic criteria eg diabetes [] Furthermore with many educational resources promoting selfmanagement of thiscondition patients with diabetes are more likely toplay an active role in managing their condition egregular selfmonitoring of blood glucose levels dietarymanagement recognising and dealing with symptomssuch as hypo and hyperglycaemia andor medication taking and are therefore more likely to selfreport accurately []There was good agreement between both measures ofmorbidity for osteoporosis and for glaucoma in the olderage group A MultiCare cohort study of primary carepatients in Germany found only moderate agreement between patientreported and GPreported osteoporosis[] A retrospective cohort study of older patients in asecondarycare setting in Canada also found moderateagreement for glaucoma between physician and patientreports [] Similar to diabetes patients are required toplay an active role in the management of osteoporosiswhile glaucoma is very often a comorbidity of diabetes[]There was moderate agreement between the measures of morbidity for asthma in the younger age cohort years Similar results have been reported for agreement between selfreported asthma and medical recorddata in older hospitalised patients [] There was alsomoderate agreement for high cholesterol in both agecohorts and for angina and Parkinsons disease in the 0cMannion BMC Geriatrics Page of Table Agreement kappa statistic and standard error between selfreported morbidity in TILDA and RxRiskV algorithm ¥ yearsTILDASelfreported morbidityDiabetes or high blood sugarRxRiskV Pharmacy claimsMedicationbased Morbidity ATC KappaκNStandardErrorN Diabetes A10AB01A10BG03 A10BH A10BX Glaucoma S01EA01S01EB03 S01EC03S01EX OsteoporosisPagets disease M05BA01M05BB09 M05BX03Pain taking pain medication Pain Opioids N02AA01N02AX02 GlaucomaOsteoporosisParkinsonAnginaHigh CholesterolManic depressionHigh blood pressure orHypertensionG03XC01 A12AX92Parkinsons disease N04AA01N04BX02 Angina C01DA02C01DA14 C01DX16 C01EB17C01EB18 Hyperlipidaemia C10AA01C10BX17 Hypertension C03AA01C03BA11 C03DA01C03EA01 C09BA02Bipolar disorder N05AN01C09BA09 C09DA01C09DA09 C02AB01C02AC05 C02DB02C02KX01PainAbnormal Heart RhythmDepressionDementiaChronic lung disease such aschronic bronchitis or emphysemaCancer or a malignant tumourEmotional nervous or psychiatricproblem such as depression oranxietyPain Inflammation M01AB01 M01AH06 Pain Opioids N02AA01N02AX02Pain Inflammation M01AB01 M01AH06 Arrhythmia C01AA05 C01BA01C01BD01 C01BD07 Depression N06AA01N06AG02 N06AX Dementia N06DA02 N06DA01Chronic airways disease R03AC02R03DC03 Malignancies L01AA01L01XX31 Anxiety N05BA01 N05BA12Congestive heart failureCirrhosis or serious liver damageHeart attack including myocardialinfarction or coronary thrombosis Chronic heart failure C03CA01C03CC01 C09AA01C09AA10C09CA01 C09CA03 C09CA06C09CA07Liver disease A05AA01A05BA08 J05AF05 J05AF07 J05AF11Antiplatelet therapy B01AC04B01AC30AnxietyStomach ulcersAlcohol or substance abuseAnxiety N05BA01N05BA12 GORD Peptic ulcer A02BA A02BCAny other heart trouble Stroke cerebral vascular diseaseMinistroke or TIA Alcohol dependence N07BB01 N07BB04Ischaemic heart diseasehypertension C07AA01C07FB07C08CA01C08DB01Antiplatelet therapy B01AC04B01AC30Antiplatelet Anticoagulation therapya B01AC04B01AC30B01AA03B01AB06 B01AB10Incontinence Neurogenic Bladder Urinary Incontinence V07ANPsychotic illness N05AA01 N05AX17PsychosisATC Anatomical Therapeutic ChemicalGORD GastroOesophageal Reflux DiseaseaAnticoagulant counted if patient prescribed antiarrhythmic for Atrial Fibrillation ie if patient not in sinus rhythm [] 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskVAge yearsGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecall YesDepression YesPolypharmacyYesAgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyHypertension HeartAttack StrokeTIAHigh Cholesterol HeartTrouble Cancer EmotionalProblems Depressiononly Stomachulcers Asthma Arthritisgeneral RheumatoidArthritis only Angina Congestive HeartFailure Abnormal HeartRhythm Anxiety LungDisease 0cMannion BMC Geriatrics Page of Table Odds ratios with confidence intervals for patientlevel characteristics associated with discordance between themeasures of morbidity selfreport and RxRisk and RxRiskV ContinuedHypertension HeartAttackOsteoporosis Psychosisonly StrokeTIAHigh CholesterolHeartTroubleCancerEmotionalProblemsAnxietyIncontinence PainPain meds AgeGenderMaleFemaleMarital StatusMarriedNever MarriedSeparatedDivorcedWidowedEducationPrimaryNoneSecondaryThirdHigherLevelPoor DelayedRecallDepression YesPolypharmacyExcluded diabetes Parkinsons disease manic depression cirrhosis glaucoma alcohol or substance abuse and dementia as number of patients misreporting wassmall N p older age cohort Other studies have reported loweragreement for high cholesterol and higher agreement forangina and Parkinsons diseases [ ] Discordancehere may be explained by patients managing their cholesterol using nonpharmacological means eg lifestylemodifications[]Interestingly the prevalence of selfreported angina inTILDA was higher than the prevalence reported by RxRiskV This may reflect poor patient adherence if prescribed medications were not dispensedincluding cardioprotective dietThere was only fair agreement between both measures of morbidity for hypertension despite hypertensionbeing the most prevalentselfreported morbidityacross both age cohorts Higher agreement betweenselfreported antihypertensive drug use and pharmacyrecords has been reported in a populationbasedstudy and a cohort study of older people in theNetherlands [ ] The discordance observed hereis likely attributable to the omission of a major group[]increasingantihypertensivesofcalciumchannelblockersCCBs in the current version of the RxRisk and RxRiskV algorithms [ ] This is significant giventhat CCBs are recommended as firstline therapy inpatients aged years [] Equally since hypertension is considered to be a condition without symptomsthis may influence patient adherence toantihypertensive medications and their proclivity tofill a prescription for these medications There wasalso fair agreement for pain in the older age groupwith agreementsomewhat when selfreported pain specified taking pain medication Theprevalence of selfreported pain was higher than themedicationbased RxRiskV prevalenceand thismay be due to patients managing their pain throughnonpharmacological or lifestyle interventions such asphysiotherapy and cognitive behavioural therapy []In both age cohorts there was poor to fair agreement between selfreporting of emotional problems 0cMannion BMC Geriatrics Page of poorfoundagreementeg depression anxiety and medicationbased measures These findings are consistent with previous research whichbetweenphysician diagnosis and patient selfreports of anxiety and depression [] This low level of agreementmay be due to a potential stigmatisation bias as only of patients regularly dispensed antidepressants selfreported as having depression in theolder age cohort [ ] Equallyit may be thatcertain antidepressants eg amitriptyline are beingused for other indications such as neuropathic pain[ ] There was also poor agreement in bothage cohorts for stomach ulcers and for incontinenceand chronic airways disease COPD in the older cohort Like depression poor agreement here may bedue to gastrointestinal medications being used by patients for other indications such as preventative orsymptomatic reasons [] The poor agreementforchronic airways disease may reflect the nonspecificquestion used in TILDA to measure this selfreportedmorbidity as there is evidence in the literature thatquestionnaire design is an important determinant ofpatient recall In a US study the prevalence of selfreported COPD was found to increase when more explicit questions were asked about emphysema chronicbronchitis and COPD in combination [] The pooragreement between the two measures for incontinenceis most likely reflective of the current version of theRxRiskV which compares selfreported urinary incontinence with dispensed diapers and pads supplies []agepoordelayedincreasingA number of factors were associated with discordance between the two measures of morbidity particularlyrecalldepression and polypharmacy A study determiningthe agreement between selfreported and diagnosisbased multimorbidity in older community dwellingwomen reported similar findings where agreementwas found to decrease with decreasing cognition andeducation increasing age and fo | 2 |
Acute myeloid leukemia AML is a complex hematological disease characterized by genetic and clinical heterogeneity The identification and understanding of chromosomal abnormalities are important for the diagnosis and management of AML patients Compared with recurrent chromosomal translocations in AML t816p112p133 can be found in any age group but is very rare and typically associated with poor prognosisMethods Conventional cytogenetic studies were performed among AML patients recorded in our oncology database over the last years Fluorescence in situ hybridization FISH was carried out to detect the translocation fusion Array comparative genome hybridization aCGH was carried out to further characterize the duplication of chromosomesResults We identified three AML patients with t816p112p133 by chromosome analysis Two of the three patients who harbored an additional 1q duplication were detected by FISH and aCGH aCGH characterized a Mb and Mb gain in chromosome at band q321q44 separately in these two patients One patient achieved complete remission CR but relapsed months later The other patient never experienced CR and died years after diagnosisConclusion A 1q duplication was detected in two of three AML patients with t816p112p133 suggesting that 1q duplication can be a recurrent event in AML patients with t816 In concert with the findings of previous studies on similar patients our work suggests that 1q duplication may also be an unfavorable prognostic factor of the diseaseKeywords 1q duplication Acute myeloid leukemia t816p112p133 Prognostic factorBackgroundAcute myeloid leukemia AML is a common disease characterized by immature myeloid cell proliferation and bone marrow failure which can be subdivided into pathogenetically different subtypes [] Over the past two decades the incidence has increased by [ ] Furthermore AML has poor longterm survival with a Correspondence lzhang202003163com Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of ChinaFull list of author information is available at the end of the high relapse rate [] Therefore AML represents a substantial health problem that requires strict monitoring and innovative treatment strategies The development of newer effective treatment strategies is necessary for AML patientsTo date the detection of cytogenetic abnormalities has been regarded as a critical prognostic tool for AML treatment [] Hence it is urgently necessary to identify chromosomal rearrangements in AML patients and provide the whole spectrum of cytogenetic abnormalities for AML [] According to the World Health anization classification system updated in AML with recurrent genetic abnormalities including t821q22q22 The Authors Access 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 0cLiu a0et a0al Mol Cytogenet Page of t1517q24q21 t1517PMLRARA t11q23MLL inv16p131q22 and t1616p131q22 has been identified [ ] Nonrandom chromosomal abnormalities such as deletions and translocations have been detected in approximately of all adult AML patients Moreover chromosomal abnormalities have been recognized as genetic events that can cause and promote this disease [] Certain cytogenetic abnormalities including t821q22q22 t1517q24q21 and inv16p131q22 are associated with longer remission and survival while alterations of chromosomes 11q23 and complex karyotypes are associated with poor response to therapy and shorter overall survival [] Chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBMYH11 and t11q23MLL are usually found in AML patients [ ] However AML with t816p112p133KAT6ACREBBP is a very rare AML subtype and can be found in any age group from infancy to the eighth decade of life with a female predominance [] A majority of adult patients with t816p112p133 are therapy related [] and pediatric patients tend to be de novo [] There are approximately cases reported in the literature [] and the first t816p112p133 in an infant was described in [] Some AML patients with t816 p112p133 have a bleeding tendency and disseminated intravascular coagulopathy which are overlapping clinical features that mimic acute promyelocytic leukemia APL [] Unlike APL AML with t816p112p133 has an unfavorable treatment response and outcome [ ] As a sole chromosomal anomaly t816p112p133 is found in more than of reported cases and one or more additional chromosomal anomalies can be seen in the remaining cases [] The most common secondary chromosomal anomalies are total or partial trisomy and monosomy or deletion of the long or short arm of chromosome [ ] Comparatively the gain of 1q in variable sizes has also been frequently noticed in patients with t816p112p133 in these large studies [ ]Recurrent cytogenetic abnormality t816p112p133 is seldom associated with AML and the 1q duplication in AML patients with t816p112p133 has never been discussed In the present study a total of de novo or treatmentrelated AML patients were collected from our laboratory oncology database Among them three patients were detected with t816p112p133KAT6ACREBBP and two of these three showed an additional copy of partial chromosome 1qMethodsPatientsThis study was approved by the Institutional Review Board IRB of Oklahoma University IRB Number A total of AML patient samples were studied cytogenetically from to at the Genetics Laboratory of Oklahoma University Health Sciences Center Bone marrow samples were obtained from three of the patients who had t816p112p133Conventional cytogenetic analysisShortterm cultures of unstimulated bone marrow samples were established and harvested according to standard laboratory protocols Karyotype analysis was performed using Giemsa and trypsin techniques for Gbanding The cytogenetic abnormalities were described according to the International System for Human Cytogenetic Nomenclature ISCN Fluorescence in a0situ hybridization analysisFluorescence in a0 situ hybridization FISH assays were performed according to the manufacturers instructions in combination with our established laboratory protocols A PMLRARA dualcolor dualfusion translocation probe Abbott Molecular Inc Des Plaines IL USA subtelomerespecific probes for chromosome parm and qarm and whole chromosome painting WCP probes for chromosomes and were purchased from Cytocell Ltd NY USA A spectrum greenlabeled probe mapping to the 8p1121 region and a spectrum orangelabeled probe mapping to the 16p133 region were created in house with the following BACPAC clones RP11642I24[chr8 4167633641856494hg19] and RP11589C21[chr8 4187370242036222hg19] RP11619A23[chr16 37200763914571hg19] and RP1195J11[chr16 38603744025510hg19] Childrens Hospital Oakland Research Institute Oakland CA USA The KAT6A gene located on 8p1121 and the CREBBP gene located on 16p133 were covered by the greenlabeled and redlabeled homebrewed probes respectively All probes were validated before use Chromosome spreads were counterstained with 46diamidino2phenylindole DAPI4 in antifade medium Vector Laboratories Inc CA USA Digital images carrying specific hybridization signals were captured and processed on CytoVision version Applied Spectral Imaging Carlsbad CA USAaCGH analysisGenomic DNA was extracted from each of the three patients bone marrow pellets according to the standard operating procedure using the phenol and chloroform method with a commercially available DNA extraction kit Puregene blood kit Qiagen Valencia CA or Nucleic Acid Isolation System QuickGene610L FUJIFILM Corporation Tokyo Japan Two aCGH platforms NimbleGen and Agilent were used in this study For the 0cLiu a0et a0al Mol Cytogenet Page of NimbleGen aCGH platform human reference genomic DNA was purchased from Promega Corporation Promega Corporation Madison WI USA The patients DNA and the reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming and then equal quantities of both labeled products were mixed and loaded onto a a0K oligonucleotide chip Roche NimbleGen Inc Madison WI USA to hybridize at a0 °C for a0 h in a MAUI hybridization system BioMicro Systems Salt Lake City UT according to the manufacturers protocols with minor modifications The slides were washed with washing buffers Roche NimbleGen Inc after hybridization and scanned using a Roche Scanner MS Microarray Scanner Roche NimbleGen Inc Images were analyzed using NimbleScan software version and SignalMap software version Roche NimbleGen Inc The genomic positions were determined using GRCh36hg18 UCSC Genome Browser For the Agilent aCGH platform human reference genomic DNA was purchased from Agilent Corporation Agilent Corporation Santa Clara CA USA The patients DNA and the purchased reference DNA were labeled with either Cyanine Cy3 or Cyanine Cy5 by random priming Agilent Corporation Patient DNA labeled with Cy3 was combined with a normal control DNA sample labeled with Cy5 of the same sex and hybridized to an Agilent à a0K oligo microarray chip Agilent Technologies by incubating in an Agilent Microarray Hybridization Oven Agilent Technologies After a0h of hybridization at a0°C the slides were washed and scanned using the NimbleGen MS Microarray Scanner Roche NimbleGen Inc Agilents CytoGenomics software Agilent Technologies was applied for data analysis The genomic positions were determined using GRCh37hg19 UCSC Genome BrowserCase presentationCase An 82yearold male presented with anemia was referred to us for AML evaluation His subsequent lab results and hospital records were not available in our clinical databaseCase A 28yearold female presented with disseminated intravascular coagulopathy was referred to rule out APL Her complete blood examination and bone marrow aspirate smears were not available Flow cytometry revealed monocytic cells positive for CD4 CD11b partial CD13 bright CD14 partial CD15 CD33 bright and HLADR partial but negative for CD3 CD7 CD34 CD117 MPO and TdT consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient achieved hematological CR on day and cytogenetic CR on day after induction chemotherapy and then relapsed a0months laterCase A 69yearold female with a medical history of breast cancer after lumpectomy chemotherapy and radiation presenting with generalized weakness pancyt ia and fever was referred to us for disease progression evaluation A complete blood examination showed a white blood cell count of à 109L with blasts a hemoglobin count of a0 gL and a platelet count of à 109L Her bone marrow aspirate smear demonstrated over myeloblasts Flow cytometry revealed that of the blast cells expressed CD45 moderate CD34 dim CD38 HLADR CD13 CD15 and CD33 and were negative for CD117 consistent with a diagnosis of AML with monocytic differentiation subtype M5 The patient started consolidation chemotherapy but had spontaneous regression and died a0years after AML diagnosisResultsIn case routine chromosome analysis detected an abnormal karyotype with a translocation between the short arms of chromosomes and Fig a01a in of cells consistent with a diagnosis of AML with t816p112p133 The nomenclature of the cytogenetic findings in patient was t816p112p133[]46XY[] No other consistent karyotypic aberrations were detected Thus this male patient was excluded from subsequent FISH and aCGH analysesIn case chromosome analysis demonstrated the same chromosome rearrangement between and in all cells Besides of these cells showed an extra chromosome segment attached to chromosome Fig a01b The karyotypes in patient were described as 46XXt816p112p133 add14p112[]46XY[] Negative FISH t1517q24q21PMLRARA further ruled out a diagnosis of APL data not shown Metaphase FISH analysis confirmed the t816p112p133KAT6ACREBBP fusion and demonstrated a part of chromosome on chromosome Fig a02a and b In addition to characterizing the extrachromosomal material aCGH was carried out aCGH confirmed the FISH findings and detected a a0Mb gain from chromosome at bands q321q44 a0bp GRCh36hg18 USCS Genome Browser Fig a03aIn case t816p112p133 with a gain of a similar chromosome segment on the long arm of chromosome was detected in of cells by karyotyping analysis Fig a0 1c FISH confirmed the KAT6ACREBBP fusion and revealed additional chromosome material Fig a02c and d Loss of the end portion of the chromosome long arm was not found by FISH Fig a03e aCGH further detected a gain from chromosome at bands 1q321q44 results for 0cLiu a0et a0al Mol Cytogenet Page of a Patient b Patient Fig Representative abnormal karyotypes of three patients with t816p112p133 a Karyotype of patient showing 46XYt816p112p133 as the sole abnormality b and c Karyotypes of patients and showing 46XXt816p112p133 and an additional chromosome segment attached to the short arm of chromosome and the long arm of chromosome respectively Translocated derivatives and are indicated by black arrows and derivatives and are indicated by red arrows 0cLiu a0et a0al Mol Cytogenet Page of c Patient Fig continued a0 bp GRCh37hg19 UCSC Genome Browser Fig a0 3b The molecular size was a0MbDiscussionAML is one of the most common diseases characterized by the proliferation of blast cells in bone marrow or peripheral blood which accounts for approximately of adult leukemia cases As reported previously common chromosomal translocations such as t821RUNX1RUNX1T1 inv16CBFBfrequently observed and numerous MYH11 are uncommon chromosomal aberrations also exist in AML [] The detection of these fusion transcripts is important for the diagnosis and progression monitoring of AML patients []t1517PMLRARA and In previous large studies approximately AML cases with t816p112p133 have been reported [] Among them cases showed a gain by 1q of variable sizes [ ] As an uncommon entity t816 accounts for of all cases of AML [] In our study three patients with t816p112p133 were identified one man and two women The two women were both diagnosed with AML subtype M5 and showed an extra copy of 1q at the same bands q321q44 which were different from the nine reported cases above The clinical features and cytogenetic data of the cases of AML with t816p112p133 and 1q duplications are summarized in Table a0 To the best of our knowledge this is the first study of the delineation of 1q duplication by aCGH in AML patients with t816p112p133AML patients with this abnormality often show unique clinical and biological characteristics [] Compared with the current categories t1517 t821 inv16 and t11q23 in AML t816 is clustered closer to t11q23 and shares commonly expressed genes [] Xie et a0 al reported adult AML cases with t816p112p133 indicating that t816p112p133 commonly exhibits monoblastic or myelomonocytic differentiation and arises in patients with a history of cytotoxictreated cancer Patients with de novo AML with t816 or treatmentrelated AML with t816 without adverse prognostic factors have a good outcome [] Identifying adverse prognostic factors is of importance to the choice of therapy and evaluation of survival in AML patients with t816 0cLiu a0et a0al Mol Cytogenet Page of CREBBPKAT6A fusionKAT6A8p1121CREBBPKAT6A fusionKAT6ACREBBP fusionCREBBP16p133CREBBP16p133KAT6ACREBBP fusiona KAT6A8p1121c WCP14WCP1WCP14b WCP1WCP1TelVysion 3q WCP1WCP1WCP3WCP1d WCP3TelVysion 3p TelVysion 3p TelVysion 3q e Fig Metaphase FISH of patient a and c showing KAT6ACREBBP fusion signals WCP FISH indicating the extra chromosomal materials on chromosome and chromosome were both from chromosome b and d No loss of the end portion of the chromosome long arm was indicated eOver the past a0 years cytogenetic and molecular technologies have largely promoted the efficiency of the identification and characterization of this disease [] Compared with conventional cytogenetic analysis and FISH methods aCGH is an attractive method for the investigation of cancer genomes [] aCGH has higher resolution simplicity high reproducibility shorter turnaround time and precise mapping of aberrations Most importantly it avoids the need for cell culture and dividing cells [] Furthermore aCGH chromosomal analysis facilitates rapid detection and duplication of cytogenetic abnormalities previously undetectable by conventional cytogenetics [] In our investigation we applied aCGH to characterize the additional chromosome materials in patients and and interestingly found that the two patients 0cLiu a0et a0al Mol Cytogenet Page of revealed the same extra copy of 1q at bands q321q44 Patients with 1q duplication have also demonstrated a wide range of multiple malformations such as intellectual disability macrocephaly large fontanels prominent foreheads broad flat nasal bridges higharched palates retrognathia lowset ears and cardiac defects [ ] More recent studies have shown that a 1q gain is also related to a portion of solid tumors For instance the gain of 1q is well known as a poor prognostic biomarker of Wilms tumor [] and it plays an important role in predicting poor clinical outcome in patients with thyroid carcinoma as well [] In addition patients with a 1q duplication showed worse survival and high risk in acute leukemia Burkitt lymphoma and myeloproliferative neoplasms [] The outcomes of 1q duplication in the nine reported AML patients with t816p112p133 are summarized in Table a0 Seven patients data were available These seven patients two adult and five pediatric all received induction chemotherapy and six achieved CR At the time of last followup two adult patients and three of five pediatric patients had died Only two pediatric patients were alive We reported two adult patients here patient achieved CR but relapsed a0 months later and patient had spontaneous regression and died a0 years after diagnosis Taken together the findings suggest that 1q duplication might be associated with adverse outcomes in AML patients with t816p112p133 However the significance of the 1q duplication in AML with t816 needs to be further investigated Since such changes have been seldom reported the pathogenic effects of 1q duplication in AML patients with t816p112p133 require more studies to be delineatedConclusionThree patients were detected with t816p112p133 from an AML patient database Two female patients were identified with a 1q duplication by FISH and aCGH analyses Combining our investigation with the findings of published studies we conclude that 1q duplication is a recurrent finding in AML patients with t816 Our data also suggest that 1q duplication might be associated with unfavorable prognosis in these cases The understanding of cytogenetic data would contribute to the diagnosis and treatment evaluation of AMLFig aCGH results of patient and patient showing partial 1q gain duplicated 1q regions are indicated by red frames 0cLiu a0et a0al Mol Cytogenet Page of Table The previously reported AML cases with a0t816p112p133 and a01q duplicationSex Age years FAB type Karyotype1q BandsOutcome yearsLast stateCase Case FFHaferlach et al FM5M5M5aDiab et alM M4Diab et alDiab et alDiab et alDiab et alFFFFXie et alM Brown et alM Brown et alFM45M4M4M5M4M4M446XXt816p112p133 add14p112[]46XX[]46XXt816p112p133[]46idemadd3q27[]45XXt816p11p13der1013q10q10[]46XXder7t17q21q35t816p11p13[]46XX[]46XY1del1p22t816p11p1310der14t1014q112p112[]47XYdel1q11der1t18p11q112x2i5p10810der14t1014q112p112der16t8165XXt816p11p1318der21t121q12p13[]46XX[]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110q11p11[]46idemadd7p21[]46XX[46XYt816p11p13der14t114q31p11[]46XderXtX1q26q23t816p11p13der11t1111p11q1346XYder3t38q27q13del6p22t816p112p133del10q21q25add13p112del16p12del20p112del20q112q133[]46idemdel1p35p363del15q23add19p131[]46XYt816q27q13del12q21q241del13q21q3116der19t119q32p133mar[]46XYdel6p22t816p112p133[cp2]46XY[]47XderYtY1q12q21 6t816 p11p13[]47idemdel13q3q3 [checked with CAD data]46XXt816p11p13[]46idemder10t110q11p11[]46idemadd7p21der10t110 q11p11 []46idemadd7p21 []46XX []1q321q441q321q44CR after inductionRelapsed months laterspontaneous regression1q21NAPartial 1q gain CR for 1q121q111q311q23CR for CR for CR for NA1q32CR for monthsAliveDiedNADeadAliveDiedAliveNADead1q21No CRDied month after treatment1q11Early remission after course Relapsed at months and months after diagnosisDiedAML acute myeloid leukemia FAB FrenchAmericanBritishh M male F female NA not available CR complete remissionAbbreviationsAML Acute myeloid leukemia aCGH Array comparative genomic hybridization FISH Fluorescence in situ hybridization APL Acute promyelocytic leukemia WCP Whole chromosome painting CR Complete remissionAcknowledgementsNot applicableAuthors contributionsM Liu and YR gathered clinical information and drafted the manuscript YR YK and M Liu performed routine cytogenetic analysis and participated in the interpretation of the results M Li performed FISH analysis and participated in the interpretation of the results XL supervised the FISH analysis and helped draft the manuscript XW performed CGH array analysis and helped draft the manuscript LZ and SL conceived the study participated in its design and 0cLiu a0et a0al Mol Cytogenet Page of extensively reviewed and revised the manuscript All authors have read and approved the final manuscriptFundingThis study has received no funding supportAvailability of data and materialsAll data generated or analyzed during this study are included in this published Ethics approval and consent to participateThis study was approved by University of Oklahoma Institutional Review Board for the Protection of Human SubjectsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Hematology The First Hospital of China Medical University Nanjing North Street Shenyang Liaoning Peoples Republic of China Department of Pediatrics University of Oklahoma Health Sciences Center Oklahoma City OK USA Department of Neurology The Second Hospital of Jilin University Jilin Peoples Republic of China Received April Accepted August References Braess J Acute myeloid leukemia Dtsch Med Wochenschr Luppi M Fabbiano F Visani G Martinelli G Venditti A Novel agents for acute myeloid leukemia Cancers Basel Cancer Research UK Acute myeloid leukaemia AML incidence statistics https wwwcance rrese archu khealt hprofe ssion alcance rstati stics stati stics bycance rtypeleuka emiaamlincid ence Accessed Aug Jung J Cho BS Kim HJ Han E Jang W Han K Lee JW Chung NG Cho B Kim M Kim Y Reclassification of acute myeloid leukemia according to the WHO classification Ann Lab Med Murphy T Yee KWL Cytarabine and daunorubicin for the treatment of acute myeloid leukemia Expert Opin Pharmacother Byrd JC Mrózek K Dodge RK et al Pretreatment cytogenetic abnormalities are predictive of induction success cumulative incidence of relapse and overall survival in adult patients with de novo acute myeloid leukemia results from Cancer and Leukemia Group B CALGB Blood Lindsley RC Mar BG Mazzola E et al Acute myeloid leukemia ontogeny is defined by distinct somatic mutations Blood Vardiman JW Thiele J Arber DA et al The revision of the World Health anization WHO classification of myeloid neoplasms and acute leukemia rationale and important changes Blood Saultz JN Garzon R Acute myeloid leukemia a concise review J Clin Med Döhner H Weisdorf DJ Bloomfield CD Acute myeloid leukemia N Engl J Med Strickland SA Shaver AC Byrne M et al Genotypic and clinical heterogeneity within NCCN favorablerisk acute myeloid leukemia Leuk Res Yang JJ Park TS Wan TSK Recurrent cytogenetic abnormalities in acute myeloid leukemia Methods Mol Biol Coenen EA Zwaan CM Reinhardt D et al Pediatric acute myeloid leukemia with t816p11p13 a distinct clinical and biological entity a collaborative study by the InternationalBerlinFrankfurtMunster AMLstudy group Blood Xie W Hu S Xu J Chen Z Medeiros LJ Tang G Acute myeloid leukemia with t816p112p133KAT6ACREBBP in adults Ann Hematol Haferlach T Kohlmann A Klein HU et al AML with translocation t816p11p13 demonstrates unique cytomorphological cytogenetic molecular and prognostic features Leukemia Gervais C Murati A Helias C et al Acute myeloid leukaemia with 8p11 MYST3 rearrangement An integrated cytologic cytogenetic and molecular study by the groupe francophone de cytogénétique hématologique Leukemia Diab A Zickl L AbdelWahab O et al Acute myeloid leukemia with translocation t816 presents with features which mimic acute promyelocytic leukemia and is associated with poor prognosis Leuk Res Schouten TJ Hustinx TW Scheres JM Holland R de Vaan GA Malignant histiocytosis Clinical and cytogenetic studies in a newborn and a child Cancer Brown T Swansbury J Taj MM Prognosis of patients with t816p11p13 acute myeloid leukemia Leuk Lymphoma Barrett R Morash B Roback D et al FISH identifies a KAT6ACREBBP fusion caused by a cryptic insertional t816 in a case of spontaneously remitting congenital acute myeloid leukemia with a normal karyotype Pediatr Blood Cancer Schumacher J Szankasi P Kelley TW Detection and quantification of acute myeloid leukemiaassociated fusion transcripts Methods Mol Biol DÃazBeyá M Navarro A Ferrer G et al Acute myeloid leukemia with translocation 816p11p13 and MYST3CREBBP rearrangement harbors a distinctive microRNA signature targeting RET protooncogene Leukemia Veigaard C Nørgaard JM Kjeldsen E Genomic profiling in high hyperdiploid acute myeloid leukemia a retrospective study of cases Cancer Genet Yasar D Karadogan I Alanoglu G et al Array comparative genomic hybridization analysis of adult acute leukemia patients Cancer Genet Cytogenet van der Veken LT Buijs A Array CGH in human leukemia from somatics to genetics Cytogenet Genome Res Laskowska J Szczepanek J StyczyÅski J Tretyn A Array comparative genomic hybridization in pediatric acute leukemias Pediatr Hematol Oncol Mehrotra M Luthra R Ravandi F et al Identification of clinically important chromosomal aberrations in acute myeloid leukemia by arraybased comparative genomic hybridization Leuk Lymphoma Kulikowski LD Bellucco FTS Nogueira SI et al Pure duplication 1q41qter further delineation of trisomy 1q syndromes Am J Med Genet A 2008146A2663 Nowaczyk MJM Bayani J Freeman V Watts J Squire J Xu J De novo 1q32q44 duplication and distal 1q trisomy syndrome Am J Med Genet A 2003120A229 Cone EB Dalton SS Van Noord M Tracy ET Rice HE Routh JC Biomarkers for wilms tumor a systematic review J Urol Xu B Ghossein R Genomic landscape of poorly differentiated and anaplastic thyroid carcinoma Endocr Pathol Fournier A Florin A Lefebvre C Solly F Leroux D Callanan MB Genetics and epigenetics of 1q rearrangements in hematological malignancies Cytogenet Genome Res Lancman G Tremblay D Barley K et al The effect of novel therapies in highmolecularrisk multiple myeloma Clin Adv Hematol Oncol Bacher U Schnittger S Grüneisen A Haferlach T Kern W Haferlach C Inverted duplication dup1q32q21 as sole aberration in lymphoid and myeloid malignancies Cancer Genet Cytogenet Marcellino BK Hoffman R Tripodi J et al Advanced forms of MPNs are accompanied by chromosomal abnormalities that lead to dysregulation of TP53 Blood Adv Beach DF Barnoski BL Aviv H et al Duplication of chromosome [dup1q21q32] as the sole cytogenetic abnormality in a patient previously treated for AML Cancer Genet Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c' | 2 |
"autophagy is an evolutionary cellular program that serves for thebreakdown of cytoplasmic components within lysosomes [] inidentified autophagy in's electron microscopic studies firstmammalian cells but the molecular pathways were not understooduntil the discovery of autophagy genes atgs in yeast by performinggenetic screening it is a cytoprotective rather than a selfdestructive process it is extensively accepted as a main regulator of innateand adaptive immune mechanisms the change in which completelyimpact the pathogenesis of disease and the processes that are influencedby autophagy includes the regulation of inflammation antigen presentation and bacterial clearance moreover autophagy aids in themaintenance of fundamental anelle populations such as mitochondria which is necessary for cellular bioenergetics and homeostasis homeostasis of the cell is been accomplished by maintaining the biosynthesis and turnoverthere are two broader protein degrading systems in eukaryoticcells first is the ubiquitinproteasome system responsible for the selective breakdown of most shortlived proteins and second is the lysosomalsystem the primary anelle called lysosome ineukaryotes is known for degradation through its acid hydrolases inunfavourable nutrient deprivation condition autophagy arbitrates aregulated phagocytosis via lysosomes autophagy is mediated byautophagosome that is thought to be an on selective degradation systemas it engulfs some of the cytoplasmic contents the ubiquitinproteasome system concedes only ubiquitinated proteins for degradation andit marks a remarkable contrast to autophagosome process autophagosomes a doublemembered vesicle that engulfs durableproteins impaired anelles intracellular pathogenic anisms andtransports it to the lysosomes that are fused to form autolysosome andthe inner vesicle along with its cargo is been degraded at the time ofstarvation the remaining macromolecules are again recycled to thecytosol for reuse the precise mechanism in cargo recognition isuncertain but this process involves ubiquitination autophagicprocess is separated into distinct steps which includes induction recognition selection of cargo formation of vesicle then occurs the fusion of autophagosomevacuole followed by the degradation of thecargo and release into the cytosol various atg proteins are indulged inthis process and it consists of the central autophagic machinery autophagy encompasses different process by which cells delivercytoplasmicaredegradation theylysosomalsubstratesfor corresponding author at department of biotechnology psg college of arts and science civil aerodrome post coimbatore indiaemail address rashmiffrajangmailcom rr rasmi all authors deserve contribution equally101016jlfs2020118308received june received in revised form august accepted august available online august elsevier inc all rights reserved 0cs vishnupriya life sciences macroautophagy chaperonemediated autophagy cma and microautophagy all the three processes of autophagy are morphologically distinct in microautophagy the lysosomal membrane invaginations or protrusions are needed to seize cargo once thecargo is captured the uptake directly happens at the limiting membrane of the lysosome that includes intact anelles cma uses chaperones to sequester cargo proteins that have a pentapeptide motifthese proteins are unfolded and translocated directly across the lysosomal membrane via lamp2a receptor macroautophagy involves requisition of cargo vesicle formation and its subsequenttransport to the lysosome in recent years deletion of the autophagy related genes atg invarious model anisms has proved that autophagy plays decisive rolein adaptive responses to stress cellular differentiation and development an oncogenic event may establish by the partial minimization inthe autophagic capacity atg6beclin one of the phylogeneticallypremeditated autophagy genes is often subdued at one locus in humancancers studies in mice have shown that beclin is a haplo insufficienttumor suppressor autophagic programmed cell death was primarily depicted in actively developing tissues several conjugationsystems comprising of the atg genes are available that take part inautophagasomal elongation one such system is the lc3 microtubule associated protein light chain and atg8 conjugation system lc3 is the mammalian conjugative protein ortholog of yeastprotein atg8 the lipid derivative phosphatidylethanolamine bindsto lc3 to form lc3ii an important molecular marker of autophagylc3ii remains on the mature autophagosome until it fuses with thelysosomes fig the beclin complex gives rise to an incipientautophagosome membrane and it assemble around cargo in a vesiclethat combines with a lysosome forming autolysosome that is degradedby acid hydrolases present in the lysosomes lung injury clinical implicationsthe primary an of gas exchange is the pair of lungs theymediate inspiration of oxygen and elimination of mono and dioxides ofcarbon lung also serves as an attractive target for the entry of thepathogens regulation of the pulmonary functions is mediated by intricate cells of endothelial and epithelial lining dendritic cells alveolarmacrophages and fibroblasts all these pulmonary cells are highlyheterogeneous in nature they together in association respond to thelung injury by provoking inflammatory and immune responses airway epithelial cells express pattern recognition receptors prrsalong with toll like receptors ctype lectins rigi and inflammasome components which are involved in the innate response against microbes microbial cell wall constituents likelipopolysaccharide are sensed by prrs and induce an inflammatoryresponse alveolar epithelium comprises type i and type ii alveolarcells apart from these the mucus layer and the physical barrier madeby the epithelium contribute to the first line of defense lung injury is described as any damage to the associated tissues and compartments of the pulmonary system the concept of lung injury influenced by the microenvironment can be demonstrated via series ofchanges in cell deformability and manifestation of intercellular adhesive molecules the major causes of lung injury are atelectasisalveolar instability volutrauma barotrauma infections and oxygentoxicity the pathogenicity of acute and chronic lung injury iscorrelated with the release of proteases free radicals and growth regulatory proteins by the alveolar macrophages neutrophil takescare of the extreme ranges of lungs searching for pathogens therebyfig mechanism of autophagy ulk1pi3kmtor signalling pathway binds to endoplasmic reticulum and activates dcedp1 that initiates beclin and atg512conjugation system which forms isolation membrane this is followed by the sequestration process that leads to autophagosomal formation the autophagosomefuses with lysosomes to form autolyososome that leads to degradation 0cs vishnupriya eliminating via phagocytosis they undergo transendothelial andtransepithelial migration primary host defense mechanism oflungs include the immunity conferred by surfactant proteins spnamely spa and spd they link both innate and adaptive immunity byregulating the responses of innate immune cells antigen presentingcells apcs and tcells they can act as potential biomarkers of lunginjury the implication of autophagy over lung injury is tortuousas its function varies highly with the cell types specific to the pulmonary disorders it provides both defensive and injurious outcomes of lung injury lung injury types and associated pathologies acute lung injury ali and acute respiratory distress syndromeardsacute lung injury and acute respiratory distress syndrome arecommon grievous diseases among critically illpatients and are evidential sources of mortality and morbidity they are expressed alongwith hypercapnia and hypoxemia ards is outlined as the most seriousform of ali ali is characterized by inflammation of lung surfacesresulting in the disruption of alveolarcapillary membrane followed bytransmigration of neutrophils significant event in the procession of aliand ards and outbreak of cytotoxic mediators immune responsemediated by several components like tcells macrophages naturalkiller nk cells chemokines and proinflammatory cytokines also has apredominant role in the progression of the acute injury and results inimmune reconstitution inflammatory syndrome iris immune cellsinvolved in the immune response like macrophages and monocytes arethought to be involved in the pathophysiology of iris elevatedlevels of cd14 cd16 monocyte population and an resulting increase in the proinflammatory cytokines il6 tnfα and c reactiveprotein crp are also observed in tuberculosis tbiris patients lung endothelial biomarkers like vwf and epithelial biomarkers likespd are the diagnostic targets of ali sepsis pancreatitis pneumoniatrauma transfusions aspiration and inhalation of toxic gases are someof the clinical factors of ali and ards histological patterns ofali and ards have the chances of demonstrating diffuse alveolar damage alveolar haemorrhage eosinophilic pneumonia and acute fibrinous pneumonia associated with ards may also result frompathogens like fungal viral bacterial and parasitic most commonpathogen strains include streptococcus pneumoniae respiratory virusesstaphylococcus aureus fungal pathogens like pneumocystis jirovecii andaspergillus fumigatus legionella pneumophila and enteric gramnegativeanisms among bacteria the virulence capability of pseudomonas aeruginosa is one of the prime determinant of the severity of thelung injury disease progress takes place in three degrees namelyacute phase exudative subacute phase proliferative and chronicphase fibrotic radiological features during the acute phasesignify twosided patchy groundglass densities relating to interstitialedema and hyaline membranes the important constituent of innateimmune system the pattern recognition receptors prrs are affiliatedwith the advancement of ali and ards the ligands of prrs includepathogenassociated molecular patterns pamps which induce inflammatory signalling events and the damageassociated molecularpatterns damps which induce neutrophilmediated tissue damageincreased incidences of mitochondrial damps result in high mortalityrates a common type of ali is the transfusionrelated acute lunginjury trali trali is defined as adult respiratory distress syndromeoccurring with transfusionsis manifested by pulmonary insufficiency severe hypoxia and dyspnea it is often reported as theneutrophil pmnmediated syndrome a particular study has reported that the potential risk factors for ali and ards may be associated with larger tidal volume vt and higher airway pressure pawduring one lung ventilation olv in postpneumonectomy aliardspatients and also in patients who developed aliards longtermeffects after acute lung injury are related to neurological impairmentsitlife sciences namely neuromuscular dysfunction neurocognitive dysfunction andneuropsychological dysfunction some of the minor impacts includeheterotopic ossification stiae and frozen joints chronic lung injury cli and bronchopulmonary dysplasia bpdchronic lung injury is characterized by a condition called pleuroparenchymal fibroelastosis ppfe in which a rapid multiplication ofsubpleural intestinal elastic fibres majorly in the upper lobes predominates along with other clinicopathological conditions following chronic lung injury cells undergoing apoptosis is cleared by aprocess called efferocytosis it is a type of phagocytosis confined only tothe cells that undergo apoptosis to maintain the cellturnover in pulmonary airways carried out mainly by the macrophages while immature dendritic cells mesenchymal cells and epithelial cells can alsoperform efferocytosis recently sarscov2 manifested as severerespiratory infection resulted in number of deaths worldwide commonrisk factors associated with death in sarscov2 patients identified arehypertension diabetes cardiovascular disease or chronic lung disease the most prevailing chronic lung injury ofinfants is thebronchopulmonary dysplasia bpd occurs when preterm infants suffering from various respiratory syndromes like meconium aspirationsyndrome and neonatal pneumonia are subjected to treatment withsupplemental oxygen and extensive mechanical ventilators it is proventhat the common risk factors of bpd increases with a fall in birthweight prematurity and gestational period apart from theabove other perinatal risk factors with which bpd is associated with areintrauterine growth restrictions race or ethnicity chorioamnionitis and genetic risk factors it is characterized bysaccular formation and elastic fibre aggregation of distal air spacesinflation and edema barotrauma and pulmonary oxygen toxicity arepathologies of bpd bpd is multifactorial in nature studiesdeclare that surviving patients of bpd have established pulmonarydysfunction incidence adults with bpd history are strictly prohibitedto cigarette smoking another form of bpd called the new bpdwhen surfactants are used as treatment new bpd is depicted by pulmonary hypertension and abnormalities in vasculopulmonary development glucocorticoids and tgfbeta are the efficient modulators thatinitiate bpd injury bpd infants have significantly lower levels ofvascular endothelial growth factor and platelet endothelial cell adhesion molecules chronic obstructive pulmonary disease copd and asthmacopd is a general illness worldwide it is defined as the completeirreversible state of airflow limitation characterized by weak inflammatory response of the lungs emphysema chronic obstructivebronchiolitis fibrosis blocking and narrowing of airways deprivationof lung parenchyma and elasticity immune cells like tlymphocyteswith cd8 dominance blymphocytes macrophages and neutrophilsare the regulators of copd acute provocation of copd is definedas the continuous deterioration from steady state facilitating an alteration in typical medication for rudimentary copd systemicinflammation responses are a result of leukotriene b4 tnfalpha interleukin il8 and proteases a decrease in the ratio of cd4 to cd8is a typical feature of pulmonary inflammatory responses additionalimpacts of copd are cardiovascular diseases nervous effects and osteoskeletal effects smoking is an important cause of systemicoxidative stresses immoderate inflammatory responses and emphysema as manifested as copd implications copd being an hetergoenousdisease patients with exacerbations are found to be associated withbacterial infections like moraxella catarrhalis streptococcus pneumoniaand haemophilus influenza apart from bacteria other microbes likevirus and fungi also comtribute to the lung microbiota and pathogenesisof lung microbiota through initiation of chronic inflammation it hasbeen found that bacteria and viruses fungi can promote local andsystemic inflammation that may contribute to the pathogenesis ofcopd specific biomarkers of copd observed are creactive 0cs vishnupriya protein crp il6 il10 and ccl18parc skeletal muscle losstakes place in copd wasting of the cell mass is evidenced to be theresult of tnfα participation in the pathogenesis of copd muscleglutamate reduction is linked with lactic acidosis in copd patientsending in muscle wastage similar to copd asthma is characterized by pulmonary obstruction and similar immune responses onlyvariation from copd is that the airway obstruction in asthma is reversible and it does not affect lung parenchyma dendritic cells are themodulators of th2 cells playing an important immune response ofasthma severe asthma is equal to the effects of copd illustrated by theincrease in neutrophils tumor necrosis factor tnf cxcl8 and decreased reception to corticosteroids while reversible copd is potentially to have subsequent asthma and copd ventilatorinduced lung injury vili and ventilatorassociated lunginjury valithe prevailing lung injury cases with ards when given ventilatorassistance mechanically in a clinical setup may develop additional lunginjuries ending up in ventilatorassociated lung injury vali similarlyin experimental models lung injury can be provoked by external application of injurious ventilation procedures contributing to ventilatorinduced lung injury vili thus vali can adversely aggravate thehealth of the ards patients at low lung volumes of ventilation atelectrauma occurs at high lung volumes of ventilation barotrauma andpulmonary edema occur biomarkers studied via experimental modelsof vali include several proinflammatory molecules like tnfα il1βil8 and antiinflammatory molecules like il10 il6 and stnfr1 respectively [] positive endrespiratory pressure peep and tidalvolume applied have direct influence on vali and are to be studiedspecifically during ali and edema it is reported that peep when provokes overinflation the extent of edema also increases mechanism ofsurfactant inactivation is seen in alveolar microvessels with an increasein fluid filtration further greater the lung volume higher is thetransmural pressure in them a retrospective cohort study revealedthat height and gender of the patients should be taken into considerations along with establishing limitations to large tidal volumes beforesetting up a ventilator experiments explain that decrease in thepeak pulmonary arterial pressure or respiratory frequency can lessenthe grimness effects of vali it is proven that hypercapnic acidosisaffords protection to vili addressable implications of vali arebarotrauma volutrauma atelectrauma biotrauma and oxytoxic effects cellular pathology includes physical disruption of cells and tissuesand activation of cytotoxic responses leucocytes are raised to likelyinteract with the endothelium as the increasing intraalveolar pressurefastens the transit time of them inferences for current medicalpractices involve lungprotective ventilation survival of patients can beencouraged by prone positioning future clinical practices involveprecisioned ventilationindividualized tidal volumes using drivingpressure individualized peep and extracorporeal strategies pulmonary fibrosis pf and cystic fibrosis cfidiopathicpulmonary fibrosis ipf is a degenerative interstitial fibrosing disease prevalent worldwide it is also termed as cryptogenicfibrosing alveolitis a typical honeycomblike structure of asymmetricairspaces covered by dense fibrosis is observed ipf is followed by interstitial pneumonia which is featured by deficit inflammation and withabsence of homogeneous participation of lung tissues studies havebeen made since ages which revealed several inherited forms of pulmonary fibrosis mutations in various genes were associated to pfnamely sftpc sftpa2 tert terc and muc5b incidence withrheumatoid arthritis and scleroderma are more likely to form pf thereis a chance of clearance of alveolar basement membrane and occurrenceof hyperplastic epithelial cells ipf ensures migration of fibroblastsinto the fibrinrich exudates thus a chemoattractant activity is createdin the airspaces after lung injury this process is proven to be regulatedthe proby lysophosphatidic acid experiment proves thatlife sciences inflammatory cytokines il1β directly regulates the initiation of acuteand chronic inflammation making it a valuable target of ipf cystic fibrosis is characterized as a most common autosomal recessivegenetic disorder caused by the mutation in cftr gene transmembraneprotein genecystic fibrosis transmembrane conductance receptor pathology of the disease involves bronchiolitis obstruction of pathways endobronchiolar infection impaired ciliary actions atelectasisfinally leading to secondary alveolar injury bacterial infections of saureus p aeruginosa and h influenzae causes cf it is the pulmonarymacrophages and polymorphonuclear neutrophils that forms the defense against infections while lymphocytemediated mucosal injury isobserved in cf patients cf patients have increased flow of tnfαil8 and il1β submucosal glands of the lungs are the crucialhosts of cftr genes as the number expressed are high hence cfcontributes to epithelial airway lining abnormalities with respect to thechanges taking place in the submucosal glands respectively the macromolecular secretions of the submucosal glands have changes in theircomposition viscosity and greatly impacts on the mucociliary clearance radiationinduced lung injury riliseveral complications of the lung malignancies require treatmentsinvolving radiation therapy rt extensive reports claim that rt maylead to a state of rili the threedimensional dosimetric predictors canbe emphasized for the risk of symptomatic rili the alveolarcapillary subunit forms the most radiosensitive complex of the lungs thusthe rili is also called as the diffuse alveolar damage radiation exposure provokes the production of reactive oxygen species creatinghigh toxicity levels in the lung parenchyma this may ultimately lead tolung fibrosis which develops one to six months after rt accompaniedby dyspnea in addition to fibrosis rili also develops radiationinduced pneumonitis gradually after months to years almost all thepatients undergoing rt have the chance of developing fibrosis radiationinduced pneumonitis is characterized by cough occasional fevernonspecific symptoms of dyspnea and chest pain with or without deformities in pulmonary functional tests while radiationinduced pulmonary fibrosis is characterized by cough differential levels of dyspnea chest pain or symptomless and stable scarring of the lung tissueswhen detected radiographically it has been stated that the incidence and occurrence of rili is directly influenced by dose and volume determinants of rt several studies provide information onrili that result in provoking inflammatory responses a biphasicmanifestation of cytokines is observed in the lung tissues once exposedto rt one such study carried out to assess the cytokine productionin c3hhen irradiated mice revealed a spatial change in the expression of proinflammatory cytokines among various cellular compartments of the lungs further it was noted that the bronchoalveolar lavage cells responded immediately while the interstitial cells contributedonly in the later stages during pneumonitis profiling of cytokinesnamely the interleukins interferons monocyte chemotactic protein1tumor necrosis factors macrophage inflammatory proteins and granulocyte colonystimulating factors can be performed to analyse the developmental risks of symptomatic radiationinduced lung injury in vitro studies experiments state that the application of melatonin andcarnosine compounds reduced the reactive oxygen species and inflammatory cytokines produced following rili regulators of autophagy in lung injurynumerous regulators of autophagy play a vital part in the development of lung injury with regard to autophagy the following are theimportant regulators fig the mechanism by which these autophagy regulators act is shown in the table lc3biithe microtubuleassociated protein light chain lc3 is the 0cs vishnupriya life sciences fig figure depicts the autophagy regulators in regard to lung injury various conditions like hypoxia starvation environmental stress and cigarette smoke leadsto autophagy that in turn causes lung injury activation of class ipi3kmtor pathway takes place under hypoxic and starvation conditions while environmentalstress results in provoking beclin 1vps34 pathway that induces the sequestration of the phagophore formation cigarette smoke induces ros accumulation which inturn causes egr1 e2f signalling to activate lc3ii along with sqtm1p62 and atg512 conjugation system the ros generated may also leads to apoptosis theautophagosome fuses with lysosomes and forms autophagolysosome that causes pulmonary arterial hypertension and lung injuryprinciple autophagic protein expressed on the doublemembraned autophagosome nearly eight homologues of lc3 proteins are studied inmammals the amino acid composition of these homologues classifiesthem into two subfamilies first one comprising of lc3a splicingvariants lc3b and lc3c taking part in autophagosomal membraneelongation while the second one comprising of gabarap gabarapl1 gabarapl2 and gabarapl3 taking part in the maturation ofautophagosome generally lc3b occurs in cytosol as lc3bi by theproteolytic cleavage of cterminal of lc3b which then unites withphosphatidylethanolamine to form lc3bii to assemble on the autophagosomal membrane conjugation of phosphatidylethanolamine withlc3bii can be revoked by the activity of atg4 thus it is clear thatoccurrence of lc3bii is crucial in the process of autophagy lc3bii levels are analysed through immunoblotting while limitationsare degradation of lc3bii by autophagy itself and the nonindication ofautophagic flux at distinct time points this can be overcome by comparison studies with lc3bi and using lysosomal protease inhibitors antilc3b antibodies can be applied to detect autophagy invarious cell types application of antilc3b to glioblastoma tissuesdemonstrated a positive detection of lc3b levels both in vitro and invivo suggesting a latent monitoring system of lc3b severalstudies state that hyperoxic conditions can result in ali and ards thisfurther triggers the morphological biomarker of autophagy lc3bii toaccumulate thereby determining the fate of cell clearance experimentsperformed in hyperoxiainduced human bronchial epithelial cells andcultured epithelial cells beas2b clearly stated that the expression oflc3bii was high mediated by the apoptotic regulators similarexperiment carried out in the hyperoxiainduced lung injury in c57bl mice inferred the involvement of apoptotic pathways in the activationof lc3bii interaction of lc3bii with fas proteins was observedmarking the importance of lc3bii in the management of ali pulmonary hypertension is a main cause of copd manifestation inlungs that affects vascular architecture when chronic hyperoxia wasinduced in the lung tissue extracts of patients with pulmonarytable regulators of autophagy in lung injury and their mechanismregulatorslc3biibeclin p62hif1bnip3mtormechanismelongation of autophagosome and its maturation requires atg8map1lc3 protein lc3i combines with phosphatidylethanolamine forming lc3ii essential for autophagosome formationthe initiation of the isolation membrane that forms autophagosome after the sequestration process is regulated by beclin and pi3kp62 along with sqstm1 is the receptor for polyubiquitinated substrates it helps in the transportation of cargo into the autophagosome by bindingwith lc3ii for degradationduring hypoxic conditions hif1 induces bnip3 that in turn brings about cell survival through autophagy and provoke cell death by apoptosisthe mammalian homologue atg13 is phosphorylated by mtor that binds ulk1 proteins fip200 phosphorylates ulk and initiates the isolationmembrane formation in autophagyreference no 0cs vishnupriya hypertension the upregulation of lc3b prevailed indicating the regulatory role of lc3b in vascular cell proliferation and mediatingadaptive cellular responses smoking results in various implications of lung injury in due course a multimeric protein complexcomprising of lc3bcaveolin1fas occurs under basal state extensivestudies reveal that smoking triggers lc3b to initiate the dissociation ofcaveolin1 from fas protein thus facilitating apoptotic pathways accordingly emphysema a destructive expression of copd isworsened by cigarette smoking in vitro studies in lung tissues of miceon exposure to cigarette smoking ensured the driving role of lc3b inregulating apoptotic mechanisms and finally developing emphysemarespectively all these experiments prove the comprehensive bridgebetween autophagy and apoptosis highly regulated by the expressionlc3b lc3bi and lc3bii bind to microtubule associated protein1b map1b wherein overexpression of map1b decreases the levels oflc3bii protein kinase c is known to cease autophagy by interferingwith autophagosomal formation both in vitro and in vivo studiesconfirmed that the lc3b phosphorylation by protein kinase c takesplace consistently in lungs the emergence of autophagy either asa protective role or maladaptive response due to sepsis was studied in acecal ligation and puncture clp induced septic mice it was ensuredautophagy to be a protective response yet an overexpression of lc3bii in the later stages of sepsis leads to ali describing a maladaptive role lung injury can be provoked by ischemiareperfusion in whichautophagy is stated as the safeguarding mechanism by moderatelymaintaining the level of lc3bii also the ischemiareperfusioninduced lung injury is positively governed by the erk12 signallingpathway that regulates the cellular expression of lc3bii respectively nanoparticles of zinc oxide on exposure to lungs may induceali further zinc oxide nanoparticles resulted in the raise of autophagosomal structures followed by the accumulation of lc3bii proteinsthus zno nanoparticlesinduced ali is autophagy dependent 3methyladenine a classical autophagy inhibitor reduced the manifestationof lc3bii and lowered the release of zinc particles thereby stoppingzno nanoparticlesrelated toxicity of lungs similarly the artificially synthesized polyamidoamine dendrimers pamam used as aneffective drugdelivery system may sometimes result in pamam nanoparticlesinduced ali the levels of lc3bii biomarkers were highindicating the autophagic responses beclin beclin was first identified by beth levine as becn1atg6 inchromosome 17q21 in the year and it is the major autophagyregulating gene it is a coiledcoil protein of molecular weight kda comprising of amino acids and acts together with bcl2an antiapoptotic protein beclin is an indispensable autophagypromoting gene that is homologue to the mammalian yeastatg6 gene which regulates cell survival of different types and is involved in the constitution of autophagosomes the initiation ofthe anization of autophagosomes is regulated by class iii phsophoinositide 3kinase pi3k and autophagy related gene beclin beclin has got a novel bcl2 homology region3 bh3 domainthe bh3 domain in beclin1 can bind to bcl family proteins that initiateapoptotic signalling and prevents the beclin 1mediated autophagy byremoving beclin from hvps34 either phosphorylation or ubiquitination of beclin or bcl2 can disunite bcl2 from beclin andincrease the activity of vps34 kinase which brings about increase infunction during autophagy autophagyassociated protein beclin1 binds to lc3i that adapts to its membranebound form lc3iiand it cooperates with the ubiquitinbinding protein p62sequestosome sqstm1 the first an that fails during sepsis is lungs thefamiliar complications of sepsis are ali and ards ali activated various autophagy related proteins like lc3ii beclin and lysosomerelated protein lamp2 and rab7 expressions in sepsisinduced ali to evaluate the function of autophagy in severe sepsis an experiment is carried out using endotoxemia that frequently uses septiclife sciences shock and clp which is a clinical polymicrobial sepsis model herebecn1 mice was susceptible to clpinduced sepsis in cysticfibrosis transmembrane conductance regulator cftr autophagy bybeclin overexpression cystamine or antioxidants and the restorationof beclin recovers the localization of beclin to endoplasmic reticulum and regresses the cf airway phenotype both in vitro and in vivoin scnn1btransgenic and cftr f508del homozygous mice and also inhuman cf nasal biopsies in lpsinduced ali there are threedistinct complexes of beclin1vps34 have been identified the firstcomplex contains beclin1 vps34 vps15 and atg14l second complexcontains beclin1 vps34 vps15 and ultraviolet irradiation resistanceassociated gene uvarag and the final complex contains beclin1vps34 vps15 uvrag and rubicon among these complex the onethat contains atg14l is concerned in the formation of autophagosomewhile others are in the autophagosome and endosome maturationbeclin forms the bridge in the recruitment of inducers and suppressorsof autophagy and simultaneously behaves as a key modulator in autophagosome formation mesenchymal stem cells mscs increases the translation level of beclin but not its transcription ratemscs might alleviate lpsali via downregulation of mir142a5p thatpermits pulmonary epithelial cells pecs to proceed with beclin mediated cell autophagy in lung disease the bacterial stu | 0 |
coronaviruses covs belong to a family of envelopedviruses with a positive sense singlestranded rna genomecovs cause illness ranging from upper respiratory tractinfections urtis resembling the common cold to lowerrespiratory tract infections lrtis such as bronchitis pneumonia and even severe acute respiratory syndrome sarswith most serious disease outcomes in the elderly immunocompromised patients and infants [ ] hcovoc43oc43 hcov229e 229e hcovnl63 nl63 andhcovhku1 hku1 were the ï¬rst documented humancovs hcovs which usually cause urtis and less frequently are associated with ltri diseases in the lastdecades two human coronaviruses created great concernfor the world medical community due to significant diseaseand mortality [ ] in severe acute respiratorysyndromecoronavirus sarscov was characterized byacute atypical pneumonia and diï¬use alveolar damagedad in roughly patients and with almost deathsrepresenting a nearly mortality rate more recentlyin a new human coronavirus designated as middle eastrespiratory syndromecoronavirus merscov was identiï¬ed and the global ongoing outbreak of mers with over oï¬cial cases and deaths represented approximately case fatality rate to date in humans over the last few months a new strain of human coronavirus sarscov2 also known as 2019ncov hascaught the worlds seven continents attention with its rapidglobal spread aï¬ecting at least countries and territoriesinfecting more than and claiming more than lives worldwide the coronavirus pandemichas promoted isolation and uncertainly fear and panicworldwide in additionlikely lead to changes inpolitical and economic power in ways that can be determined only later it willit is important to note that there are many similaritiesamong diï¬erent coronavirus species but not in all aspects 0coxidative medicine and cellular longevitydepending on the molecular mechanism of viral inhibitionpromoted by an antiviral agent the analysis of the data andcomparison between animal and human covs must be donevery carefully in fact it is important to note that there arediï¬erences between human and animal cov receptorswhich will likely result in diï¬erent aï¬nities or unlikely interactions of an antiviral agent with the diï¬erent cov receptors howeverif the antiviral agent interferes with thereplication andor assembly of the covs there is a higherprobability of obtaining similar antiviral activity results inhuman cov tests [ ] following this line our searchin specialized literature was focused mainly on studies thatinvestigated the anticoronavirus eï¬ects of natural antioxidants by inhibiting proteases for viral replication materials and methodsthe present study was carried out based on a search of the literature of natural antioxidants and coronavirus the searchperformed in the pubmed database included studies published until march and used the following keywordscoronavirusstressmerscov sarscov 229e nl63 oc43 hku1merscov virus infection and middle east respiratorysyndrome virus the scientiï¬c publications were selectedfrom studies published in the english languageantioxidants ï¬avonoids oxidative pathogenic mechanism of coronavirusinduced cell damagethe high mortality rate associated with the three pathogenichcovs has been mainly attributed to the development ofdigestive and respiratory tract injuries observed followinginfection acute atypical pneumonia and diï¬use alveolardamage that progress to deposition of ï¬brous tissue denudedairways haemorrhage and elevated macrophage ltrationare sometimes accompanied by watery diarrhoea dehydration and vomiting [ ]despite the molecular mechanisms of coronavirusinduced intestine and lung pathogenesis not fully elucidatedand still unclear studies have suggested that lateterm diseaseprogression is unrelated to viremia it is now believed morelikely to be associated with the immunopathological mechanism [ ] viral clearance and subsequent recovery frominfection require activation of an eï¬ective host immuneresponse however many immune eï¬ector cells may alsocause injury to host tissues together with ammatoryand immune response signaling the presence of oxidativecompounds such as reactive oxygen species ros playsimportant roles in the pathogenic mechanism of cell damageinduced by covs through oxidative stress oxidative stress is deï¬ned as an interruption andorderegulation of the signaling and redox system that can becaused by an imbalance in the production of oxidant andantioxidant species among the main oxidant agentsros and reactive nitrogen species rns stand out in orderto counterbalance the oxidant species there is an antioxidantsystem formed by enzymes and nonenzymatic molecules however during pathological events such as viral infections there may be an increase in the production of oxidantspecies not neutralized by the antioxidant system resultingin oxidative stress that promotes cellular damage throughprotein denaturation changes in the functions of nucleicacids lipid peroxidation and cell death []in addition during viral infection oxidative stress contributes to viral pathogenesis through stimulating ammation loss of immune function and increased viral replicationthat may occur due to the activation of the nuclear factorkappa b nfκb transcription pathway [] currentevidence suggests that cytokine dysregulationalso calledcytokine stormcontributes to severe disease caused by thepathogenic covs [ ] the exact mechanisms are notclear yet but research on uenza a virus shows that infection causes a rapid ux of ammatory cells this isfollowed by an increase in reactive oxygen species productionand cytokine expression and release which ultimately leadsto acute lung injury in general rna viruses promotechanges in the bodys antioxidant defense system aï¬ectingenzymes such as superoxide dismutase sod and catalasecat in addition to reducing the levels of antioxidantmolecules such as ascorbic acid carotenoids and reducedglutathione gsh [] wu reported that glucose6phosphate dehydrogenasean important antioxidantenzyme that produces nadph knockdown cells were moresusceptible to infection by hcov229e than normal cells interestingly ye and colleagues have reported that theinhibition of ros production alleviates ammation causedby uenza a virus infections in an experimental model of sarsinduced acute lunginjury in mice it was noted that phospholipid oxidationdue to oxidative stress is one of the main triggering factorsof acute lung injury this happens through the activation ofthe innate immune response culminating in the activationof pulmonary macrophages via tlr4triftraf6nfκbsignaling furthermore hypoxia caused by acute lunginjury can cause myocardial injury due to the production ofros aggravating infections caused by coronavirus disease covid19 mitochondria have an essential function in energy generation and for this reason their function and integrity arestrictly regulated in order to respond to varying energyrequirements and environmental conditions mitochondria are known to function as the control point in apoptoticpathways releasing proapoptotic factors mainly ros whichfunction as a signaling molecule that may result in cell death[ ] some studies have shown a relationship betweencoronavirus infection and dysfunctional or damaged mitochondria leading to the release of ros and other proapoptotic substances [ ] in a recent study xu reportedthat ros and p53 play key roles in regulating many kindsof the cell process during coronavirus infection in vero cellsaccording to the authors coronavirus infection appears toinduce a timedependent ros accumulation which in turnis linked to regulatory mechanisms of p53 activation andapoptosis in infected cells antioxidant substances promote improvement in casesof disease caused by coronaviruses such as apolipoproteinda lipocalin that promoted a neuroprotective eï¬ect against 0coxidative medicine and cellular longevityencephalitis induced by human coronavirus oc43in micethis protective eï¬ect occurred through the reduction of oxidative stress cerebral lipid peroxidation and regulation ofammation [ ] also the treatment with antioxidantssuch as pyrrolidine dithiocarbamate or nacetylcysteine significantly inhibits moreover melatonin promotes downregulation of acute lungoxidative injury due to its antiammatory and antioxidantactions making it a possible compound in the treatment ofcovid19 based on these studies compounds thathave antioxidant actions can be helpful in the treatment ofinfections promoted by coronaviruscoronavirusinduced apoptosisin general antioxidant properties of polyphenolic compounds such as some ï¬avonoids have been associated withthe presence of aromatic phenolic rings that promote theelectron donation and hydrogen atom transfer to free radicals acting as free radical scavengers reducing agents andquenchers of single oxygen formation thus the aimof this study was to investigate the antioxidant capacity andantiviral activity of natural antioxidants against coronavirusthe compounds are illustrated in figure occurrence and antioxidant properties ofanticoronavirus compoundsquercetin can be found in plants such as rubus fruticosus land lagerstroemia speciosa l pers [ ] also quercetinshows antioxidant activity at a concentration of μmoll inhepg2 cells inhibiting oxidative stress promoted by h2o2 promotes an increase in sod cat and glutathioneperoxidase gpx and reduces lipid peroxidation in rats withchronic prostatitischronic pelvic pain syndrome moreover quercetin improves sepsisinduced acute lung injury inrats by reducing lipid peroxidation and ammation andincreasing sod and cat levels in addition quercetin glycosides with antioxidant activity such as quercetin 3βglucoside have already been isolated from plants such as passiï¬ora subpeltata ortega andchamomilla suaveolens pursh rydb [ ] the administration of quercetin 3βglucoside mgkg poinstreptozotocininduced diabetic rats promotes an increasein the levels of antioxidant enzymes sod cat andgpx and nonenzymatic antioxidants vitamins c and eand gsh and a reduction of lipid peroxidation quercetin 3βgalactoside hyperoside is found mainly in plantsof the hypericum genus such as hypericum perforatum l[ ] moreover it showed cardioprotective activity inhigh glucoseinduced injury of myocardial cells throughdecreased apoptosis and ros production and increasedsod levels quercetin 7ramnoside is also found inplants of the hypericum genus such as hypericum japonicum thunb ex murray this ï¬avonoid shows hepatoprotective activity against carbon tetrachloride in mice bydecreasing lipid peroxidation and increasing cat andgsh levels in addition to presenting values of μmand22diphenyl1picrylhydrazyldpph and ²azinobis3ethylbenzthiazoline6sulphonic acid abts assays respectively μm intheepigallocatechin gallate is present in parkia roxburghii gdon and is one of the main metabolites found in green teaand liubao tea camellia sinensis lo kuntze also gallocatechin gallate can be found in this plant [] literaturedata reveal that the administration ip of mg100 g ofepigallocatechin gallate in rats with streptozotocininduceddiabetes mellitus promotes a reduction in oxidative stressthrough reductions in parameters such as indirect nitricoxide synthesis and status total oxidative as well as anincrease in levels of cat and total antioxidant capacity ofplasma furthermore it promotes cardioprotection byantioxidant mechanisms green tea has a high antioxidant capacity due to the highlevels of catechins present he and collaborators compared the antioxidant activities of catechins and reported thatepigallocatechin gallate has greater antioxidant activity viaradical scavenging activity μm with values of ± ± and ± compared to its epimergallocatechin gallate with values of ± ± and ± in the dpph abts and ferric reducingantioxidant power frap respectively amentoï¬avone is a biï¬avonoid present in leaves ofginkgo biloba l garcinia brasiliensis l and nandinadomestica l [] this biï¬avonoid has a high antioxidantcapacity demonstrated in scavenging tests ofdpph abts superoxide and hydroxyl radicals moreover amentoï¬avone prevents acute lung injury induced bysepsis in rats by decreasing thiobarbituric acid reactive substance tbars levels and by increasing levels of sod andgsh apigenin is mainly present in ï¬owers and leaves beingabundantly found in apium graveolens l petroselinum crispum mill fuss and matricaria chamomilla l sánchezmarzo and collaborators evaluated the antioxidantcapacity of apigenin using the trolox equivalent antioxidantcapacity teac oxygen radical absorbance capacityorac and frap assays the results show that apigeninhas good antioxidant activity with values of ± μmol teammol ± μmol teammol and ± μmol fe2mmol respectively in addition oraladministration of apigenin mgkgday for days in anexperimental model of cardiotoxicity induced by doxorubicin in rats promoted cardioprotection by reducing levels ofmalondialdehyde mda increasing sod levels and preventing cardiomyocyte apoptosis luteolin is present in foods such as carrot cabbage teaand apple and is found in ugni molinae turcz [ ] datashow that luteolin μgml increases the levels of gsh theexpression of gsh synthetase and the activity of sod andcat in human colon cancer cells ht29 furthermoreluteolin attenuates the sepsisinduced acute lunginjury in mice by reducing lipid peroxidation and increasingsod and cat activity in addition to suppressing the nfκbpathway herbacetin is ubiquitous in plants of the genus rhodiolasuch as rhodiola rosea l herbacetin glycosides are alsopresent in the roots of r sachalinensis a bor and show antioxidant activity veeramani reported that theadministration of herbacetin mgkg po in mice with 0coxidative medicine and cellular longevityohohohohhoohooohohhooohoh oquercetinohohoooohohohquercetin 3ð½galactosideohohooohoohoohohhoohohohoh oquercetin 7ramnosideohooohohoohoohoohohohquercetin 3ð½glucosidehooohooohohohohohohepigallocatechin gallateohgallocatechin gallateohhooohoh ohooluteolinhooohoooohoohohoohrhoifolinhohohoohohohohohohoohoohooh oamentoflavoneohhooohohohoherbacetinhohoooohooohohohoapigeninooohooohhopectolinarinooopsoralidinhooohohohohcatechinohhoohoooh ohelichrysetinohohomyricetinohohohhohoohoohoisobavachalconeohhooohscutellareinohresveratrolfigure chemical structures of bioactive antioxidants against coronavirusobesityassociated insulin resistance promotes an increasein the activity of the enzyme glucose6phosphate dehydrogenase which is directly related to the production ofnadph pectolinarin is present in plants of the genus cirsiumsuch as cirsium setidens nakai and cirsium japonicum dcthe administration of pectolinarin and mgkg pofor two weeks in rats promotes antioxidant eï¬ects in hepatic 0coxidative medicine and cellular longevitytable antioxidant properties of natural inhibitors of coronaviruscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencetype of cells μmoll mgkg po mgkg poinhibiting oxidative stress promoted byh2o2promoted an increase in sod cat andgpx and reduced lipid peroxidationreduces lipid peroxidation and increasessod and cat levelsincreases levels of sod cat gpx mgkg povitamins c and e and gsh and reduces nmoll mgkgic50 μm dpphec50 μm abtsrats with streptozotocininduced diabetes mellitus mg100 g iprats with streptozotocinnicotinamideinduceddiabetes mellitus mgkg po μm mgkg μgml ± μmol teammol ± μmol teammol and ± μmol fe2mmolrespectively mgkg pomodelshepg2 cellsrats with chronicprostatitischronic pelvicpain syndromesepsisinduced acute lunginjury in ratsstreptozotocininduceddiabetic ratshigh glucoseinducedinjury of myocardial cellsccl4induced liver damagemodel in micedpphabtsquercetinquercetin 3βglucosidequercetin 3βgalactosidequercetin ramnosideepigallocatechingallateepigallocatechingallatedpphabtsfrapgallocatechingallateamentoï¬avoneacute lung injury inducedby sepsis in ratsdpph abts superoxideand hydroxyl radicalsteacoracfrapcardiotoxicity induced bydoxorubicin in ratshuman colon cancer cellsht29acute lung injury inducedby sepsis in micemice with obesityassociated insulinresistance inductionhepatic injury induced bydgalactosamine in ratsoracapigeninluteolinherbacetinpectolinarinrhoifolincatechinlipid peroxidationdecreases apoptosis and ros productionand increases sod levelsdecreases lipid peroxidation and increasescat and gsh levelsscavenging of free radicalsreduces indirect nitric oxide synthesis andtotal oxidative statusincreased levels of cat and totalantioxidant capacity of plasmaincreased levels of cat sod and gshreduced levels of superoxide and proteincarbonyl pco and prevented dnadamage ± ± and ± ± and ± respectively ± respectivelydecreases tbars levels and increaseslevels of sod and gshscavenging of free radicalsscavenging of free radicalsreduces levels of mda increases sodlevels and prevents cardiomyocyteapoptosis μgmlincreases levels of gsh expression of gshsynthetase and the activity of sod and mgkg ip mgkg po and mgkg poapproximately troloxequivalents μmcatreduces lipid peroxidation increases theactivity of sod and cat and suppressesthe nfκb pathwayincreases the activity of glucose6phosphate dehydrogenaseincreases levels of sod gsh glutathionereductase and glutathione stransferasescavenging of free radicalsscavenging of free radicals 0coxidative medicine and cellular longevitytable continuedtype of cellscompoundtestedassaysexperimentalconcentration doseantioxidant eï¬ectreferencemodelsabtsfrap ± mol troloxequivalentsmol ± mol trolox equivalentsmoldihydrorhodamine oxidation assayandic50 ± μmisobavachalconedpph sc50frapabts sc50psoralidinelectron spin resonancemyricetinhelichrysetinscutellareinresveratroldpphchinese hamster lungï¬broblast cells v794treated with h2o2oracdpphabtssuperoxide radicalsdpph sc50r2rats with obstructive lungdiseasehypercholesterolemicapoeko mouserespectively μm ± mmic50 μmequivalent to feso4 mm respectively·7h2o and μgml and μgml μgml ± trolox equivalents ± μm ± μm and ± μm respectivelyscavenging of free radicalsscavenging of free radicalsscavenging of free radicals and respectivelyprevents dna damage and lipidperoxidationincreases the activity of sod cat andgpxscavenging of free radicalsscavenging of free radicals μmoldmscavenging of free radicals mgkg mgkgincreases sod activity and reduces mdalevelsinhibits the activity and expression ofnadph oxidasesincreases sod gpx and cat levels injury induced by dgalactosamine by increasing levels ofsod gsh glutathione reductase and glutathione stransferase [ ]rhoifolin is found in citrus fruits such as citrus limettarisso studies have indicated that its radical peroxyl scavenging capacity is higher than trolox in orac assays approximately trolox equivalents μm [ ]meanwhile the catechin is a ï¬avonoid present inleaves of green tea wine and fruits [ ] grzesik investigated the antioxidant action of catechinthrough the abts scavenging activity and frap teststhe results show values of ± mol troloxequivalentsmol and ± mol trolox equivalentsmol respectively in addition catechin shows greaterprotective properties in the dihydrorhodamine oxida ± μm than gsh and ascorbiction assay ic50acid and μm respectively psoralidin is a prenylated coumestan which is found inplants of the fabaceae family such as psoralea corylifolia lxiao and collaborators investigating the antioxidant potential of compounds isolated from p corylifolia observed thatpsoralidin shows the best antioxidant activity by the methodof electron spin resonance spectroscopy with an ic50 value of μm the compound isobavachalcone has been isolated fromplants of the fabaceae and moraceae families [ ] isobavachalcone shows a strong antioxidant activity in dpphsc50 frap and abts sc50 assays with values of μm ± mm equivalent to feso4·7h2o and mm respectively in addition the compound has beenreported to inhibit the nfκb pathway in sephadexinducedlung injury in rats [ ]helichrysetin is a chalcone that is found in plants of thehelichrysum genus such as helichrysum odoratissimum l in a study investigating the antioxidant activity of natural and prenylated chalcones vogel found that helichrysetin is the substance that shows the highest antioxidantactivity in the orac test with values of ± troloxequivalents myricetin is widely found in the plant families myricaceae and anacardiaceae and is widely used as health foodsupplement due to its antioxidant properties [ ]bennett demonstrated that myricetin reacts withoxygencentered galvinoxyl radicals more than timeshigher than vitamin e dalphatocopherol furthermoremyricetin was able to scavenge and on the dpphassay μgml and μgml respectively interestinglythe compound prevents dna damage by lipid 0coxidative medicine and cellular longevity2h2o2 2gsh2o2h2h2o2vit evit cgshnadphnonenzymatic antioxidantsgpxsodcatsemyzne tnadixoitna2h2o gssgo2 h2o2o2 h2omdapcotbarsbio m arkers of oxidative stressgeneration of rosoxidative stresscell damagenaturalantioxidants sesadixohpdan2o2 nadphnadp 2o2 hfigure the main antioxidant mechanisms of natural compounds reported in this review dashed line inhibition full line activationperoxidation and increasing the activity of sod cat andgpx in chinese hamster lung ï¬broblast cells v794treated with h2o2 scutellarein is found in scutellaria barbata d don andpolygonum viscosum buchham [ ] liu investigated the antioxidant activity of scutellarein through thedpph abts and superoxide scavenging assays they notedthat the compound shows good antioxidant activity withvalues of ± μm ± μm and ± μmrespectively while the trolox a standard antioxidant compound presented ± μm ± μm and ± μm respectively resveratrol is found in grapes peanuts and blueberriesand can be isolated from veratrum grandiï¬orum o loes literature shows that resveratrol has good antioxidantvalues of μmoldmactivity with dpph sc50r2moreover it is able to reduce the production of ros by inhibiting the activity and expression of nadph oxidases byeliminating oxidant agentsincluding radical hydroxylsuperoxide hydrogen peroxide and peroxynitrite the treatment of resveratrol mgkg po in ratsreduces oxidative stress in obstructive lung disease byincreasing sod activity and reducing mda levels indicatinga decrease in lipid peroxidation table shows themain actions of natural antioxidants discussed in this studyand figure illustrates these activities effect of natural antioxidants incoronavirus infectionsthis review focused on studies reporting on the anticoronavirus activity of natural antioxidants based on exclusioncriteria data from nineteen compounds were discussedthe oxidative stress pathway could potentially be a keyelement in coronavirusinduced apoptosis and pathogenesis for this reason it is interesting to investigate the useof antioxidants as potential therapeutic toolseither as analternative or as an adjuvant to conventional therapiesinthe treatment of coronavirus infections among the antioxidant compounds evaluated as for coronavirus infectionsare the ï¬avonoids which are compounds widely found infruits vegetables and certain beverages in fact researchgroups have reported that antioxidant ï¬avonoids includingcatechin luteolin apigenin quercetin and quercetin rhamnosideinhibit ros accumulation and apoptosis ofcells infected with diï¬erent coronavirus including porcineepidemic diarrhoea coronavirus pedv and transmissiblegastroenteritis coronavirus tgev []as shown with the recent covid19 pandemic thesearch for alternative or new antiviral therapies for theremainstreatment of coronavirus diseasesimportantbased on the literature antioxidanttherapies oï¬er anattractive option 0coxidative medicine and cellular longevitytable natural antioxidants tested in in vitro coronavirus infection models and their main results and mechanism of actionantioxidanttype of cells testedconcentrationic50antiviral eï¬ectmechanism of actionreferencecatechintgevinfected st cellscatechin μminhibition of tgevinduced apoptosissuppression of the tgevinducedbcl2 reduction baxredistribution cytochrome crelease and caspase3 activation resveratrolmersinfected vero e6cellsresveratrol μmquercetinepigallocatechingallategallocatechingallate gcgquercetin rhamnosideq7rrecombinant 3clprowas expressed in pichiapastoris gs115quercetin μmepigallocatechingallate μmgallocatechingallate μmpedvinfectedvero cellsq7r μminhibition ofmersinducedreduction of the expression ofinfectionapoptosis andnucleocapsid n protein essential prolonged cellular survivalfor merscov replicationafter virus infectioninhibition of coronavirusreplicationreduction of the formationof a visible cytopathiceï¬ect cpe without dnafragmentationgcg displayed a binding energyof kcal mol1 to the active siteof 3clpro and the galloyl moietyat 3oh position was required for3clpro inhibition activitynot speciï¬city amentoï¬avoneapigeninluteolinquercetinquercetin3βgalactosideherbacetinrhoifolinpectolinarinsarscov 3clproinhibition usingï¬uorescence resonanceenergy transfer analysismolecular dockingsprfretbasedbioassays andmutagenesistryptophanbasedï¬uorescence methodherbacetinisobavachalconequercetin3βdglucosidehelichrysetintryptophanbasedï¬uorescence methodamentoï¬avone3βgalactoside μmapigenin μmluteolin μmquercetin μmquercetin μmherbacetin μmrhoifolin μmpectolinarin μmherbacetin μmisobavachalcone μmquercetin3βdglucoside μmhelichrysetin μminhibition of sarscovreplicationflavonoids exhibited sarscov3clpro inhibitory activity[ ]inhibition of merscovreplicationflavonoids exhibited merscov3clpro inhibitory activity isobavachalconepsoralidinlineweaverburk anddixon plotsmyricetinscutellareinsprfretbasedbioassaysisobavachalcone μmpsoralidin μmmyricetin μmscutellarein μminhibition of sarscovreplicationisobavachalcone and psoralidinexhibited sarscov papainlikeprotease inhibitory activitymyricetin and scutellareininhibition of sarscovpotently inhibit the sarscovreplicationhelicase protein in vitro byaï¬ecting the atpase activity the high number of deaths and clinical complicationsobserved in sars and merscov epidemics motivatedthe search for eï¬ective therapeutic agents this was necessitated when many of the tested conventional drugs andtherapies proved ineï¬ective in treating sarsantiviralcov infections for exampletreatment ofthe initial 0coxidative medicine and cellular longevitycell culture coronavirusnaturalantioxidants suppress bcl2 reductionsuppress bax redistributionsuppress cytochorome c releasesuppress caspase3 activationreduce formation of a visiblecytopathic effect without dnafragmentationreduce nucleocapsid n protein expressioninhibit 3clike proteaseinhibit 3clike proteaseinhibit papainlike proteaseinhibit helicase protein by affectedatpase activitytgevpedvmerscovsarscovfigure inhibitory actions of natural antioxidants against coronavirussarscov with antiviral agents such as ribavirin and corticosteroids did not achieve very satisfactory resultsmainly because corticosteroids exertimmunosuppressoreï¬ects on the humoral and cellular immune systems[ ] other drugs such as pentoxifylline were considered for the treatment of sars due to its interestingtherapeutic propertiesantiammatoryantiviral immunomodulatory and bronchodilatory eï¬ectshowever it too was not successful in the clinical treatment of sarscov infection includethatinhibition ofmany antioxidant compounds show antiviral activityagainst sarscov the antiviral activity has been mainlyattributed to thethe 3clike protease3clpro of sarscov a vital enzyme for sarscov replication as an example multiples studies havereported that quercetin and quercetinderived compoundssuch as quercetin 3βgalactoside display potent 3clproinhibitory e5ï¬ect and consequent reduction of sarscovreplication other antioxidants such as epigallocatechin gallate gallocatechin gallate amentoï¬avone apigeninluteolin herbacetin rhoifolin and pectolinarin are alsofound to eï¬ciently block the enzymatic activity of sarscov 3clpro [ ]moreover some natural antioxidants exhibit promisingantiviral activity against sarscov infection by interferingwith diï¬erent targets involved in sarscov replication inparticular the sarscov papainlike protease plpro andsarscov helicase protein kim reported that isobavachalcone and psoralidin inhibit plpro in a dosedependentmanner with ic50 ranging between and μm previously yu reported that myricetin and scutellareinpotently inhibit the sarscov helicase protein in vitro byaï¬ecting the atpase activity merscov is another zoonotic coronavirus transmitted between animals and human beings that causes severemorbidity and mortality no antiviral medicines with satisfactory eï¬cacy for the treatment of merscovinfectedpatients have been identiï¬ed to date similar to sarscov natural antioxidant libraries have been probed forpotentialinhibitory compounds against merscov 3clike protease jo showed that herbacetin isobavachalcone quercetin 3βdglucoside and helichrysetinfourcompounds with recognized antioxidant activity can blockthe enzymatic activity of merscov 3clpro using atryptophanbased ï¬uorescence method furthermore theexperimental and computational studies show that ï¬avonoland chalcone are favourite scaï¬olds to bind with the catalytic site of merscov 3clpro in a study performed by lin the antiviral activitiesof resveratrol were investigated in mersinfected vero e6cells the authors reported a significantinhibition ofmerscov infection and prolonged host cell survival aftervirus infection which they speculate was promoted by resveratrol in addition they also found that the expression ofthe nucleocapsid n protein which is essential for merscov replicationis decreased after resveratrol treatment it is important to mention that in vitro models of coronavirus infection also show antiviral activity of ï¬avonoidsextracted from ï¬owering cherry cultivars and black tea[ ] finally antioxidants such as resveratrol alsoare able to block infection produced by herpesvirus the discovery of antiviral compounds from a bioactivecompound against other viruses is an interesting strategy forobtaining new antiviral drugs table shows the mainactions of the natural antioxidants against the coronavirusand figure summarizes these activities 0c conclusionsin conclusion this review shows that antioxidant compounds prominently ï¬avonoids exhibit antiviral action inmodels of coronavirus infections in general the antiviralactivity might be attributed at least in part to the inhibitoryeï¬ect on the enzymatic activity of targets involved in coronavirus replication including sarscov 3clpro sarscovpapainlike protease plpro sarscov helicase proteinand merscov 3clpro in addition some studies provideevidence that the reduction of ros accumulation retardsthe coronavirusactivated apoptotic signaling thereforethe mechanisms of oxidative stress could be the key elementto be studied in coronavirus infectionsincluding thoserelated to ammatory processes arising from the action ofthis virus obviously further investigations are needed toelucidate other pharmacological mechanisms by which natural antioxidants play an antiviral eï¬ect despite the ï¬ndingsreported in this reviewthey cannot be generalized tocovid19 however the data provided support to the investigation of natural antioxidants as a potential therapeuticapproach in the treatment for covid19 and its severe clinical complications either as an alternative or as an adjuvantto conventional therapies and contribute to the search fornew prototypes in the development of drugs against coronavirus infectionsabbreviations229e3clproabtshuman coronavirus229e3clike protease²azinobis3ethylbenzthiazoline6sulphonic acidcoronavirusescatalasediï¬use alveolar damage22diphenyl1picrylhydrazylferric reducing antioxidant powerreduced glutathioneglutathione peroxidasehuman coronaviruseshuman coronavirushku1lower respiratory tract infectionsmalondialdehydecovscovid19 coronavirus disease catdaddpphfrapgshgpxhcovshku1lrtismdamerscov middle east respiratory syndromecoronavirusnfκbnuclear factor kappa bhuman coronavirusnl63nl63human coronavirusoc43oc43oxygen radical absorbance capacityoracprotein carbonylpcoporcine epidemic diarrhoea coronaviruspedvplpropapainlike proteasereactive nitrogen speciesrnsreactive oxygenated speciesrossarssevere acute respiratory syndromesarscov severe acute respirator | 0 |
"In vivo transthoracic HIFU application in pigs caused a mean peak temperature increase up to 53.7°C in a simulated lesion (BioGlue®) deep inside the flooded lung. HIFU energy penetrated through the pleura and flooded lung into the target lesion. The simulated lesion which was located at a 6 cm depth below the transducer was heated by HIFU. The temperature increase was highly variable and inconstant. The mechanism of heat generation in the BioGlue®-simulated lesion was unclear. The lesion consisted of purified BSA and glutaraldehyde. Although this simulated lesion was not a true human tumour the acoustic and thermal properties are similar to those of human cancerous tissue due to similar densities and high protein contents. In addition movements of the heart and mediastinum may be transmitted to the target lesion bringing them out of the focal zone. Lung flooding seems to be ideal for HIFU application because sonographic imaging is possible and because of other advantages. Compared to other human tissues a flooded lung has an ideally suitable beam path because water has a very low attenuation. In contrast to flooded lung tissue tumour tissue converts acoustic energy into a therapeutic thermal dose and enables selective heating of the cancer mass. Consequently damage to healthy lung tissue is minimised. In addition to acoustic advantages there are other favourable conditions for tumour ablation in a flooded lung. There is no pulmonary blood flow in a flooded lung [22]. Perfusion reduces heat and is not desirable with locoregional thermal therapy. In addition ischaemia-related acidosis sensitises tumour tissue to heat. In comparison to a ventilated lung lung flooding reduces tumour movements caused by breathing. The following limitations existed in the current study. The risk of lung flooding in patients with limited lung function is unclear. Further studies on patients with limited lung function should be performed to examine the influence of unilateral lung flooding on haemodynamics and gas exchange. Ongoing studies in pigs showed that lung flooding of only one lobe is feasible. Transcutaneous application was not applied for the in vivo or ex vivo studies. One rib had to be resected to apply the HIFU applicator which consisted of a HIFU transducer and sector-array probe to the chest of a pig. Technical improvements in the application system are necessary to ensure that the HIFU focal zone is aimed precisely at the target lesion with the guidance of sonography. Transthoracic HIFU application is difficult despite resection of the ribs because the intercostal spaces in the animal model are narrow. A method and transducers have been developed to avoid the shielding of therapeutic ultrasound by the human rib cage [23]. Different HIFU exposure schemas (ten seconds ex vivo versus one second on/off in vivo) and transducers were applied during this study. Ultrasound imaging is disturbed if HIFU is continuously on. Therefore the intermittent one second on/off in vivo schema was needed to control the focal alignment with the thermocouple during the one second off interval. For HIFU application to human tumours resected lung lobes were used. The gas-free filling occurs at 71.4% of the resected lobes. This limitation is due to residual air in non-collapsed bronchi that can only solved by resorption under in vivo conditions [11]. Lung flooding was performed with cooled saline at 15°C for the ex vivo HIFU application to ensure that hypoxic damage did not occur within the tumour and lung tissues. This was important because the NADPH-diaphorase staining method is based on mitochondrial vitality which is very sensitive to ischaemia. A hyperechoic area was found within the tumour tissue immediately following HIFU exposure. Grayscale changes have been shown to be a useful marker for HIFU-induced tissue destruction [8]. However it is unclear whether the area of grayscale changes corresponds with the ablated area such that a hyperechoic sonolesion represents irreversibly damaged tissue. B-mode ultrasound imaging is probably not the best method for monitoring tissue response. Currently the most important problem associated with ultrasound-guided HIFU ablation is the lack of reliable thermometry and lesion production monitoring [6]. In addition to ultrasound imaging real-time magnetic resonance thermometry could be important for ascertaining the extent of tumour destruction [24-26]. Conclusions In combination with lung flooding high-intensity focused ultrasound produced a thermal effect in an ex vivo model of human lung carcinoma and in simulated lung tumours in an in vivo porcine model. High-intensity focused ultrasound is a potential strategy for treating lung cancer. Further studies will examine HIFU therapy in animal models of lung cancer. Abbreviations BSA: Bovine serum albumin; FIO2: Fraction of inspired oxygen; HIFU: High-intensity focused ultrasound; H&E: Hematoxylin and eosin; NSCLC: Non-small cell lung cancer; NADPH-diaphorase: Nicotinamide adenine dinucleotide phosphate-diaphorase. Competing interests The authors declare that they have no financial or non-financial competing interests. Authors contributions FW was responsible for HIFU technique and temperature measurement. He collected and evaluated the data and wrote the manuscript. HS and SB performed the anaesthesia. CB performed the histopathological and enzyme histochemistry examinations. TGL co-wrote and revised the manuscript and discussed the results with the authors. All authors read and approved the manuscript. Acknowledgments This work was funded by the SRH Foundation Heidelberg Germany. The funder had no role in the study design or in the collection analysis and interpretation of data. The authors would also like to thank Uwe Leder for his anizational support and Mrs. Petra Dobermann for active help with the animal experiments. DKFZ Atlas of Cancer Mortality 2007 Heidelberg: German Center of Cancer Research Drings P Dienemann H Wannenmacher M Management of Lung cancer 2002 Berlin Heidelberg New York: Springer Colice GL Rubins J Unger M Follow-up and surveillance of the lung cancer patient following curative-intent therapy Chest" | 1 |
Radial shock waves prevent growth retardation caused by the clinically used drug vismodegib in ex vivo cultured bonesSowmya Ramesh123 Lars Svendahl245 Vrisha Madhuri135 farasat Zaman2In childhood medulloblastoma patients the hedgehog antagonist vismodegib is an effective anticancer treatment but unfortunately induces irreversible growth arrests and growth impairment limiting its use in skeletally immature patients We hypothesized that radial shock wave treatment rSWT may protect druginduced growth impairment owing to its osteogenic effects Fetal rat metatarsal bones were exposed to vismodegib day nM andor rSWT single session other bones from day were continuously exposed to a Gli1 antagonist GANT61 µM andor rSWT single session Control bones were untreated The bone length was measured at intervals histomorphometric analysis and immunostaining for PCNA Gli1 and Ihh were performed on the sectioned bones Bones treated with vismodegib showed impaired bone growth reduced height of the restingproliferative zone and reduced hypertrophic cell size compared to control In vismodegib treated bones a single session of rSWT partially rescued bone growth increased the growth velocity hypertrophic cell size and restored growth plate morphology Bones exposed to GANT61 showed impaired bone growth and disanized growth plate while when combined with rSWT these effects were partially prevented Locally applied rSWT had a chondroprotective effect in rat metatarsal bones and suggest a novel strategy to prevent growth impairment caused by vismodegibHedgehog Hh proteins are wellknown to be overexpressed in paediatric medulloblastoma1 Mutations that occur in the family of Hhpathway genes such as patched1 suppressor of fuse and smoothened leads to an increased level of the gliomaassociated oncogene Gli1 a downstream transcription factor of Hh2 In the clinic hedgehog inhibitors are used to decrease the Hhactivity and thereby impede tumor progression3 However stable expression of the Hhgene is essential to maintain chondrocyte proliferation and hypertrophy during bone growth6 A recent study reported that prolonged exposure to vismodegib a Hhantagonist in children with medulloblastoma resulted in irreversible growth plate fusion causing growth arrest of long bones78 Preclinical studies in young mice exposed to a Hhantagonist also showed growth arrests and bone growth defects9 Mechanistic studies revealed that even brief exposure to a Hhinhibitor was enough to damage the growth plates by diminishing the numbers of reserve and proliferative chondrocytes9 These findings further imply that it may not be possible to arrive at a dose that selectively targets tumor growth with no sideeffects on bone development Therefore in children a protective strategy for growth plate shielding without interfering with the desired anticancer effects of vismodegib in the neural tissue is highly desiredIn vitro studies using cultured rat metatarsal bones10 and in a0vivo studies in rabbits11 and humans12 have shown that radial shock wave treatment rSWT a noninvasive modality used in the clinic have stimulatory effects on bone growth10 Furthermore the observed stimulation of chondrocyte proliferation and hypertrophy induced by rSWT was partially linked to local upregulation of Gli1 in cultured metatarsal bones10 Interestingly previous in a0vitro studies revealed that highenergy shock wave treatment increased the uptake of chemotherapy agents13 1Department of Paediatric Orthopaedics Christian Medical College Vellore India 2Division of Paediatric Endocrinology Department of Womens and Childrens Health Karolinska Institutet Solna Sweden 3Centre for Stem Cell Research A Unit of inStem Bengaluru Christian Medical College Bagayam Vellore India 4Paediatric Endocrinology and Metabolism Astrid Lindgren Children²s Hospital Karolinska University Hospital Solna Sweden 5These authors contributed equally Lars Svendahl and Vrisha Madhuri email sowmyarameshkiseScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on vismodegib treated bones a Fetal rat metatarsal bones cultured exvivo were treated with the Hhinhibitor vismodegib a0nM for a0days n dotted line a single session of highenergy rSWT a0Hz a0mJ n or both n and thereafter followed for a0days The graph shows increases in bone length over time a0from day All error bars indicate SD Twoway ANOVA was applied b Graph shows the increase in the growth velocity on day of control vismodegib rSWT and vismodegib rSWT treated bones All error bars indicate SD Representative images of metatarsal bones stained with Alcian blue c untreated control d vismodegib e rSWT and f vismodegib rSWT Magnification 10x g height measurements of R P zone and h hypertrophic cell size Quantification of immunostaining for i Gli1 and j Ihh using the ImageJ software p p p thereby lowering the dose of the drug when applied to cell lines derived from human osteosarcoma14 human colorectal adenocarcinoma15 and anaplastic thyroid cancer16 The effect was mediated by a transient increase in cell membrane permeability allowing passage of a higher concentration of the drug16A recent report suggested that locally applied rSWT can promote longitudinal bone growth of rat metatarsal bones cultured exvivo in the absence of serumgrowth factors10 Based on these findings we hypothesized that rSWT may also have the capacity to prevent growth failure caused by Hhinhibitors We aimed to investigate the potential for rSWT to prevent growth retardation caused by two different Hhantagonists vismodegib and the Gli1 antagonist GANT61 in a wellestablished model of cultured fetal rat metatarsal bonesResultsEffect of vismodegib on bone growth and rescuing effects of rSWT To allow transient inhibition of Hhactivity cultured fetal rat metatarsal bones were treated with vismodegib for a0days whereafter growth was monitored for another a0days without any treatment Bones treated with vismodegib grew less than untreated controls and the difference was significant when measured on day ± vs ± bone length increase from day respectively p Fig a01aTo address the primary objective of this study if rSWT can prevent growth failure caused by hedgehog inhibition we first studied if a single exposure to rSWT can rescue metatarsal bone growth after transient exposure to a0days treatment with vismodegib Indeed bones treated with rSWT single exposure on day in combination with vismodegib day grew significantly better than bones treated with vismodegib alone when measured Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Rescuing effects of rSWT on GANT61 treated bones a Fetal rat metatarsal bones cultured exvivo treated with the Hhinhibitor GANT61 a0µM n a single session of rSWT a0Hz a0mJ n or both n and followed for a0days The graph shows increases in bone length a0from day All error bars indicate SD Twoway ANOVA was applied Representative images of metatarsal growth plates stained with Alcian blue b untreated control c GANT61 d rSWT and e GANT61 rSWT Magnification 10x f Height measurements of R P zone and g hypertrophic cell size Quantification of immunostaining for h PCNA and i Gli1 using the ImageJ software p p on day ± vs ± respectively p Fig a01a Also the growth velocity tended to be higher in rSWT vismodegib compared to vismodegib alone Fig a01bGrowth plate morphology Histological evaluation revealed severe disruption in the growth plate morphology in the bones treated with vismodegib compared to control Fig a01cd The disrupted growth plate morphology in vismodegib treated bones was found to be more normal in bones receiving rSWT alone Fig a01e and rSWT vismodegib Fig a01f When comparing vismodegib treated bones to controls we found reduced height of the resting proliferative R P zone ± a0µm vs ± a0µm respectively p Fig a01g The height of the R P zone tended to be increased in bones exposed to rSWT vismodegib when compared to vismodegib alone ± a0µm vs ± a0µm respectively p ns Fig a01g Histomorphometric analysis showed decreased size of hypertrophic chondrocytes in the bones treated with vismodegib compared to control ± a0µm vs ± a0µm respectively p Fig a01h In bones treated with rSWT vismodegib the hypertrophic chondrocytes were significantly larger compared to vismodegib alone and similar in size as in untreated control bones ± a0µm ± a0µm and ± a0µm respectively p vs vismodegib p ns vs control Fig a01h Immunostaining for Gli Fig a01i and Ihh Fig a01j did not show any significant effects of vismodegib andor rSWTShock wave treatment prevents bone growth retardation caused by GANT61 To study the effects of continuous inhibition of Hhactivity fetal rat metatarsal bones were treated with the Gli1 antagonist GANT61 from day of culture until the termination of the experiment on day Already on day GANT61 induced a pronounced suppression of the bone growth ± ± p vs control which remained at endpoint on day ± ± p vs control Fig a02aNext we asked if a single exposure to rSWT can rescue from growth retardation caused by continuous Hhinhibition induced by GANT61 Indeed metatarsal bones exposed to rSWT on day were rescued from GANT61induced growth retardation already from day of culture ± ± p vs GANT61 alone which remained until endpoint ± ± p vs GANT61 alone Fig a02aGrowth plate morphology Compared to control in the bones continuously treated with GANT61 growth plate morphology was found to be disturbed with disanized chondrocyte columns on day Fig a02b c Bones treated with rSWT alone showed anized growth plate morphology Fig a02d The growth rescuing Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ceffect of rSWT in GANT61 treated bones was accompanied by a normalization of growth plate morphology including anized chondrocyte columns Fig a02eHistomorphometric analyses showed no significant differences in the height of the R P zone between GANT61 treated bones with or without rSWT ± a0µm ± a0µm p ns Fig a02f There was an increased size of hypertrophic chondrocytes in rSWT GANT61 group when compared to GANT61 alone ± a0µm ± a0µm p Fig a02g Furthermore the number of proliferative chondrocytes tended to be lower in GANT61 treated bones when compared to control ± cells ± cells p ns Fig a02h The number of proliferative PCNA positive cells tended to be higher in the rSWT GANT61 group compared to GANT61 alone ± cells ± cells p ns Fig a02h Immunoexpression of Gli1 tended to be reduced in GANT61 treated bones p ns vs control whereas bones treated with rSWT GANT61 tended to have higher Gli1 expression compared to GANT61 alone p ns Fig a02iDiscussionWe aimed to investigate the potential for locally applied rSWT to prevent bone growth impairment caused by the hedgehog inhibitor vismodegib a therapeutic investigational drug using a wellestablished model of ex a0vivo cultured fetal rat metatarsal bones Herein we report that a single session of rSWT partially prevented growth retardation caused by both transient and continuous Hhinhibition induced by vismodegib and GANT61 respectively The growth rescuing effects by rSWT were accompanied by preservation of growth plate morphology disrupted by the Hhinhibitors Altogether our data suggest that rSWT has the potential to noninvasively protect bones from growth retardation caused by vismodegibBone growth is majorly dependent on the preservation of a unique anization of chondrocytes in the growth plate17 Recent reports have demonstrated that long term exposure to vismodegib the first Hhantagonist approved in the US by FDA led to permanent growth impairment in children with medulloblastoma78 To date no successful strategy that targets tumor cells with no adverse effect on longitudinal bone growth has been described Previous reports have shown that rSWT a treatment modality that is already used in children for musculoskeletal indications18 can stimulate longitudinal bone growth locally in exvivo cultured metatarsal bones even in the absence of any systemic growth factors10 Proinflammatory cytokines are also known to impair bone growth19 and interestingly shockwave treatment has been shown to reduce inflammation and apoptosis while stimulating the regeneration of various tissues2021 These findings encouraged us to expand this knowledge and further investigate the potential for rSWT to prevent bone growth impairment caused by HhinhibitorsVismodegib at a0nM concentration has shown to impair bone growth in exvivo cultured metatarsal bones22 and decrease proliferation of the precursors of a0cerebellar granule neurons23 while in a0vivo studies in a model of medulloblastoma have also shown that vismodegib inhibits Gli1 at a IC50 of a0nM24 a similar range of concentration as used in the present study In young mice transient inhibition of the Hh pathway has been reported to cause permanent defects in bone and growth plate structure9 Similar to the previous in a0vivo observations in young mice9 our histomorphometric growth plate data suggest that partial loss of Hhactivity may result in the breakdown of chondrocyte columnar anization and reduced size of hypertrophic chondrocytes Also the disrupted growth plate ultrastructure caused by Hhinhibition explains the observed growth deficit in our study model system Besides undesired apoptosis of stemlike cells within the growth plate is another wellknown contributing factor linked to growth retardation caused by anticancer drugs2526Our key finding is that a single administration of rSWT not only prevented bone growth retardation caused by transient exposure to the Hhinhibitor vismodegib but also rescued bone growth under a condition of continuous Hhinhibition induced by another Hhinhibitor GANT61 Furthermore rSWT also improved growth velocity and restored growth plate morphology in bones exposed to vismodegib or GANT61 Thus our findings highlight the potential for shock wave technology to be developed as a new and safe treatment strategy to minimize deleterious effects of Hhinhibitors selectively in the growth plates of treated childrenHedgehog signaling drives chondrocyte proliferation and hypertrophy in the growth plate cartilage6 From in a0vitro studies we know that Hhinhibitors decrease the expression of Gli1 and induce cell cycle arrest in prostate cancer cells27 Despite rSWT rescued bones from Hhinhibitor impaired bone growth we did not see significant alterations in the expression of Gli1 and Ihh suggesting a crosstalk between hedgehog signaling and other pathways28 We speculate that the bone rescuing effect of rSWT is more evident if there is any ongoing disturbance within growth plate chondrocytes Indeed it was interesting to note that despite continuous exposure to a Hhinhibitor a single session of rSWT could partially rescue the bone growthOur study has several limitations Firstly the bone growth rescuing effects of rSWT were documented in an exvivo bone culture model and we do not know if this will be applicable under in a0vivo conditions Nevertheless in a0vivo studies in rats or mice are of limited value when it comes to exploring the potential for rSWT to rescue from vismodegib induced bone growth impairment as their growth plates do not normally fuse29 Secondly we only applied a single dose of rSWT while multiple sessions could potentially be even more efficient when it comes to preventing growth impairment caused by Hhinhibitors Thirdly the concentration of vismodegib used in the present study is different from the plasma concentration a0µM achieved in children30 Nevertheless mimicking a gradual decline in bone growth in order to test the rescuing effect of rSWT is more important in our experimental setting We therefore claim a protective effect and not a clinical effect which will require more rigorous testing Consequently our proof of concept finding s up a window of opportunity to explore the potential for locally applied rSWT to prevent bone growth impairment caused by vismodegib as it may not be possible to extrapolate the doses used for preclinical studies to a clinical setting3132In summary we here present a novel treatment strategy based on clinically used rSWT to locally prevent bone growth impairment caused by vismodegib a promising anticancer drug used in children with medulloblastoma Scientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A hypothetical schematic diagram showing how extracorporeal radial shockwave treatment rSWT may be administered to lowhigh growth velocity areas a0encircled selectively to restore vismodegibinduced bone growth impairment in children with medulloblastomaFig a0 Before any clinical studies our promising ex a0vivo findings need to be validated in an in a0vivo animal model like the rabbit where growth plate fusion normally occurs just like in humansMethodsThe experiments were approved by the local institutional review board Min No and the institutional animal ethics committee Min No at Christian Medical College Vellore Animal care compiled with the Guide for the Care and Use of Laboratory Animals A a0radial shock wave machine from Radialspec Medispec Gaithersburg MD USA was used for the studyBone an culture system Metatarsal bones were microdissected from the hind limbs of Sprague Dawley rat fetuses sacrificed on day of gestation Ex a0vivo cultures were performed as previously reported33 Briefly each bone was transferred to a 24well plate and cultured in medium containing DMEMF12 a0mM betaglycerophosphate ascorbic acid a0µgml and gentamycin The medium was replenished every two days Figure a0 shows the experimental overviewExposure to Vismodegib Vismodegib was purchased from Selleck Chemicals Houston Texas USA The experimental setup consisted of four groups Metatarsal bones were cultured for a0days and were either a treated with vismodegib a0nM n from day to b a single exposure to rSWT impulses a0Hz a0mJ n on day c vismodegib rSWT n or d were left untreated control n The bone length was measured on days and Exposure to GANT61 GANT61 was purchased from Sigma Aldrich St Louis Missouri USA Metatarsal bones were treated with the a Hhinhibitor GANT61 a0µM from day to n b a single exposure Scientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Experimental overview Middlethree metatarsals from the hind limb of embryonic rat at a 1920th day of gestation were dissected and b cultured exvivo c Bones were either exposed to vismodegibGANT61 a single session of highenergy radial shock wave treatment rSWT vismodegibGANT61 with rSWT or left untreated During this time d total bone length measurements at different intervals are performedto rSWT on day impulses a0Hz a0mJ n c GANT61 rSWT or d were left untreated control n The bone length was measured on days and Bone length measurement Digital images were captured for bone length measurements using an inverted microscope Leica Microsystems All measurements were performed by one of the investigators blinded to the nature of the group using an inbuilt measurement tool Bone growth is expressed as percent bone length increase from day Bowed bones were measured in two parts added togetherQuantitative histology and immunostaining After termination of the culture the metatarsal bones were fixed with paraformaldehyde embedded in paraffin and fivemicrometer sections were cut along the longitudinal axis proximal to distal followed by staining with SafraninO and Alcian blue The microscopic description of the growth plate morphology included an assessment of the anization of the chondrocyte column Histomorphometric analysis was performed to measure the height of the restingproliferating zone at five different regions of the growth plate and the size of hypertrophic chondrocytes Hypertrophic cells were defined by a height along the longitudinal axis greater than a0µm Eight hypertrophic chondrocytes from the proximal and distal growth plate were measuredImmunostaining was performed as previously described34 Antigen retrieval was performed in citrate buffer at ° Celsius and endogenous peroxidase activity was quenched with H2O2 in methanol for a0min followed by a wash with PBS For immunostaining sections were blocked with bovine serum albumin for a0h incubated with primary antibodies dilution Gli1 Abcam Cambridge MA USA and Ihh mouse monoclonal antibody Santa Cruz Biotechnology Dallas TX USA overnight at ° Celcius After incubation for a0h with secondary polyclonal antimouse or antirabbit biotinylated antibody DakoCytomation Glostrup Denmark dilution sections were incubated with ABC solution and developed with diaminobenzidine Sections were counterstained with Alcian blue Nonimmune immunoglobin G IgG of the same species as the primary antibodies were used as negative controls Three to five bones per group were analyzed To quantify immunostaining the ImageJ software National Institutes of Mental Health Bethesda MD USA was used and the percentage of DAB positivity was calculated digitally using a plugin IHC profilerStatistical analysis All statistics were carried out using GraphPad Prism GraphPad Software Inc La Jolla CA USA Data were summarized using means ± SD for the bone length measurements and histomorphometric assessments A twoway ANOVA with Dunnett multiple comparisons test35 was performed to examine the change in bone length in terms of treatment and days Pairwise comparisons were done corrected for the alpha levels Margin plots with SD were presented to visualize the change in bone length KruskalWallis and ManWhitney U test were performed when the data were not normally disturbed A p value of was considered to indicate a significant differenceData availabilityAll data generated or analysed during this study are included in this manuscriptScientific RepoRtS 101038s41598020699040Vol1234567890wwwnaturecomscientificreports 0cReceived March Accepted July References Gerber N et al Recent developments and current concepts in medulloblastoma Cancer Treat Rev Gorlin R J Neurocutaneous Disorders Phakomatoses and Hamartoneoplastic Syndromes Springer Berlin Gajjar A J Robinson G W Medulloblastomatranslating discoveries from the bench to the bedside Nat Rev Clin Oncol Kool M et al Molecular subgroups of medulloblastoma an international metaanalysis of transcriptome genetic aberrations and clinical data of WNT SHH Group and Group medulloblastomas Acta Neuropathol Kieran M W Targeted treatment for sonic hedgehogdependent medulloblastoma Neurooncology Ohba S Hedgehog signaling in endochondral ossification J Dev Biol Robinson G W et al Irreversible growth plate fusions in children with medulloblastoma treated with a targeted hedgehog pathway inhibitor Oncotarget Kieran M W et al Phase I study of oral sonidegib LDE225 in pediatric brain and solid tumors and a phase II study in children and adults with relapsed medulloblastoma 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Drug Development Springer Berlin ReaganShaw S Nihal M Ahmad N Dose translation from animal to human studies revisited FASEB J Chagin A S Chrysis D Takigawa M Ritzen E Svendahl L Locally produced estrogen promotes fetal rat metatarsal bone growth an effect mediated through increased chondrocyte proliferation and decreased apoptosis J Endocrinol Bov©e J V van den Broek L J CletonJansen AM Hogendoorn P C Upregulation of PTHrP and Bcl2 expression characterizes the progression of osteochondroma towards peripheral chondrosarcoma and is a late event in central chondrosarcoma Lab Investig Vincent K et al Aging of mouse intervertebral disc and association with back pain Bone AcknowledgementSR would like to personally acknowledge Dr Sumith K Mathew Assistant Professor Clinical Pharmacology Christian Medical College for his input on calculating for clinically relevant dose This study was supported by internal grants received from Christian Medical College Vellore Centre for Stem Cell Research Swedish Research Council Swedish Childhood Cancer Foundation HKH Kronprinsessan Lovisas f¶rening Ake Wibergs Stiftelse and Karolinska Institutet Sweden access funding provided by Karolinska InstituteAuthor contributionsSR LS VM FZ designed the study SR carried out data collection and statistical analysis SR LS VM FZ analysed data SR wrote the initial draft of the manuscript FZ VM and LS contributed to the revision of the manuscript All authors contributed to the interpretation of resultsScientific RepoRtS 101038s41598020699040Vol0123456789wwwnaturecomscientificreports 0ccompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to SRReprints and permissions information is available at wwwnaturecomreprintsPublishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Access This article is licensed under a Creative Commons 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the periparturient period is one of the most challenging periods in dairy cows and encompasses the a0wk prior to and a0wk after parturition the nutrient requirements of dairy cows change greatly during this time largely due to the exponential growth of the gravid uterus and fetus followed by the demands of lactation nrc inflammation oxidative stress and adipose tissue mobilization lead to a reduction in dry matter intake dmi during the periparturient period this reduction in dmi leads to a negative nutrient balance nnb with a shortfall in the nutrient availability for the cow and fetus ingvartsen and andersen additionally this reduction in dmi also increases the risk of metabolic ketosis fatty liver milk fever and immunerelated disorders the risk of these diseases poor reproduction and low efficiency is greatly impacted by the degree and length of time during which these systems metabolism and immune response remain out of balance loor et a0al 2013a much of the research over the last decade have examined these biological interactions to identify the mechanisms behind the metabolic physiologic and immune adaptations that occur during the periparturient period loor et a0al 2013a 2013b roche et a0al bradford et a0al it is now known that nutrients such as amino acids aa serve functional roles outside of their use as building blocks for proteins and have immunomodulatory properties and interact through common biochemical pathways eg 1carbon metabolism figure a0 this concept has been well explored in nonruminant species li et a0 al sikalidis with nutritional management during the periparturient period continuing to be an active area of research it is important to develop a system understanding the potential immunometabolic role that dietary aa may play during this period thus the objective of this review is to provide an overview of physiological adaptations during the periparturient period the immune system and methods to assess immune function and oxidative stress this will be followed by a more specific discussion of the immunometabolic roles of specific aa and their potential effects in dairy cow during the periparturient period the potential effects of enhanced aa supply during the preweaning period will also be discussed brieflybiological adaptations in the transition cowa brief overviewduring the nnb associated with the periparturient period biological mechanisms coordinate the mobilization of body reserves in order to support fetal growth and milk production bauman and currie insulin concentrations are reduced and the response of hormonesensitive lipase in adipose tissue eg low insulin high growth hormone and catecholamines or high glucocorticoid concentrations is greater to facilitate lipid mobilization this periparturient period is also characterized by a state of inflammation encompassing an increase in hepatic production of positive acutephase proteins app such as haptoglobin and serum amyloid a a0saa and a decrease in the production ingvartsen the authors published by oxford university press on behalf of the american society of animal sciencethis 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 cited for commercial reuse please contact spermissionsoupcoms175 0cs176 of animal science vol no suppl abbreviationsaa akt app asct2 bcaa bcat bcka bhmt bmec bsa cbs csad cth dmi gator1 gcl gclc gpx gsh gsr gss il inos kegg klh lps mpo mtor mtorc1 mtr nfe2l2 nfκb nnb nos pc pmn pmnl pmtor rns rom ros rpm saa sahh sam samtor sell shmt slc1a1 slc1a5 sod stat tag th tlr tnfα vldl amino acidsprotein kinase bacutephase proteinsalanineserinecysteine transporter branchedchain amino acidsbranchedchain amino transferasesbranchedchain αketoacidsbetaine homocysteine methyltransferasebovine mammary epithelial cellsbovine serum albumincystathionine synthasecysteine sulfinic acid decarboxylasecystathionine gammalyasedry matter intakegtpaseactivating protein activity towards rags glutamate cysteine ligaseglutamate cysteine ligase catalyticglutathione peroxidaseglutathioneglutathione reductaseglutathione synthaseinterleukininducible noskyoto encyclopedia of genes and genomeskeyhole limpet hemocyaninlipopolysaccharidemyeloperoxidasemechanistic target of rapamycinmtor complex 5methyltetrahydrofolatehomocysteine methyltransferasenuclear factor erythroid 2like2nuclear factor kappa bnegative nutrient balancenitric oxide synthasephosphatidylcholinepolymorphonuclear neutrophilspolymorphonuclear leukocytesphosphorylated mtorreactive nitrogen speciesreactive oxygen metabolitesreactive oxygen speciesrumenprotected metserum amyloid asadenosyl homocysteine hydrolasesadenosyl methioninesadenosylmethionine sensor upstream of mtorlselectinserine hydoxymethyltransferasesolute carrier family member solute carrier family member superoxide dismutasesignal transducer and activator of transcriptiontriacyglycerolthelpertolllike receptorstumor necrosis factorαverylowdensity lipoproteinsof negative app such as albumin bertoni et a0 al it has been well established that these responses are mediated by the proinflammatory cytokines interleukin il6 il1 and tumor necrosis factorα tnfα kindt et a0al additionally oxidative stress also occurs during this period and is driven by the imbalance between the production of reactive oxygen metabolites rom reactive nitrogen species rns and the neutralizing capacity of antioxidant mechanisms in tissues and blood some of the wellestablished cellular antioxidants include glutathione gsh taurine superoxide dismutase sod and vitamins a a0and e bernabucci et a0al when oxidative stress overwhelms cellular antioxidant capacity rom induce an inflammatory response that is controlled via changes in mrna abundance of transcription regulators eg signal transducer and activator of transcription [stat3] nuclear factorkappa b [nfκb] the increase in oxidative stress and inflammation during this period is also negatively associated with a reduction in liver functionality and measurement of app can provide a useful tool to assess liver function as well as inflammation bertoni and trevisi during the periparturient period aa metabolism is also altered with moderate carcass protein losses reported even when animals are fed to their predicted metabolizable protein requirements bell et a0 al additionally circulating aa concentrations change dramatically with favorable circulating profiles of many aa not being restored until d postpartum zhou et a0al 2016b furthermore the total concentration of aa in plasma reaches a nadir around day postpartum zhou et a0al 2016b which corresponds to a peak in total disease incidence during early lactation ingvartsen this decrease in circulating aa is likely associated with the increased use of aa for gluconeogenesis as well as for hepatic production of app thus it is important to investigate how supplemental aa during the periparturient period may modulate metabolism and immune responses to promote production and reduce susceptibility to diseasegeneral overview of the immune a0systemthe immune system consists of both the adaptive and innate immune responses which are linked together through signaling molecules such as cytokines the innate immune system is the first line of defense and includes physical barriers such as epithelial cell layers that express tight cell junctions and the mucus layer of the respiratory gastrointestinal and genitourinary tracts chaplin cells involved in the innate immune response include macrophages polymorphonuclear neutrophils pmn dendritic cells mast cells eosinophils natural killer cells and natural killer t cells turvey and broide the focus of the present review is on the effects of aa on phagocytic cells that is cells that engulf and kill such as macrophages and pmn macrophages not only phagocytose invading pathogens but also produce cytokines such as ils and tnfα which initiate innate and adaptive immune responses and recruit pmn to the site of infection chaplin the adaptive immune response consists of t lymphocytes b lymphocytes and humoral factors marshall et a0 al there are two types of t lymphocytes cytotoxic t cells cd8 cells and thelper th cells cd4 cells marshall et a0al cytotoxic t cells detect and eliminate infected cells while th cells produce ils and interferonγ ingvartsen and moyes the b lymphocytes are cells that produce antibodies that bind to antigens on the surface of pathogens to mark them for destruction marshall et a0al 0ccoleman et a0al s177aa immune function and oxidative a0stressduring the periparturient period the metabolic status is associated with the inflammatory regulation of peripheral blood mononuclear cells with a more pronounced inflammatory response in those cells during the nnb immediately postpartum agrawal et a0 al mann et a0 al this period is also associated with altered signaling of nutrientsensing kinases in immune cells such as the protein kinase b akt and mechanistic target of rapamycin mtor pathway which may modulate cytokine production mann et a0al aa can directly and indirectly alter the immune system besides being used in energy metabolism reactions and the synthesis of proteins aa are critical for the synthesis of other functional molecules such as the antioxidants gsh and taurine no histamine and hydrogen peroxide li et a0 al thus this section of the review will focus on the role that aa play in modulating immune function and oxidative stress in dairy cows during the periparturient period a a0special focus will be placed on aa involved in 1carbon metabolism as this represents an interconnected route through which aa could impact molecular events such as epigenetic regulation protein synthesis via mtor energy metabolism and antioxidant synthesis a a0summary of studies investigating the effects of aa on immune function and oxidative stress in dairy cows is provided in table a0 a a0summary model of how aa might alter immune function and oxidant status is depicted in figure a0methioninemethionine not only is essential for protein synthesis but also serves as a functional nutrient that is needed for the production of the antioxidants gsh and taurine atmaca and provision of methyl groups finkelstein these features are exemplified by the central role of met in 1carbon metabolism figure a0 in these pathways met is used to synthesize sadenosyl methionine sam which can be used to methylate dna and to support phosphatidylcholine pc and carnitine synthesis for fatty acid metabolism vance et a0al during the periparturient period triacyglycerol tag accumulate in the liver leading to mitochondrial dysfunction inflammation and reduced liver function li et a0al du et a0al pc is the main phospholipid component of verylowdensity lipoproteins vldl vance thus enhancing pc synthesis through greater met supply may help improve vldl synthesis and reduce hepatic tag accumulationas part of 1carbon metabolism homocysteine can be remethylated to met using betaine or folate as methyl donors bhmt and via betaine homocysteine methyltransferase 5methyltetrahydrofolatehomocysteine methyltransferase mtr respectively homocysteine is also used to synthesize cystathionine via cystathionine synthase cbs in the first reaction of the transsulfuration pathway banerjee et a0 al cystathionine is used to make cys which is utilized to synthesize the antioxidants taurine or gsh cbs is allosterically figure interrelationships among components of the 1carbon metabolism pathway methionine cycle folate cycle and transsulfuration pathway 5mthf 5methyltetrahydrofolate amd1 adenosylmethionine decarboxylase arg arginase b12 cobalamin b2 riboflavin b6 pyridoxal ²phosphate cbs cystathionine betasynthase dcsam decarboxylated sadenosylmethionine dhfr dihydrofolate reductase dnmt dna methyltransferase dtmp thymidine monophosphate dump deoxyuridine monophosphate ftcd formimidoyltransferase cyclodeaminase gnmt glycine nmethyltransferase mat methionine adenosyltransferase mthfr methylenetetrahydrofolate reductase mtrr 5methyltetrahydrofolatehomocysteine methyltransferase reductase odc1 ornithine decarboxylase pemt phosphatidylethanolamine nmethyltransferase sahh sadenosylhomocysteine hydrolase shmt serine hydroxymethyltransferase sms spermine synthase srm spermidine synthase tdh threonine dehydrogenase thf tetrahydrofolate tyms thymidylate synthetase 0cs178 of animal science vol no suppl activated by sam banerjee et a0 al therefore enhanced sam production with increased met supply can help enhance the flux of the transsulfuration pathway increasing taurine and gsh production to help reduce oxidative a0stressin dairy cattle low levels of serum met postpartum are associated with severe hepatic lipidosis shibano and kawamura and other than his met is the only aa for which net uptake by the liver increased pre and postpartum larsen and kristensen therefore enhancing postruminal met supply during the periparturient period is of interest to increase circulating concentrations of met for its functional roles in the body the work from dalbach et a0 al demonstrated that rumenprotected met rpm can be used to increase serum concentrations of met in the first 2wk postpartum which will enhance the availability of met for protein synthesis and metabolism via the 1carbon pathways in terms of production supplementation of met during the peripartal period concomitantly increases milk yield milk protein and milk fat soon after calving ordway et a0 al osorio et a0 al these responses are in large part driven by enhancing met availability and by the additional flux of met through the met cycle in the liver which consequently increases the production of downstream compounds such as sam pc gsh and taurine additionally work feeding rpm during the periparturient period has detected positive responses in maintaining consistent rates of dmi prepartum last d and faster and greater rates of dmi during the first to d after calving osorio et a0al zhou et a0 al 2016c batistel et a0 al the same milk production response was also observed when met was supplemented postpartum as the isopropyl ester of 2hydroxy4methylthiobutanoic acid stpierre and sylvester as described earlier the transient inflammatorylike status around parturition appears to be a normal aspect of the adaptations to lactation as cows approach parturition those with greater but still subclinical concentrations of circulating cytokines have greater inflammation and oxidative stress and lower liver function along with lower milk yield and lower postpartum dmi bertoni et a0 al in addition to their function in the immune system cytokines interferons and tnfα also elicit pathophysiological effects leading to sickness table summary of studies in dairy cows investigating the effects of supplemental aa on immune function oxidative stress and inflammation tissuecellstreatmentmain outcomeplasmarpm for d prepartum and d postpartumimprovements in plasma biomarkers indicating reduced oxidative stress and inflammation and enhanced liver function increased neutrophil phagocytosis and oxidative burstreferencebatistel et a0al plasmaabomasal infusion of glu for first infusions of gln increased the abundance of cd4 tcells on day doepel et a0al d postpartumpostpartum and increased the abundance of monocytesmammary rpm for d prepartum and d methionine supply upregulated expression of genes involved in han et a0al glandplasmapostpartumantioxidant metabolism and increased activation of nfe2l2intravenous infusions of gln for glutamine infusion decreased plasma haptoglobin and increased jafari et a0al d postcalvinglpsbinding protein and saa subcutaneous rpm for d prepartum and d enhanced met supply increased mrna and protein abundance of liang et a0al 2019aadiposepmnlpostpartumenzymes related to gsh metabolismincubation with met andor supplemental met coupled with adequate choline enhanced gene lopreiato et a0al cholineexpression of pathogen recognition mechanisms methionine ameliorated the increased inflammation and oxidative stress observed when cells were incubated without choline plasma and protected gln for d postpartum increased total blood protein and albumin decreased plasma nemati et a0al milkwhole bloodrpm for d prepartum and d aspartate aminotransferase and milk somatic cell countincreased whole blood neutrophil phagocytosis on day osorio et a0al 2013apostpartumpostpartum with supplemental metliverrpm for d prepartum and d methionine supply altered flux through 1carbon metabolism via osorio et a0al 2014aliver and plasmaplasmapostpartumchanges in mrna to support antioxidant and met synthesisrpm for d prepartum and d methionine increased liver gsh and decreased concentrations of osorio et a0al 2014bpostpartum plasma biomarkers of inflammationrpm for d during midlactation increased proliferative ability of peripheral blood t lymphocytes soder and holden with supplemental metplasmarpm for d prepartum and increased antioxidant capacity of plasma and cd4cd8 t sun et a0al postpartumlymphocyte ratio with met supplywhole bloodrpm for d prepartum and d methionine damped the hyperresponse of il1 during an lps vailatiriboni et a0al plasmajugular infusion of arg and lps in arginine alleviated lpstriggered inflammation by decreasing il6 zhao et a0al 2018apostpartumchallenge through improvements in oxidative stressserumjugular infusion of arg and lps in infusion of arg promoted antioxidant mechanisms during lpszhao et a0al 2018bmidlactation cowsinducible nos and lpsbinding proteinmidlactation cowsrpm for d prepartum and d postpartumrpm for d prepartum and d triggered inflammation by increasing total antioxidant capacity and gsh peroxidase activity and decreasing malondialdehyde increased hepatic gsh and improved plasma biomarkers of liver function and inflammation with met neutrophil phagocytosis capacity and oxidative burst were also increased with metenhanced met supply increased mrna expression of genes zhou et a0al 2016azhou et a0al 2017bpostpartumassociated with pc and antioxidant synthesisrpm for d prepartum and d decreased expression of genes related to inflammation and zhou et a0al 2018bliver and plasmaliver pmnlpostpartumoxidative stress 0ccoleman et a0al s179figure the theoretical model of cellular aa utilization amp adenosine monophosphate atp adenosine triphosphate ctp cytidine triphosphate dttp deoxythymidine triphosphate gmp guanosine monophosphate imp inosine monophosphate no nitric oxide r5p ribose 5phosphate tca cycle tricarboxylic acid cycle ump uridine monophosphate utp uridine5²triphosphatebehaviors whose primary manifestation is satiety larson and dunn an example of this behavior in dairy cows is the reduction in dmi around calving in mice these cytokines have been shown to reduce meal size and duration as well as decrease meal frequency and prolong intermeal intervals platasalamán furthermore cytokines directly affect the hypothalamus il1 and ifn act directly and specifically on the glucosesensitive neurons in the brain satiety and hunger sites platasalamán in addition to increases in dmi and milk production rpm supplementation during the periparturient period has been associated with positive health responses across four studies osorio et a0al 2014b sun et a0al zhou et a0al 2016a batistel et a0 al there have been consistent improvements in the concentrations of plasma biomarkers of inflammation where il1 and haptoglobin have decreased and albumin has increased summarized in table a0 improvements in biomarkers of oxidative stress have also been observed with enhanced met supply during the periparturient period in the study by batistel et a0al plasma concentrations of ferricreducing antioxidant power carotene tocopherol and total and reduced gsh were increased with rpm while rom were lower sun et a0al also observed an improvement in blood antioxidant status with rpm increasing total antioxidant capacity glutathione peroxidase gpx activity and vitamin e a0a a0similar effect was observed in liver tissue by osorio et a0 al 2014b where cows fed rpm had greater hepatic concentrations of total and reduced a0gshfrom a mechanistic standpoint changes in the mrna abundance of sadenosyl homocysteine hydrolase sahh mtr sod1 glutamate cysteine ligase catalytic gclc subunit and dna methyltransferase 3a suggested alterations in flux through 1carbon metabolism osorio et a0al 2014a importantly sahh the enzyme that makes homocysteine from sadenosylhomocysteine was upregulated with met which would support a supply of homocysteine to be used for antioxidant and met synthesis furthermore in the study by zhou et a0al 2016a greater met supply compared with rumenprotected choline increased antioxidant concentration in liver tissue despite a lower concentration of pc those responses were due to the greater abundance of phosphatidylethanolamine methyltransferase the enzyme that utilizes sam and phosphatidylethanolamine to make pc and cbs zhou et a0al 2017b additionally enhanced met supply during the periparturient period was observed to increase mrna and protein abundance of enzymes related to gsh metabolism in subcutaneous adipose tissue suggesting greater activation of those pathways liang et a0al 2019a a a0greater dietary supply of choline did not change the mrna abundance of bhmt and mtr in cows zhou et a0al 2017ba positive effect of met supplementation on mammary gland antioxidant mechanisms was observed by han et a0 al mrna abundance of gpx1 gclc glutamate cysteine ligase gcl modifier subunit malic enzyme ferrochelatase and ferritin heavy chain and genes involved in gsh and iron metabolism were upregulated protein abundance of phosphorylated nuclear factor erythroid 2like2 nfe2l2total nfe2l2 was also increased han et a0al nfe2l2is a regulator of transcription of antioxidant genes hence an increase in phosphorylated nfe2l2 suggested greater activation of antioxidant systems and is likely one of the mechanisms behind the changes in mrna abundance lastly across studies there has also been a consistent improvement in the concentrations of plasma biomarkers of liver function such as increases in paraoxonase and cholesterol with rpm osorio et a0al 2014b zhou et a0al 2016a batistel et a0 al which is likely linked to the reduction in inflammation and oxidative stress thus the consistent changes across studies in metabolites and plasma biomarkers as well as mrna abundance across tissues indicate that enhanced met supply during the periparturient period reduces oxidative stress and inflammation however more work is needed to verify the exact mechanisms behind the observed changes 0cs180 of animal science vol no suppl enhanced postruminal supply in the form of rpm during the periparturient period has been associated with improvements in immune cell function when rpm was provided for d prepartum and d postpartum whole blood neutrophil phagocytosis was increased compared with control cows at d postpartum osorio et a0 al in the study by zhou et a0 al 2016c rpm supplementation from day prepartum to day postpartum increased neutrophil phagocytosis capacity and oxidative burst activity zhou et a0 al 2016a this same improvement in neutrophil immune function was observed when rpm was supplied from day prepartum to day postpartum batistel et a0 al furthermore an increase in the proliferative ability of peripheral blood t lymphocytes was observed when rpm was supplemented to midlactation cows soder and holden zhou et a0 al 2018b isolated polymorphonuclear leukocytes pmnl and observed lower abundance of genes related to inflammation il1b tlr2 nfκb and stat3 and oxidative stress cbs gpx1 glutathione synthase [gss] and sod2 as well as an increase in plasma taurine with supplemental met suggesting a better redox and inflammatory status of those cells additionally those same cows were used for an ex vivo whole blood challenge with lipopolysaccharide lps to further investigate immune cell responses during this challenge a table summary of additional beneficial effects of feeding rpm during the transition period and early lactationbiomarkerresponse12sitebiological functionmetabolism carnitine cholesterolonecarbon metabolism cystathionine betasynthase activity cystathionine cystine homocysteineinflammation il1beta haptoglobin albumin oxidative stress romliveroxidation of fatty acidsplasmalipoprotein metabolismliverplasmaplasmaplasmaantioxidant synthesish2s biosynthesis redox statusredox statusmethylation reactionsplasmaproinflammatory cytokineplasmainflammation signalplasmaacutephase responseplasmaperoxides gsh taurine antioxidant capacity paraoxonasesuperoxide ohradicalsliver blood antioxidantantioxidantplasmaplasmatotal antioxidants in bloodplasmaantioxidant enzyme beneficial increase beneficial decrease no change in concentration2relative to a control or rumenprotected choline supplemented diet osorio et a0al 2014b zhou et a0al 2016a sun et a0al batistel et a0al vailatiriboni et a0al hyperresponse in il1 was observed around parturition which likely arose from oxidative stress vailatiriboni et a0 al however rpm supplementation dampened this hyperresponse likely through improvements in oxidative stress vailatiriboni et a0al the recent work by lopreiato et a0 al investigated the effects of incubating bovine pmnl with met andor choline and observed that supplemental met coupled with adequate choline enhanced gene expression of tlr2 and lselectin sell which are pathogen recognition mechanisms in the same experiment cells incubated without choline had greater mrna abundance of il1b il6 il10 and myeloperoxidase mpo glutathione reductase gsr gss cystathionine gammalyase cth and cysteine sulfinic acid decarboxylase csad indicating greater inflammation and oxidative stress this effect however was ameliorated by supplementing additional met lopreiato et a0 al thus the increased dmi and milk production observed when feeding rpm can be partly explained by a reduction in inflammation as it directly at the hepatic level and by dampening the immune cell overresponse and indirectly reducing oxidative stress decreases circulating proinflammatory cytokinesenhanced met supply during the periparturient period has also been associated with changes in mtor signaling mtor is a serinethreonine kinase that plays a central role in integrating environmental cues from growth factors nutrients particularly aa and energy powell et a0al in dairy cattle mtor has traditionally been studied in the context of its role in regulating protein synthesis however work from humans and nonruminant species has indicated that mtor is an important regulator of immune responses powell et a0 al jones and pearce when activated by aa mtor directs an activation of anabolic metabolism which allows growth proliferation and development this makes the activation of mtor in immune cells particularly important for maintaining proliferation and without proper activation cells may enter periods of growth arrest jones and pearce methionine specifically may interact with mtor via the production of sam specifically sam can bind to sadenosylmethionine sensor upstream of mtor samtor a protein that inhibits mtor complex mtorc1 by interacting with gtpaseactivating protein activity towards rags gator1 gu et a0al when sam binds to samtor it inhibits the association of samtor and gator1 allowing mtorc1 to be activated gu et a0al to our knowledge there is only one study investigating the expression of mtor signaling proteins in immune cells in dairy cattle and work is also lacking in nonruminant species in periparturient cows the activation of aktmtor signaling in immune cells was reduced postpartum compared with prepartum mann et a0al importantly agrawal et a0al also identified the expression of aa transporters and the kyoto encyclopedia of genes and genomes kegg pathways related to 1carbon metabolism and mtor in pmnl from peripartal cows a a0 list of these transporters and kegg pathways related to aa and 1carbon metabolism are summarized in table a0 together these studies support the potential importance of aa for nutrient signaling in immune a0cellsrecent work indicating that enhanced met supply activates mtor signaling in the mammary gland supports its role in enhancing protein synthesis for example in vitro enhancing met supply to immortalized bovine mammary epithelial cells bmec by varying the ratio of lys to met increased the concentration and utilization of essential aa particularly branchedchain aa bcaa dong et a0 al this change 0cwas potentially mediated by alterations in aa transporters controlled by mtor dong et a0al other studies with bmec have also revealed a potential for greater mtor activation when met supply is enhanced nan et a0al sala ma et a0al in vivo the effects of supplemental met during the periparturient period on mtor signaling were explored using cows from the study by batistel et a0al in the mammary gland cows receiving rpm had lower protein abundance of total mtor and phosphorylated mtor pmtor compared with control cows on day postpartum but the ratio of pmtortotal mtor was not different suggesting that there was no difference in mtor activation between treatments ma et a0 al however changes in aa transporters and insulin signaling indicated that insulin sensitivity of the mammary gland was enhanced with supplemental met ma et a0al in subcutaneous adipose tissue the mrna and protein abundance of some aa transporters and pmtor were upregulated with enhanced met supply while changes in insulin signaling and plasma glucose also indicated that met helped improve insulin sensitivity liang et a0 al 2019b thus enhancing met supply during the periparturient period may lead to mtor activation in immune cells as well as improved nutrient uptake which could help to support proliferation and development additional work in ruminants and nonruminants is needed to understand whether met modulates mtor signaling in immune cellscysteinecysteine can be synthesized endogenously from homocysteine as described earlier and is needed to synthesize gsh and taurine gsh is synthesized via two enzymes gcl and gss lushchak to synthesize taurine cysteine sulfinic acid is first synthesized from cysteine by cysteine dioxygenase and can then be utilized by csad to produce taurine park et a0al dietary cys has been explored as a way to improve health in nonruminant species and humans under a variety of conditions including type2 diabetes cardiovascular disease and liver disease to name a few yin et a0 al across these studies increased dietary cys increased the concentrati | 0 |
"The greater than 2-fold increase in affinity is indicative of multivalent binding of the peptide to the cell surface and not merely an increase in the number of peptide units. Attachment of the peptides to the CB-TE2A chelator does not affect the affinity of the peptide compared to the parental dimer of H2009.1-10mer. Interestingly the two divalent conjugates H2-(M10)2 and H2-D10 have the same affinity for H2009 cells. Thus displaying the peptide off a lysine core or the divalent CB-TE2A core does not significantly affect the specific cell binding. Of note CB-TE2A conjugates of the A20FMDV peptide have also been reported 48. However in the modification one of the carboxylate groups was consumed for the peptide conjugation which might compromise the stability of the Cu(II) complex moiety. In addition it adds an extra positive charge to the peptide conjugate when labeled with 64Cu which might contribute to the high kidney uptake. In contrast all our peptide conjugates maintain an intact CB-TE2A core to form the neutral Cu(II)-CB-TE2A complex with non-compromised stability. Towards the goal of utilizing the multivalent effect for ?v?6-integrin PET imaging we conducted non-invasive imaging evaluation studies in two SCID mouse models bearing H2009 (?v?6+) and H460 (?v?6-) tumors. The H2009 (?v?6+) tumor was clearly visualized by all four probes monovalent (64Cu-M10) and divalent (64Cu-(M10)2 64Cu-D10 and 64Cu-AcD10) at 1 h p.i. In contrast much lower tumor uptake was observed for all the probes in H460 (?v?6-) tumor while their distribution profiles in the other tissue were almost identical to those observed in the H2009 tumor-bearing mice. This result demonstrates the ?v?6 binding specificity of the H2009.1-10mer peptide. However though the H2009 tumor was visualized by 64Cu-M10 at 1 h p.i. its imaging contrast became barely visible at 4 and 24 h p.i. The low tumor contrast of 64Cu-M10 is likely caused by its suboptimal in vivo kinetics receptor binding and stability. These drawbacks can be potentially resolved or mitigated by multimeric presentation of the peptide on a scaffold. Indeed the divalent probes (64Cu-(M10)2 64Cu-D10 and 64Cu-AcD10) showed an approximate 3-fold tumor uptake increase as compared to 64Cu-M10 which clearly demonstrates the role of the multivalent effect in imaging signal amplification. In addition the tumor PET signal retention of the three divalent probes stayed above 0.70 %ID/g even at 24 h p.i. While the increased affinity of the dimeric probes contributes to the increased tumor accumulation increased stability and improved in vivo kinetics are likely to play a role as well. Interestingly no significant tumor uptake difference was observed for the three divalent probes out to 24 h p.i. indicating the two multivalent presentation approaches afforded virtually the same result. While this observation was somewhat to our surprise it may result from the flexible PEG spacer that has been incorporated into our probe design which provides no spatial constraints to the ligand-receptor binding. All three divalent probes showed a similar biodistribution profile in the H2009 and H460 tumor models with elevated uptake (< 3 fold) in major ans other than kidneys as compared to the monovalent one. This is likely caused by the molecular weight induced difference of in vivo kinetics. Although we observe relatively high tumor uptake compared to background accumulation in the mediastinum region we recognize the need to further increase accumulation of radiolabel in the tumor. Improved tumor uptake is anticipated to increase sensitivity of detection. One advantage of our bifunctional chelator design is that we can layer the valency moving rapidly from monomeric to octomeric peptides. As the valency increases we anticipate an increase in affinity for ?v?6 and improved in vivo kinetics 30. Additionally the design allows for facile PEGylation of both the peptide ligand and the chelator which may improve tumor uptake by increasing circulation time of the probe 49. Defined length PEG linkers are available allowing for controlled empirical testing. Rapid kidney uptake and efficient clearance is a desired feature of targeted PET imaging probes. However high kidney uptake is unwanted even for lung cancer imaging because it renders the kidneys to unnecessary radiation exposure. Given the high kidney uptake and retention observed for both 64Cu-(M10)2 and 64Cu-D10 ( ) they may not find practical applications in PET imaging of ?v?6-integrin. By immunocytochemical staining we found negligible expression of ?v?6 integrin in the kidneys of H2009 tumor bearing mice (data not shown) indicating the non-specific nature of the observed high kidney accumulation. Further as shown in the PET imaging results of 64Cu-CB-TE2A-(c(RGDyK))2 in both H2009 and H460 tumor models the CB-TE2A-based construct is not the culprit of the high kidney uptake. Therefore we reasoned that the high kidney uptake and retention resulted from the peptide itself. Two factors may play an important role - the peptide's stability and charge in vivo. As such we introduced an acetyl group to cap the N-terminus of the H2D10 conjugate which led to H2AcD10. The acetylation reduces the overall charge from +4 (H2D10) to +2 (H2AcD10) while limiting proteolysis of the peptide by N-terminal peptidases 36. As anticipated 64Cu-AcD10 showed a drastic decrease in kidney uptake and retention and the level of tumor uptake was maintained compared to its non-acetylated counterpart 64Cu-D10 (). In addition a significant uptake reduction was also observed in other non-target ans including lung and liver. The improved tumor-to-lung contrast is of great importance to the imaging detection of NSCLCs. " | 1 |
"The objective response rate (ORR) for patients with adenocarcinoma (primary endpoint) was 5% (2 partial responses; 1?sided P?=?.372 for null hypothesis [H0]: ORR ? 5%) and 6% (1 partial response) for patients with nonadenocarcinoma. Responders included: 2 of 25 EGFR mutation?positive tumors; 1 of 3 EGFR wild?type with HER2 amplification. Median progression?free survival was 12 weeks overall (n?=?66) and 18 weeks (n?=?26) for patients with EGFR mutation?positive tumors. Common treatment?related adverse events were of grade 1 or 2 severity manageable with standard supportive care and included diarrhea (grade 3 [G3] 12%) acneiform dermatitis (G3 6%) exfoliative rash (G3 3%) dry skin (G3 0%) fatigue (G3 3%) and stomatitis (G3 2%). Six patients (9%) discontinued due to treatment?related adverse events. By patient report NSCLC symptoms of dyspnea cough and pain (chest arm/shoulder) showed improvement first observed after 3 weeks on therapy. S Dacomitinib demonstrated preliminary activity and acceptable tolerability in heavily pretreated patients and may offer benefit in molecularly defined patient subsets. Cancer 2014;120:11451154. 2014 The Authors. Cancer published by Wiley Periodicals Inc. on behalf of American Cancer Society. This study investigated the efficacy and safety of dacomitinib in advanced refractory nonsmall cell lung cancer (NSCLC) selecting for patients with KRAS wild?type tumors to exclude those least likely and simultaneously enrich for those most likely to benefit from therapy. Although the observed response rate was low a number of patients experienced prolonged disease control accompanied by rapid and durable lung cancer symptom relief suggesting that dacomitinib has relevant activity against KRAS wild?type NSCLC. dacomitinib PF?00299804 nonsmall cell lung cancer erlotinib adenocarcinoma nonadenocarcinoma source-schema-version-number 2.0 component-id cncr28561 cover-date 15 April 2014 details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_NLM version:4.0.5 mode:remove_FC converted:21.05.2014 We thank all of the participating patients and their families as well as the global network of investigators research nurses study coordinators and operations staff. Medical writing support was provided by Christine Arris at ACUMED (Tytherington UK) with funding from Pfizer Inc. Introduction Following failure of chemotherapy and erlotinib treatment options are limited for patients with advanced nonsmall cell lung cancer (NSCLC). Reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib selectively target EGFR/HER1 one of the members of the human epidermal growth factor receptor (HER) family and are most effective in cancers harboring EGFR mutations. The remaining members of the HER family comprise HER2 and HER4 tyrosine kinases and the kinase?null HER3.1 HER family members act together via hetero? and homodimerization to enable downstream signaling pathways modulating a range of cellular activities including growth proliferation differentiation and migration.1 In contrast to patients with EGFR?mutation?positive tumors patients with KRAS?mutant NSCLC are unlikely to respond to gefitinib or erlotinib and do not have an improved progression?free survival (PFS) compared with those who have placebo following erlotinib therapy.2 Dacomitinib (PF?00299804) is a potent irreversible oral small?molecule inhibitor of HER1/EGFR HER2 and HER4 tyrosine kinases with antitumor activity in both gefitinib?sensitive and gefitinib?resistant including EGFR T790M preclinical NSCLC models.3 Dacomitinib demonstrated encouraging antitumor activity against NSCLC in Western and Japanese patients in phase 1 studies5 further supported by preliminary data from phase 2 NSCLC studies conducted in Asian patients with KRAS wild?type refractory disease7; unselected patients previously treated with chemotherapy8; and patients with EGFR?mutant disease (first?line treatment).9 This phase 2 trial (ClinicalTrials.gov identifier NCT00548093) assessed the efficacy safety and impact on health?related quality of life (HRQoL) of dacomitinib in patients with KRAS wild?type NSCLC who progressed after 1 or 2 chemotherapy regimens and erlotinib. Materials and Methods Patient Population Main inclusion criteria were age??18 years histologically or cytologically confirmed advanced NSCLC progression on erlotinib and 1 or 2 regimens of chemotherapy confirmation of KRAS wild?type tumor or known EGFR exon 19 deletion or EGFR exon 21 mutation (previously documented EGFR mutation was accepted when insufficient tissue was available for KRAS testing) and Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 to 2. Exclusion criteria included chemotherapy radiotherapy biological or investigational agents or surgery within 4 weeks of study entry; EGFR inhibitors within 2 weeks of study entry; intolerance to erlotinib; prior investigational EGFR?targeted therapy without written agreement of the study sponsor; and uncontrolled or significant cardiovascular disease. Trial Design and Treatment This was a multicenter open?label phase 2 trial. To address differences in the expected response rates between tumors of different histologies10 2 cohorts comprising patients with adenocarcinoma and those without adenocarcinoma (nonadenocarcinoma) were enrolled. Patients received 45 mg of dacomitinib once daily on an empty stomach (2 hours before or after dacomitinib intake) on a continuous basis during a 21?day cycle. Dose interruptions of?<2 weeks without discontinuation from the study were allowed for toxicity; 2 dose attenuation levels of 30 mg and then 20 mg were allowed. Treatment was discontinued for disease progression intolerance (grade 3 or 4 toxicity or intolerable grade 2 toxicity that does not resolve to grade 1 or baseline after 2 weeks' interruption) global deterioration of health?related symptoms protocol noncompliance or patient withdrawal. The primary endpoint was best overall response (BOR) according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.012 for patients with tumors of adenocarcinoma histology. Secondary efficacy endpoints included: BOR in patients with tumors of nonadenocarcinoma histology duration of objective response PFS PFS at 6 months (PFS6M) overall survival (OS) and OS at 6 (OS6M) and 12 (OS12M) months. Other secondary endpoints were safety; patient?reported outcomes (PROs) of HRQoL" | 1 |
" the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour and patient characteristics treatment efficacy and tolerability and quality of life better patient selection might lead to improved outcomesmethods this post hoc exploratory analysis examined the effect of prognostic factors on outcomes in the randomized double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with mcrc refractory to standard chemotherapies recourse trial patients were redivided by prognosis into two subgroups those with metastatic sites at randomisation low tumour burden and ¥ months from diagnosis of metastatic disease to randomisation indolent disease were included in the good prognostic characteristics gpc subgroup the remaining patients were considered to have poor prognostic characteristics ppcresults gpc patients n386 had improved outcome versus ppc patients n414 in both the trifluridinetipiracil and placebo arms gpc patients receiving trifluridinetipiracil n261 had an improved median overall survival vs months hr ci to p00001 and progression free survival vs months hr ci to p00001 than ppc patients receiving trifluridinetipiracil n273 improvements in survival were irrespective of age eastern cooperative oncology group performance status ecog ps kras mutational status and site of metastases at randomisation in the trifluridinetipiracil arm time to deterioration of ecog ps to ¥ and proportion of patients with ps0 discontinuing treatment were longer for gpc than for ppc patients vs months and vs respectively low tumour burden and indolent disease were factors of good prognosis in late line mcrc with patients experiencing longer progression free survival and greater overall survivalintroductioninclusion of new therapeutic options into the current treatment landscape in metastatic colorectal cancer mcrc has led to an increased survival in the last couple of key questionswhat is already known about this subject º the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour characteristics and patient factors as well as treatment characteristics such as tolerability efficacy and quality of life effects trifluridinetipiracil is indicated in pretreated patients with mcrc based on results of the pivotal randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial which demonstrated significantly improved overall survival os compared with placebo with a manageable safety profilewhat does this study add º in recourse classification of patients as having good prognostic characteristics gpc defined as those with low tumour burden metastatic sites at randomisation and less aggressive disease ¥ months from diagnosis of first metastasis at randomisation identified a subgroup of patients with improved os and progression free survival with trifluridinetipiracil compared with patients with poor prognostic characteristics treated with trifluridinetipiracil and gpc patients treated with placebohow might this impact on clinical practice º low tumour burden and indolent disease were shown to be factors of good prognosis in late line mcrc with these patients experiencing longer time on treatment and greater os this suggests that these patients could be candidates to receive further lines of therapy post trifluridinetipiracildecades1 first line treatment of patients typically involves the use of vascular endothelial growth factor vegf or epidermal growth factor receptor egfr targeted agents eg bevacizumab cetuximab panitumumab to fluoropyrimidine based fluorouracil or tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesscapecitabine chemotherapy regimens depending on the presence or absence of ras mutation positive disease2 in the usa immunotherapies nivolumab±ipilimumab or pembrolizumab are also recommended for the treatment of patients with mismatch repair deficient or microsatellite instability high disease4 in the second line setting vegf targeted treatments eg aflibercept ramucirumab can also be used in combination with chemotherapy2 the optimal chemotherapeutic regimen for use beyond third line remains unclear where resistantrefractory disease and residual toxicity potentially limit the treatment options with only two possible candidates at present5the general condition and performance status of a patient are strong prognostic and predictive factors for mcrc treatment2 fitter patients are typically assigned to a more intensive treatment approach ie a combination of cytotoxic agents with a biological agent than less fit patients2 the choice of treatment in the metastatic setting is generally influenced by tumour characteristics tumour burden localisation and biology patient characteristics age eastern cooperative oncology group performance status ecog ps an function and comorbidities and treatment characteristics efficacy toxicity profile administration and quality of life qol effects2the proportion of patients with mcrc receiving active treatment decreases from line to line leaving more than half of patients who received an active treatment in the first line without treatment in the third line setting even in randomised clinical trials in folfiri plus cetuximab versus folfiri plus bevacizumab as first line treatment for patients with metastatic colorectal cancer only of patients reached third line6 data from the usa indicate that only of patients receiving a first line of treatment move into the second line move to the third line and only will receive a fourth line of treatment7 being unable to receive a subsequent line of treatment therefore appears to have a negative impact on the patients survivalis trifluridinetipiracil ftdtpi lonsurf indicated for the treatment of adult patients with mcrc who have been previously treated with or are not considered candidates for available therapies including fluoropyrimidine based oxaliplatin based and irinotecan based chemotherapies anti vegf agents and anti egfr agents for eligible patient ras wild type combination of tipiracil hydrochloride with the nucleoside metabolic inhibitor trifluridine improves its bioavailability by inhibiting its catabolism by thymidine phosphorylase8 the relatively limited non haematological toxicity of trifluridinetipiracil makes it a good option in the third line and refractory settings2 in the pivotal phase iii randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial conducted in patients with mcrc eligible for treatment in the third line and beyond treatment with trifluridinetipiracil versus placebo extended overall survival median os vs months hr p0001 and progression free survival median pfs vs months hr p000110 this effect was shown in all subgroups regardless of age ecog ps geographical region race and kras mutational status10 furthermore trifluridinetipiracil was well tolerated with few serious adverse events aes reported haematological toxicities were the most frequently observed aes10 also time to deterioration of ecog ps to ¥ was significantly improved median vs months hr p000110 with of patients treated with trifluridinetipiracil remaining at ps at discontinuation11 remaining at ecog ps is important as it could allow patients to further benefit from subsequent therapy and potentially extend their survival in recourse and of patients treated with trifluridinetipiracil remained alive at and months respectively in the refractory setting in the post hoc analysis described here we set out to explore other factors that could extend survival in the recourse population for the purposes of our exploratory analysis we defined the characteristics of good prognosis as low tumour burden metastatic sites by response evaluation criteria in solid tumors recist evaluation at randomisation and less aggressiveindolent disease ¥ months from diagnosis of first metastasis to randomisation which are known to be strong prognostic factors in patients with mcrc with good ecog ps12 our ultimate aim is to explore how clinicians can better predict individual treatment outcomes and support treatment selection through the continuum of carematerials and methodsstudy design and patientsthe study design and methodology of the recourse trial clinicaltrials gov number nct01607957 have been previously published10 in brief recourse was a phase iii randomised double blind placebo controlled study comparing the efficacy and safety of trifluridinetipiracil plus best supportive care with those of placebo plus best supportive care10 this study included patients with metastatic biopsy provendocumented adenocarcinoma of the colon or rectum who were previously treated with ¥ standard chemotherapy regimens or who had tumour progression within months of their most recent chemotherapy or who had clinically significant aes precluding readministration of standard chemotherapies patients were randomised to trifluridinetipiracil mgm2 two times a day on days and every weeks or matching placebo10 randomisation was stratified according to kras mutation status wild type vs mutant time from diagnosis of first metastasis to randomisation vs ¥ months and geographical region japan vs usa european union and australia10 all patients had adequate an function and were ecog ps tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0cof at inclusion10 the primary endpoint of the study was os and secondary endpoints included pfs objective response rate clinical benefit rate and safety10patient subgroupsin examining the effects of prognostic factors on treatment outcomes in the current analysis several subgroups of recourse patients were considered patients from recourse n800 were divided according to good prognostic characteristics gpc and poor prognostic characteristics ppc good prognosis was considered to be defined by low tumour burden metastatic sites by recist tumour evaluation at randomisation and less aggressiveindolent disease ¥ months from diagnosis of first metastasis to randomisation12 of the gpc subgroup n386 patients received trifluridinetipiracil and received placebo the remaining patients were included in the complementary ppc subgroup n414 of these received trifluridinetipiracil and received placeboanalysis outcomesos and pfs in the gpc subgroup were compared with those in the ppc subgroup these subgroups were then analysed according to other tumour and patient characteristics that is metastatic site at randomisation for those sites present in of the population liver lung lymph or peritoneum ecog ps vs kras mutation status wild type vs mutant and age vs ¥ years os and pfs with trifluridinetipiracil were compared with placebo and were analysed according to prognostic subgroups within each of the two arms finally the effect of prognostic classification of patients on ecog ps deterioration was analysed for all patients and subgroupsstatistical methodsdemographic and baseline characteristics of patients were summarised by treatment arm and subgroups using descriptive statistics n mean sd median minimum and maximum andor frequency distributions as appropriatethe differences in os pfs and time to ecog ps deterioration between trifluridinetipiracil and placebo patients or between subgroups of patients in a specific arm of treatment were assessed using the stratified log rank test stratification factors used for the randomisation from a cox proportional hazards model for each arm or each subgroup survival was summarised using kaplan meier curves and was further characterised in terms of the median with the corresponding two sided cisresultspatientsbaseline patient demographics and clinical characteristics were generally similar between gpc and ppc patients table in the trifluridinetipiracil arm slight imbalances were seen in ecog ps more gpc than ppc open accesspatients had an ecog ps of and kras status more gpc than ppc patients were kras wild type also more gpc than ppc patients had received ¥ prior regimens among the ppc group treated with trifluridinetipiracil of patients had ¥ months from diagnosis of first metastasis to randomisation but had ¥ metastatic sites and of patients had metastatic sites but months from diagnosis of first metastasis similar differences were observed in the placebo arm with the exception of kras status which was comparable in the gpc and ppc subgroupstreatmentamong trifluridinetipiracil treated patients those in the gpc group received more treatment cycles mean sd compared with patients in the ppc group mean sd online supplementary table s1 a higher proportion of gpc patients than ppc patients receiving trifluridinetipiracil had a dose delay vs respectively or dose reduction vs respectively which is consistent with a longer duration of treatment online supplementary table s1 however median dose intensity in the first four cycles was high ¥ and did not differ markedly between the groups cycle in the gpc group and the ppc group cycle and respectively cycle and respectively cycle and respectivelythe effect of good versus poor prognosis classifications on survivalsurvival curves for the gpc versus ppc subgroups are shown in figure median os was longer in the gpc subgroup than the ppc subgroup for both trifluridinetipiracil vs months hr ci to p00001 figure 1a and placebo vs months hr ci to p00001 figure 1b rates of month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively and month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively were also higher in gpc subgroups compared with ppc subgroups median pfs with trifluridinetipiracil was also longer in the gpc subgroup versus the ppc subgroup vs months hr ci to p00001 respective values for gpc versus ppc in the placebo arm were versus months hr p00699 pfs at and months in the ppc subgroup was and for trifluridinetipiracil and and for placebo respectively in the gpc subgroup these were and with trifluridinetipiracil and and with placebo respectivelyeffects of good prognostic factors on the relative efficacy of trifluridinetipiracilmedian os was prolonged with trifluridinetipiracil versus placebo in both subgroups but to a greater tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable baseline patient demographics and clinical characteristics according to prognosistrifluridinetipiracilplacebogpc subgroup n261 ppc subgroup n273 gpc subgroup n125ppc subgroup n141 females male asian other years to years ¥ yearsmedian age yearspatient age n gender n ethnicity n ecog ps n kras status n time since diagnosis of metastasis n number of prior regimens n number of metastatic sites n site of metastatic lesion n primary site of disease n liver lung lymph peritoneum ¥ ¥ mutant wild type months ¥ months colon rectum defined as metastatic sites and ¥ months since first metastasis only those in more than of the intent to treat population are included liver lung lymph and peritoneumecog ps eastern cooperative oncology group performance status gpc good prognostic characteristics ppc poor prognostic characteristicsextent in the gpc subgroup than in the ppc subgroup figure 2a similarly median pfs was prolonged with trifluridinetipiracil versus placebo in both subgroups with the greatest magnitude of benefit observed in the gpc patients figure 2banalysis of prognostic factorsthe effect of various prognostic factors on median os and pfs is shown in table their effect on month and month os and month month and month pfs is shown in online supplementary tables and for both tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessfigure overall survival os for the good prognostic characteristics gpc and poor prognostic characteristics ppc subgroups in patients receiving a trifluridinetipiracil or b placebo ap0001 one sided bp0001 two sided ftdtpi trifluridinetipiracil mos median overall survival nr not reachedtrifluridinetipiracil and placebo the gpc subgroup had better median os and pfs than the ppc subgroup irrespective of patient age ¥ vs years ecog ps vs kras mutation status mutant vs wild type and liver metastases yes vs nowhen analysing the gpc subgroup the absence of liver metastasis at randomisation n153 representing of the gpc and of the intent to treat population was found to be the best factor of prognosis further information on this group of patients is available in online tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessa overall survival os b progression free survival pfs and c time to eastern cooperative oncology group figure performance status ecog ps ¥ with trifluridinetipiracil versus placebo in the good prognostic characteristics gpc n386 and poor prognostic characteristics ppc n414 subgroups ftdtpi trifluridinetipiracil mos median overall survivalsupplementary table s4 and online supplementary figures s1 s3 among gpc patients treated with trifluridinetipiracil median os was months longer in patients with no liver metastases compared with those with liver metastases vs months table the month os rate in gpc patients treated with trifluridinetipiracil was in those without liver metastases and in those with liver metastases corresponding month os rates in these groups were and respectively online supplementary table s2 median os was also longer in patients with no liver metastases compared with those with liver metastases in the trifluridinetipiracil ppc subgroup vs months and both the gpc and ppc subgroups of the placebo arm vs months and vs months respectively table in the group of ppc patients treated with trifluridinetipiracil the month and month os rates were and respectively in those without liver metastases compared with and respectively in those with liver metastases online supplementary table s2 for the trifluridinetipiracil and placebo arms patients with baseline ecog ps had higher median os compared with ecog ps patients in both the gpc and ppc subgroups table in the trifluridinetipiracil arm age or ¥ years and kras status did not seem to affect the treatment outcome table similar results were found for pfs with an effect for all trifluridinetipiracil gpc and ppc subgroups with median pfs values ranging from to months table among gpc patients treated with trifluridinetipiracil the month pfs rate was in those with no liver metastases compared with in those with liver metastases corresponding month pfs rates in the ppc group of patients treated with trifluridinetipiracil were and respectively online supplementary table s3 no such effect was observed in the placebo arm with values ranging months whatever the prognosis at the outset for almost all subgroups median tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0ctable the effect of various prognostic factors on median overall survival os and progression free survival pfsnumber of patientsftdtpi placebomedian survival monthshr cinumber of patientsftdtpiplacebomedian survival monthshr ciopen accessosgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgrouppfsgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroup vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs vs vs to to to to to to to to to to to to to to to to to to to to to to to to to to to to liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age¥ yearsn124n53n110n65kras mutantn119n64n153n71liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age ¥ yearsn124n53n110n65kras mutantn119n64n153n71 vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to good prognostic characteristics gpc were defined as metastatic sites at randomisation and ¥ months from first metastasis to randomisationftdtpi trifluridinetipiracil ppc poor prognostic characteristics ecog ps eastern cooperative oncology group performance statustabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable effects of prognostic classification of patients on eastern cooperative oncology group performance status ecog psmedian time to deterioration to ecog ps ¥ monthsftdtpiplaceboitt population n80011good prognosis patients n386poor prognosis patients n414ftdtpi trifluridinetipiracil itt intent to treatpfs was longer and all hrs favoured treatment with trifluridinetipiracil table effects of prognostic classification of patients on ecog psdata relative to the effect of ecog ps are presented in table the proportion of gpc patients treated with trifluridinetipiracil with an ecog ps of at treatment discontinuation was among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of at treatment discontinuation similarly among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of at treatment discontinuation the median time to deterioration of ecog ps to ¥ in patients receiving trifluridinetipiracil was months in the gpc subgroup and months in the ppc subgroup figure 2ctolerability and safetythe most common aes in patients receiving trifluridinetipiracil were nausea anaemia neutropenianeutrophil count decrease diarrhoea fatigue and reduced appetite online supplementary table s5 the most common grade ¥ aes experienced by patients receiving trifluridinetipiracil were haematological anaemia neutropenianeutrophil count decrease white blood cell count decrease there was no evidence of a higher incidence of aes in patients with ppc versus gpc in the group receiving trifluridinetipiracil but there was a trend towards a higher incidence of aes in placebo recipients with ppc compared with gpc online supplementary table s5discussionthe results of our analysis show that patients in the gpc subgroup consistently performed better than those in the ppc subgroup in both the trifluridinetipiracil and placebo arms within the same subgroups patients treated with trifluridinetipiracil performed better than placebo trifluridinetipiracil has consistently been shown to provide a significant survival benefit to patients with mcrc refractory to standard therapy with a well tolerated safety profile in three large scale randomised clinical trials10 a previous subanalysis of recourse showed that trifluridinetipiracil was more effective than placebo in patients irrespective of region age racialhr ci to to to p valueecog ps at treatment discontinuation ftdtpiplaceboethnic differences or kras mutation status17 in the current analysis further categorisation of patients as having good prognosis using the criteria of metastatic sites by recist tumour evaluation at randomisation and ¥ months from diagnosis of first metastasis to randomisation12 identified a subgroup of patients with improved os and pfs with trifluridinetipiracil compared with poorer prognosis patients ie those with ¥ metastatic sites and months from first metastasis pfs and os were also improved in gpc patients treated with trifluridinetipiracil compared with gpc patients who received placebopatients with gpc received more cycles of treatment than patients with ppc because progression was delayed in this group which may have contributed to the better survival outcomes the difference cannot be explained by a difference in dose intensity since this was high and similar in both the ppc and gpc subgroups of patients receiving trifluridinetipiracil in addition there was no evidence for higher toxicity in the ppc than the gpc group in fact the haematological aes occurred at a slightly higher rate in gpc patients than in ppc patients who received trifluridinetipiracil which probably reflects a longer exposure to treatment in the gpc group more patients in the gpc than in the ppc subgroup had dose delays which suggests that grade ¥ haematological aes were appropriately managed during treatmentit is thought that the availability of more treatment options for mcrc has contributed to an improvement in os over the last years3 indeed a retrospective study in elderly patients aged ¥ years a patient population more prone to comorbidities poor performance status and the development of treatment related toxicity reported a correlation between os and the number of treatment lines received18 thus maintaining the general condition and performance status of a patient throughout the continuum of care is of great importance especially beyond the second line to ensure patients remain fit with good qol5 our analysis showed that the majority of patients in the gpc subgroup discontinued treatment with an ecog ps of at the time of disease progression suggesting that these patients could be candidates to receive further lines of therapy post trifluridinetipiracil this is important when sequencing through the continuum of care this is in line with other tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0canalyses indicating preservation of health related qol on treatment of patients with mcrc with trifluridinetipiracil19 while the post hoc nature of this analysis limits it to an exploratory analysis the relatively large number of patients analysed make these data a good tool to estimate the expected outcomes when treating patients with refractory mcrc with trifluridinetipiracil the smaller size of some of the subgroups may limit the s that can be drawn thus preventing an evaluation of other parameters that might impact on outcomes such as lactate dehydrogenase levels the exact definition of good and poor prognostic factors12 may require further validation in a prospective cohortthe current analysis shows that compared with poor prognosis patients treated with either trifluridinetipiracil or placebo and good prognosis patients treated with placebo patients with gpcs treated with trifluridinetipiracil adequate an function ecog ps metastatic sites by recist tumour evaluation at randomisation and ¥ months from diagnosis of first metastasis have an increased survival in terms of median os and month and month survival rates treatment with trifluridinetipiracil is effective and provides the majority of patients the opportunity to maintain ecog ps and the possibility to receive further treatment options through the continuum of careauthor affiliations1vall dhebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall dhebron institute of oncology uvic ucc iob quironmedical oncology vall d'hebron hospital barcelona catalunya spain3medical oncology ospedale policlinico san martino istituto di ricovero e cura a carattere scientifico per l'oncologia genova liguria italy4department of medical oncology university hospital centre besançon besancon bourgogne franche comté france5kashiwa national cancer center hospital east kashiwa chiba japan6department of medical oncology dana farber cancer institute boston massachusetts usa7centre of excellence methodology and valorization of data centex mvd institut de recherches internationales servier suresnes france8global medical affairs les laboratoires servier sas suresnes île de france france9digestive oncology ku leuven university hospitals leuven leuven flanders belgiumacknowledgements the authors would like to thank andrea bothwell who wrote the first draft of this manuscript on behalf of springer healthcare communications this medical writing assistance was funded by institut de recherches internationales servier suresnes francecontributors jt and srmv contributed to the conception and design of the study all authors were involved in the acquisition analysis and interpretation of data and in writing andor revising drafts of the manuscript all authors have read and approved the final draft of the manuscript and accept responsibility for the finished article and the decision to submit the manuscript for publicationfunding the recourse study was funded by taiho oncology and taiho pharmaceutical co this analysis was funded by servier in partnership with taihocompeting interests jt has received personal fees from array biopharma astrazeneca bayer ag beigene boehringer ingelheim chugai genentech open accessgenmab as halozyme imugene limited inflection biosciences limited ipsen kura oncology eli lilly and company merck menarini merck serono merrimack pharmaceuticals merus molecular partners novartis peptomyc pfizer pharmacyclics proteodesign sl rafael pharmaceuticals f hoffmann la roche sanofi seattle genetics servier symphogen taiho pharmaceutical vcn biosciences biocartis foundation medicine haliodx sas pharmaceuticals and roche diagnostics ga has had an advisory role or received honoraria or travel grants from hoffmann la roche merck serono amgen sanofi bayer servier and bristol myers squibb afs has had an advisory role for amgen bayer celgene roche merck serono sanofi and servier and has attended a speakers bureau for amgen astrazeneca bayer bristol myers squibb celgene lilly merck serono roche sanofi and takeda evc has received research funding from amgen bayer boehringer ingelheim celgene ipsen lilly merck merck kga novartis roche sanofi and servier and has attended advisory boards for astellas astrazeneca bayer bristol myers squibb celgene lilly merck sharp dohme merck kgaa novartis roche and servier cb has attended advisory boards for roche servier and sanofi and has received a research grant from roche ao has received honoraria from ono bms chugai taiho eisai and amgen and has received research funding from bristol myers squibb an immediate family member of ao has been employed by celgene rjm declares no conflicts of interest lv and srmv are employees of servierpatient consent for publication not requiredethics ap | 0 |
"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 | 0 |
craigpattersontelecaretoj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per loncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatientssaï¬f al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset ogorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseandimplicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 commonduringadversetheoutbreakconï¬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 crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c' | 0 |
colorectal cancer crc is one of the most common malignanttumors in china chen crc is one of the ï¬veleading causes of cancer death and its incidence is graduallyincreasing owing to obesity and lifestyle changes du chen postoperative treatments includingchemotherapy and radiotherapy are important for longer patientsurvival traditional chinese medicine tcm has become anoption for preventing crc metastasis and enhancing the eï¬ectsof chemotherapy shi xu xie tcm is used as an alternative or supplementary treatmentin the united states and europe and has been widely used totreat various diseases in asia especially in china wang tcm has also been widely investigated in asia for eï¬ectiveand lowtoxicity monomer compounds to develop new drugs forcancer therapy and to counteract drug resistance sui zheng xie in china patients usually choose tcm for adjuvant therapyafter curative resection xu the eï¬ectiveness oftcm has been proven in multiple cancers including breastcancer lee hepatocellular carcinoma chen pancreatic cancer kuo and crc shi xu in crc tcm significantlyimproved diseasefree survival in stage ii and iii crc in aretrospective cohort study including patients shi in a multicenter prospective cohort study including patients with stage ii and iii crc postoperative tcmtreatment was associated with better diseasefree survival andoverall survival compared to those of the untreated groupxu certain active ingredients in tcm herbsmay have stronger activity in inhibiting cell proliferation andpromoting cell apoptosis tan huang and hu for example bufalin an active component of the tcmchan su can reverse multidrug resistance by inhibiting theprotein expression and eï¬ux function of abcb1 yuan cinobufagin another cardiotonic steroid isolated fromchan su suppresses tumor neovascularization by disrupting theendothelial mtorhif1α pathway to trigger reactive oxygenspeciesmediated vascular endothelial cell apoptosis li of the frequently used tcm treatments the most eï¬ectivesingle herbs are ginseng radix ren shen hedyotis diï¬usa willdbai hua she she cao scutellaria barbata ban zhi lian andastragali radix huang qi lee wu however the underlying mechanisms of these remedies remainunknown network pharmacology can eï¬ciently and quicklyidentify the interactions between drugs and target proteinsproviding a foundation for tcm application zhang fufang yiliu yin fyy is a tcm formula that has beenused in clinical practice for cancer treatment our previousstudy found that fyy inhibited cell proliferation migration andinvasion and promoted apoptosis in hepatocellular carcinomayang fyy contains eight herbs astragali radixhuang qi ganoderma lucidum ling zhi semen armeniacaeamarum ku xing ren h diï¬usa willd bai hua she she caoaconiti lateralis radix praeparata fu zi glycyrrhiza glabralinne gan cao radix panacis quinquefolii xi yang shenfyy inhibits colorectal cancer progressionand platycodi radix jie geng of these herbs radix panacisquinquefolii ginseng radix h diï¬usa willd and astragaliradix are commonly used in anticancer formulas lee wu g lucidum and platycodi radix alsoreportedly have anticancer eï¬ects radix astragali jung g lucidum dai platycodi radix park andlee and h diï¬usa willd zhang inhibitcancer cell proliferation polysaccharides in g lucidum inhibitthe proliferation of crc cells upregulating the expression of p21protein and blocking cells at the g2m phase na in the current study we investigated the anticancer eï¬ectof fyy on crc cells in vitro and in vivo and a networkpharmacology analysis was performed to explore the potentialmolecular mechanisms the information obtained in this studywill aid in elucidating the previously unavailable mechanismsof action of fyy in crc and developing fyy as an adjuvanttherapy for crcmaterials and methodspreparation of fyy and cell culturethe components of fyy conformed to the provisions stated bythe chinese pharmacopoeia fyy was prepared at the weifanghospital of traditional chinese medicine shandong chinayang fyy mgml was stored at ¦c untiluse and was further diluted to the required concentrations insubsequent cell experiments human crc cell lines hct116and sw480 were purchased from the cell resource center of theshanghai institutes for biological sciences chinese academy ofsciences shanghai china hct116 cells were grown in rpmi medium rpmi1640 hyclone united states and sw480cells were grown in dulbeccos modiï¬ed eagles medium dmemhyclone united states containing fetal bovine serumfbs gibco brl united states and penicillinstreptomycinsigmaaldrich st louis mo united states in co2 at ¦cin a humidiï¬ed incubatorcell viability and colony formationassayscells per well were seeded into 96well plates andincubated overnight at ¦c co2 in a humidiï¬ed incubatorwhen the cells adhered to the wall hct116 and sw480 cellswere treated with or mgml of fyy or pbs asa control for and h cell viability was measured using acell counting kit8 cck8 beyotime institute of biotechnologyinc shanghai china ten microliters of the cck8 solutionwas added to each well and then samples were incubated at ¦cfor h finally the absorbance value at nm was determinedusing a multiskantm fc microplate photometer thermo fisherscientiï¬c inc united stateshct116 and sw480 cells were treated with or mgmlof fyy or pbs as a control for h the cells perwell were then cultured in sixwell plates and the medium waschanged every days for days cell colonies were ï¬xed with paraformaldehyde and then stained with giemsa beyotimeinstitute of biotechnology inc shanghai china for minfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiona colony formation assay was performed to count viable colonies cells per colonycell cycle analyseshct116 and sw480 cells were treated with or mgmlfyy or pbs as a control for h the collected cells were ï¬xed in cold ethanol and stored at ¦c overnight the nextday the cells were washed twice with cold pbs then µlrnase a µgml and µl propidium iodide µgmlsigma aldrich st louis mo united states were added to eachsample and incubated for min in the dark measurements weretaken using a ï¬ow cytometer facscan bd biosciences bedfordma united states and the data were analyzed using flowjo software tree star inc ashland or united statescell apoptosis analysescell apoptosis was detected using an apoptosishoechst staining kit beyotime biotechnology shanghai chinasamples were ï¬xed with paraformaldehyde atroomtemperature for min and stained with mgml hoechst at room temperature for min then ï¬uorescencewas detected under an olympus ix50 microscope olympuscorp tokyo japan at magniï¬cation apoptotic cellswere identiï¬ed using an alexa fluor annexin vdead cellapoptosis kit invitrogentmmolecular probes eugene orunited states after centrifugation at g for min the celldensity was counted and diluted in annexinbinding buï¬erto obtain cellsml µl per assay cells were stainedwith µl of annexin vfitc and µl propidium iodide atroom temperature for min in the dark and then µl ofbinding buï¬er was added measurements were taken using a ï¬owcytometer and the data were analyzed using flowjo softwarenetwork pharmacologyactive fyy compounds were screened using the traditionalchinese medicine systems pharmacology database tcmsp1ru with the pharmacokinetic information retrievalï¬lter based on the tcmsp platform the oral bioavailabilityand druglikeness were set to ¥ and ¥ to obtainqualiï¬ed herbal compounds the chemical structures of thecompounds were drawn using chembiooï¬ce kerwin crc targets were predicted and screened using thegenecards database2 stelzer and omim platform3amberger and hamosh venny venny wasused to screen for common targets between fyy and diseaserelated targetsdrug compounddiseasetarget networks were built usingcytoscape v software shannon and themerge function was used to analyze the core compoundsprotein interaction networks of the common fyy and crcrelated targets were built using the string database platform1httptcmspwcomtcmspphp2httpswwwgenecards3httpswwwomim4httpbioinfogpcnbcsicestoolsvennywith medium conï¬dence and rejecting the target proteinindependent of the network szklarczyk gene ontology go analysis and kyoto encyclopediaof genes and genomeskegg pathway analysis wereperformed using metascape zhou enrichedgo terms and relevant pathways with pvalues wereselected for better prediction and veriï¬cation of the biologicalprocess and mechanismwestern blot analysisthe following primary antibodies obtained from cell signalingtechnology inc danvers ma united states were used inthe immunoblotting analysis pi3k p110α akt pan pakt ser473 bcl2 bclxl bax p21 cmyc andgapdh total proteins were extracted fromcells and tissues using ripa lysis buï¬er cwbio beijing chinaequal amounts of protein from each sample were separatedby sdspage electrophoresis and then transferred onto045µm pvdf membranes biorad laboratories herculesca united states subsequently the membranes were blockedwith milk in pbs plus tween pbst for minincubated with primary antibodies overnight at ¦c and thenincubated with goat antirabbit horseradish peroxidases abcamcambridge ma united states or goat antimousehorseradish peroxidases abcam cambridge ma united states for h at room temperature finallythe bandwas detected using an enhanced chemiluminescence reagentand visualized with a fusion fx7 system vilber lourmatfrance imagej software was used to calculate the intensity grayvalue of each protein band and gapdh served as a controlfor normalizationtumor xenografts in nude miceten male balbc nude mice weeks old ± gwere purchased from beijing vital river laboratory animaltechnology co ltd beijing china the mice were housedat ± ¦c under a 12h lightdark cycle with free accessto food and water all animal experiments were completedat the speciï¬cpathogenfree medical animal laboratory of theaï¬liated hospital of qingdao university and approved bythe animal ethics committee of the aï¬liated hospital ofqingdao university ahqu20180310a hct116 cells cells per tumor were subcutaneously injected into the rightarmpit of the nude mice seven days after tumor inoculationthe tumor size was measured using a vernier caliper andthe mice were divided into two groups the fyy treatmentgroup and a control group n mice per group thefyy group was intragastrically administered ml10 g bodyweight daily in a primary concentration of mgml thecontrol group was intragastrically administered an equivalentvolume of pbs tumor sizes were measured every daysand calculated using the following formula tumor volumemm3 length width2 the nude mice werekilled by cervical dislocation on day and the tumorswere excised weighed and photographed finallytumorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontissue and liver tissue were stored in formalin or at¦c for subsequent immunohistochemistry or western blotanalyses respectivelyimmunohistochemistrytumor and liver tissues of the nude mice were ï¬xed with paraformaldehyde for h and then embedded in paraï¬nembedded paraï¬n sections were dewaxed in xylene andrehydrated in ethanol antigen retrieval was performed in m citrate buï¬er ph using a pressure cooker followed byincubation for min samples were then washed thrice with pbsand ï¬xed in ethanol for min ki67 antibody novuscolorado united states was stained with a streptavidinperoxidase detection kit zsgbbio beijing china accordingto the kit instructionsstatistical analysisdata analysis was performed using graphpad prism softwaresan diego ca united states all experimental data wereexpressed as the mean ± sd the statistical signiï¬cance of theresults was analyzed by oneway analysis of variance anovafor multiple group comparisons and students ttest for two groupcomparisons a value of p was considered statisticallysignificant all experiments were performed in triplicateresultsfyy inhibited proliferation and promotedapoptosis of crc cells in vitrofufang yiliu yin significantly inhibited the growth of hct116and sw480 cells in a dosedependent manner figure 1a thecolony formation assay showed that the number of the coloniesin the fyy group and mgml was lower than that of thecontrol group figure 1bcolony formation ability was significantly inhibited by mgml p and mgml p fyy forhct116 and for sw480 cells respectively the cell cycle analysisshowed no significant diï¬erence in the percentage of cells ins p for mgml and g2m phases p for mgml in hct116 however a significant increase in g0g1phase was found after treatment with increasing concentrationsof fyy p for mgml figure 2a in hct116similar results were obtained for sw480 cells fyy blockedcell cycle at the g0g1 phase in a concentrationdependentmanner fyy inhibited the expression of cmyc p for mgml and promoted the expression of p21 protein p for mgml figure 2b in hct116 similar results wereobserved in sw480 cells this indicated an inhibitory eï¬ect oncell proliferationcell apoptosis as shown by hoechst staining increased afterfyy treatment figure 3a flow cytometry analysis showedthat the early p for mgml and late apoptosisp for mgml of hct116 cells were significantlypromoted figure 3b by fyy treatment similar results wereobtained for sw480 cells figure 3bnetwork pharmacological analysis offyy targeting crca total of compounds from fyy were retrieved oralbioavailability ¥ and drug likeness ¥ from the tcmspdatabase supplementary table a total of genes related tothese compounds and genes related to crc were screenedout using venny figure 4a common targets wereobtained supplementary table data imported into cytoscape to construct compounddiseasetarget networks figure 4a showed that of the fyy compounds may aï¬ect disease targets the top core compounds were screened based on the topologicalproperties of degree as shown in table quercetin kaempferolluteolin betasitosterol isorhamnetin formononetin calycosinjaranol acacetin and naringenin were the top active fyyingredients against crc the other active compoundsare listed in supplementary table two networks wereconstructed for the top core compounds and the remaining active compounds figure 4a the proteinprotein interactionnetwork built using string software used to investigatethe mechanisms of fyy provided common targets aftersetting the conï¬dence level above figure 4b theprioritization of key targets was analyzed according to thedegree of the node exported from the string database andthe top ï¬ve targets were cyclind1 mapk8 egfr myc andesr1 figure 4cbiological function and pathwayenrichment of fyy on crcthe biologicalfunctions and signaling pathways from allcore targets were enriched the top biological enrichmentresults are shown in figure 4d fyy aï¬ected crc throughmultiple go biological processes including apoptotic signalingpathway response to steroid hormone and response to inanicsubstance kegg analysis results included cancer prostatecancer apoptosis and pi3kakt signaling pathwayswe further investigated how the fyy mechanism promotedapoptosis using rtpcr and western blot analysis of hct116and sw480 cells fyy inhibited the relative expression ofpi3k mrna p figure 5a fyy downregulated theexpression of pi3k pakt bcl2 and bclxl and upregulatedthe expression of bax p figures 5bc takentogether these data support the idea that fyy induces crccell apoptosis by modulating the pi3kakt pathway and bcl2family proteinsfyy inhibited tumor growth and cellproliferation in vivothe hct116 cell xenograft model used to investigate theantitumor eï¬ect of fyy showed that fyy significantlyinhibited tumor growth compared to the control figure 6athe average tumor volumesafter days oftreatmentwere ± mm3in the control group and ± mm3 in fyytreated group figure 6b whiletumor weights were ± and ± mgrespectively ki67 significantly decreased in the fyytreatedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited colorectal cancer cell proliferation a cck8 assay indicated that fyy inhibited the proliferation of hct116 and sw480cells in a dose and timedependent manner after and h of treatment pbs was used for the control treatment n per group b colony formation abilitydecreased after treatment with different concentrations of fyy for both hct116 and sw480 n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy significantly inhibited the colorectal cancer cell cycle a fyy significantly inhibited the cell cycle progress of hct116 and sw480arresting them at the g2m phase as shown by ï¬ow cytometry assay n per group b the expression of cmyc decreased and p21 increased with fyytreatment n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained usinganovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy promoted colorectal cancer cell apoptosis a hoechst staining analysis indicated that fyy promoted apoptosis includingchromatin condensation and nuclear fragmentation in hct116 and sw480 cells magniï¬cation b flow cytometry indicated that fyy promoted the earlyand late apoptosis of hct116 and sw480 cells n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure network pharmacological analysis and biological functional enrichment analysis of fufang yiliu yin fyy a venn diagram showed common targetsof fyy in colorectal cancer crc compounddiseasetarget networks of fyy against crc b proteinprotein interactions identiï¬ed by string software c thepredicted key targets of fyy treatment of crc d go and kegg pathway enrichment analysesfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontable the top bioactive compounds of fufang yiliu yin are listed below according to the degree of similarity of the compounddiseasetarget networkspubchem cidmolecule namequercetinformulac15h10o7ob dlkaempferolc15h10o6luteolinc15h10o6degreestructurebetasitosterolc29h50oisorhamnetinc16h12o7formononetinc16h12o4calycosinc16h12o5jaranolc17h14o6acacetinc16h12o5naringeninc15h12o5glycyrolc21h18o67methoxy2methyl isoï¬avonec17h13no5continuedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong table continuedpubchem cidmolecule name7omethylisomucronulatolformulac18h20o5ob dllupiwighteonec20h18o5glyasperin fc20h18o6fyy inhibits colorectal cancer progressiondegreestructureob oral bioavailability dl druglikenesscrc tumor xenograft group figure 6b the expression ofpi3k pakt bcl2 and bclxl followed the same trend asthe in vitro study results figure 6cdiscussionboth retrospective and prospective studies have proven theanticancer eï¬ects of tcm on crc shi xu here we reported the anticancer eï¬ect of the fyyformula which contains eight ingredients fyy significantlyinhibited cell proliferation and promoted crc cell apoptosisin vitro fyy also inhibited xenograft tumor growth in vivousing a network pharmacology analysis we found that fyymay act on crc through active compounds targeting crcrelated genes that regulate the apoptosis and pi3kaktsignaling pathwaysto better understand the complementary eï¬ects of fyyformula ingredients we retrieved a total of compounds fromthe tcmsp database supplementary table compounddiseasetarget networks showed that of the compoundsmay aï¬ect crcrelated targets by searching pubmed wethe top compounds table exhibit antifound thatcrc eï¬ects mainly by promoting apoptosis and inhibiting cellproliferation for example quercetin was mostly related toprotective eï¬ects against crc and is found in three of the eightremedies in fyy astragali radix huang qi h diï¬usa willdbai hua she she cao and g glabra linne gan cao quercetininhibits crc progression by promoting cell apoptosis andautophagy as well as inhibiting angiogenesis and inï¬ammationdarband quercetin induces apoptosis by inhibitingdiï¬erent signaling pathways including the mapkerk pi3kaktand nfκb signaling pathways zhang xavier it also inhibits the migration and invasion of crc cells viaregulating the tolllike receptor 4nfκb signaling pathway han further kaempferol induces crc cell apoptosischoi while isorhamnetin formononetin andnaringenin show anticancer eï¬ects by inhibiting cell proliferationli abaza the similarity of theeï¬ects provided by fyy compounds may provide a mutualenhancement eï¬ect but this must be further tested using singleor mixed compoundsfufang yiliu yin induced cell cycle arrest in crc cells at theg0g1 phase and promoted apoptosis in hct116 and sw480cells to explain the mechanism by which fyy inhibits cellproliferation and promotes apoptosis we performed proteinprotein interaction network kegg and go pathway analysesproteinprotein interaction network analysis indicated the topï¬ve targets were cyclind1 mapk8 egfr cmyc and esr1biological functional analysis indicated apoptosis and cancerrelated pathways including the pi3kakt signaling pathwaythen our experimental study conï¬rmed the activation ofthe pi3kakt pathway and bcl2 family proteins as well ascmyc expressiontraditional chinese medicine formulas reportedly inhibitcancer progression by diï¬erent signaling pathways a tcmformula jianpi jiedu inhibits crc tumorigenesis and metastasisvia the mtorhif1αvegf pathway peng another tcm formula huang qin ge gen tang enhances the ï¬uorouracil anticancer eï¬ect by regulating the e2f1ts pathwayliu the zhi zhen fang formula reverses multidrugresistance mediated by the hedgehog pathway in crc sui these formulas as well as fyy all contain astragaliradix huang qi h diï¬usa willd bai hua she she caog glabra linne gan cao and radix panacis quinquefolii xiyang shen however there have been no reports regarding theanticancer eï¬ect of tcm formulas acting through the apoptosisand pi3kakt pathways in crc figure in the current studywe found that fyy decreased the transcription and protein levelof pi3k figure and further inhibited the phosphorylationof akt in both the cells and tumor tissues figures accumulating evidence indicates that the pi3kakt pathwayplays an important role in tumor development pi3k can partiallyactivate akt at the thr308 or ser473 sites by inducing thetranslocation of akt to the cell membrane via phosphoinositidedependent kinase akt inhibition is usually indicated by afrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy modulated the expression of the pi3kakt signaling pathway and bcl2 family proteins relative pi3k mrna expression wasaltered by fyy treatment in hct116 and sw480 cells a n per group expression levels of pi3k akt pakt bcl2 bclxl and bax were altered by fyytreatment in hct116 b and sw480 cells c n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited tumor growth in vivo a subcutaneous xenograft tumors after days demonstrated that fyy inhibited xenograft tumrowth n per group b tumor volume was significantly smaller after days of fyy treatment n per group ihc analysis of ki67 expression infyytreated tumor and liver tissues magniï¬cation the pvalues were obtained using anova c protein expression levels of pi3k akt pakt bcl2bclxl and bax in tumor tissues n per group values are shown as the mean ± sd p and p vs control group the pvalues were obtainedusing students ttestdecrease in the pakt ser473 level and is mostly achievedby inhibiting pi3k using pi3kspeciï¬c inhibitors ly294002or wortmannin reener and marti the regulation ofpi3kakttranscription and protein expression by a tcmtreatment has been previously reported tcm interventiondecreased pakt levels following the concentration gradientof the tcm treatment while the total overall akt level wasunchanged gu zhao calycosina component of astragali radix reportedly inhibits crcproliferation through the erβmediated regulation of the igf1rand pi3kakt signaling pathways zhao quercetinkaempferol and rutin in h diï¬usa willd also exhibit anticancereï¬ects in crc by regulating the pi3kakt signaling pathwaycai frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure schematic representation of the proposed pi3kakt signalinginduced cell cycle arrest and apoptosis triggered by fufang yiliu yin fyy by combiningthe network pharmacological analysis and our results we hypothesized that fyy activates the pi3kakt signaling pathway and modulates the expression of p21cmyc and bcl2 family proteins thereby inducing cell cycle arrest and apoptosiscellapoptosisandinhibited metastasiswe previously found that fyy inhibited cell proliferationofpromotedhepatocellular carcinoma yang fyy may havea similar eï¬ect on diï¬erent types of cancer although wedemonstrated both the anticancer eï¬ects of fyy and the actionmechanism by which it operates limitations of this study includethe following ï¬rst we did not investigate the antimetastaticeï¬ect of fyy on crc a migration and invasion assay andcrc liver metastasis model should be used to investigate thissecondfurther studies should investigate whether mutualenhancement eï¬ects exist between the applications of fyyand regular chemotherapy and also examine its eï¬ect ondrug resistancein conclusion our study ï¬ndings showed that fyy inhibitedproliferation and promoted apoptosis in crc cells by modulatingthe pi3kakt signaling pathway and bcl2 family proteins webelieve that fyy could be a promising adjuvant therapy for crcethics statementthe animal study was reviewed and approved by animalethics committee of the aï¬liated hospital of qingdaouniversity ahqu20180310a written informed consent wasobtained from the ownerstheiranimals in this studyfor the participation ofauthor contributionsbd and cz obtained funding conducted the research andprepared the manuscript zy and qj performed the experimentssz prepared and provided the fyy formula yw and hzperformed the network pharmacology analysis cs designed thestudy and interpreted the data all authors contributed to the and approved the submitted versiondata availability statementall data presented in thissupplementary materialstudy areincluded in thefundingthis work was supported by the china postdoctoral sciencefoundation grant numbers 2016m602098 and 2018m640615the taishan scholars program ofshandong provincefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiongrant number the shandong higher educationyoung science and technology support program grant number2020kjl005 the qingdao postdoctoral science foundationgrant number and the national natural sciencefoundation of china grant number supplementary materialthe supplementary material for this can be found onlineat httpswwwfrontiersins103389fcell202000704fullsupplementarymaterialreferencesabaza m s orabi k y alquattan e and alattiyah r j growthinhibitory and chemosensitization eï¬ects of naringenin a natural ï¬avanonepuriï¬ed from thymus vulgaris on human breast and colorectal cancer cancercell int doi 101186s1293501501940amberger j s and hamosh a searching online mendelian inheritance inman omim a knowledgebase of human genes and genetic phenotypes currprotoc bioinformatics doi 101002cpbi27cai q lin j wei l zhang l wang l zhan y hedyotis diï¬usawilld inhibits colorectal cancer growth in vivo via inhibition of stat3 signalingpathway int j mol sci doi 103390ijms13056117chen q shu c laurence a d chen y peng b g zhen z j eï¬ect of huaier granule on recurrence after curative resection of hcca multicentre randomised clinical trial gut doi 101016s0618chen w zheng r baade p d zhang s zeng h bray f cancerstatistics in china ca cancer j clin doi 103322caacchoi j b kim j h lee h pak j n shim b s kim s h reactive oxygen species and p53 mediated activation of p38 and caspases iscritically involved in kaempferol induced apoptosis in colorectal cancer cellsj agric food chem doi 101021acsjafc8b02656dai s liu j sun x and wang n ganoderma lucidum inhibitsproliferation of human ovarian cancer cells by suppressing vegf expressionand upregulating the expression of connexin bmc complement alternmed doi darband s g kaviani m youseï¬ b sadighparvar s pakdel f g attari j s quercetin a functional dietary ï¬avonoid with potential chemopreventive properties in colorectal cancer j cell physiol doi 101002jcp26595du z x jia x y and lin k colorectal cancer mortality characteristicsand predictions in china asian pac j cancer prev doi 107314apjcp201516177991gu z f zhang z t wang j y and xu b b icariin exerts inhibitoryeï¬ects on the growth and metastasis of kyse70 human esophageal carcinomacells via pi3kakt and stat3 pathways environ toxicol pharmacol doi 101016jetap201706004han m song y and zhang x quercetin suppresses the migrationand invasion in human colon cancer caco2 cells through regulating tolllikereceptor 4nuclear factorkappa b pathway pharmacogn mag s237s244doi huang c and hu g shikonin suppresses proliferation and inducesapoptosis in endometrioid endometrial cancer cells via modulating mir106bptenaktmtor signaling pathway biosci rep 38bsr20171546 doi101042bsr20171546jung y jerng u and lee s a systematic review of anticancer eï¬ectsof radix astragali chin j integr med doi 101007s116550152324xkerwin s m chembiooï¬ce ultra suite j am chem soc doi 101021ja1005306kuo y t liao h h chiang j h wu m y chen b c chang c m complementary chinese herbal medicine therapy improves survivalof patients with pancreatic cancer in taiwan a nationwide populationbasedcohort study integr cancer ther doi lee y w chen t l shih y r v tsai c l chang c c liang h h adjunctive traditional chinese medicine therapy improves survival inpatients with advanced breast cancer a populationbased study cancer doi 101002cncr28579li c yang x chen c cai s and hu j isorhamnetin suppresses coloncancer cell growth through the pi3kaktmtor pathway mol med rep doi 103892mmr20141886li x chen c dai y huang c han q jing l cinobufaginsuppresses colorectal cancer angiogenesis by disrupting the endothelialmammalian target of rapamycinhypoxiainducible factor 1α axis cancer sci doi 101111cas13988liu h liu h zhou z parise r a chu e and schmitz j c herbalformula huang qin ge gen tang enhances 5ï¬uorouracil antitumor activitythrough modulation of the e2f1ts pathway cell commun signal doi101186s1296401802181na k li k sang t wu k wang y and wang x anticarcinogeniceï¬ects of water extract of sporodermbroken spores of ganoderma lucidumon colorectal cancer in vitro and in vivo int j oncol doi103892ijo20173939park j c and lee y j in vivo and in vitro antitumor eï¬ects of platycodind a saponin puriï¬ed from platycodi radix on the h520 lung cancer cell evidbased complement alternat med doi peng w zhang s zhang z xu p mao d huang s jianpi jiedudecoction a traditional chinese medicine formula inhibits tumorigenesismetastasis and angiogenesis through the mtorhif1alphavegf pathwayj ethnopharmacol doi 101016jjep201805039reener m c and marti f the balancing act of akt in t cells front biol doi 101007s1151501212026ru j li p wang j zhou w li b huang c tcmsp adataba | 0 |
" Bryant et al. Overall survivalAverage Za0.370.00011Beer (N = 442) Overall survivalAverage Zb0.620.00003Fisher0.500.00068GSEA0.650.00001Euclidean0.650.00001Mahalanobis0.670.00001GSE8894 (N = 61) Recurrent free survivalAverage Zb0.900.01163Fisher0.910.01076GSEA0.780.02899Euclidean0.870.01544Mahalanobis0.680.05485aDerived from mean and SD of all cancer samples in the dataset bDerived by mean and SD of the nRef.Prognostic gene expression signatures for Stages II and III colon cancers have been reported in seven papers yielding 207 genes in total (Bandres et al. 2007; Barrier et al.2006 2007; Eschrich et al. 2005; Kopetz and Abbruzzese 2009; Lin et al. 2007; Wang et al. 2004). The genes are enriched in 32 REACTOME pathways (False Discovery Rate (FDR) < 0.05 pathway size < 96). We assumed the 32 pathways were valid as ground truth to be identified and analyzed in the colon cancer dataset GSE39585 (Stages II and III were only considered). Average Z provided best performer (sensitivity = 0.88) with 28 pathways deemed as significant. GSEA Fisher Euclidean and Mahalanobis gave the following values0.780.66 0.06 and 0.03 respectively.These results satisfied us that our approach captures the fundamental knowledge of cancer thus it is reasonably considered as iPAS.To investigate which of the candidates for iPAS most robustly reflect phenotype association we evaluated the proposed methods by determining whether survival-associated pathways are validated in datasets never used for discovery using LUAD and colon cancer [LUAD: Beers set n = 442 for discovery GSE8894 (n = 61) GSE3141 (n = 58) for validation; colon cancer: GSE39582d (n = 443) for discovery GSE39582v (n = 123) and GSE17536 (n = 109) for validation logrank P < 0.05 comparing tumors in the top 50th percentile of aberrance scores to those in the bottom 50th percentile]. Validation rates varied depending on the dataset and these were possibly affected by the small sample size compared with that of the discovery set. In these cases we were not able to determine a superior method that outperformed the others. Average Z gave the highest validation rate in three of four dataset with validation rates of GSE8894 (43.6% 92/211) GSE3141 (13.3% 28/211) and GSE17536 (10.7% 24/224). When validation rates from four datasets are averaged Average Z gave the highest validation rate (21.9% Fig. 2 blue bars). Pathways validated as significantly associated with patient survival for each cancer are listed in the Supplementary Materials (Supplementary Tables S1 and S2). Fig. 2.Averaged validation rate of discovered survival-related pathway at four datasets. Proposed approach using nRef (blue) versus conventional approach that standardizes individual sample by mean and SD of entire cohort dataset (red)We also investigated the validation rate of iPAS candidates under the conditions where the same data are not standardized by the nRef but instead standardized by the mean and SD of the cohort dataset which consists of only cancers (Fig. 2 red bars). It is noteworthy that use of the nRef increased the validation rate for every iPAS candidate investigated. This implies that the strategy of using accumulated normal samples as a reference is beneficial in terms of pathway-based survival analysis.3.2 Identification of clinical importanceCluster analysis of using Average Z as the iPAS method on Beers data identified 12 pathway clusters (denoted by 1?12 in Fig. 3) and 3 sample clusters (S2?S4; S1 is from the nRef; Fig. 3). Sample clusters S2 and S4 represent well the differentiation status of LUAD (Fisher exact test P < 4.65 10?15). Well-differentiated adenocarcinoma resembles the normal glandular structure; therefore it is a reasonable result that cluster S2 is close to the nRef. The survival outcome of S2 and S4 are significantly different (P < 0.0028) and this assures us that unbiased clustering-based iPAS has enough sensitivity to capture clinically important associations. This finding is concordant with prior knowledge that well-differentiated LUAD patients are likely to have better prognosis (Barletta et al. 2010). Pathway cluster P9 is distinguished as commonly upregulated in tumor samples. The pathways are transfer RNA aminoacylation amino acid or purine synthesis DNA elongation and the extension of telomeres. Fig. 3.Clustered iPAS of LUAD dataset. Pathways (n = 583) and samples (n = 442) are clustered according to iPAS. Normal samples are clustered at left (S1). Tumors (S2?S4) deviate from normal in both up- and downregulated directions (darker red and blue respectively). Sample clusters are well-representing histopathological differentiation status (S2: for well-differentiated LUAD P < 4.65 10?15) and overall survivalUnbiased pathway-based clustering of colon cancer data also captures clinically important associations by revealing sample clusters that are survival related (S2 and S3 P = 0.0037 Supplementary Fig. S1). It is important to note that iPAS is not only sensitive enough to identify clinically meaningful substructure of patients but also reveals common characteristics of a cancer at the same time. For example pathways commonly up- or downregulated in all cancer samples for example P9 or P2 would have not been discovered if the analysis had been performed by a conventional approach that does not make use of nRef (Supplementary Fig. S2).3.3 Pathway-based identification of cancerCancer develops unique mechanisms for malignancy. Therefore it is reasonable to believe that identifying the unique molecular aberrances of cancer will aid in cancer diagnosis. Our empirical study of iPAS-based clustering of LUAD revealed several pathways commonly up- or downregulated in all of the cancer samples. Further analysis was performed to determine whether iPAS could be successfully used in the accurate identification of cancer. We tested this in a simple unsupervised way by judging whether an unknown sample is significantly different against the nRef as a tumor if not as normal. We performed a 5-fold cross-validation one hundred times with the LUAD dataset which consisted of 120 cancers and 120 normal samples. Microarray data from the normal samples was randomly divided into five groups and four of the five served as the reference group. The remaining group was used as the true normal set for the test of pathway-based identification of cancer. To build true cancer set for the test the same number of cancer sample was randomly picked. We considered 583 pathways in REACTOME giving 293 500 (583 pathways 5-fold 100 repeats) AUCs and accuracy values. We averaged AUCs and accuracies from the five candidate methods for iPAS and used this as a representative AUC and accuracy of a given pathway.By ranking the pathways by AUC top pathways that marked averagely high performance by all iPAS candidates are listed (Supplementary Table S3). The amino acid synthesis and interconversion and transamination pathway showed the highest classification performance. Unsurprisingly this pathway was one of the commonly upregulated pathways in the analysis of the Beers data (Fig. 3 pathway cluster P9). Among the tested iPAS candidates for this pathway Mahalanobis yielded the highest AUC (0.980) while Average Z gave 0.936 and Fishers exact test gave the lowest value (0.914). The standardized gene expression pattern for this pathway differed between tumor and normal. Many of the genes deviated from mean of the nRef by more than two orders of sigma contributing to its best performance out of all iPAS candidate methods including ORA method like Fishers exact test (Fig. 4a). Fig. 4.(a) Expression pattern of genes in the pathway. Each line represents sample. (gray: normal red: tumor). Dashed line represents expression value deviated 1.96? from the mean expression value of normal tissues. (b). Performance of classification of cancer by amino acid synthesis and interconversion and transamination. AUC of 0.980 has marked in discovery set (95% confidence interval provided as error bar) independent validation set results AUC of 0.982 (Validation 1: normal samples in validation set served as reference) and 0.982 (Validation 2: normal samples in discovery set served as reference)We also analyzed the influence of using the subset of normal samples as nRef. We compared the pathway-based cancer identification results using the full set of normal samples (n = 120) against 100 different runs using 75% (n = 90) 50% (n = 60) of randomly chosen normal samples. Among the pathways that marked averagely high performance in the identification of cancer the best and the second best pathways are considered amino acid synthesis and interconversion and transamination and unwind of DNA respectively. The result shows little loss of performance even though only a half of normal samples were used for the test (Fig. 5a and b). Fig. 5.Performance of pathway-based identification of cancer (AUC)" | 1 |
" In another study by the same group a set of different miRNAs could be used to differentiate hepatocellular carcinomas from metastatic tumors in the liver [25]. miRNA expression differs between tumor types within the same tumor type in different patients and between primary tumors and metastases. Hence it may not be surprising to find miR-182 to have divergent impact in different stages of NSCLC. Increasing evidence demonstrate that adenocarcinomas and SCC of the lung are separate lung cancer entities have dissimilar features and may respond differently to therapy. Targeted drugs with specific effects in certain histological subgroups have been developed. Certain miRNA-signatures can differentiate SCC from non-SCC and may facilitate the distinction between them [26]. Demonstrating a significant prognostic effect by miR-182 in SCC and not in adenocarcinomas underscores the diversity between the histological subgroups. In a previous published paper from our group [27] we explored the impact of miR-155 in the same cohort. We found this miRNA to be very stage- and tissue specific with a significant impact on survival only in node positive SCC patients. miR-182 has been regarded as an oncogene in most contexts. In a cohort of 253 glioma patients high miR-182 expression was found to be a negative prognostic factor [12]. In melanoma cell lines Segura and coworkers showed that high miR-182 expression stimulated migration and survival. The same group treated liver metastases in mice with anti-miR-182 and obtained a lower tumor burden and a lower mir-182-level than in untreated mice [1328]. Also in breast tumors and cervical cancers miR-182 seems to have an oncogenic impact [2930]. There are other studies that have identified miR-182 as a tumor suppressor. Kong et al. found miR-182 to be underexpressed in human gastric cancer cell lines. They showed that the oncogene cAMP responsive element binding protein 1 (CREB1) is a target of miR-182 and that high levels of miR-182 leads to lower levels of CREB1 and suppressed gastric adenocarcinoma cell growth [31]. In melanoma cell lines Poell et al. found miR-182 to be a strong inhibitor of cell proliferation [14]. Yan and coworkers found similar effects in uveal melanoma cells where they identified MITF BCL2 and cyclin D2 as potential targets of miR-182. Transfection of miR-182 into cultured uveal melanoma cells led to a significant decrease in cell growth migration and invasiveness [16]. In lung cancer data on miR-182 have been conflicting regarding its prognostic role. In 70 lung cancer tissue samples Zhu and coworkers observed an association between high expression of the members of the miR-183 family (miR-96 miR-182 and miR-183) and poor overall survival [11]. In contrast two in vitro studies using cell lines did not support the notion of miR-182 exerting an oncogene role in lung cancer. Sun et al. found miR-182 through regulation of RGS17 to suppresses lung tumorigenesis [15]. Consistently Zhang and coworkers reported miR-182 to inhibit proliferation and invasion of human lung adenocarcinoma cells via its effect on human cortical actin-associated protein (CTTN) [32]. miR-182 has a number of target genes and it is evident that the regulation of these genes can result in both inhibition and stimulation of tumorigenesis. In NSCLC our results suggest that tumor inhibiting miR-182 features dominate and thus make this miRNA a favorable prognostic factor. Based on the association with angiogenesis suggested from the GSEA [17] we investigated the correlation between miR-182 and a set of angiogenesis-related protein markers. There was a negative correlation between miR-182 and FGF2. Our group has published data on FGF2 which identify this marker as an independent negative prognostic factor in lung cancer cells [22]. Fibroblast growth factor receptor substrate 2 (FRS2) is a downstream mediator of the fibroblast growth factor pathway and is a target gene of miR-182. FRS2 is thought to induce tumor progression through stimulation of angiogenesis [1733]. In our total NSCLC cohort the coexpression between miR-182 and FGF2 showed an independent significantly worse prognosis for low miR-182/high FGF2 than for high miR-182/low FGF2 (P?=?0.015 ). A correlation was also detected between miR-182 and MMP-7. In a previous paper our group found high MMP-7 expression to be an independent favorable prognostic factor in this same NSCLC cohort [23]. When examining coexpression of the two variables those with high miR-182 and high MMP-7 expression had an independently better survival than those with low miR-182/low MMP-7 expression (HR 0.49 P?=?0.015). When stratifying on histology the SCC patients with high/high expression had a remarkably better prognosis than the rest of the groups (HR 0.26 P?=?0.012 ). To our knowledge there are no published data linking miR-182 and MMP-7. Few studies have described the connection between FGF2 and MMP-7 [3435]. Based on our strong results from the co-variations between miR-182 and particularly MMP-7 it would be interesting to see functional studies exploring potential relations between these two markers. In our previous pilot study on miRNA signatures [17] miR-182 appeared as an oncogene since it was up-regulated in short vs long term NSCLC survivors and in NSCLC vs normal tissues. In our large unselected NSCLC cohort presented herein we surprisingly observed that high miR-182 expression is associated with improved survival at least in subgroups of patients with NSCLC. It has to be kept in mind that the explorative study was based on a small sample only 20 NSCLC cases and 10 normal lung tissues. Hence the contrasting results may be due at least in part to selection bias in the explorative study. Besides in the present study the favorable prognostic impact by miR-182 was seen in subgroups of NSCLC patients and assessments were tissue specific (only in tumor cells) using in situ hybridization and not real time qPCR as in the pilot study [17]. When using qPCR a contribution from the stromal compartment will influence the result and the stromal expression of miR-182 may be different from that of the tumor cells. Conclusion In miR-182 tended to be a favorable prognostic factor in the total NSCLC cohort. Moreover in stage II and in SCC patients we found miR-182 to have tumor suppressor properties. Nevertheless our study must be regarded as hypotheses generating and needs to be confirmed in other cohorts and functional studies. We found a weak but significant association between mir-182 and the angiogenesis related markers FGF2 and MMP-7. It would be interesting to see further studies exploring these associations. Competing interests The authors declare that they have no competing interests. Authors contributions HS participated in the design of the study contributed to the clinical and demographic database did the statistical analysis and drafted the manuscript. TD SA and SAS contributed to the clinical and demographic database and SAS in making the TMAs. TD and SA contributed to the statistical analysis. SAS and HS scored the cores. RMB and LTB supervised and participated in the study design result interpretation and writing. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/14/138/prepub Acknowledgements The study was solely funded by the Northern Norway Regional Health Authority (Helse Nord RHF) which is responsible for the public hospitals in northern Norway. The funders had no role in study design data collection and analysis decision to publish or preparation of the manuscript. The authors thank the engineers Mona I Pedersen and Marta Uso who carried out the ISH-procedures. Jemal A Siegel R Xu J Ward E Cancer statistics 2010 CA Cancer J Clin 2010 60 277 300 10.3322/caac.20073 20610543 Drilon A Rekhtman N Ladanyi M Paik P Squamous-cell carcinomas of the lung: emerging biology controversies and the promise of targeted therapy Lancet Oncol " | 1 |
Genetic alterations in the 3q263132 locus conferan aggressive prostate cancer phenotypeBenjamin S SimpsonSusan Heavey Jason Pitt5 Caroline M Moore6 Hayley C Whitaker Niedzica Camacho234 Hayley J Luxton Hayley Pye Ron Finn1Largescale genetic aberrations that underpin prostate cancer development and progressionsuch as copynumber alterations CNAs have been described but the consequences ofspeciï¬c changes in many identiï¬ed loci is limited Germline SNPs in the 3q2631 locus areassociated with aggressive prostate cancer and is the location of NAALADL2 a gene overexpressed in aggressive disease The closest gene to NAALADL2 is TBL1XR1 which is implicated in tumour development and progression Using publiclyavailable cancer genomic datawe report that NAALADL2 and TBL1XR1 gainsampliï¬cations are more prevalent in aggressivesubtypes of prostate cancer when compared to primary cohorts In primary disease gainsampliï¬cations occurred in CI and CI for NAALADL2 and TBL1XR1 respectively increasing in frequency in higherGleason grade and stage tumours Gainsampliï¬cations result in transcriptional changes andthe development of a proproliferative and aggressive phenotype These results support apivotal role for copynumber gains in this genetic region Molecular Diagnostics and Therapeutics Group Research Department of Targeted Intervention Division of Surgery Interventional Science UniversityCollege London London UK Human Oncology and Pathogenesis Program Memorial Sloan Kettering Cancer Center New York NY USA MarieJoseand Henry R Kravis Center for Molecular Oncology Memorial Sloan Kettering Cancer Center New York NY USA Department of Pathology MemorialSloan Kettering Cancer Center New York New York for Genomics Research Discovery Sciences Biopharmaceutical RD AstraZeneca Cambridge UK Cancer Institute of Singapore National University of Singapore Singapore Singapore Department of Urology UCLH NHS Foundation Trust London UKemail HayleyWhitakeruclacukCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xProstate cancer PCa is the most common noncutaneouscancer in developed countries12 and is deï¬ned by dynamicgenome alterations and both its pathological and geneticheterogeneity3 An important pathological predictor of prostatecancer aggressiveness is Gleason grade used to assess risk ofprogression and stratify patients for treatment however theunderlying genomic changes which accompany more aggressivetumours remains incompletely deï¬nedOverall copynumber alteration CNA burden has been linkedto poorer prognosis in prostate cancer associating with Gleasongrade biochemical recurrence and prostate cancer speciï¬c deathhowever the exact mechanism driving these prognostic changes isunknown and thought to be primarily driven by general chromosomal instability4 Changes in speciï¬c loci have also beenlinked to aggressiveness in particular gains in proliferative geneseg MYC 8q24 and loss of tumour suppressors PTEN 10q23and NKX31 8p2178 Many genetic alterations have been linkedwith prostate cancer such as point mutations in SPOP FOXA1and IDH19 Largescale oncogenic structural rearrangementstranslocations and copynumber changes are also common oftenleading to the coordinated dysregulation of multiple elements forexample the loss of 21q which is associated with the TMPRSSERG fusion rearrangement and the subsequent rearrangement ofSMAD410 Improved understanding of the mechanisms governing disease pathogenesis and progression may allow for bettertherapeutic exploitation for example genetic alterations in theDNA repair machinery have been linked to susceptibility toPARP inhibitors in a range of tumour types and alterations in ARconfer sensitivity or resistance to androgen deprivation therapy inmetastatic castrate resistant prostate cancer mCRPC11NAALADL2 is located on 3q2631 and is a member of theglutamate carboxypeptidase II family along with the widely studied PCa marker PSMA NAALAD112 and its expression haspreviously been associated with prostate tumour stage andgrade13 with expression predicting poor survival following radicalprostatectomy13 A large genomewide association study GWASof prostate cancer cases found rs78943174 a SNP withinthe 3q2631 NAALADL2 locus was associated with high Gleasonsum score14 A further rs10936845 SNP was identiï¬ed within aGATA2 motif that increases NAALADL2 expression in prostatecancer patients where increased expression also predicted biochemical reccurence15 The same study showed even higherbinding preference to HOXB13 and FOXA1 to this site suggestingcooccupancy by these important transcription factors both ofwhich have been shown to be involved in AR cistromereprogramming1516functionsAdjacent to NAALADL2 in the genome is TBL1XR1 a corecomponent of nuclear receptor corepressor NCoR complex thatacts as a coregulator of nuclear receptors uencing severalcellularandammation17 TBL1XR1 is also an androgen receptor AR coactivator18 Expression of TBL1XR1 has been associated withpoor prognosis in several cancers predicting poor overall survivaland lymph node metastasis in gastric19 and ovarian cancers20 andrecurrence in colorectal21 breast22 and liver cancers23antiapoptosisincludinggrowthHere we utilise largescale publicly available genomic data tobetter characterise the broad somatic copynumber changesoccurring within the 3q263132 locus particularly centredaround gainsampliï¬cations in NAALADL2 and TBL1XR1 andlinking them to the clinical characteristics of aggressive prostatecancerResults3q263132 gain frequency is increased in aggressive PCaCopynumber alterations often alter the expression of the gene inwhich they occur with gene dosage known to correlate withmRNA expression Genetic structural variants are also known toalter transcriptional regulation by altering cisregulatory elementssuch as promotors and enhancers resulting in differentialexpression2425 Increased NAALADL2 and TBL1XR1 expressionhave previously been linked to poor prognosis in cancers leadingus to examine the frequency of somatic copynumber gains inthese genes across various prostate cancer subtypes19Alteration frequency was assessed using data from cBioportalFig 1a and all study data was processed using a standardisedpipeline to ensure comparable results Alteration frequency wasassessed in a total of patients samples in nonoverlapping studies Appendix eleven studies focused onprimary prostate cancer four on metastatic prostate cancer andone on neuroendocrine and castrateresistant cancers Signiï¬cantcopynumber increases above a derived background thresholdwere categorised as gains and copynumber decreases as deletions Overall the distribution of NAALADL2 and TBL1XR1alterations were significantly different between disease subtypesto that which is expected Chisquared goodnessofï¬ttestFig Somatic alteration frequency of NAALADL2 and TBL1XR1 across prostate cancer subtypes in publically available genomic studies n a NAALADL2 genetic alteration frequency across different subtypes of prostate cancer b TBL1XR1 genetic alteration frequency across differentsubtypes of prostate cancer P primary prostate cancer M metastatic prostate cancer NE neuroendocrine prostate cancer and castrate resistantprostate cancer CRPC All annotations were assigned using Genome Nexus and CNAs are called using GISTIC or RAE algorithms Pvalues show theresults of a Chisquared goodnessofï¬t test to determine if the number of observed patients with each alteration type is different from that which isexpected across each cancer subtype Results detailed in Supplementary data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xp and p Fig 1a b with gains beingmost frequent in castrateresistant prostate cancer and respectively followed by neuroendocrine and metastatic and then primary prostatecancer and Fig 1a b3q263132 gains extend across an oncogenerich region ofChr3 As CNAs are known to associate with more aggressivesubtypes of prostate cancer we investigated their association withclinical characteristics to establish if changes can be detected earlyin the life history of cancer predicting more aggressive diseaseWe utilised copynumber data from primary an conï¬neddisease from both the UK and Canadian International CancerGenome Consortium ICGC cohorts and The Cancer GenomeAtlas TCGA These studies use intermediatehigh risk prostatecancer patients with no treatment prior to radical prostatectomyTo allow comparisons between the studies data were reanalysedusing the Genomic Identiï¬cation of Signiï¬cant Targets in Cancer GISTIC2 methodfavoured by the broad institute andTCGA27 as it distinguishes between lowlevel copy numberincreases gains and highlevel copynumber increases ampliï¬cations Within the three cohorts we found that copynumbergains across both genes were frequent with gains in NAALADL2ranging from Canada to UK and between for ampliï¬cations Table TBL1XR1 had an almostidentical CNA frequency of between UK to Canada Table We ï¬tted a randomeffects model to more accurately estimatethe frequency of NAALADL2 and TBL1XR1 gainampliï¬cationscombining the data from all three cohorts which estimated thetrue frequencies to be CI and CI for NAALADL2 and TBL1XR1 respectively Supplementary Fig Leaveone out analysis and adiagnostic plots revealed that the ICGC Canada study was asignificant source of heterogeneity thereforethe study wasremoved and the model reï¬t The ï¬nal estimated frequency ofNAALADL2 and TBL1XR1 gainsampliï¬cations was CI and CI respectivelyin primary prostate cancerDue to their close proximity in the genome we investigated ifgainsampliï¬cations in NAALADL2 and TBL1XR1 cooccurred inthe same patients using a genomewide Fishers exact test with afalse discovery rate correction NAALADL2 and TBL1XR1significantly coampliï¬ed in all three cohorts ICGC UK p ICGC Canada p 158e and TCGA p testingconï¬rmed that widespanning gainsampliï¬cations occurred inneighbouring regions in the majority of patients In the ICGC UKSupplementary Fig Additionallycohort n there was a significant cooccurrence of somaticcopynumber gainsampliï¬cations in NAALADL2 with TBL1XR1FDRcorrected Fishers exact test Fig 2a Gainsin this region also significantly correlated with two regionsspanning chromosomes and both gains previously describedas being abundant in prostate cancer Supplementary Data The Canadian cohort n showed a similar pattern of cooccurrence with gainsampliï¬cations spanning the region surrounding NAALADL2 and TBL1XR1 3p253 to 3q29 Fig 2bThere was also a significant cooccurrence with gains in thebeginning of chromosome as well as some sporadic cooccurrence across the genome Fig 2b Supplementary Data These results were supported by the outcome of the same analysisin the TCGA cohort n although several large spikes ofcooccurrence were also observed in regions nottoNAALADL2 and TBL1XR1 as these spikes were not present inthe other two cohorts they most likely represent artefacts Fig 2cSupplementary Data Overall across the three cohorts therewere was a consistent coampliï¬cation in region spanning genes between 3p141 and 3q29 While a number of patients hadmultiple CNAs we found no consistent cooccurrence withcommon CNAs such as MYC gain FGFR1 gain PTEN loss RB1loss or NKX31 loss FDRcorrected Fishers exact test p The 3q26 region where NAALADL2 and TBL1XR1 are locatedis rich in oncogenes such as PIK3CA SOX2 ECT2 and PRKCIwhich may act to drive tumorigenesis29 We determined thenumber of known oncogenes within this deï¬ned region bycomparing the overlapping genes that coampliï¬ed withNAALADL2 and TBL1XR1 in alltheNetwork of Cancer Genes database30 This revealed that of genes are known oncogenes including BCL6 ATRand PI3K family members Supplementary Data These resultsconï¬rm that a high proportion of prostate cancer patientsdevelop large copynumber gains across multiple oncogenes inthis genetic regionthree cohorts againstlocalGains in 3q263132 associate with adverse clinical featuresCommon prostate cancer CNAs such as those in MYC andPTEN are known to associate with higher Gleason grade31Consistent with these ï¬ndings we also found NAALADL2 andTBL1XR1 ampliï¬cations were highly correlated with GradeGroup GG showing that the frequency of NAALADL2 andTBL1XR1 gains tripling between GG1 and GG2 lesions and morethan doubling between GG2 and Table A Chisquaredgoodnessofï¬t test showed that the distribution of gainsampliï¬cations between Grade groups was significantly different to thedistribution of diploid patientsfor both NAALADL2 andTBL1XR1 p and p WhenTable Alteration frequency of NAALADL2 and TBL1XR1 called via the GISTIC2 method in three nonoverlapping primary anconï¬ned radical prostatectomy cohorts from the International Cancer Genome Consortium ICGC and The Cancer GenomeAtlas TCGAICGC UKICGC CANADATCGANAALADL2TBL1XR1NAALADL2TBL1XR1NAALADL2TBL1XR1AlterationDeep DelShallow DelDiploidGainAmpliï¬cationTotalnnnnnnThe degree of copy number alteration is discretised into ï¬ve categories ampliï¬cation gain representing low and high level copy number increase diploid no significant CNA and shallow and deepdeletion representing low and high level copy number lossCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xand compared to those without and Moreover ofthe patients who had their lymph nodesexamined the percentage of patients with lymph node positivitydeï¬ned through positivity on haematoxylin and eosin stainingHE was more than double in patients with NAALADL2 orTBL1XR1 gains and compared to those withouta gain and Chisquared goodnessofï¬t test p and p Finally while only one man in the cohorthad evidence of positive ï¬ndings in his bone scan we did observea significant between the number of equivocal bone scans inpatients with gains and compared to and in those patients without gainsChisquaredgoodnessofï¬t test p and p for NAALADL2and TBL1XR1 respectively however the number of expectedcases in each of these categories was less than adding someuncertainty to this result We found no significant difference inthe mean age between patients with different copynumbers ofNAALADL2 or TBL1XR1 KruskallWallis rank sum test p and As gainsampliï¬cations in NAALADL2 and TBL1XR1 coincidewith a cluster of known oncogenes and coincide with clinicalvariables linked to more aggressive disease we also compareddiseasefree survival Comparing patients with gainsampliï¬cations in NAALADL2 and TBLXR1 to those with diploid copies weobserved no significant association in the ICGC UK cohort n although there was a trend towards reduced diseasefreesurvival Supplementary Fig 4A In the larger TCGA cohortn there was a significant reduction in diseasefree survivalin patients with a gain in either NAALADL2 Logrank MantelCox p or TBL1XR1 Logrank MantelCox p Supplementary Fig 4BUnivariable Cox regression conï¬rmed that carrying a gainampliï¬cation in NAALADL2 and TBL1XR1 in the TCGA cohortresulted in reduction in diseasefree survival hazard ratio HR CI p Forreference weperformed a similar analysis of patients with PTEN deletion orMYC gains two common copy number alterations with provenin prostate cancer3233association with diseasefree survivalWhen patients were stratiï¬ed solely by CNA status and survivalcompared using the KaplanMeier method those patients withMYC gain or PTEN deletion homo or hemizygous showed nosignificant difference in diseasefree survival Logrank MantelCox p and p respectively while those stratiï¬ed byNAALADL2 gain TBL1XR1 gain or both NAALADL2 andTBL1XR1 gain showed significant differences in survival Logrank MantelCox p Supplementary Fig 5AE Univariable Cox regression estimated the hazard ratios for thesecopynumber alterations as CI CI and CI for MYCPTEN and NAALADL2TBL1XR1 respectively We also comparedthe diseasefree survival of patients with only a copynumberalteration in each of the four genes where each group wasmutually exclusive Supplementary Fig 5F G This showed thaton the whole patients with CNAs in NAALADL2TBL1XR1 hadreduced or equal diseasefree survival as those with either onlyMYC gain or only PTEN loss Patients with copy number gains inboth had a worse prognosis All clinical data is available inSupplementary Data Fig Genomewide cooccurrence with NAALADL2 and TBL1XR1 gainsampliï¬cations The Y axis shows log10 qvalues from a Fishers exact testbetween gainampliï¬cations in NAALADL2 and cooccuring genes Thedotted line represents the threshold for statistical signiï¬cance aftercorrection for multiple testing a Signiï¬cantly cooccurring gains across thegenome in the ICGC UK cohort b Signiï¬cantly cooccurring gains acrossthe genome in the ICGC Canada cohort c Signiï¬cantly cooccurring gainsacross the genome in the TCGA cohort NAALADL2 and TBL1XR1 cytobandpositions are labelled All Fisher tests use NAALADL2 gain or ampliï¬cationas the altered group Full results are detailed in Supplementary Data compared to common CNAs such as PTEN loss and MYC gainthe alteration frequency of NAALADL2 and TBL1XR1 was morecorrelated with higher Gleason grade groups Spearmans rho was p p for NAALADL2 and TBL1XR1and p for PTEN and MYC respectivelySupplementary Fig 3AMoreover we also noted the same pattern ofincreasingfrequency of gains with T stage Chisquared goodnessofï¬t testp and p respectively Table Patients with gains exhibited differences in the location of thetumour within the prostate with and of thosewith NAALADL2 and TBL1XR1 gains having tumoursinoverlapping and multiple zones compared to just and for those without gains Chisquared goodnessofï¬t testp and p There was also an increased relativenumber of positive surgical margins Chisquared goodnessofï¬ttest p and p in patients with gains As CNAs in NAALADL2 and TBL1XR1 were associated withclinical characteristics such as Gleason grade group and T stagewe used multivariable Cox regression models to conï¬rm that anychanges in survival were driven by these associations and foundthat copy number gains in NAALADL2 and TBL1XR1 were nolonger significant once corrected for Gleason grade and T stagep Supplementary Data These results suggest thatthe differences in diseasefree survival seen when stratiï¬ed byCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xTable The frequency of NAALADL2 and TBL1XR1 gainampliï¬cations by Gleason Grade Group in the TCGA cohortGrade groupGG1ObservedExpected within GGGG2ObservedExpected within GGGG3ObservedExpected within GGGG4ObservedExpected within GGGG5ObservedExpected within GGTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalGrade groups deï¬ned as Grade Group Gleason score ¤ Grade Group Gleason score Grade Group Gleason score Grade Group Gleason score Grade Group Gleason scores and Displayed are the numbers of patients observed with gain or without diploid a gainampliï¬cation in this region in each Grade Group Additionally the expected number ofpatients estimated to be within each category is also shown along with the percentage of each Grade Group which is made up by patients with or without a gain Bold values indicate the overallpercentage of the group with a given copynumber state All clinical data detailed in Supplementary Data Table The frequency of NAALADL2 and TBL1XR1 gainampliï¬cations by T stage in the TCGA cohortT stageT2aObservedExpected within T stageT2bObservedExpected within T stageT2cObservedExpected within T stageT3aObservedExpected within T stageT3bObservedExpected within T stageT4ObservedExpected within T stageTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalDisplayed are the numbers of patients observed with gain or without diploid a gainampliï¬cation in this region in each T stage Additionally the expected number of patients estimated to be withineach category is also shown along with the percentage of each T stage which is made up by patients with or without a gain Bold values indicate the overall percentage of the group with a given copynumber state All clinical data detailed in Supplementary Data COMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xgainampliï¬cation status are driven by strong association withthese clinical variablesIn the ICGC cohortsindividuals with somatic singlebasealterations in NAALADL2 also associated with reduced diseasefree survival in a combined ICGC cohort as well as associatingwith reduced diseasefree and overall survival in an early onsetprostate cancer cohort ICGC EOPC Denmark Singlebasesubstitutions in TBL1XR1 were only associated with diseasefreesurvival in the ICGC EOPC cohort Supplementary Fig Singlebase alterations did not occur with a frequency greater than onein any single base in NAALADL2 or TBL1XR13q263132 gains cooccur with proproliferative transcriptionTo determine the potentialfunctional consequences of gainswithin the NAALADL2 and TBL1XR1 amplicon mRNA expression proï¬les were explored using the TCGA RNAseq dataDESeq2 was used to determine differentially expressed genesbetween patients with copynumber gains for both NAALADL2and TBL1XR1 compared to those without For NAALADL2 therewere differentially expressed genes DEGs and DEGsfor TBL1XR1 when the two groups were compared FDR Supplementary Data Our previous study on NAALADL2identiï¬ed nine genes which were reciprocally regulated by overexpression or knockdown of NAALADL226 Of these nine wefound that three cancer antigen XAGE1B adhesionmotiliy regulator SPON2 and AR regulator HN1 were significantly differentially expressed p in patients with a NAALADL2 gainand in the same direction as the overexpression model2634When comparing the DEGs between patients with and withoutin either NAALADL2 or TBL1XR1 wegainsampliï¬cationsobserved that of the DEGs overlapped between NAALADL2 and TBL1XR1 Fig 3a of the geneswere located within the locus we identiï¬ed as coampliï¬ed withNAALADL2 and TBL1XR1 and were differentially expressedconsistent with a mechanism of selfregulating expression2425TBL1XR1 was one ofsignificant overlapping DEGsNAALADL2 was just on the boundary of statistical signiï¬canceFDR corrected Wald test p Supplementary Fig theNAALADL2 has been shown to be coexpressed with numberof androgen regulated proteins and contains a number of ARbinding sites and TBL1XR1 is an AR coactivator and may beinvolved in AR cistrome reprogramming18263738 We thereforelooked at overlap between androgen regulated genes with ARbinding sites full or partial and genes demonstrated to beandrogen regulated following R1881 stimulation in at least twoindependent studies3739 shared genes were differentiallyexpressed in patients with NAALADL2 and TBL1XR1 gainsampliï¬cations that contained AR binding sites and demonstratedandrogen regulation by R1881 genes had either aAR binding motif were androgen regulated in two or morestudies or both Fig 3bOf the overlapping DEGs a total of were knownoncogenesSupplementary Data which may drive anaggressive clinical phenotype Of note was PI3K family membersPIK3C2G PIK3CA PIK3CB PIK3R4 Mucin family membersMUC1 MUC4 and MUC6 and other prostate cancer associatedgenes such as SMAD4 SOX9 and SPOP794041 Additionallyseveral genes which form commercial prognostic assays were alsodifferentially expressed such as the Decipher assay NFIB LASP1ZWILCH THBS2 COL1A2 and COL5A142 Oncotype DX assaySFRP4 COL1A1 KLK2 TPX24344 and the Prolaris assayASPM BUB1B CENPF and FOXM145We inspected of the top most significant shared DEGs usingunsupervised hierarchal clustering Fig 3b SupplementaryData DEGs mostly displayed upregulation consistent with agenedosage effect Fig 3b24 Enrichment for biological processes was assessed by Geneset enrichment analysis GSEA forNAALADL2 and TBL1XR1 gainsseparately and by overrepresentation analysis ORA on the shared DEG list usingWebGestalt46GSEA on the individual lists of DEGs showed that despite a largeoverlap the enriched biological processes did differ between the twogenes patients with a gain in NAALADL2 showed enrichment inprocesses related to NADH dehydrogenase complex assemblyFDR mitochondrial respiratory chain complex assemblyFDR translational initiation FDR cytochromecomplex assembly FDR protein localisation toendoplasmic reticulum FDR and cytoplasmic translationFDR Supplementary Data Patients with a gain inTBL1XR1 showed enrichment in mitotic cell cycle phase transitionchromosome segregation actin ï¬lamentbased movement microtubule cytoskeleton anisation involved in mitosis regulation ofcell cycle phase transition cell cycle G1S phase transition FDR as well as a number of other processes SupplementaryData To understand the combined effect of gainsampliï¬cation inthese genes we investigated overrepresentation of processes in theDEGs which were common to both NAALADL2 and TBL1XR1In the shared DEG list the significantly enriched Gene OntologyGO biological processes were all involved in the cell cycle cyclepathway including mitotic regulation and chromosome segregation Fig 3c Supplementary Data These ï¬ndings support ahypothesis whereby gains in NAALADL2 and TBL1XR1 concomitantly bring about mRNA expression changes which supportan aggressive proproliferative phenotype in primary prostatecancerDiscussionIn this study we present evidence that somatic copynumber gainsin NAALADL2 and TBL1XR1 are more frequent in high gradeand aggressive forms of prostate cancer These results are bolstered by studies which have identiï¬ed CNAs in this region inmCRPC however to our knowledge this is the ï¬rst time thesegains have been reported in neuroendocrine disease47 We alsodemonstrate that NAALADL2 and TBL1XR1 gains occur in anearlier setting cooccurring with gains in neighbouring genes Amajor barrier to the adoption of CNA based tests in the clinic isthe reliance on expensive NGS approaches as well as sufï¬cientsequencing depth and coverage to assess overall copynumberburden The discovery of smaller clinically significant loci couldallow for cheaper quicker targeted approaches particularly if asingle loci can elude to gainsampliï¬cations in a larger regionsurvivalto diseasefreeIn primary prostate cancer Gainsampliï¬cations in this regionassociated with Gleason grade tumour stage number of positivelymph nodes bone scan results and as these variables contributetostratiï¬ed byNAALADL2TBL1XR1 status also have altered diseasefree survival times Our work is supported by previous studies that haveeluded to the clinical signiï¬cance of this locus particularly asgermline SNPs within this locus have been associated with higherGleason grade tumours and more aggressive disease14 This alsosupports smaller studies such as those by HeselmeyerHaddadet al who identiï¬ed two out of seven patients with gains inTBL1XR1 in recurrent prostate cancer48 However these studiesinvestigated these genes in isolation na¯ve to the larger context inwhich these alterations occur Here we have found that gainsampliï¬cations atthis locus not only coamplify with otherdescribed oncogenes but associate with much larger transcriptional changes which are consistent with the observed aggressiveclinical phenotypetimepatientsCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xFig Transcriptomic changes in patients with NAALADL2TBL1XR1 gains a Venn diagram showing the number and percentage of overlapping DEGsbetween patients with NAALADL2 gainampliï¬cation and TBL1XR1 gainampliï¬cation overlap b Venn diagram showing the number ofNAALADL2 and TBL1XR1 DEGs and genes with identiï¬ed AR binding sites determined through ChIPSeq and AR knockdown and genes shown to beandrogen regulated following R1881 stimulation c Unsupervised hierarchal clustering of the top most significant DEGs bar beneath upper dendrogramshows copynumber status of patients where red is patients with a gain in both NAALADL2 and TBL1XR1 and grey represents those without gainampliï¬cation in these genes Heatmap represents meancentred z scores derived from RKPM values d Chord diagram showing significantly overrepresented GO biological processes and key genes within these processes All clinical data detailed in Supplementary DataCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xOverall changes in copynumber burden have been shown to beindicative of genetically unstable tumours and predict prostatecancer relapse5 Many single CNAs have already been describedthat predict PSA recurrence after radical prostatectomy includingPTEN loss cooccurrence of PTEN FAS 10q2331 and PAPSS210q23210q2331 loss a loss of 16q with or without a loss ofPTEN a loss within 6q 13q gains in MYC 11q1317 7q and aconcurrent loss of 8p22 with a gain of 8q2487 Compared towellknown CNAs such as PTEN loss and MYC ampliï¬cation wehave observed that Gainsampliï¬cations in NAALADL2TBL1XR1equally or better segregate patients who will have reduced diseasefree survivalThe gainsampliï¬cations in NAALADL2TBL1XR1 also corresponded to a significantincrease in both NAALADL2 andTBL1XR1 mRNA supporting previous studies that have described upregulation of these genes and linked them to poor prognosis in various cancers19 This suggests that gains in thesegenes may cause increased expression of NAALADL2 andTBL1XR1 in cancers We also noted a number of the differentiallyexpressed genes between patients with and without a gainampliï¬cation in NAALADL2TBL1XR1 have been shown to beandrogen regulated however further work is required to determine if gainsampliï¬cations in this region cause changes in ARtranscriptional regulation through cis regulatory elements or as adirect consequence of the genes altered in this region1837In those patients with these gains we noted transcriptionalchanges in several genes associated with aggressive prostatecancer including differential expression of genes appertaining toprognostic assays such as Decipher Oncotype DX and Prolaris aswell as families such as mucins50 This may explain theaggressive clinical phenotype observed in these patients We alsoobserved that when weighted individually there were differencesin enrichment of biological processes between those with NAALADL2 gains and TBL1XR1 gains suggesting that each generesults in some unique cellular changesOur ï¬nding that gains in the 3q26 locus result in concurrentexpression of oncogenes located within this region and theirdownstream targets identiï¬es multiple potential therapeutic avenues warranting further investigations This study centred aroundtwo genes NAALADL2 and TBL1XR1 both of which areattractive therapeutic targets with TBL1XR1 previously suggestedas a potential cancer target operating via the TGFβ signallingpathway and potentially regulating AR signalling5354 Additionally the tumour speciï¬city of NAALADL2 and basal membranouslocalisation makes it potentially accessible using antibodydrugconjugates13 This approach may be feasible if like other familymembers such as PSMA antibody binding results in subcellularinternalisation12 Moreover several of the oncogenes in whichgains cooccur as well as the downstream oncogenes activatedfrom gains in the 3q26 region such as ATR PI3K family members PIK3C2G PIK3CA PIK3CB PIK3R4 MUC4 BCL6 SOX9can be therapeutically targeted or have been suggested as therapeutic targets in cancer5155 In the PI3K pathway PIK3CBspeciï¬c inhibitors may have utility in patients with mutationsampliï¬cations andor fusion of this gene59 These ï¬ndings mayhave clinical relevence as it has been reported by de Bono et althat many individuals who had durable year responses toPIK3CBspeciï¬c inhibition harboured activating mutation orampliï¬cation in PIK3CB60 and phase II trials of ipatasertib anAkt inhibitor targeting the PI3KAkt axis has shown promise inlate stage mCRPC61 Together our results suggest that largescalegenomic gainsampliï¬cations occur in the 3q26 region in a hi | 2 |
" nlr plr and lmr have been associated with pancreatic ductal adenocarcinoma pdac survivalprognostic value and optimal cutpoints were evaluated to identify underlying significance in surgical pdac patientsmethods nlr plr and lmr preoperative values were available for pdac patients who underwent resectionbetween and os rfs and survival probability estimates were calculated by univariate multivariable andkaplanmeier analyses continuous and dichotomized ratio analysis determined bestfit cutpoints and assessed ratiocomponents to determine primary driversresults elevated nlr and plr and decreased lmr represented and of the cohort respectively osp and rfs p were significantly decreased in resected pdac patients with nlr ¥ compared to thosewith nlr optimal prognostic os and rfs cutpoints for nlr plr and lmr were and respectivelylymphocytes alone were the primary prognostic driver of nlr demonstrating identical survival to nlrs nlr is a significant predictor of os and rfs with lymphocytes alone as its primary driver weidentified optimal cutpoints that may direct future investigation of their prognostic value this study contributes tothe growing evidence of immune system influence on outcomes in earlystage pancreatic cancerkeywords neutrophil lymphocyte ratio platelet lymphocyte ratio lymphocyte monocyte ratio pancreatic cancerbiomarker correspondence mokengemalafamoffitt1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usafull 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 0cpointer bmc cancer page of pancreatic ductal adenocarcinoma pdac is the thirdleading cause of cancerrelated death in the us with anestimated deaths in and a 5year overall survival os rate of among newly diagnosed pdacpatients only to present with resectable diseasewith resection as the only chance for cure prognosis isgenerally poor with reported 5year os of afterresection [] ajcc tnm staging is the only widelyaccepted indicator of prognosis for resectable pancreaticcancer however its performance in earlystage diseasehas been questioned additionally controversy regarding initial treatment of earlystage pancreatic cancerpersists yielding no uniform treatment algorithm giventhe wide variation in the biological behavior of pdacand treatment algorithms for this disease there is an unmet need for enhanced prognostic biomarkers biomarkers derived from easily obtainable laboratory valueshave shown potential to meet this need and may help tostratify patients with earlystage pancreatic cancer andguide future treatment plansconventionally survival outcomes among cancer patients have been determined by the disease stage and receipt of treatment more recently howeverincreasedattention has been directed toward the role of inflammation and immune response in the tumor microenvironment and their effects on tumor behavior quantifyingthe systemic inflammatory response by creactive protein and various nutritional parameters has shown prognostic significance in gastrointestinal gynecological andthoracic cancers additionally inflammatory indicesand immunologic ratios including ratios comprised ofintratumoral or circulating neutrophils plateletslymphocytes and monocyte counts have been proposed tobe prognostic biomarkers for a wide range of malignancies []the neutrophil to lymphocyte ratio nlr platelet tolymphocyte ratio plr and lymphocyte to monocyte ratio lmr are among the many surrogate biomarkers forinflammation that have been associated with outcomesin gastrointestinal cancers although these ratios havebeen reported to have promising prognostic value fewstudies have examined the effect of these inflammatoryratios in us surgical cohorts [] moreover manysingleinstitution studies have reported inconsistentprognostic outcomes for these surrogate biomarkers wepreviously reported an inverse association between survival and nlr in patients with borderline resectable disease to expand the scope of our previous analysiswe evaluated the prognostic significance of the nlrplr and lmr in a cohort of patients with resectedpdac who were treated at a highvolume cancer centerfurthermore we aimed to establish optimal nlr plrand lmr cutpoints for determining os and recurrencefree survival rfs and define the primary factor drivingthe prognostic value of these ratios for survival outcomes we hypothesized that preoperatively increasednlr and plr and decreased lmr were associated withworse os in patients with resectable pdacmethodsa retrospective review was conducted using our institutional prospective pancreatic cancer database as part ofour ongoing outcomebased study the study was approved by our institutional review board mcc16446and patient consent was unable to be obtained as thisstudy was conducted retrospectively on deidentified dataposing less than minimal risk patients diagnosed withpdac who underwent curativeintent resection for thetreatment of their disease were identified resectable andborderline resectable pdac patients were defined and included on the basis of the nccn guidelines applied at thetime of diagnosis pancreatic resection included open orminimally invasive pancreaticoduodenectomy total pancreatectomy and distal pancreatectomy performed at ourinstitutionpatient characteristics were summarized using descriptive statistics including median and range for continuous measures and proportions and frequenciesforcategorical measures kaplanmeier plots were made todetermine os and rfs for the nlr plr and lmrsurvival probability estimates were calculated using thekaplanmeier method univariate and multivariable coxproportionalhazard models for os and rfs were runfor each ratio as continuous predictors and dichotomized forms the nlr plr and lmr were calculatedby dividing the absolute neutrophil count by thelymphocyte count the platelet count by the lymphocytecount and the lymphocyte count by the monocytecount respectively dichotomized analyses included neutrophil and lymphocyte counts and percentages whichwere defined as the proportion of neutrophils or lymphocytes to all white blood cells in the sample valuesused for these calculations were part of the last completeblood count and differential obtained after neoadjuvanttherapy and before operative intervention cutpoints of and were used for nlr plr and lmr respectively nlr cutpoints were determined on the basisof values used in previously published studies [ ]cutpoints for plr and lmr were not well establishedtherefore the medians of the observed data were usedoptimal nlr plr and lmr cutpoints for the prediction of os and rfs were determined using maximallyselected rank statistics based on the logrank method the resulting cutpoint for each ratio provided thebest separation of the responses into groups in whichthe standardized rank statistics take their maximumthe p value approximation was based on the improved 0cpointer bmc cancer page of bonferroni inequality variables were evaluated inrelation to os and rfs for predetermined cutpoints andnewly identified bestfit cutpoints all analyses were performed using r software version resultsa total of patients treated at our institution between and were eligible for this study two hundredseventyseven patients with complete data met the inclusion criteria and were included in the analysis the meanage was ± years of whom were maletwentyfive percent of patients had a charlson comorbidity index cci ¤ had a cci of to and had a cci ¥ medicare with a private supplement wasthe largest represented insurance provider among patients sixtyfour percent of our cohort was classified as resectable and treated with upfront resection and received neoadjuvant systemic therapy marginnegative r0 resection was achieved in of our patients with and demonstrating lymphovascularand perineural invasion respectively table mean preoperative nlr plr and lmr was ± ± and ± respectively additional file using the predetermined cutpoints described above and of patients demonstrated preoperative nlr ¥ plr ¥ and lmr ¤ respectivelyos was significantly shorter among patients with annlr ¥ than patients with an nlr in univariatehr [ ci ] p and multivariable hr [ ci ] p analysestable neither the plr nor lmr demonstrated a significant association with os table and fig patients with a high nlr also demonstrated significantlyworse rfs in univariate hr [ ci ]p and multivariable hr [ ci ] p analyses table and fig this wasnot observed with plr or lmr in multivariable analyses pathologic t stage presence of grade complications cci ¥ nlr node positivity and perineuralinvasion were found to be significant predictors of osand rfs tables and maximally selected rank analyses of nlr plr andlmr were performed to identify optimal cutpoints forpredicting os and rfs os optimal cutpoints for nlrplr and lmr were and respectively forrfs cutpoints were and respectively because neutrophil percentage is highly correlated with nlrwe found the corresponding cutpoint for determining ahigh neutrophil percentage to be resulting in patients being above the cutpoint similarly lymphocytepercentage was highly negatively correlated with nlrwith a corresponding cutpoint percentage of thecomponents of nlr was analyzed separately to evaluatetheir prognostic importance the lymphocyte percentagealone yielded a survival curve that was identical to that ofthe nlr whereas the neutrophil percentage km plot wasnot statistically significant additional file discussionwe demonstrated a statistically significant associationbetween preoperative nlr and both os and rfs inpdac patients who underwent curativeintent resectionat a highvolume cancer center plr and lmr failed todemonstrate any correlation with survival in additionwe identified optimal cutpoints for immunologic ratiosurvival analyses on the basis of our cohort data finallywe identified the lymphocyte component of nlr to bethe primary driver of survival prognosis to our knowledge this is the largest us cohort utilized to analyzeimmunologic ratio biomarkerassociated outcomes andperform dichotomized analyses for the purpose of identifying the prognostic driver of the nlr in surgical pdacpatientsinflammation and the inflammatory response have beendiscussed extensively in the literature in relation to tumorigenesis progression and metastasis furthermorelinkshave been established between the inflammatory responseand oncogenic signaling pathway interactionstumormicroenvironment analyses and use of immunetargetedtherapies surrogate biomarkers of inflammation haveproven useful in predicting disease progression recurrenceand overall prognosis across a wide range of malignancies[ ] in a metaanalysis evaluating the role of thesystemic immuneinflammation index zhong showedthat an elevated systemic immuneinflammation index isassociated with worse os in hepatocellular carcinomaurinary cancers gastrointestinal cancers and smallcell lungcancer in a review of patients with gastrointestinalmalignancies nora demonstrated nlr and plr to besignificant predictors of lymph node positivity metastaticdisease and recurrence especially when used in combination the use of the nlr plr and lmr have shownpromise in pancreatic adenocarcinoma demonstratingprognostic value in both resectable and palliative populations [ ]the nlr has shown substantial potential for prognostic utility in pancreatic adenocarcinoma patients in alarge retrospective analysis of surgical pdac patients alow nlr was associated with longer median survival vs months p and an nlr ¥ independently predicted poor prognosis hr [ ci] p giakoustidis further explored pretreatment nlr in surgical pdac patients andidentified decreased os rates to be associated with a highnlr in univariate analyses which maintained independent prognostic significance in multivariable analyses two recent metaanalyses including a total of patients have also suggested an association between 0cpointer bmc cancer page of table descriptive statistics of study cohortsnlr demographicsn overalln age median range ynlr ¥ n plr n pvalue plr ¥ n lmr ¤ n pvalue lmr n sex no femalemalerace no blackotherwhite bmi median range cci no ¥ tumor sizepathologic stage no t0t1 no t2 no t3preoperative resectabilityno neoadjuvant therapy nonoyesmargin no negativepositivelymphovascular invasionno pvalue borderlineresectable noyes perineural invasion no noyes complication 34a no noyes completion of adjuvanttherapy no 0cpointer bmc cancer page of table descriptive statistics of study cohorts continuednlr ¥ demographicsn nlr n overalln nopvalueplr n plr ¥ n pvaluelmr ¤ n lmr n pvalueyes aclaviendindo classification of surgical complicationsabbreviations bmi body mass index nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratio cci charlesoncomorbidity indexnlr and os in which elevated nlr carried poor prognoses zhou found elevated nlr to be associatedwith shorter rates of os hr [ ci ]p and diseasefree survival hr [ ci] p evaluating os alone mowbray also demonstrated that significantly shorter rates ofos were associated with elevated nlr hr [ci ] p we corroborated these results in our own resected pdac patients and similarlydemonstrated that decreased rates of os were associatedwith an nlr ¥ in multivariable analyses additionallywe showed a significant association between hightable univariate and multivariate cox proportional hazard models for overall survivalvariablep valueunivariate analysishr cimultivariable analysishr ciap valuegenderfemalemaleage¤ pathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananananana reference reference reference nanananananananananananananacomplication grade 4bpositive nodesamodel includes age gender pathologic stage cci complication score nlr nodal and perineural invasion status b claviendindo classification ofsurgical complicationsabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte rationananana reference reference reference reference reference nananana reference nananana 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating overall survival in a nlr b plr and c lmrpreoperative nlr and a decrease in rfs our study further supports the nlr as a valid prognostic biomarkerfor earlystage pdacalthough a cutpoint of has been widely used to define highlow nlr variations in cutpoints exists withsome groups using values ranging from to [ ] with no clearly defined cutpoint we chose to perform a continuous analysis to identify an optimal cutpoint for the nlr in relation to survival based solely onthe data from our cohort optimal cutpoints of foros and for rfs were obtained our study supportsthe prognostic value of the commonly used nlr cutpoint of as the nlr was the only significant ratio inour cohort we elucidated its prognostic driver by analyzing the components of the ratio the denominatorthe lymphocyte count percentage alone yielded a survival curve identical to the nlr whereas the numeratorthe isolated neutrophil count percentage was not statistically significant suggesting that lymphocyte count percentages have equal prognostic value and perhaps offera simpler alternative to the nlr biomarker this findingis supported by those from previous studies that showedlow lymphocyte counts to be poor prognostic indicatorsin pancreatic and colorectal cancers [] the finding also has immunotherapeutic implications which corroborate basic science findings on a population level[]in contrast to our study other studies have found noprognostic significance of the nlr in some pdac patient populations recently chawla described a cohort of resectable pdac patients whose nlr atdiagnosis did not correspond to os jamieson similarly reported patients who underwent pdacresection and found no relationship between nlr and 0cpointer bmc cancer table univariate and multivariate cox proportionalhazard models for recurrencefree survivalvariablep valueunivariate analysishr cimultivariable analysishr ciapage of p value reference reference reference reference nananana reference nanagenderfemalemalepathologic staget0t1t2t3ccinlr ¥ plr ¥ lmr ¥ perineural invasionnoyesnananananananana reference reference reference nanananananananacomplication grade 4bpositive nodesabbreviations cci charlson comorbidity score nlr neutrophil to lymphocyte ratio plr platelet to lymphocyte ratio lmr lymphocyte to monocyte ratioa model includes age gender pathologic stage cci complication score nlr nodal and perineural invasion statusb claviendindo classification of surgical complicationsnanananasurvival similar findings have been reported byother groups [ ] the reasons for this variability include diverse patient populations differences in ratiocutpoints timing of blood collections and receipt ofneoadjuvant therapy in the current study of patients received neoadjuvant therapy before pancreatic resection which may havecellpopulationsinfluenced immuneincreased monocyte presence in the tumor microenvironment or in circulation has been implicated inangiogenesis tumor growth and poor prognosis in cancer patients circulating monocytes are commonlyquantified by the lmr which has demonstrated an inverse association with survival and prognosis in solidtumor malignancies few studies have investigatedthis parameter in surgical pdac patients in a large review and metaanalysis of patients li reported a favorable prognosis associated with elevatedlmr in pooled analyses hr [ ci ]p although this study included a range oflmr cutpoints and both resected and nonoperablepdac patients a prognostic value of the lmr was observed in surgical patients in subgroup analyses sierzega reported a series of resectable pdacpatients demonstrating prolonged median survival vs months p in the lmr ¥ group anlmr was an independent predictor of poor prognosis hr [ ci ] p in contrastaldemonstrated no association between lmr and os ordiseasefree survival in a large retrospective analysis ofthe prognostic effects of patientspecific nutritional andimmunologic factors in resected pdac patients we also did not show a prognostic value of lmr in ouranalyses of resected pdac patients differences in prognostic outcomes were likely due to the paucity of dataevaluating lmr and survival inconsistency in evaluatedpatient cohorts and variation of cutpoint delineationto studies previously discussed abeet 0cpointer bmc cancer page of fig kaplanmeier plot demonstrating recurrencefree survival in a nlr b plr and c lmrwe used mean values for lmr cutpoints in our analysesbecause of the variation of cutpoints reported in the literature an optimal cutpoint analysis of lmr for osand rfs was performed to clarify the reporting of lmrassociated outcomessurvival outcomes have similarly been linked to elevated plr in solid tumor malignancies comparedto other commonly described ratios the application ofplr to pdac is less clear with mixed outcomes reported giakoustidis also investigated pretreatmentplr in surgical pdac patients and identified decreasedos with high plr in univariate analyses the plrdid not maintain independent prognostic significance inmultivariable analysis interestingly patients with concurrently high nlr and plr experienced significantlydecreased os when compared to those with normalnlr and plr or those with an elevation of either ratio respectively p in a subsequentanalysis of resected and inoperable pdac patients stotz found no association between os hr [ci ] p and plr hr [ ci] p in either cohort similarly nodemonstrable association between plr and os was observed in several separate resected pdac patient series[ ] consistent with the literature discussedabove our study did not find a significant correlation between survival os or rfs and plr in resected pdacpatientshowever some authors have demonstrated the plr tobe an important predictor of survival smith and 0cpointer bmc cancer page of watanabe reported elevated plrs as the most significant determinant of survival in their resected pdaccohorts of and patients respectively [ ]reasons for inconsistent results may have included differing plr cutpoint values small patient cohorts andvariations in multidisciplinary treatments of these patients with complex pdac furthermore the plr wassynthesized using surrogates that are fundamental tomany biologic functions ie coagulation cascade whichmay explain the variability of correlation in oncologicoutcomes in our study mean values were initially usedfor plr cutpoints because of the variation reported inthe literature again an optimal plr cutpoint analysiswas performed to provide clarity and consistency in thereporting of plrassociated factorsthereforsettingis potentialthe limitations of this study include those inherent inreviewing retrospective data although our data set wasrobust and associated with an electronic medical recordthe potential for selection bias exists additionally although all blood specimens were collected in the preoperativevariationregarding the date and time blood draws were done inrelation to the surgery date the present study did notstratify patients based on receipt of neoadjuvant therapythis stratification was previously investigated by ourgroup who reported significantly decreased rates of osamong patients with increased nlr after neoadjuvanttherapy when compared to those with stable nlr finally we did not analyze pretreatment immunologicratios in patients who received neoadjuvant chemotherapy therefore we were not able to determine whetherchemotherapy significantly altered preoperative valuesthere continues to be little doubt about the importanceof inflammation and immunity in cancer biology thenlr and other immunologic ratios are derived from easily obtainable standard laboratory values with littleadded expense when obtained in the preoperative setting the nlr is a biomarker with the potential to guidetreatment algorithms in earlystage pdac patients andprovide clarity on common unresolved management dilemmas routinely debated today given their demonstrable poor outcomes patients with high nlr maybenefitfrom neoadjuvant systemic therapy variationmore detailed preoperative staging or stratification inclinical trials additionally consistent with the findingsof developing research on the tumor microenvironmentand immunotherapy lymphocytes alone may be significant drivers of survival in the context of improving outcomes ourtargeting inflammatorypathways may be relevant in chemoprevention prospective trials would serve to elucidate the provided prognostic information and provide insightinto alternativesuggestresultstreatment algorithms that can improve outcomes amongpatients with pdacsupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12885020071829additional file summary statistics of immunologic ratiosadditional file kaplanmeier plot demonstrating overall survival osin dichotomized nlr values a neutrophil and lymphocyte bpercentageabbreviationscci charlson comorbidity index lmr lymphocyte to monocyte rationlr neutrophil to lymphocyte ratio os overall survival pdac pancreaticductal adenocarcinoma plr platelet to lymphocyte ratio r0 marginnegative resection rfs recurrencefree survivalacknowledgmentseditorial assistance was provided by the moffitt cancer centers scientificediting department by dr paul fletcher daley drucker no compensationwas given beyond their regular salaries this work was presented as a posterat the ahpba meeting and the pancreas club meeting theabstract of this work was previously published in hpb journalauthors contributionsdp conception and design acquisition of data analysis and interpretation ofdata drafting of original critical revision gave final approval ofcompleted manuscript dr conception and design acquisition of dataanalysis and interpretation of data drafting of original critical revisiongave final approval of completed manuscript bp conception and designacquisition of data analysis and interpretation of data critical revision gavefinal approval of completed manuscript gm conception and designacquisition of data critical revision gave final approval of completedmanuscript se conception and design acquisition of data critical revisiongave final approval of completed manuscript zt statistical analysis andinterpretation of data critical revision gave final approval of completedmanuscript ms statistical analysis and interpretation of data critical revisiongave final approval of completed manuscript ph conception and designanalysis and interpretation of data critical revision gave final approval ofcompleted manuscript jp conception and design analysis andinterpretation of data critical revision gave final approval of completedmanuscript jf conception and design analysis and interpretation of datacritical revision gave final approval of completed manuscript mmconception and design primary investigator supervision analysis andinterpretation of data critical revision gave final approval of completedmanuscriptfundingthis work was supported by the h lee moffitt cancer center researchinstitute nci cancer center support grant p30ca076292 the funders hadno role in study design data collection and analysis decision to publish orpreparation of the manuscriptavailability of data and materialsthe data that support the findings of this study are available from thecorresponding author upon reasonable requestethics approval and consent to participatethis study was approved by the moffitt cancer center institutional reviewboard mcc because of the retrospective nature of this studypatient consent was not required no personally identifiable data for anypatients were included the study was performed in accordance with thedeclaration of helsinkiconsent for publicationthis study was approved by the moffitt cancer center institutional reviewboard mcc due to the retrospective nature of this study patientconsent was not required 0cpointer bmc cancer page of competing intereststhe authors have no conflicts of interest to declareauthor details1department of gastrointestinal oncology h lee moffitt cancer center andresearch institute usf magnolia dr tampa fl usa2department of surgery university of texas southwestern dallas tx usa3department of biostatistics and bioinformatics h lee moffitt cancer centerand research institute tampa fl usareceived april accepted july referencessiegel rl miller kd jemal a cancer statistics ca cancer j clin ryan dp hong ts bardeesy n pancreatic adenocarcinoma n engl j medkatz mh wang h fleming jb longterm survival aftermultidisciplinary management of resected pancreatic adenocarcinoma annsurg oncol neoptolemos jp palmer dh ghaneh p comparison of adjuvantgemcitabine and capecitabine with gemcitabine monotherapy in patientswith resected pancreatic cancer espac4 a multicentre openlabelrandomised phase trial lancet oettle h neuhaus p hochhaus a adjuvant chemotherapy withgemcitabine and longterm outcomes among patients with resectedpancreatic cancer the conko001 randomized trial jama chen dt davisyadley ah huang py prognostic fifteengenesignature for early stage pancreatic ductal adenocarcinoma plos one2015108e0133562helm j centeno ba coppola d histologic characteristics enhancepredictive value of american joint committee on cancer staging inresectable pancreas cancer cancer proctor mj morrison ds talwar d a comparison of inflammationbased prognostic scores in patients with cancer a glasgow inflammationoutcome study eur j cancer bindea g mlecnik b tosolini m spatiotemporal dynamics ofintratumoral immune cells reveal the immune landscape in human cancerimmunity hong x cui b wang m yang z wang l xu q systemic immuneinflammation index based on platelet counts and neutrophillymphocyteratio is useful for predicting prognosis in small cell lung cancer tohoku jexp med zhong jh huang dh chen zy prognostic role of systemic immuneinflammation index in solid tumors a systematic review and metaanalysisoncotarget templeton aj mcnamara mg seruga b prognostic role of neutrophiltolymphocyte ratio in solid tumors a systematic review and metaanalysisj natl cancer inst 20141066dju124 giakoustidis a neofytou k costa neves m identifying the role ofneutrophiltolymphocyte ratio and plateletstolymphocyte ratio asprognostic markers in patients undergoing resection of pancreatic ductaladenocarcinoma ann hepatobiliary pancreatic surg glazer es rashid om pimiento jm hodul pj malafa mp increasedneutrophiltolymphocyte ratio after neoadjuvant therapy is associated withworse survival after resection of borderline resectable pancreatic ductaladenocarcinoma surgery sierzega m lenart m rutkowska m preoperative neutrophillymphocyte and lymphocytemonocyte ratios reflect immune cellpopulation rearrangement in resectable pancreatic cancer ann surg oncolli w tao l zhang l xiu d prognostic role of lymphocyte to monocyteratio for patients with pancreatic cancer a systematic review and metaanalysis oncotargets ther abe t nakata k kibe s prognostic value of preoperative nutritionaland immunological factors in patients with pancreatic ductaladenocarcinoma ann surg oncol quigley da dang hx zhao sg genomic hallmarks and structuralvariation in metastatic prostate cancer cell e759 halazun kj aldoori a malik hz elevated preoperative neutrophil tolymphocyte ratio predicts survival following hepatic resection for colorectalliver metastases eur j surg oncol lausen b schaumacher m maximally selected rank statistics biometricslausen b sauerbrei w schumacher v classification and regression treescart used for the exploration of prognostic factors measured on differentscales in university of essex research repository p mantovani a allavena p sica a balkwill f cancerrelated inflammationnature giakoustidis a neofytou k khan az mudan s neutrophil to lymphocyteratio predicts pattern of recurrence in patients undergoing liver resectionfor colorectal liver metastasis and thus the overall survival j surg oncolli c wen tf yan ln postoperative neutrophiltolymphocyte ratioplus platelettolymphocyte ratio predicts the outcomes of hepatocellularcarcinoma j surg res nora i shridhar r huston j meredith k the accuracy of neutrophil tolymphocyte ratio and platelet to lymphocyte ratio as a marker fastrointestinal malignancies j gastrointest oncol ye s bai l comparison and validation of the value of preoperativeinflammation markerbased prognostic scores in resectable pancreaticductal adenocarcinoma cancer manag res zhou y wei q fan j cheng s ding w hua z prognostic role of theneutrophiltolymphocyte ratio in pancreatic cancer a metaanalysiscontaining patients clin chim acta mowbray ng griffith d hammoda m shingler g kambal a alsarirehb a metaanalysis of the | 0 |
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 | 0 |
colorectal cancer crc is one of the most common malignanttumors in china chen crc is one of the ï¬veleading causes of cancer death and its incidence is graduallyincreasing owing to obesity and lifestyle changes du chen postoperative treatments includingchemotherapy and radiotherapy are important for longer patientsurvival traditional chinese medicine tcm has become anoption for preventing crc metastasis and enhancing the eï¬ectsof chemotherapy shi xu xie tcm is used as an alternative or supplementary treatmentin the united states and europe and has been widely used totreat various diseases in asia especially in china wang tcm has also been widely investigated in asia for eï¬ectiveand lowtoxicity monomer compounds to develop new drugs forcancer therapy and to counteract drug resistance sui zheng xie in china patients usually choose tcm for adjuvant therapyafter curative resection xu the eï¬ectiveness oftcm has been proven in multiple cancers including breastcancer lee hepatocellular carcinoma chen pancreatic cancer kuo and crc shi xu in crc tcm significantlyimproved diseasefree survival in stage ii and iii crc in aretrospective cohort study including patients shi in a multicenter prospective cohort study including patients with stage ii and iii crc postoperative tcmtreatment was associated with better diseasefree survival andoverall survival compared to those of the untreated groupxu certain active ingredients in tcm herbsmay have stronger activity in inhibiting cell proliferation andpromoting cell apoptosis tan huang and hu for example bufalin an active component of the tcmchan su can reverse multidrug resistance by inhibiting theprotein expression and eï¬ux function of abcb1 yuan cinobufagin another cardiotonic steroid isolated fromchan su suppresses tumor neovascularization by disrupting theendothelial mtorhif1α pathway to trigger reactive oxygenspeciesmediated vascular endothelial cell apoptosis li of the frequently used tcm treatments the most eï¬ectivesingle herbs are ginseng radix ren shen hedyotis diï¬usa willdbai hua she she cao scutellaria barbata ban zhi lian andastragali radix huang qi lee wu however the underlying mechanisms of these remedies remainunknown network pharmacology can eï¬ciently and quicklyidentify the interactions between drugs and target proteinsproviding a foundation for tcm application zhang fufang yiliu yin fyy is a tcm formula that has beenused in clinical practice for cancer treatment our previousstudy found that fyy inhibited cell proliferation migration andinvasion and promoted apoptosis in hepatocellular carcinomayang fyy contains eight herbs astragali radixhuang qi ganoderma lucidum ling zhi semen armeniacaeamarum ku xing ren h diï¬usa willd bai hua she she caoaconiti lateralis radix praeparata fu zi glycyrrhiza glabralinne gan cao radix panacis quinquefolii xi yang shenfyy inhibits colorectal cancer progressionand platycodi radix jie geng of these herbs radix panacisquinquefolii ginseng radix h diï¬usa willd and astragaliradix are commonly used in anticancer formulas lee wu g lucidum and platycodi radix alsoreportedly have anticancer eï¬ects radix astragali jung g lucidum dai platycodi radix park andlee and h diï¬usa willd zhang inhibitcancer cell proliferation polysaccharides in g lucidum inhibitthe proliferation of crc cells upregulating the expression of p21protein and blocking cells at the g2m phase na in the current study we investigated the anticancer eï¬ectof fyy on crc cells in vitro and in vivo and a networkpharmacology analysis was performed to explore the potentialmolecular mechanisms the information obtained in this studywill aid in elucidating the previously unavailable mechanismsof action of fyy in crc and developing fyy as an adjuvanttherapy for crcmaterials and methodspreparation of fyy and cell culturethe components of fyy conformed to the provisions stated bythe chinese pharmacopoeia fyy was prepared at the weifanghospital of traditional chinese medicine shandong chinayang fyy mgml was stored at ¦c untiluse and was further diluted to the required concentrations insubsequent cell experiments human crc cell lines hct116and sw480 were purchased from the cell resource center of theshanghai institutes for biological sciences chinese academy ofsciences shanghai china hct116 cells were grown in rpmi medium rpmi1640 hyclone united states and sw480cells were grown in dulbeccos modiï¬ed eagles medium dmemhyclone united states containing fetal bovine serumfbs gibco brl united states and penicillinstreptomycinsigmaaldrich st louis mo united states in co2 at ¦cin a humidiï¬ed incubatorcell viability and colony formationassayscells per well were seeded into 96well plates andincubated overnight at ¦c co2 in a humidiï¬ed incubatorwhen the cells adhered to the wall hct116 and sw480 cellswere treated with or mgml of fyy or pbs asa control for and h cell viability was measured using acell counting kit8 cck8 beyotime institute of biotechnologyinc shanghai china ten microliters of the cck8 solutionwas added to each well and then samples were incubated at ¦cfor h finally the absorbance value at nm was determinedusing a multiskantm fc microplate photometer thermo fisherscientiï¬c inc united stateshct116 and sw480 cells were treated with or mgmlof fyy or pbs as a control for h the cells perwell were then cultured in sixwell plates and the medium waschanged every days for days cell colonies were ï¬xed with paraformaldehyde and then stained with giemsa beyotimeinstitute of biotechnology inc shanghai china for minfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiona colony formation assay was performed to count viable colonies cells per colonycell cycle analyseshct116 and sw480 cells were treated with or mgmlfyy or pbs as a control for h the collected cells were ï¬xed in cold ethanol and stored at ¦c overnight the nextday the cells were washed twice with cold pbs then µlrnase a µgml and µl propidium iodide µgmlsigma aldrich st louis mo united states were added to eachsample and incubated for min in the dark measurements weretaken using a ï¬ow cytometer facscan bd biosciences bedfordma united states and the data were analyzed using flowjo software tree star inc ashland or united statescell apoptosis analysescell apoptosis was detected using an apoptosishoechst staining kit beyotime biotechnology shanghai chinasamples were ï¬xed with paraformaldehyde atroomtemperature for min and stained with mgml hoechst at room temperature for min then ï¬uorescencewas detected under an olympus ix50 microscope olympuscorp tokyo japan at magniï¬cation apoptotic cellswere identiï¬ed using an alexa fluor annexin vdead cellapoptosis kit invitrogentmmolecular probes eugene orunited states after centrifugation at g for min the celldensity was counted and diluted in annexinbinding buï¬erto obtain cellsml µl per assay cells were stainedwith µl of annexin vfitc and µl propidium iodide atroom temperature for min in the dark and then µl ofbinding buï¬er was added measurements were taken using a ï¬owcytometer and the data were analyzed using flowjo softwarenetwork pharmacologyactive fyy compounds were screened using the traditionalchinese medicine systems pharmacology database tcmsp1ru with the pharmacokinetic information retrievalï¬lter based on the tcmsp platform the oral bioavailabilityand druglikeness were set to ¥ and ¥ to obtainqualiï¬ed herbal compounds the chemical structures of thecompounds were drawn using chembiooï¬ce kerwin crc targets were predicted and screened using thegenecards database2 stelzer and omim platform3amberger and hamosh venny venny wasused to screen for common targets between fyy and diseaserelated targetsdrug compounddiseasetarget networks were built usingcytoscape v software shannon and themerge function was used to analyze the core compoundsprotein interaction networks of the common fyy and crcrelated targets were built using the string database platform1httptcmspwcomtcmspphp2httpswwwgenecards3httpswwwomim4httpbioinfogpcnbcsicestoolsvennywith medium conï¬dence and rejecting the target proteinindependent of the network szklarczyk gene ontology go analysis and kyoto encyclopediaof genes and genomeskegg pathway analysis wereperformed using metascape zhou enrichedgo terms and relevant pathways with pvalues wereselected for better prediction and veriï¬cation of the biologicalprocess and mechanismwestern blot analysisthe following primary antibodies obtained from cell signalingtechnology inc danvers ma united states were used inthe immunoblotting analysis pi3k p110α akt pan pakt ser473 bcl2 bclxl bax p21 cmyc andgapdh total proteins were extracted fromcells and tissues using ripa lysis buï¬er cwbio beijing chinaequal amounts of protein from each sample were separatedby sdspage electrophoresis and then transferred onto045µm pvdf membranes biorad laboratories herculesca united states subsequently the membranes were blockedwith milk in pbs plus tween pbst for minincubated with primary antibodies overnight at ¦c and thenincubated with goat antirabbit horseradish peroxidases abcamcambridge ma united states or goat antimousehorseradish peroxidases abcam cambridge ma united states for h at room temperature finallythe bandwas detected using an enhanced chemiluminescence reagentand visualized with a fusion fx7 system vilber lourmatfrance imagej software was used to calculate the intensity grayvalue of each protein band and gapdh served as a controlfor normalizationtumor xenografts in nude miceten male balbc nude mice weeks old ± gwere purchased from beijing vital river laboratory animaltechnology co ltd beijing china the mice were housedat ± ¦c under a 12h lightdark cycle with free accessto food and water all animal experiments were completedat the speciï¬cpathogenfree medical animal laboratory of theaï¬liated hospital of qingdao university and approved bythe animal ethics committee of the aï¬liated hospital ofqingdao university ahqu20180310a hct116 cells cells per tumor were subcutaneously injected into the rightarmpit of the nude mice seven days after tumor inoculationthe tumor size was measured using a vernier caliper andthe mice were divided into two groups the fyy treatmentgroup and a control group n mice per group thefyy group was intragastrically administered ml10 g bodyweight daily in a primary concentration of mgml thecontrol group was intragastrically administered an equivalentvolume of pbs tumor sizes were measured every daysand calculated using the following formula tumor volumemm3 length width2 the nude mice werekilled by cervical dislocation on day and the tumorswere excised weighed and photographed finallytumorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontissue and liver tissue were stored in formalin or at¦c for subsequent immunohistochemistry or western blotanalyses respectivelyimmunohistochemistrytumor and liver tissues of the nude mice were ï¬xed with paraformaldehyde for h and then embedded in paraï¬nembedded paraï¬n sections were dewaxed in xylene andrehydrated in ethanol antigen retrieval was performed in m citrate buï¬er ph using a pressure cooker followed byincubation for min samples were then washed thrice with pbsand ï¬xed in ethanol for min ki67 antibody novuscolorado united states was stained with a streptavidinperoxidase detection kit zsgbbio beijing china accordingto the kit instructionsstatistical analysisdata analysis was performed using graphpad prism softwaresan diego ca united states all experimental data wereexpressed as the mean ± sd the statistical signiï¬cance of theresults was analyzed by oneway analysis of variance anovafor multiple group comparisons and students ttest for two groupcomparisons a value of p was considered statisticallysignificant all experiments were performed in triplicateresultsfyy inhibited proliferation and promotedapoptosis of crc cells in vitrofufang yiliu yin significantly inhibited the growth of hct116and sw480 cells in a dosedependent manner figure 1a thecolony formation assay showed that the number of the coloniesin the fyy group and mgml was lower than that of thecontrol group figure 1bcolony formation ability was significantly inhibited by mgml p and mgml p fyy forhct116 and for sw480 cells respectively the cell cycle analysisshowed no significant diï¬erence in the percentage of cells ins p for mgml and g2m phases p for mgml in hct116 however a significant increase in g0g1phase was found after treatment with increasing concentrationsof fyy p for mgml figure 2a in hct116similar results were obtained for sw480 cells fyy blockedcell cycle at the g0g1 phase in a concentrationdependentmanner fyy inhibited the expression of cmyc p for mgml and promoted the expression of p21 protein p for mgml figure 2b in hct116 similar results wereobserved in sw480 cells this indicated an inhibitory eï¬ect oncell proliferationcell apoptosis as shown by hoechst staining increased afterfyy treatment figure 3a flow cytometry analysis showedthat the early p for mgml and late apoptosisp for mgml of hct116 cells were significantlypromoted figure 3b by fyy treatment similar results wereobtained for sw480 cells figure 3bnetwork pharmacological analysis offyy targeting crca total of compounds from fyy were retrieved oralbioavailability ¥ and drug likeness ¥ from the tcmspdatabase supplementary table a total of genes related tothese compounds and genes related to crc were screenedout using venny figure 4a common targets wereobtained supplementary table data imported into cytoscape to construct compounddiseasetarget networks figure 4a showed that of the fyy compounds may aï¬ect disease targets the top core compounds were screened based on the topologicalproperties of degree as shown in table quercetin kaempferolluteolin betasitosterol isorhamnetin formononetin calycosinjaranol acacetin and naringenin were the top active fyyingredients against crc the other active compoundsare listed in supplementary table two networks wereconstructed for the top core compounds and the remaining active compounds figure 4a the proteinprotein interactionnetwork built using string software used to investigatethe mechanisms of fyy provided common targets aftersetting the conï¬dence level above figure 4b theprioritization of key targets was analyzed according to thedegree of the node exported from the string database andthe top ï¬ve targets were cyclind1 mapk8 egfr myc andesr1 figure 4cbiological function and pathwayenrichment of fyy on crcthe biologicalfunctions and signaling pathways from allcore targets were enriched the top biological enrichmentresults are shown in figure 4d fyy aï¬ected crc throughmultiple go biological processes including apoptotic signalingpathway response to steroid hormone and response to inanicsubstance kegg analysis results included cancer prostatecancer apoptosis and pi3kakt signaling pathwayswe further investigated how the fyy mechanism promotedapoptosis using rtpcr and western blot analysis of hct116and sw480 cells fyy inhibited the relative expression ofpi3k mrna p figure 5a fyy downregulated theexpression of pi3k pakt bcl2 and bclxl and upregulatedthe expression of bax p figures 5bc takentogether these data support the idea that fyy induces crccell apoptosis by modulating the pi3kakt pathway and bcl2family proteinsfyy inhibited tumor growth and cellproliferation in vivothe hct116 cell xenograft model used to investigate theantitumor eï¬ect of fyy showed that fyy significantlyinhibited tumor growth compared to the control figure 6athe average tumor volumesafter days oftreatmentwere ± mm3in the control group and ± mm3 in fyytreated group figure 6b whiletumor weights were ± and ± mgrespectively ki67 significantly decreased in the fyytreatedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited colorectal cancer cell proliferation a cck8 assay indicated that fyy inhibited the proliferation of hct116 and sw480cells in a dose and timedependent manner after and h of treatment pbs was used for the control treatment n per group b colony formation abilitydecreased after treatment with different concentrations of fyy for both hct116 and sw480 n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy significantly inhibited the colorectal cancer cell cycle a fyy significantly inhibited the cell cycle progress of hct116 and sw480arresting them at the g2m phase as shown by ï¬ow cytometry assay n per group b the expression of cmyc decreased and p21 increased with fyytreatment n per group values are shown as the mean ± sd p p and p vs control group the pvalues were obtained usinganovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy promoted colorectal cancer cell apoptosis a hoechst staining analysis indicated that fyy promoted apoptosis includingchromatin condensation and nuclear fragmentation in hct116 and sw480 cells magniï¬cation b flow cytometry indicated that fyy promoted the earlyand late apoptosis of hct116 and sw480 cells n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure network pharmacological analysis and biological functional enrichment analysis of fufang yiliu yin fyy a venn diagram showed common targetsof fyy in colorectal cancer crc compounddiseasetarget networks of fyy against crc b proteinprotein interactions identiï¬ed by string software c thepredicted key targets of fyy treatment of crc d go and kegg pathway enrichment analysesfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiontable the top bioactive compounds of fufang yiliu yin are listed below according to the degree of similarity of the compounddiseasetarget networkspubchem cidmolecule namequercetinformulac15h10o7ob dlkaempferolc15h10o6luteolinc15h10o6degreestructurebetasitosterolc29h50oisorhamnetinc16h12o7formononetinc16h12o4calycosinc16h12o5jaranolc17h14o6acacetinc16h12o5naringeninc15h12o5glycyrolc21h18o67methoxy2methyl isoï¬avonec17h13no5continuedfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong table continuedpubchem cidmolecule name7omethylisomucronulatolformulac18h20o5ob dllupiwighteonec20h18o5glyasperin fc20h18o6fyy inhibits colorectal cancer progressiondegreestructureob oral bioavailability dl druglikenesscrc tumor xenograft group figure 6b the expression ofpi3k pakt bcl2 and bclxl followed the same trend asthe in vitro study results figure 6cdiscussionboth retrospective and prospective studies have proven theanticancer eï¬ects of tcm on crc shi xu here we reported the anticancer eï¬ect of the fyyformula which contains eight ingredients fyy significantlyinhibited cell proliferation and promoted crc cell apoptosisin vitro fyy also inhibited xenograft tumor growth in vivousing a network pharmacology analysis we found that fyymay act on crc through active compounds targeting crcrelated genes that regulate the apoptosis and pi3kaktsignaling pathwaysto better understand the complementary eï¬ects of fyyformula ingredients we retrieved a total of compounds fromthe tcmsp database supplementary table compounddiseasetarget networks showed that of the compoundsmay aï¬ect crcrelated targets by searching pubmed wethe top compounds table exhibit antifound thatcrc eï¬ects mainly by promoting apoptosis and inhibiting cellproliferation for example quercetin was mostly related toprotective eï¬ects against crc and is found in three of the eightremedies in fyy astragali radix huang qi h diï¬usa willdbai hua she she cao and g glabra linne gan cao quercetininhibits crc progression by promoting cell apoptosis andautophagy as well as inhibiting angiogenesis and inï¬ammationdarband quercetin induces apoptosis by inhibitingdiï¬erent signaling pathways including the mapkerk pi3kaktand nfκb signaling pathways zhang xavier it also inhibits the migration and invasion of crc cells viaregulating the tolllike receptor 4nfκb signaling pathway han further kaempferol induces crc cell apoptosischoi while isorhamnetin formononetin andnaringenin show anticancer eï¬ects by inhibiting cell proliferationli abaza the similarity of theeï¬ects provided by fyy compounds may provide a mutualenhancement eï¬ect but this must be further tested using singleor mixed compoundsfufang yiliu yin induced cell cycle arrest in crc cells at theg0g1 phase and promoted apoptosis in hct116 and sw480cells to explain the mechanism by which fyy inhibits cellproliferation and promotes apoptosis we performed proteinprotein interaction network kegg and go pathway analysesproteinprotein interaction network analysis indicated the topï¬ve targets were cyclind1 mapk8 egfr cmyc and esr1biological functional analysis indicated apoptosis and cancerrelated pathways including the pi3kakt signaling pathwaythen our experimental study conï¬rmed the activation ofthe pi3kakt pathway and bcl2 family proteins as well ascmyc expressiontraditional chinese medicine formulas reportedly inhibitcancer progression by diï¬erent signaling pathways a tcmformula jianpi jiedu inhibits crc tumorigenesis and metastasisvia the mtorhif1αvegf pathway peng another tcm formula huang qin ge gen tang enhances the ï¬uorouracil anticancer eï¬ect by regulating the e2f1ts pathwayliu the zhi zhen fang formula reverses multidrugresistance mediated by the hedgehog pathway in crc sui these formulas as well as fyy all contain astragaliradix huang qi h diï¬usa willd bai hua she she caog glabra linne gan cao and radix panacis quinquefolii xiyang shen however there have been no reports regarding theanticancer eï¬ect of tcm formulas acting through the apoptosisand pi3kakt pathways in crc figure in the current studywe found that fyy decreased the transcription and protein levelof pi3k figure and further inhibited the phosphorylationof akt in both the cells and tumor tissues figures accumulating evidence indicates that the pi3kakt pathwayplays an important role in tumor development pi3k can partiallyactivate akt at the thr308 or ser473 sites by inducing thetranslocation of akt to the cell membrane via phosphoinositidedependent kinase akt inhibition is usually indicated by afrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy modulated the expression of the pi3kakt signaling pathway and bcl2 family proteins relative pi3k mrna expression wasaltered by fyy treatment in hct116 and sw480 cells a n per group expression levels of pi3k akt pakt bcl2 bclxl and bax were altered by fyytreatment in hct116 b and sw480 cells c n per group values are shown as the mean ± sd p p and p vs control groupthe pvalues were obtained using anovafrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure fufang yiliu yin fyy inhibited tumor growth in vivo a subcutaneous xenograft tumors after days demonstrated that fyy inhibited xenograft tumrowth n per group b tumor volume was significantly smaller after days of fyy treatment n per group ihc analysis of ki67 expression infyytreated tumor and liver tissues magniï¬cation the pvalues were obtained using anova c protein expression levels of pi3k akt pakt bcl2bclxl and bax in tumor tissues n per group values are shown as the mean ± sd p and p vs control group the pvalues were obtainedusing students ttestdecrease in the pakt ser473 level and is mostly achievedby inhibiting pi3k using pi3kspeciï¬c inhibitors ly294002or wortmannin reener and marti the regulation ofpi3kakttranscription and protein expression by a tcmtreatment has been previously reported tcm interventiondecreased pakt levels following the concentration gradientof the tcm treatment while the total overall akt level wasunchanged gu zhao calycosina component of astragali radix reportedly inhibits crcproliferation through the erβmediated regulation of the igf1rand pi3kakt signaling pathways zhao quercetinkaempferol and rutin in h diï¬usa willd also exhibit anticancereï¬ects in crc by regulating the pi3kakt signaling pathwaycai frontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressionfigure schematic representation of the proposed pi3kakt signalinginduced cell cycle arrest and apoptosis triggered by fufang yiliu yin fyy by combiningthe network pharmacological analysis and our results we hypothesized that fyy activates the pi3kakt signaling pathway and modulates the expression of p21cmyc and bcl2 family proteins thereby inducing cell cycle arrest and apoptosiscellapoptosisandinhibited metastasiswe previously found that fyy inhibited cell proliferationofpromotedhepatocellular carcinoma yang fyy may havea similar eï¬ect on diï¬erent types of cancer although wedemonstrated both the anticancer eï¬ects of fyy and the actionmechanism by which it operates limitations of this study includethe following ï¬rst we did not investigate the antimetastaticeï¬ect of fyy on crc a migration and invasion assay andcrc liver metastasis model should be used to investigate thissecondfurther studies should investigate whether mutualenhancement eï¬ects exist between the applications of fyyand regular chemotherapy and also examine its eï¬ect ondrug resistancein conclusion our study ï¬ndings showed that fyy inhibitedproliferation and promoted apoptosis in crc cells by modulatingthe pi3kakt signaling pathway and bcl2 family proteins webelieve that fyy could be a promising adjuvant therapy for crcethics statementthe animal study was reviewed and approved by animalethics committee of the aï¬liated hospital of qingdaouniversity ahqu20180310a written informed consent wasobtained from the ownerstheiranimals in this studyfor the participation ofauthor contributionsbd and cz obtained funding conducted the research andprepared the manuscript zy and qj performed the experimentssz prepared and provided the fyy formula yw and hzperformed the network pharmacology analysis cs designed thestudy and interpreted the data all authors contributed to the and approved the submitted versiondata availability statementall data presented in thissupplementary materialstudy areincluded in thefundingthis work was supported by the china postdoctoral sciencefoundation grant numbers 2016m602098 and 2018m640615the taishan scholars program ofshandong provincefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cdong fyy inhibits colorectal cancer progressiongrant number the shandong higher educationyoung science and technology support program grant number2020kjl005 the qingdao postdoctoral science foundationgrant number and the national natural sciencefoundation of china grant number supplementary materialthe supplementary material for this can be found onlineat httpswwwfrontiersins103389fcell202000704fullsupplementarymaterialreferencesabaza m s orabi k y alquattan e and alattiyah r j growthinhibitory and chemosensitization eï¬ects of naringenin a natural ï¬avanonepuriï¬ed from thymus vulgaris on human breast and colorectal cancer cancercell int doi 101186s1293501501940amberger j s and hamosh a searching online mendelian inheritance inman omim a knowledgebase of human genes and genetic phenotypes currprotoc bioinformatics doi 101002cpbi27cai q lin j wei l zhang l wang l zhan y hedyotis diï¬usawilld inhibits colorectal cancer growth in vivo via inhibition of stat3 signalingpathway int j mol sci doi 103390ijms13056117chen q shu c laurence a d chen y peng b g zhen z j eï¬ect of huaier granule on recurrence after curative resection of hcca multicentre randomised clinical trial gut doi 101016s0618chen w zheng r baade p d zhang s zeng h bray f cancerstatistics in china ca cancer j clin doi 103322caacchoi j b kim j h lee h pak j n shim b s kim s h reactive oxygen species and p53 mediated activation of p38 and caspases iscritically involved in kaempferol induced apoptosis in colorectal cancer cellsj agric food chem doi 101021acsjafc8b02656dai s liu j sun x and wang n ganoderma lucidum inhibitsproliferation of human ovarian cancer cells by suppressing vegf expressionand upregulating the expression of connexin bmc complement alternmed doi darband s g kaviani m youseï¬ b sadighparvar s pakdel f g attari j s quercetin a functional dietary ï¬avonoid 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" african americans aa are at high risk for colorectal cancer crc studies report a increasein crc risk with physical inactivity obesity and metabolic syndrome activation of the wntcatenin ctnnb1signaling pathway plays a critical role in colorectal carcinogenesis accumulating evidence also indicates a role ofwntctnnb1 signaling in obesity and metabolic diseasesaim to examine the association between obesity catenin expression and colonic lesions in african americansmethods we reviewed the pathology records of colorectal specimens from to crcs advanced adenomas and normal colon tissues tissue microarrays tma were constructed from these samplesimmunohistochemistry ihc for ctnnb1 catenin clone catenin1 was performed on the constructed tmasthe ihc results were evaluated by pathologists and the nuclear intensity staining was scored from to bmisex age location of the lesion and other demographic data were obtainedresults positive nuclear staining in normal advanced adenoma and crc was and respectively p crc was asso ciated with positive status for nuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma nuclearstaining percentage has a fair diagnostic ability for crc with an auc of 95ci overweightobese patients bmi did not show a significant difference in p nuclear ctnnb1 staining positive in normal weight vs positive in overweightobese the association between nuclear intensityand crc was not different between normal and overweight patients p for interaction the positive nuclearctnnb1status in crc stage iii and iv of all crc was not different from stage i and ii vs respectively p continued on next page correspondence b_shokranihowardedu hashktorabhowardedu1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usafull 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 0cshokrani bmc gastroenterology page of continued from previous page in our study advanced adenoma and crc were associated with activation of catenin in physically fitoverweight and obese patients thus obesity and wntcatenin pathway seem to be independent in africanamerican patients wntcatenin signaling pathway has a potential to be used as an effector in coloncarcinogenic transformation whether or not bmi is a modifier of this pathway needs to be investigated furtherkeywords catenin colorectal cancer advanced adenoma african americans colorectal cancer crc is one of the most commoncancers in the industrialized world lifestyle and epidemiological factors associated with an increased risk ofcrc include physical inactivity obesity and metabolicsyndrome in the united states approximately twothirds of the adult population are overweight or obesewhich represents a putative risk factor for multiple target an malignancies including crc there is evidence to suggest that excess adiposity is associated with up to greater risk of crc comparedwith normal weight individuals and that physical activity may decrease colorectal cancer risk althoughexcessive accumulation of white adipose tissue wat isthe key feature of adiposity obesity is clinically definedby a bmi over kgm2 which does not take fat contentinto account it is also known that most crcs arise froma genetic and morphological adenoma to carcinomatransition also it is widely accepted that both crcsand colorectal adenomas cras share similar etiologicalcauses which explains why cras which are amongstthe mostfindings in all crcscreening participants are present in more than ofgeneral asymptomatic populations consequentlyrisk algorithms have been applied to use bmi as a predictor variable to stratify individuals according to theirrisk of colorectal neoplasia however the underlyingmechanisms that might explain the association and themagnitude ofthe connection between excess bodyweight and crc remain unclearfrequent pathologicalin the obesitycancer relationship multiple biologicalprocesses including insulininsulinlike growth factorigf1 insulin resistance sexual hormones estrogensand proinflammatory cytokines tnfα il6 and crpactively participate all these elements create a favorable environment for carcinogenesis and a decrease inapoptosisas a separate molecular pathway activation of thewnt signaling pathway plays a critical role in colorectalcarcinogenesis wnt ligands are a family of proteinsfor normal cell development that are importantcatenin ctnnb1 is a major mediator of the wntpathway that is traditionally classified into canonical catenindependent and noncanonical cateninindependent wnt canonical pathway utilizes a group ofcell surface receptors called frizzled frz to activateseveral pathways the most important one involving catenin and apc in the absence of wnt signalingapc causes degradation of catenin preventing its accumulation in the cytoplasm by forming a complex withcatenin which leads to the phosphorylation and eventually destruction of catenin by the proteasome signaling by wnt blocks this process allowing cateninto migrate from the cytoplasm to the nucleus once inthe nucleus catenin upregulates cmyc cyclin d1and other genes which increase cellular proliferation therefore continuous wnt signaling can be seenin cells with loss of apc metabolic syndromeassociated conditionssuch asobesity and type ii diabetes are influenced by geneticand functional variations in the wnt signaling pathway wnt signaling when activated represses the terminal differentiation during adipogenesis whereby preadipocytes take on the characteristics of mature adipocytes a cascade of transcriptional events like the induction of catenin ensues which in turn inducesenhancer binding proteinα cebpa and peroxisomeproliferatoractivated receptorÎ pparg the excessive accumulation of wat features adiposity butobesity does not take fat content into account recently genetic factors linked to fat mass and adipositywere reported to be associated with increased obesityrisk in young obese individuals wholeexome sequencing revealed rare gainoffunction mutations inctnnb1catenin the cateninregulated transcription of an adipocytederived chemokine calledserum amyloid a3 saa3 leads to the formation of a catenintcf complex in mature adipocytes that promotes the proliferation of preadipocytes in wat andthereby increases obesity and the risk for metabolic syndrome other data also suggest that obesity and lack ofphysical activity are associated with a higher risk forcolorectal cancer [ ] these findings have importantimplications especially in the obese and physically inactive african american population that may haveunderlying predisposing mutations to colorectal cancer the aim of this study is to assess the catenin expression profile in colorectal premalignant and malignant lesions in correlation with obesity as determined by 0cshokrani bmc gastroenterology page of body mass index bmi or waist circumference wc inafrican american populationmethodspatients and clinical datacolorectaltissue samples submitted to the surgicalpathology laboratory at howard university hospitalfrom january to december were retrieved from the pathology archive system powerpath¢a total of samples were included in the study consisting of tissue samples from crc n advancedadenoma n and normal colon n patientsdata included age sex height weight and waist circumference body mass index bmi was calculated in thestudy table the protocol of this study was approvedby the howard university institutional review boardirbtissue microarray tma constructionhematoxylineosin stained slides he slides weremade from paraffinembedded blocks the he slideswere reviewed by two pathologists to confirm the pathological diagnosis and to mark areas of interest multipleareas from more than one block were marked to ensurea good representability of the sample on the tma fivetma paraffin blocks each containing cores of mmin diameter each and mm distance from each otherwere constructed tissuespecific and an system controls were included in each tmaimmunohistochemical ihc analysis of ctnnb1 cateninthe constructed tmas were stained for catenin theimmunostaining was carried out as follows dako monoclonal mouse antihuman betacatenin cateninc1intended for laboratory application to identify qualitatively by light microscopy catenin positive cells in normal and neoplastic tissues was used at a dilution of using the envision dab code k4006 detectionsystem the deparaffinized tmas were treated prior tothe ihc staining procedure target retrievalinvolvedimmersion of tissue sections in a preheated buffer solution and maintaining heat in a water bath °cfor greater adherence of tissue sections to glass slidessilanized slides dako code s3003 were used targetretrieval solution code s1700 or 10x concentratecode s1699 using a 20min heating protocol was usedthe cellular staining pattern of anticatenin is mainlymembranous especially at the cellcell boundaries positive nuclear staining and diffuse cytoplasmic staining arealso reported in cancer cells fig evaluation and assessment of the catenin expressiontwo pathologists interpreted the ihc slides cateninexpression status was assessed based on the pattern ofstaining nuclear cytoplasmic and membranous intensity to and percentage of staining to the staining would be considered negative if there wasweak or no nuclear expression or positive if there wasmoderate or strong nuclear expressionstatistical analysisdistribution of continuous and categorical variables weretested by kruskalwallis and chisquare test betweendifferent groups respectively we used logistic regression analysis to test association between the staining andrisk of crc after adjusting for age and gender areaunder the curve auc was calculated for variables withsignificant association with crc using receiver operative characteristics curve all statistical analyses wereperformed by stata statacorp college station txresultsepidemiological characteristics and bmi in normaladvanced adenoma and crcthe bmi was calculated for individual patients and normal subjects as represented in table crc patientswere older p while our healthy normal population was mostly overweight higher bmi was moreclosely associated with advanced adenoma and crchowever the differences were not significant table advanced adenomas and crcs were associated withpositive nuclear ctnnb1we assessed whether alterations in wntctnnb1 catenin signaling plays any role colon carcinogenesispositive catenin nuclear stains were seen in normaladvanced adenomas and crcs were and respectively p table based on the designationtable epidemiological characteristics and bmi in normal advanced adenoma and crc patientsage median iqrmale n bmi kgm2 median iqroverweight n normaln advanced adenoman crcn p value 0cshokrani bmc gastroenterology page of fig immunostain for catenin in three individuals normal a à advanced adenoma b à and cancer c d à and à respectively showing membranous staining in the normal cytoplasmic and membranous staining in adenoma with no evidence of nuclearexpression arrow showing lack of nuclear staining and nuclear and cytoplasmic staining in cancer arrow showing nuclear stainingof n intensity which is associated with higher risk ofcancer crcs were associated with positive status fornuclear ctnnb1 intensity age gender adjusted or 95ci p for positive nuclearstaining compared to noncrc samples normal or advanced adenoma fig nuclear staining percentagehas also a fair diagnostic ability for crc with an auc of 95ci table fig overweight and obese patients show a trend withpositive nuclear ctnnb1 expressionpositive nuclear ctnnb1 staining was in normalweight and in overweightobese bmi patientsthis difference pointed to trend that was not statisticallysignificantassociation between nuclear intensity and crc in normaland overweight patientsthe association between nuclear intensity and crcwas not statistically significant different between normal weight and overweight patientsinteraction tables and the positive nuclearctnnb1 status in crc stage iii and iv of allcrc was not different from stage i and ii vs respectively p p fortable catenin nuclear and cytoplasmic expression tabulated as intensity and percentage in normal advanced adenoma andcrcnormaln cnc intensity n intensity advanced adenoma n crcn c cytoplasmic n nuclearc intensity and n intensity mean intensity and aboveoverall p valuep value for advanced adenoma vs normalp value for crc vs other 0cshokrani bmc gastroenterology page of fig catenin nuclear and cytoplasmic expression in normal advanced adenoma and crcdiscussionone of the important risk factors in colorectal cancer isobesity [ ] catenin is an ecadherin binding proteinthat mediates cellcell adhesion and plays a role inthe canonical wnt signaling pathway that controls thecoordinated expansion and differentiation of the intestinal crypt stem cells degradation of catenin byphosphorylation followed by alteration of destructioncomplex apc gsk3 and axin results in inactivation if wnt pathway in our study we found thatwas associated with an increased adjusted or of 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenomathe gatekeeper gene apc is a negative regulator of catenin and is mutated in approximately of sporadic and hereditary colon cancers there are severalmutations that can cause an accumulation of cateninin tumor cells such as mutations of the apc gene pointmutations in gsk3 or mutations in catenin gene itself []our positive nuclear staining in crc and its association with the positive status for nuclear ctnnb1intensity compared to noncrc samples are in contrastto a study by brabletz which showed that catenin is localized in the cytoplasm and membrane ofthe tumor cells whereas in our study it was mainly concentrated in the cytoplasm and the nucleus they alsomentioned that there was positive nuclear staining at theinvasive front as catenin is involved in tumor progression such is not the case in our studyindeed evenwhen considering nuclear staining in our specimensthere was no statistically significant differences betweenstage iiiiv cases staining versus stages iii crc caseslevels of staining the fact that catenin is expressedearly in the african american specimens analyzed heremight partially explain the aggressive nature of crc inthis population in addition we showed that there is uniform membranous staining in normal and increasingcytoplasmic and nuclear staining in advanced adenomasand crcs this confirms that the decrease in membranous staining begins with dysplastic changes leading to atable association of bmi with catenin nuclear intensity in advanced adenoma casesadvanced adenoma with catenin expression n in intensity and no nuclear intensity n bmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p value 0cshokrani bmc gastroenterology page of table association of bmi with catenin nuclear intensity in crc casescrc with catenin expression with high nuclear intensity and without negativebmi median interquartileoverweight n nuclear intensity negativen nuclear intensity n p valueprogressive disappearance atcrcsthe membrane levelinas we mentioned above a major risk factor for crc isobesity which continues to expand as a pandemicworldwide the american cancer society cancerprevention study ii states that there is an increased incidence of crc esophageal adenocarcinoma and othercancers with obesity in our study we showed that of advanced adenoma patients and of crc patients were overweight with bmi in comparison toadvanced adenoma the percentage was lower in cancerperhaps due to the late stage of cancer and weight lossin the interim table there are several mechanismsby which obesity is believed to promote crc this includes increase in leptin levels that cause an increase ingrowth and proliferation of colon cancer cells altered adipokine levels altered gut microbiome apartfrom increased steroid hormones and growth factors insulin is however the established biochemical linkand the main pathway involved is pik3aktmtorpathway elevated igf1 and insulin act through the insulin receptors and phosphotidylinositide3 kinase in addition to the above findings we also found thatoverweight and obese patients bmi did not showa significantly increased expression p of nuclearctnnb1 positive in normal weight vs positive in overweightobese morikawa found that inobesepositivity wasnuclear ctnnb1patientsassociated with significantly better cancerspecific survival suggesting that wnt signaling acts as a switch andwhen it is on adipogenesis is repressed kennell demonstrated that activated frz1 frizzled promotes catenin stabilityinhibits apoptosis and adipogenesisross also showed that wnt signaling acts as a molecular switch that controls adipogenesis upregulationof wnt signaling maintains preadipocytes in an undifferentiated state and when wnt signaling is prevented theydifferentiate into adipocytes [ ]although in our study there was no association between nuclear intensity and crc between normal andoverweight patients p for interaction there is accumulating evidence to show that the state of chronicinflammation incited by obesity might play a role in promoting colorectal carcinogenesis [ ] of the manymarkers tnfα is important [ ] as it activateswnt signaling through the induction of gsk3 phosphorylation resulting in increased nuclear localization ofcatenin in addition to tnfα other humoralagents associated with obesity might also be contributingto the activation of wnt signaling like il1 and adiponectin which is decreased in the obese state and is notan inflammatory cytokine that can modulate gsk3catenin signaling pathway although multiplemechanisms may be operating in parallel and contributing to the protumorigenic milieu wnt is a pivotaltumorigenic pathway aberrations of which isfig the putative relationship between obesity and colorectal cancer evolution pathways by cellular ctnnb1 status based on the data by thecurrent study our study suggests that there is no association between obesity and ctnnb1 expression 0cshokrani bmc gastroenterology page of important in the evolution of most sporadic crc insummary there is positive nuclear staining in crcs which was associated with the positive status fornuclear ctnnb1 intensity adjusted or 95ci p for positive nuclear staining compared to noncrc samples normal or advanced adenoma this shows that advanced adenomas and crcswere associated with activation of catenin in physically fit overweight and obese patients fig advanced adenoma and crc were associated with activation of catenin in physically fit overweight andobese patients thus participation of obesity and wntpathway seem to be independent crc factors in africanamerican patientsinflammationdriven activation ofwnt signaling as a potential pathway linking obesity tothe development of crc needs to be investigated furtherin the african american population this might provideinsights into the identification of new therapeutic targetsto reduce the burden of obesityassociated crcabbreviationscrc colorectal cancer wat white adipose tissue aa african americansacknowledgementsnot applicableauthors contributionsconceived and designed experiments bs ha performed experiments bsel ha hb analyzed data ha hb mn th aa and zs contributed reagentsmaterialsanalysis tools zs ha hb bs and el wrote and edited manuscriptbs and ha provided statistical analysis mn all authors have read andapproved the manuscriptfundingthis project was supported in part by grant from the national institute onminority health and health disparities of the national institutes of healthunder award numbersg12md007597 the funder had no role in designing or execution of thisstudyavailability of data and materialsall data generated or analyzed during this study are included in thispublished ethics approval and consent to participatethis retrospective and chart review study was conducted according to theworld medical association declaration of helsinki and was approved by theinternal review board of howard university since the chart review was donethrough unidentifiable approach no consent form needed for this studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests related to thismanuscriptauthor details1department of medicine department of pathology and cancer centerhoward university college of medicine geia avenue nwwashington dc usa 2division of pulmonary allergy and criticalcare medicine university of pittsburg pittsburg pa usareceived april accepted august referencesresearch iafco estimated cancer incidence mortality and prevalenceworlwide in lyon globocan haggar fa boushey rp colorectal cancer epidemiology incidencemortality survival and risk factors clin colon rectal surg oxentenko as body size and incident colorectal cancer a prospectivestudy of older women cancer prev res phila ma y obesity and risk of colorectal cancer a systematic review ofprospective studies plos one 201381e53916slattery ml physical activity and colorectal cancer sports med wong mc lam ty tsoi kk hirai hw chan vc ching jy chan fk sung jja validated tool to predict colorectal neoplasia and inform screening choicefor asymptomatic subjects gut sung jjy wong mcs lam tyt tsoi kkf chan vcw cheung w ching jyla modified colorectal screening score for prediction of advanced neoplasiaa prospective study of subjects jgastroenterolhepatol liu z dietinduced obesity elevates colonic tnfalpha in mice and isaccompanied by an activation of wnt signaling a mechanism for obesityassociated colorectal cancer j nutr biochem najdi r holcombe rf waterman ml wnt signaling and coloncarcinogenesis beyond apc j carcinog macdonald bt tamai k he x wntbetacatenin signaling componentsmechanisms and diseases dev cell denysenko t wntbetacatenin signaling pathway and downstreammodulators in low and highgrade glioma cancer genomics proteomics willert k nusse r betacatenin a key mediator of wnt signaling curr opingenet dev vella a camilleri m pharmacogenetics potential role in the treatment ofdiabetes and obesity expert opin pharmacother christodoulides c adipogenesis and wnt signalling trendsendocrinol metab world health anization [httpwwwwhointmediacentrefactsheetsfs311en] chen m lu p ma q cao y chen n li w zhao s chen b shi j sun yshen h sun l shen j liao q zhang y hong j gu w liu r ning g wangw wang j ctnnb1betacatenin dysfunction contributes to adiposity byregulating the crosstalk of mature adipocytes and preadipocytes sci adv20206eaax9605 morikawa t kuchiba a yamauchi m meyerhardt ja shima k nosho kchan at giovannucci e fuchs cs ogino s association of ctnnb1 betacatenin alterations body mass index and physical activity with survival inpatients with colorectal cancer jama morikawa t prospective analysis of body mass index physical activityand colorectal cancer risk associated with betacatenin ctnnb1 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environment proc natlacad sci u s a 0cshokrani bmc gastroenterology page of berger na obesityassociated gastrointestinal tract cancer from beginningto end cancer calle ee the american cancer society cancer prevention study iinutrition cohort rationale study design and baseline characteristicscancer frezza ee wachtel ms chirivainternati m influence of obesity on the riskof developing colon cancer gut berger na obesity and cancer pathogenesis ann n y acad sci guo s insulin signaling resistance and the metabolic syndrome insightsfrom mouse models into disease mechanisms j endocrinol 20142202t1t23 ross se inhibition of adipogenesis by wnt signaling science kennell ja macdougald oa wnt signaling inhibits adipogenesis throughbetacatenindependent and independent mechanisms j biol chem liu z yingka y inflammation driven activation of wnt pathway a potentialmechanism responsible for obesity associated colorectal cancer obes resopen j tilg h moschen ar adipocytokines mediators linking adipose tissueinflammation and immunity nat rev immunol fischerposovszky p wabitsch m hochberg z endocrinology of adiposetissue an update horm metab res oguma k activated macrophages promote wnt signalling throughtumour necrosis factoralpha in gastric tumour cells embo j wang y adiponectin modulates the glycogen synthase kinase3betabetacatenin signaling pathway and attenuates mammary tumorigenesis ofmdamb231 cells in nude mice cancer res renehan ag roberts dl dive c obesity and cancer pathophysiologicaland biological mechanisms arch physiol biochem publishers notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c" | 0 |
" in head and neck cancer hnc the relationship between a delay in starting radiotherapy rt andthe outcome is unclear the aim of the present study was to determine the impact of the amount of time beforetreatment intervention tti and the growth kinetics of individual tumors on treatment outcomes and survivalmethods two hundred sixtytwo hnc patients with primary tumors treated with definitive chemo rt wereretrospectively analyzed the tti was defined as the time interval between the date of histopathologic diagnosisand the first day of the rt course volumetric data on tumors were obtained from diagnostic and rt planningcomputer tomography ct scans in order to calculate the tumor growth kinetic parametersresults no significant association between locoregional control or causespecific hazards and tti was found thelog hazard for locoregional recurrence linearly increased during the first days of waiting for rt although this wasnot significant the median tumor volume relative increase rate and tumor volume doubling time was 32dayand days respectively and neither had any impact on locoregional control or causespecific hazards the association between a delay in starting rt and the outcome is complex and does not harm allpatients waiting for rt further research into imagingderived kinetic data on individual tumors is warranted inorder to identify patients at an increased risk of adverse outcomes due to a delay in starting rtkeywords head and neck cancer radiotherapy waiting time treatment delay outcome with new cases and deaths reported in head and neck cancer hnc is the eighth mostcommon and most lethal cancer in men worldwide in addition to surgery and systemic therapy radiotherapy rt is one of the cornerstones for treatment of thiscancer owing to the rising cancer incidence rate in ageing populations and the widening list of indications forirradiation the demands for rt have increased dramatically over the past decades [ ] the increasing complexity of pretreatment diagnostics and rt technology correspondence pstrojanonkoisi1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia4chair of oncology faculty of medicine university of ljubljana ljubljanasloveniafull list of author information is available at the end of the has led to delays in treatment decisionmaking and thereduction in linear accelerator throughputthat hasresulted in a significant imbalance between the demandfor rt and the availability of rt capacities in manypublically funded health systems this is also the case inslovenia [ ]due to obvious ethical reservations the only way tostudy the impact of delays in starting rt on treatmentoutcomes are retrospective observational analyses of cohorts from different institutions or countries intuitively one would expect that the prolongation of thetime taken before treatment intervention tti is harmful to patients both the likelihood of tumor growth andthe acquisition of a metastatic phenotype increases as afunction of time furthermore advanced tumors aremore difficult to treat than smaller tumors 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 0cžumer radiation oncology page of indeed a systematic review of pertinent literature fromthe period by chen showed an increasein the risk of local hnc recurrence of for everymonth of delay in definitive rt however certainstudies included in chens metaanalysis and also somemore recent reports negated the association betweenthe delay in definitive rt and the increased risk of treatment failure [] several different biases inherent inretrospective analyses either related to the quality ofdiagnostic procedures and treatment or to the inhomogeneity of the studied population as well as a selectionbias ie patients with fasttumor progression or ahigher burden of symptoms receive priority in treatment and significant variability in the kinetics of individual hnc cases may abolish the effect of tti inoutcomes [] however if patients with advancedor fastgrowing tumors have to wait longer the magnitude of this effect may be overestimated furthermore no compelling relationship between treatmentdelay and prognosis was found in some other cancertypes []in order to determine what would be an acceptabletti in hnc patients treated with definitive rt or concurrent chemoradiation we aimed to analyze the impactof tti and growth kinetics of individual tumors on theoccurrence of localregional failure distant metastasisand survival in the present study of a cohort of slovenepatients with hncmethodsin a retrospective study patients with oral cavity oropharyngeal hypopharyngeal or laryngeal squamous cellcarcinoma scc who were treated with curativeintentdefinitive rt with or without concurrent chemotherapybetween january and december at the institute of oncology ljubljana slovenia were includedpatients with t1n0 or t2n0 glottic cancer were left outof this cohort the period was chosen because of fluctuations in the waiting time for irradiationas a result of intensive renovations and expansion in thedepartment of radiotherapy that took place over thistime span from patients medical and rt charts wecollected information on clinical gender age onset ofsymptoms date and type of disease recurrence anddeath tumor histology site of origin tnm stage andtreatment characteristics rt technique regimen anddose duration of rt type of concurrent chemotherapy[cct] and the number of cycles administered thetnm stage was determined according to the 7th editionof the uicc classification systemfor analysis of the impact of tumor growth kinetics ontreatment outcomes the volumes ml of primary tumors and neck nodal metastases as marked on diagnostic and rt planning computer tomography ct scanswere compared patients with the same basic clinicaldisease and treatment characteristics as indicated abovebut with both sets of ct scans available were selectedfor this part of the study diagnostic ct scans were performed through the acquisition of mm thin ct sections whereas planning ct scans had a slice thicknessof mm both with intravenous iodine contrast enhancements sets with extensive artefacts were excluded forthe purposes of rt planning patients were positionedsupine on the flat tabletop and a fivefixation pointthermoplastic mask was used lymph nodes were considered positive if the smallest diameter was more than cm andor the necrotic center or extracapsular extension was seen if available segmentation was guided bymagnetic resonance imaging mri sets and the resulting contours around the primary tumors and metastaticneck nodes represented a consensus between two radiation oncologists and a radiologist volumes of primarytumors and metastatic neck nodes were separately calculated by a computer software program used for rt planning xio computerized medical systems inc stlouis usa eclipse varian medical systems inc paloalto usa the end points in this part of the study werechanges in the tumornodal volume and tnm stage thecalculation of the primary tumor volume doubling timeand their impact on the treatment outcomestatisticsthe study protocol was approved by the republic ofslovenia national medical ethics committee no for retrospective studies a written consentis deemed unnecessary according to national regulationsbasic descriptive statistics were reported with meansstandard deviations and ranges for numerical variablesand as percentages for categorical variables in patientswith two simultaneous hncs some characteristics werereported in regards to patients while others were reported in regards to tumorsthe survival curves were computed using the kaplanmeier estimator and the aalenjohansen estimator wasused to estimate the cumulative probabilities of competing risks the effect of covariates was analyzed using amultiple cox regression analysis with all the analysesthe data were censored at a fiveyear followupwhen focused on the survival of patients the analyseswere completed with patients as the units and the timewas measured from the first day of therapy until deaththe overall survival os regardless of the cause ofdeath and the absolute risk cumulative probability ofdying due to index cancer were reported in the cox regression only the index cancer deaths causespecifichazard csh were considered to be events of interestin the analyses where locoregional control lrc wasof primary interest the calculations were performed in 0cžumer radiation oncology page of regards to tumors excluding the nonresponders to rtie those with residual local or regional tumors at weeks after rt completion for the latter group weconcluded that it is the radioresistance of tumor cellsthat are responsible for the persistence of the diseaseafter therapy and not that patients had to wait for rtthe followup time was calculated from the last day oftherapy the estimated cumulative probability of localandor regional recurrence distant metastases lrcprobability of being still alive and without local andorregional recurrence and diseasefree survivaldfsprobability of being still alive and without events locoregional and distant failure and deaths were the events ofinterest were reported all the analyses were conductedin regards to the tumors as independent units this assumption was checked in the sensitivity analysis andallowed for gamma frailtythe assumptions of the cox regression were checkeda nonlinear effect a spline with degrees of freedomwas allowed for the numerical variables and the proportional hazards assumption was tested using schoenfeldresidualsthe tti was defined as the time interval between thedate of histopathologic diagnosis and the first day of thert course tumor growth kinetics was expressed as thetumor volume relative increase rate per day and as thetumor volume doubling time in daysthe tumor volume relative increase rate per day was 13 calculated as 12v t 2ðþþv t 1ðt2 t1where vt1 gross tumor volume at time t1 ieon diagnostic ct scans and vt2 gross tumor volume at time t2 ie on planning ct scans it was reported as the percentage increase was reported as per day the tumor volume doubling time was calculated asðv t 2ðv t 1ðln 2ð þ t t þþlnþsince a onetoone relationship existed between thetwo only the tumor volume relative increase rate whichrequires no extrapolation was considered for modellingall analyses were performed using r statistical software version and a pvalue below was considered statistically significantresultsimpact of time to treatment initiationbetween and patients with oral cavity oropharyngeal hypopharyngeal or laryngeal primarysccs were treated with definitive chemo radiotherapywith curative intent there were men and women aged years mean the majority oftumors originated in the oropharynx tumors in patients and were tnm stage iv tumors in patients the tti ranges from to days with mean days the distribution of the tti is only slightlyasymmetric with median and interquartile rangeiqr fig patients were irradiated with dimensionaltechnique or 3dimensionalconformal isocentric technique to the median rt dose gy iqr delivered in gy dailyfractions iqr concurrently to rt patientsreceived chemotherapy consisted of platinbased monochemotherapy patients or mitomycinecbleomycin combination patients the characteristics of patients and their tumors are shown in table treatment outcome and survivalclinical andor radiologic assessment at weeksposttherapy confirmed disease persistence locally andor regionally in cases ie in patients and thesepatients were excluded from further analyses of lrcand dfs thus patients with tumors were analyzed and during followup a local andor regional relapse was recorded in cases with a mediantime from treatment completion of months range months the two and fiveyear probability of localrecurrence after the end of treatment was and respectively whereas the regional relapse was and respectively after threeyears posttherapy we only observed a small increase ie inlocal recurrence probability whereas the probability ofregional relapse was stable after the third year followupthe probability of occurrence of distant metastases at years was and at years it was at the second and fifthyear followup the lrc was confidence interval [ci] and ci respectively and the dfs was ci and ci respectivelyin the fifth year posttreatment out of patients had died the index cancer was the reason in of the cases during the first years posttherapythe probability of cancerrelated death steeply increasedfrom zero to but later the changes were less pronounced at the second and fifth year posttreatment theos independent of the cause of death was ci and ci respectivelyeffect of covariatesin the univariate analysis the following parameters weretested for lrc and csh age gender stage of diseasetype of treatment rt vs rt cct and tti for rtas continuous variable results are presented in table 0cžumer radiation oncology page of fig distribution of ttitable clinical characteristics of patients and their tumorscharacteristicsgenderno femalemaleage yearsatumor locationboral cavityoropharynxhypopharynxlarynxtumor stagebstage istage iistage iiistage ivtreatmentbradiotherapyconcurrent chemoradiotherapyrt duration daysatime to treatment interventiona ± ± ± the same parameters were also included in the multivariate model table the occurrence of locoregional recurrence was significantly related to the diseasestage p whereas the relationship with agewas only of marginal significance p higherage had a lower hazard no significant associationwith the type of treatment could be found p table the log hazard for locoregional recurrenceseemed to linearly increase during the first days oftable effect of covariates on locoregional control and indexcancerspecific hazard n patients with tumors anunivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherap cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt a mean ± sd rangeb eleven patients had two simultaneous primary tumorsci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapy 0cžumer radiation oncology page of table impact of tti on locoregional control and indexcancerspecific hazard n patients with tumors amultivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt ci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapywaiting for rt although the association between thehazard and tti wasregardless ofwhether we made an allowance for nonlinearity ornot fig 2ainsignificantthe hazard of dying due to the index cancer ie cshwas found to be favorably associated with a lower disease stage p and the addition of cct to rtp whereas age gender and tti did not reachthe level of statistical significance table fig 2bassumptions and sensitivity analysisproportional hazards for all included variables and thelinearity assumption for age were analyzed and presented graphically some indication of nonproportionalhazards could be found in gender but allowing for nonproportional hazards did not change the interpretationof the other covariates as a part of the sensitivity analysis the assumption of the dependence of the tumorsbelonging to the same individual was relaxed but nochanges in the interpretation of the results could be observed as an additional confounder the timedependentcovariate duration of rt was considered but its effectdid not prove to be importantimpact of tumor growth kineticsdiagnostic and planning ct scans median interval days range days were available from patientsfive of these patients had two primary tumors the majority of patients were males with a meanage of years range and the patients hadprimary tumors located in the oropharynx table when two ct sets were compared the original tstage of the primary tumor was increased ie upgradedin two cases and the nodal stage was increased in six patients due to the limited number of npositive casesn only volumes of primary tumors werecompared between the two ct sets for the calculationof the tumor volume relative increase rate per dayand tumor doubling time the absolute increase intumor volume ranged from to cm3 per daymedian no increase in volume was observed infive patients ct scans in these five patients were takenfig trend of the hazard for locoregional recurrence a and for index cancerspecific death b 0cžumer radiation oncology page of table characteristics of patients with available diagnostic andplanning ct scanscharacteristicsgenderno femalemaleage yearsatumor locationboropharynxhypopharynxlarynxstagebstage istage iistage iiistage ivtherapybradiotherapyconcurrent chemoradiotherapytumor volume relative increase rate dayatumor volume doubling time daysaa mean ± sd rangeb five patients had two simultaneous primary tumors from days to days apart median days the median tumor volume relative increase rate was perday median range and the mediantumor volume doubling time was days range days no difference was observed when these two parameters were compared between different tumor stagestreatment outcome and survivalamong the tumors the time taken for local andorregional relapse and the occurrence of distant metastaseswere assessed for tumors in which treatment resultedin clinicalradiological eradication of the disease at weeks posttherapy local andor regional relapse wasrecorded in nine cases with a median time fromtreatment completion of months range monthsthe two and fiveyear probability of local recurrenceafter the end of the treatment was and respectively whereas for the regional relapse it was and respectively at years posttherapy only anincrease in the local recurrence probability of wasobserved whereas the probability of regional relapseremained stable the two and fiveyear probability ofdistant metastases was at two and years thelrc was ci and ci respectively and the dfs was ci and ci respectivelyin the yearperiod after the start of treatment outof patients died the index cancer was thereason in cases at two and years posttreatment the os rates were ci and ci respectivelyeventseffect of covariatesdue to the low number of events local andor regionalrecurrence ninecancerspecific deaths events only univariate regression models were fittedthe following covariates were tested in the modelsoverall disease stage initial tumor volume tumor doubling time and relative increase rate day of primarytumor volume measures of tumor kinetics did not showany impact on lrc or csh only an inverse relationshipbetween the initial primary tumor volume and csh wasobserved hr for index cancerspecific death perevery cm3 increase in the volume of primary tumorstable there was no difference in the value of dfsbetween patients with a primary tumor volume relativeincrease rate 1day and 1day p fig discussionwhile it is intuitively anticipated and confirmed by theresults of the metaanalysis that a delay in starting rtwould have a negative impact on the treatment of patients with hnc this association was not confirmed inour study we only found a statistically insignificantupward trend in the risk of locoregional recurrence forthe first days of rt delay in addition the differencesin the growth kinetics between individual tumors whichwere studied in a smaller group of patients were considerable but did not appear to be of significance for theprediction of treatment outcomesobviously the relationship between tti and diseaseprognosis in patients with hnc is more complex than itmight seem at first glance the first negative impact ofwaiting for treatment to begin is the risk that the tumorwill increase in size andor metastasize during this timemaking it harder to treat or resulting in it becoming untreatable [ ] however the time for a tumor to growis only one of the factors that determines prognosis andthe absence of a statistically significant association between tti and the treatment outcome in our study andis thus not surprising []many other reportsmoreover no obvious methodological differences couldbe found between these negative studies and the positive studies that confirmed the association between ttiand treatment outcomes [] in both groups thereare individual studies that have similar sample sizestumor sitestage mix and periods covered speaking infavor of comparable quality of diagnostics therapy andstatistics across the studies 0cžumer radiation oncology page of table effect of stage and tumor kinetics on locoregional control and overall survivalparameterstnm stageiv vs iiiivtuper cm3vtu relative increase rateper dayvtu relative increase rate¤ vs 1dayvtu volume of primary tumorlocoregional controlhr cipvaluecause specific hazardhr cipvalueon the contrary in more recently reported analyses ofthe national cancer registries data an adverse effect ofwaiting for radiotherapy was clearly established []however the results from this type of analysis should betaken with caution not only due to the limitations inherent in the tumor registry data unmeasured confounding selection biasincomplete data and codingerrors but also because the effect of delaying rt oncancerspecific outcomes was not evaluated in additionpatients irradiated in postoperative and definitive settings were not analyzed separately overall the researchmethodology and interpretation used in these studieswere criticized and the magnitude of the effect that theysupposedly demonstrated was questioned in the present study a linear increase in the log hazardfor locoregional recurrence was found during the first days of waiting for rt although it was not statisticallysignificant it is possible that unknown confounders thatwere not accounted in our analysis eg tumor growth kinetics reduced the statistical power of the tti the resultsfrom fortin and naghavi who reported on theincreased risk of locoregional failure with tti daysand days respectively [ ] are the most comparable to our own however optimal ttithresholdsfig impact of primary tumor volume relative increase rate to diseasefree survival in patients with no residual disease at weeks posttherapy 0cžumer radiation oncology page of identified in different studies showed considerable variations pointing to the uncertainty of such calculations andtheir dependence on the characteristics of the analyzedpopulation [ ] the possible role of classical prognostic factors such as location of primary tumor diseasestage and addition of cct is not expected to be relevant inthis respect as in our and other similar studies the statistical significance of tti was verified by multivariateanalysisthe effect of tti on treatment outcomes however isnot only conditioned by the duration of waiting for rtbut also on the rate of tumor growth in hnc a vast heterogeneity in tumor cell kinetics has been observed conditioned by the local milieu from which it arises whichdiffers from patient to patient [ ] historicallydifferent methods were explored to evaluate tumor cellkinetics but did not succeed in providing clinically relevant kinetic parameters [ ] more recently a comparison of two sets of imaging data acquired at twodifferent time points was successfully employed for thispurpose usually volumetric data are extracted from diagnostic and rtplanning ct scans for the calculation ofdifferent parameters reflecting the rate of tumor growtheg tumor volume doubling time or absolutepercentagetumor volume increase per day despite some differencesin the calculation methodology across studies a large variation in the individual values of kinetic parameters wasseen in all of them including ours indicating that all ofthe studied populations represent a unique mix of slowand fast growing tumors [] among our patientsfive had no measurable increase in primary tumor volumeeven when the interval between ct scans was up to days the inverse relationship between kinetic parametersdetermined by the comparison of two ct datasets andtreatment outcomes was implied or even confirmed inseveral smaller studies pointing to potential clinical utilityof imagingderived kinetic data [] in our grouphowever no such association was found a small numberof patients and in contrast to other studies the inclusion of different tumor sites could contribute to the negative result as well as differences in the changes in kineticproperties triggered by rtccr in individual tumors tumor radiosensitivity may also influence the effect oftti on outcome while to some extent it may be evaluated before rt begins eg by molecular profiling identification of hypoxic cells and the determination of hpvstatus in oropharyngeal primary tumors in daily clinicalpractice it is usually not considered when planning treatment [ ] in order to diminish the impact of intrinsic tumor radiosensitivity the patients in our study withresidual disease at weeks postrt were excludedfrom the analysis of lrc we hypothesized that the inability to achieve a complete tumor response to chemoradiation was due to the radiobiological characteristicsof the disease and not the delay in starting rt howeverthe exclusion of these patients from the analysis did notaffect the end resultsour study has weaknesses which are mostly due to itsretrospective nature however for obvious ethical reasons this is an inevitable feature of studying delays inthe initiation of cancer therapy we are aware that thereis always some doubt as to the accuracy of the diagnosticprocedures the staging the quality of planning and theimplementation of rt in the case of retrospective research nonetheless the same restraint exists in the caseof other similar studies and even more so in the case ofanalyses of cancer registry datasets an important feature of our data set is that no patients were lost duringthe followup and in our opinion is free from manyhidden biases that larger studies inevitably bring withsince almost half of our patients had oropharyngeal carcinoma missing information regarding the p16 or human papillomavirus hpv tumor status could be ofimportance howeverin the cohort of oropharyngealcancer patients treated at our institution between and only had a hpvrelated tumor thus we reasonably assume that the impact of p16hpv tumor status on the study results was negligible inaddition in hpvpositive cases perni found nosignificant association between tumor growth velocitycalculated from serial pretreatment ct scanslocaland distant control or os and the same was reportedby chu who measured metabolic growth velocityusing pretreatment petct scans [ ]other drawbacks to consider include technical limitationsin ct scan acquisition the accuracy of presentation of actual tumor volume on ct images and the precision of thedelineation of gross tumor volume to minimize errors inthe estimation of comparative tumor volumes only highquality pairs of ct images were selected for the analysis oftumor kinetics in addition physical findings documentedin clinical records and when available diagnostic mriswere used for this purpose and labelled tumor volumes represented a consensus between two experienced radiationoncologists and a radiologist all dedicated to hnc management like many other studies [ ] the comorbidity burden was not registered in our patientsalthough it should be taken into account when assessingsurvival outcomes finallylags in the prebiopsyperiod were not addressed in our study including patientdelay and delays in referral and diagnostics which may addsignificantly to the total tti these are also costly and potentially fatal but can be successfully reduced by effectivecoordination between providers []sthe relationship between tti and treatment outcomesis multifaceted so the controversy of published results is 0cžumer radiation oncology page of not surprising in this study we found that delays in theonset of rt do not harm all patients as tti is a problem in many public health systems further research iswarranted and should focus on two areas evaluatinglarge population surveys with highquality data andtreatmentrelated outcomes not just os and the prognostic relevance of imagingderived kinetic data of individualtumors which appeared promising in severalsmaller and statistically underpowered studies in orderto obtain a tool to identify patients at increased risk oftreatment failure due to delays in starting rt in a situation without clear knowledge to whom waiting for irradiation is harmful the only possible recommendationcould be that the waiting time for rt should be asshort as reasonably achievable asara abbreviationshnc head and neck cancer rt radiotherapy tti time to treatmentintervention scc squamous cell carcinoma cct concurrent chemotherapyct computer tomography mri magnetic resonance imaging os overallsurvival csh causespecific hazard lrc locoregional control dfms distantmetastasisfree survival dfs diseasefree survival iqr interquartile rangeci confidence interval hpv human papillomavirusacknowledgementsthis study was financially supported by the slovenian research agencyprogram no p30307authors contributionsstudy concepts žumer b strojan p study design žumer b pohar perme mstrojan p data acquisition all authors quality control of data strojan p dataanalysis and interpretation pohar perme m strojan p statistical analysispohar perme m manuscript preparation all authors manuscript editingstrojan p manuscript review žumer b pohar perme m strojan p the authors read and approved the final manuscriptfundingthis study was financially supported by the slovenian research agencyprogram no p3availability of data and materialsthe datasets analyzed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study protocol was approved by the republic of slovenia national medicalethics committee no all patients gave consent for using theirdata for study purposes at the start of their treatment for retrospective studies awritten consent is deemed unnecessary according to national regulationsconsent for publicationthe republic of slovenia national medical ethics committee approved thestudy which was conducted in accordance with the ethical standards laiddown in an appropriate version of the declaration of helsinki theneed for consent was waived by the republic of slovenia national medicalethics committeecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia 2institute of biostatistics and medicalinformatics faculty of medicine university of ljubljana ljubljana slovenia3department of radiology institute of oncology ljubljana ljubljanaslovenia 4chair of oncology faculty of medicine university of ljubljanaljubljana sloveniareceived 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 world health anization who life expecta | 0 |
Pralatrexate is a folate analogue inhibitor of dihydrofolate reductase exhibiting high affinity for reduced folate carrier1 with antineoplastic and immunosuppressive activities similar to methotrexate Despite advances in multimodality treatment strategies the survival rates for children with highrisk neuroblastoma have failed to improve Therefore the intense research continues in order to identify the ideal novel agent or combination of chemotherapy drugs to treat highrisk neuroblastomaMaterials and Methods Four human neuroblastoma cell lines were used to determine IC50 values of select chemotherapy agents Antiproliferative effects of pralatrexate were assessed by adherent and nonadherent colony formation assays Cell cycle arrest and apoptosis were measured by flow cytometry and immunoblotting PDX tissue culture was used to assess ex vivo efficacyResults Treatment with pralatrexate in all four neuroblastoma cell lines blocked cell growth in 2D and 3D culture conditions in a timedependent manner The potency of pralatrexate was tenfold stronger than methotrexate as measured by IC50 Pralatrexateinduced apoptosis was confirmed by caspase3 activation and PARP cleavage MYCN and SLC19A1 mRNA expressions were decreased with pralatrexate in MYCNamplified neuroblastoma cellsConclusions Pralatrexate demonstrated effective inhibition of cell growth and viability The higher potency of pralatrexate compared to methotrexate a drug with high levels of toxicity suggests pralatrexate may be a safer alternative to methotrexate as an effective chemotherapeutic agent in the treatment of patients with highrisk neuroblastomaINTRODUCTIONNeuroblastoma is a pediatric tumor derived from neural crest cells It is the most common pediatric solid tumor accounting for approximately of pediatric cancer deaths [] and it typically presents as a painless abdominal mass in infants and toddlers of to months of age [] Poor prognostic factors in children with neuroblastoma include age months at time of diagnosis unfavorable histology increased vascularization and MYCN amplification [] Despite intense research focused on the biology of neuroblastoma it remains one of the most enigmatic pediatric cancers in terms of its underlying molecular pathogenesis There has been only incremental improvement in the overall survival of children with highrisk neuroblastoma necessitating the search for a novel agent or combination of chemotherapy drugs []is key Altered metabolism to cancer cell proliferation Among the various metabolic pathways that are affected folate metabolism plays an important role Folate is essential for DNA synthesis and cell growth especially in rapidly dividing cells Inhibition of folate metabolism is the basis for many chemotherapy drugs In neuroblastoma folate mediated onecarbon metabolism is associated with aggressiveness and MYCN amplification Oncotargetwwwoncotargetcomwwwoncotargetcom Oncotarget Vol No pp 0cMethotrexate is a widely used [] A study by Lau in demonstrated higher folate requirements in MYCN amplified neuroblastoma cells compared to nonMYCN amplified cells [] They also showed that the increased folate uptake is mediated by reduced folate carrier1 RFC1 which is encoded by the gene SLC19A1 [] Previous studies have demonstrated that SLC19A1 is associated with MYCN amplification in neuroblastoma and that SLC19A1 is a direct transcriptional target of Nmyc [] The association between MYCN amplification and folate metabolism suggests the potential role of antifolate drugs in the treatment of neuroblastomasinhibitor of folate metabolism It inhibits dihydrofolate reductase DHFR and therefore disrupts purine and thymidylate biosynthesis leading to inhibited DNA replication and cell death However in the 1970s methotrexate was found to have high levels of toxicity combined with low treatment response rates in neuroblastoma patients and therefore it has not been clinically used for neuroblastoma treatment [] Pralatrexate is a folate analogue inhibitor of DHFR that exhibits high affinity for RFC1 [] and folylpolyglutamate synthetase FPGS Pralatrexate demonstrates antineoplastic and immunosuppressive properties that are similar to methotrexate It was FDA approved in the United States for treatment of relapsed or refractory peripheral Tcell lymphoma in [] A study by Serova in demonstrated decreased mRNA expression of SLC19A1 and SLC25A32 a mitochondrial folate carrier with pralatrexate treatment in several cancer cell lines [] As previously discussed SLC19A1 is downstream target of Nmyc in neuroblastoma The high affinity of pralatrexate for the SLC19A1 encoded RFC1 protein may demonstrate a potential role in the treatment of MYCNamplified neuroblastomaThe development of a new chemotherapeutic regimen is a long process that can take years to enter clinical trials and subsequently into bedside therapy Identifying alterative applications for previously FDAapproved drugs is a method that allows for quicker use in clinical practice [] Therefore we sought to evaluate current FDA approved antineoplastic drugs as potential novel treatment strategies for highrisk neuroblastoma and set out to assess the inhibitory role of pralatrexate on neuroblastoma cellsRESULTSThe IC50 of pralatrexate is tenfold less than methotrexateFour human neuroblastoma cell lines including MYCNamplified BE2C CHP212 and LAN1 as well as the nonMYCN amplified cell line SKNAS were treated with methotrexate or pralatrexate Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM µM measured by Cell Titer Glo¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments As shown in A in all four cell lines the IC50 of pralatrexate was approximately tenfold less than the IC50 of methotrexate These data demonstrate that highrisk neuroblastoma cells have enhanced pralatrexate sensitivity compared to methotrexate and that pralatrexate inhibits both MYCN amplified and nonMYCN amplified neuroblastoma growth in the low nanomolar range in vitroPralatrexate inhibited neuroblastoma cell growthTo demonstrate the timing of growth inhibition we treated neuroblastoma cells with pralatrexate or nM and measured cellular viability over a time course of days B Significant cell growth inhibition was first noted by day in SKNAS and CHP212 cells and by day in LAN1 and BE2C cells This indicates that pralatrexate effectively inhibits the proliferative potential of neuroblastoma cells We further validated the effects of pralatrexate on neuroblastoma cells in vitro by quantifying colony growth in a 3D matrix hydrogel where cells grow and selfassemble into clusters 3D cultures are more physiologically relevant and better represent in vivo tissue BE2C and LAN1 cells are high colonyforming neuroblastoma cell lines [] Concurrent treatment with pralatrexate completely abolished the ability of BE2C and LAN1 cells to develop colonies in gel drops A Both BE2C and LAN1 cell lines treated with pralatrexate demonstrated a decreased colony count and a decrease in colony size compared to cells treated with DMSO Figure 2B and 2C The colonies were counted from three separate microscopic fields and their size was measured using the scale bar on each image using Image J These findings suggest that pralatrexate represses the tumorigenesis potential and tumor progression of neuroblastomaPralatrexate induced G1 phase cell cycle arrest apoptosis and decreased Nmyc expressionTo further test whether pralatrexate directly altered neuroblastoma cell proliferation we evaluated the cell cycle distribution of treated cells compared with control Cell cycle analysis was performed in BE2C cells treated with pralatrexate nM or control at days and after treatment We observed a significant but modest increase in the G1 phase of the cell cycle ranging from a increase in the G1 cell population demonstrating induction of G1 cell cycle arrest Figure 3A Given the dramatic decrease in cell viability observed between days and of pralatrexate treatment Figure 1B we hypothesized that pralatrexate may also induce apoptosis in neuroblastoma cells To confirm apoptosis Oncotargetwwwoncotargetcom\x0cin cells treated with pralatrexate Western blotting was performed BE2C and CHP212 cells were treated with increasing doses of pralatrexate and nM The protein expression of total and cleaved caspase3 as well as total and cleaved PARP were examined at each increasing dose of pralatrexate Figure 3B confirming the induction of apoptosis Apoptosis was also seen secondary to pralatrexate treatment in nonMYCN amplified cells SKNAS SKNSH and SHSY5Y Supplementary Figure Given the proliferating role of Nmyc in neuroblastoma tumorigenesis we next evaluated whether pralatrexate could alter the Nmyc expression Interestingly we found decreased Nmyc expression with increasing doses of pralatrexate in both BE2C and CHP212 cells Figure 3B demonstrating persistent defects in proliferative potential induced by pralatrexate in neuroblastomaPralatrexate decreased MYCN and SLC19A1 gene expressions compared to SLC25A32Previous studies demonstrated that the expression of SLC19A1 the gene encoding the RFC1 receptor is associated with MYCN amplification in neuroblastoma [] Pralatrexate is a folate analogue inhibitor with high affinity for RFC1 [] Therefore we sought to examine the effects of pralatrexate treatment on MYCN and SLC19A1 gene expression in BE2C and CHP212 cell lines compared to the effects in nonMYCN amplified cells BE2C and CHP212 cells were treated with and nM pralatrexate or DMSO and qPCR was performed As expected from the finding in Figure 3B pralatrexate treatment resulted in decreased MYCN expression in both BE2C and CHP212 cells Figure 4A and 4B Interestingly we also found that both BE2C and CHP212 cells treated with pralatrexate demonstrated decreased SLC19A1 expression but no difference in SLC25A32 expression compared to control cells Figure 4A and 4B Treatment of nonMYCN amplified cells SKNAS SKNSH and SHSY5Y did not affect SLC19A1 or SLC25A32 expression Supplementary Figure These findings may support the previous studies [ ] that SLC19A1 is a direct transcription target of MYCN in neuroblastomas and pralatrexate treatment affects SLC19A1 expression in MYCN amplified cells but not SLC25A32 expression In addition in both BE2C and CHP212 cells pralatrexate did not decrease FPGS mRNA expression Figure 4C and 4D Neither SLC25A32 or FPGS expression was affected in nonMYCN amplified cells treated with pralatrexate Supplementary Figure Interestingly in the BE2C cells and the nonMYCN amplified cells pralatrexate treatment led to an increase in DHFR expression This may imply treatment with pralatrexate selected for cells with increased DHFR expression and inherent pralatrexate resistance or a resultant upregulation of the DHFR gene with DHFR protein inhibition These findings would Figure IC50 of human neuroblastoma cell lines BE2C CHP212 and LAN1 MYCNamplified and SKNAS nonMYCNamplified A The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with methotrexate were and µM respectively The IC50 doses of BE2C CHP212 LAN1 and SKNAS cells treated with pralatrexate were and µM respectively Cell growth was determined after h of continuous exposure to methotrexate or pralatrexate nM µM measured by Cell Titer Glo¢ assay and the IC50 of each drug was calculated for each cell line using the Genedata Screener software Values shown are mean ± SD of three separate experiments B Cell viability was assessed using the Cell Counting Kit8 assay Pralatrexate significantly inhibited cell viability in all four neuroblastoma cell lines as compared to DMSO control groupOncotargetwwwoncotargetcom\x0cbe consistent with findings by Serova et al in which pralatrexateresistant cells demonstrated increased DHFR protein expression []Pralatrexate treatment response in ex vivo neuroblastoma growthGiven these persistent in vitro findings we next evaluated whether pralatrexate could be evaluated ex vivo to guide treatment decisions for individual patients We used a neuroblastoma PDX model where tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin The size of these fragments is comparable to the size of a standard clinical tumor biopsy specimen The fragments were then cultured for days in the presence of pralatrexate nM using standard cell culture conditions Notably tumor tissues were significantly affected by ex vivo pralatrexate treatment and showed decreased Ki67 staining compared to tissues cultured in vehicle control treatment Figure These results suggest that a simple shortterm ex vivo treatment assay of a viable tumor specimen may aid in identifying neuroblastoma patients who are likely to gain benefit from pralatrexate treatment options in the futureDISCUSSIONHighrisk neuroblastoma remains quite difficult to cure necessitating the discovery of new chemotherapy agents to be used alone or in combination therapy Previous studies have reported an increased folate in MYCNamplified neuroblastoma cells demand mediated by the RFC1 receptor [] Additionally the gene encoding the RFC1 receptor SLC19A1 is a direct transcriptional target of Nmyc in neuroblastoma cells suggesting a role for antifolate drugs in the treatment of neuroblastoma Methotrexate has previously been studied in neuroblastoma however it was found to have a prohibitive toxicity and has not been used in neuroblastoma clinically In contrast pralatrexate a folate analogue inhibitor is similar to methotrexate with a more favorable side effect profile suggesting a potential role for the use of pralatrexate as a chemotherapeutic agent against neuroblastomaThe present study sought to determine the effects of treatment with pralatrexate on in vitro and ex vivo cell growth in four human neuroblastoma cell lines The IC50 of pralatrexate was found to be 10fold less than that of methotrexate This tenfold difference between pralatrexate and methotrexate was also found in colon breast and thyroid cancer cells by Serova et al in [] The decreased IC50 of pralatrexate allows for treatment with lower doses and a more tolerable sideeffect profile compared to methotrexate independent of MYCN amplificationPralatrexate not only induced celldeath via apoptosis but it also successfully inhibited neuroblastoma in vitro cell growth and proliferation in 2D and 3D cell cultures as well as in our PDX exvivo model By inhibiting the RFC1 receptor pralatrexate decreased the amount of folate entering cells and in turn decreased Figure Pralatrexate inhibited neuroblastoma colony growth A Representative images of light microscopy à magnification for BE2C and LAN1 cells after days of treatment with pralatrexate versus control Pralatrexate treatment decreased cell growth in both BE2C and LAN1 cells compared to control scale bar μm B Colony count and colony size for BE2C cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drug C Colony count and colony size for LAN1 cells were analyzed and quantified mean ± SD p for nM pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cDNA synthesis This was demonstrated by the increased time spent in the G1phase of the cell cycle in cells treated with pralatrexate PDXs have been shown to parallel clinical outcome in various tumor types [] The major applications of neuroblastoma PDXs would be related to drug testing exploration of treatment resistance and biomarker discovery Combining PDXs and ex vivo culture will incorporate human tumor tissue in its native Figure Effects of pralatrexate treatment on caspase3 PARP and Nmyc protein expression A Cell cycle analysis with propidium iodide demonstrates enhanced G1 cell cycle arrest at and h following treatment Cell cycle analysis was completed with events per replicate mean ± SD p for nM pralatrexate treatment vs no drug B Treatment with increasing doses of pralatrexate induced apoptosis in BE2C and CHP212 cells Cells treated with pralatrexate demonstrated cleaved caspase3 protein expression when treated with and nM doses Cleaved PARP expression was noted after treatment with nM Treatment with pralatrexate decreased Nmyc protein expression in BE2C and CHP212 cells βactin was used as an internal controlOncotargetwwwoncotargetcom\x0c3D state and enable dynamic manipulation of the system minimizing animal experiments and costLau has shown that SLC19A1 is a downstream direct transcriptional target of Nmyc in neuroblastoma cells and that MYCNamplified cells have an increased folate dependence [] In our study pralatrexate also led to a decrease in expression of the RFC1 genes SLC19A1 and SLC25A32 The decrease in SLC19A1 was more pronounced compared to the mitochondrial folate receptor gene SC25A32 suggesting pralatrexate may be more specific to the cytosolic RFC1 receptor compared to the mitochondrial RFC1 receptor However further studies are necessary to investigate this relationship Meanwhile pralatrexate treatment led to a marked increase in DHFR expression in BE2C cells and a slight increase in CHP cells This is unlikely an upregulation of DHFR and more indicative of increased DHFR mRNA being harvested from pralatrexate resistance cells Similar results were found in a previous study on colon breast and thyroid cancer cells lines Serova found that pralatrexateresistant cells had increased DHFR protein expression [] The increase in DHFR expression may lead to an increase in the amount of DHFR protein requiring more than nM of pralatrexate to inhibit cell growth and proliferation However further studies surrounding the dose of pralatrexate and its relationship to DHFR gene expression are neededNeuroblastoma is a heterogenous tumor and further studies are needed to examine the effects of pralatrexate on additional cells lines Additionally the remaining cells that survived after pralatrexate treatment may represent pralatrexate resistant cells Future studies are needed to elucidate potential mechanisms of pralatrexate resistance such as increased DHFR gene expression as well as the relationship between pralatrexate and SLC19A1 versus other folate synthesis enzyme expressions Given pralatrexate is already an FDAapproved and in clinical use future clinical studies are needed to investigate the effects of pralatrexate treatment on neuroblastoma in vivoMATERIALS AND METHODSCells antibodies and reagentsThe neuroblastoma cell line LAN1 was a gift from Dr Robert C Seeger University of Southern California Los Angeles CA All other neuroblastoma cell lines BE2C CHP212 SKNAS SKNSH and SHSY5Y Figure Effects of pralatrexate treatment on MYCN SLC19A1 and SLC25A32 gene expressions After day of treatment the mRNA expression of MYCN SLC19A1 and SLC25A32 were measured by qPCR in A BE2C cells treated with nM of pralatrexate when compared with DMSO treated cells and in B CHP212 cells treated with nM of pralatrexate C D qPCR was performed on BE2C and CHP212 cells treated with pralatrexate to assess for mRNA expression of two keyenzymes in folate synthesies DHFR and FPGS in the treatment mean ± SD p for pralatrexate treatment vs no drugOncotargetwwwoncotargetcom\x0cwere purchased from the American Type Culture Collection ATCC Manassas VA Cells were maintained in RPMI with glutamine and FBS at °C in a humidified atmosphere consisting of CO2 and air Primary antibodies for Caspase3 Cat No PARP Cat No Nmyc Cat No were purchased from Cell Signaling Technology Danvers MA Ki67 Cat No was from Abcam Cambridge MA and βactin Cat No A2066 was from SigmaAldrich St Louis MO Methotrexate and pralatrexate were obtained from National Cancer InstituteDivision of Cancer Treatment and DiagnosisDevelopmental Therapeutics Program httpdtpcancergov and dissolved in dimethyl sulfoxide DMSO and further diluted in culture media to desired concentrations Neuroblastoma COGN415à patientderived xenograft PDX cells were obtained from the Childhood Cancer Repository maintained by the Childrens Oncology Group COG and Xenograft RepositoryDrug sensitivity and dose responsive curve assayFor cell viability screening cells [BE2C1500well LAN1 3000well CHP2124000well SKNAS 3000well] were plated and treated the following day with methotrexate and pralatrexate Cell growth was determined after h of continuous exposure to nM µM of methotrexate or pralatrexate using Cell Titer GloTM reagent Promega with luminescence measured using an EnVision multilabel plate reader PerkinElmer IncCell viability assayNeuroblastoma cells were seeded onto 96well plates permitted to attach overnight and were treated with either pralatrexate or nM or DMSO for days Cell viability measurements using the Cell Counting Kit8 Dojindo Molecular Technologies Inc Rockville MD were obtained daily3D colony formation assayBE2C or LAN1 cells were trypsinized embedded in μl of Cultrex® RGF BME Type matrix hydrogel Trevigen Gaithersburg MD and seeded in 48well plates cellswell RPMI medium containing FBS was added with pralatrexate treatment and incubated for days Colonies were photographed and the number and size were quantified The colonies were counted from three separate microscopic fields and their size was measured by the scale bar on each image using Image JCell cycle analysisCell cycle distribution was analyzed using flow cytometry with propidium iodide Sigma Aldrich BE2C cells were plated at equal numbers à cells and treated with either pralatrexate nM or DMSO At day and after treatment cells were washed and fixed in ethanol Fixed cells were incubated with mgmL RNAase for minutes at °C stained with propidium iodide mg mL and analyzed on a BD FACSCalibur BD Biosciences San Jose CAqPCR and immunoblottingTotal RNA was isolated and purified using a TRIzol® Reagent Thermo Scientific cDNA was synthesized using the qScript cDNA SuperMix QuantaBio RealFigure Ex vivo tissue culture model recapitulated antitumor response to pralatrexate Representative HE and Ki67 immunohistochemistry staining sections were obtained from a neuroblastoma PDX COGN415à treated ex vivo with nM pralatrexate or vehicle control for days and demonstrated poorly differentiated neuroblastoma cells and decreased Ki67 staining in pralatrexatetreated tumor compared to vehicle control à magnification scale bar μmOncotargetwwwoncotargetcom\x0creverse reverse time PCR and data collection were performed on a CFX96 instrument BioRad Data were normalized to an endogenous control βactin Specific target primers are MYCN forward 5ʹGCTTCTACCCGGACGAAGATG3ʹ reverse 5ʹCAG CTCGTTCTCAAGCAGCAT3ʹ SLC19A1 forward 5ʹAACAGGTCTGGGTTTTGTGC3ʹ 5ʹGTGCAGTATCATGCCCTGTG3ʹ SLC25A32 forward 5ʹATTGGTGGAAGCTGATTTGC3ʹ 5ʹTGGTCTGGATTTGGTCAACA3ʹ DHFR forward 5ʹCTCAAGGAACCTCCACAAGG3ʹ reverse 5ʹGTTTAAGATGGCCTGGGTGA3ʹ FPGS forward 5ʹGGGTGACCCTCAGACACAGT3ʹ reverse 5ʹGTCTTCAGGCCATAGCTTCG3ʹ Amplification was performed for cycles of s at °C s at °C and s at °C Whole cell lysates were collected using cell lysis buffer and equal amounts of protein were loaded on a NuPAGE BisTris gel followed by transfer onto PVDF membranes BioRad Hercules CA USA and probed with antibodiesEx vivo culture and immunohistochemistryCOGN415à patientderived xenograft cells were obtained from the Childhood Cancer Repository maintained by COG Clinical and genomic features of the tumors were detailed in a study by Harenza in [] Cells were suspended in Matrigel diluted with PBS and à cells were injected into the flank of NOD scid gamma mice at weeks of age UTSW Mouse Breeding Core All studies were approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center To keep cost down we used only female mice for passing PDX Mice were euthanized once tumors reached mm and tumors were dissected into 1mm3 pieces and cultured in duplicate on a presoaked gelatin sponge Johnson and Johnson in 24well plates containing µL RPMI with FBS antibioticantimycotic solution mgmL hydrocortisone and mgmL insulin SigmaAldrich Tissues were cultured at °C for days with either pralatrexate nM or vehicle control then formalinfixed and paraffin embedded COGN415à tissue sections were stained with hematoxylin and eosin or with an antibody against Ki67Statistical analysisAll results are shown as the mean value ± SD statistical analyses were performed using student ttest for comparisons between the groups A p value of was considered significant GraphPads Prism software was used for the statistical analysisAbbreviationsCOG Childrens Oncology Group DHFR dihydrofolate reductase DMSO dimethyl sulfoxide FPGS folylpolyglutamate synthetase PDX patientderived xenograft RFC1 reduced folate carrier1Author contributionsStudy concept and design RAC SL JQ DHC Acquisition of data RAC SL JQ Analysis and interpretation of data RAC SL JQ DHC Drafting of manuscript RAC SL JQ DHC Critical revision RAC SL JQ DHC RAC and SL contributed equally to this workACKNOWLEDGMENTSWe thank Karen Martin for her assistance in manuscript preparationCONFLICTS OF INTERESTThe authors declare no conflicts of interestFUNDINGThis work was supported by a grant from the National Institutes of Health R01 DK61470REFERENCES Colon NC Chung DH Neuroblastoma Adv Pediatr httpsdoiorg101016jyapd201103011 [PubMed] Park JR Eggert A Caron H Neuroblastoma biology prognosis and treatment Hematol Oncol Clin North Am httpsdoiorg101016jhoc200911011 [PubMed] Matthay KK Maris JM Schleiermacher G Nakagawara A Mackall CL Diller L Weiss WA Neuroblastoma Nat Rev Dis Primers httpsdoiorg101038nrdp201678 [PubMed] Sidarovich V De Mariano M Aveic S Pancher M Adami V Gatto P Pizzini S Pasini L Croce M Parodi F Cimmino F Avitabile M Emionite L A HighContent Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy Mol Cancer Ther httpsdoiorg10115815357163MCT170841 [PubMed] Schramm G Wiesberg S Diessl N Kranz AL Sagulenko V Oswald M Reinelt G Westermann F Eils R Konig R PathWave discovering patterns of differentially regulated enzymes in metabolic pathways Bioinformatics httpsdoiorg101093bioinformaticsbtq113 [PubMed] Lau DT Flemming CL Gherardi S Perini G Oberthuer A Fischer M Juraeva D Brors B Xue C Norris MD Marshall Oncotargetwwwoncotargetcom\x0cGM Haber M Fletcher JI et al MYCN amplification confers enhanced folate dependence and methotrexate sensitivity in neuroblastoma Oncotarget httpsdoiorg1018632oncotarget3732 [PubMed] OConnor OA Amengual J Colbourn D Deng C Sawas A Pralatrexate a comprehensive update on pharmacology clinical activity and strategies to optimize use Leuk Lymphoma httpsdoiorg101080 [PubMed] Foss FM Evaluation of the pharmacokinetics preclinical and clinical efficacy of pralatrexate for the treatment of Tcell lymphoma Expert Opin Drug Metab Toxicol httpsdoiorg10151717425255201159540 [PubMed] Visentin M Unal ES Zhao R Goldman ID The membrane transport and polyglutamation of pralatrexate a newgeneration dihydrofolate reductase inhibitor Cancer Chemother Pharmacol httpsdoiorg101007s0028001322319 [PubMed] Serova M Bieche I Sablin MP Pronk GJ Vidaud M Cvitkovic E Faivre S Raymond E Single agent and combination studies of pralatrexate and molecular correlates of sensitivity Br J Cancer httpsdoiorg101038sjbjc6606063 [PubMed] Walton JD Kattan DR Thomas SK Spengler BA Guo HF Biedler JL Cheung NK Ross RA Characteristics of stem cells from human neuroblastoma cell lines and in tumors Neoplasia httpsdoiorg101593neo04310 [PubMed] Hidalgo M Amant F Biankin AV Budinska E Byrne AT Caldas C Clarke RB de Jong S Jonkers J Maelandsmo GM RomanRoman S Seoane J Trusolino L et al Patientderived xenograft models an emerging platform for translational cancer research Cancer Discov httpsdoiorg10115821598290CD14 [PubMed] Harenza JL Diamond MA Adams RN Song MM Davidson HL Hart LS Dent MH Fortina P Reynolds CP Maris JM Transcriptomic profiling of commonlyused neuroblastoma cell lines Sci Data httpsdoiorg101038sdata201733 [PubMed]Oncotargetwwwoncotargetcom\x0c' | 2 |
several immunotherapeutic strategies that harness the exquisite speciï¬city of the immune systemto eliminate tumors have emerged during the past decade these include cancer vaccines immunecheckpoint blockade and adoptive cell therapy act with the potential to revolutionize thestandard of care for a range of malignanciesto a large extent the speciï¬city of immunotherapy is dependent on the recognition of speciï¬ctumor antigens especially neoantigens neoantigens are a kind of tumor antigen derived fromtumorspeciï¬c somatic mutations and are highly restricted to tumor cells with minimal establishedimmune tolerance neoantigenbased cancer vaccines have shown promising therapeutic eï¬ectsin the clinic in addition a growing body of evidence indicates that neoantigenspeciï¬c tcells underlie the success of the recently emergent immune checkpoint inhibitor therapy adoptive transfer of autologous in vitro expanded tumorltrating lymphocytes tils wasreported to achieve dramatic clinical responses in some metastatic cancer patients especially inthose with melanoma and cervical cancer indepth studies have revealed the criticalroles of neoantigenspeciï¬c t cells in maintaining durable responses following act in support of these ï¬ndings the adoptive transfer of selected tils targeting neoantigens led tosignificant tumor regression increasing research attention has been shifted to identifyingand selecting neoantigenspeciï¬c t cells however such a precise targeting strategy posesa great challenge in terms of the identiï¬cation and isolation of neoantigenspeciï¬c t cells methodshave been proposed and developed for this purpose here we attempt to summarize the knownstrategies for isolating neoantigenspeciï¬c t cellsedited bycyrille j cohenbarilan university israelreviewed bymanel juanhospital clnic de barcelona spainrodabe n amariauniversity of texas md andersoncancer center united statescorrespondencezhenyu dingdingzhenyuscueducnspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in oncologyreceived december accepted june published august citationli q and ding zy the ways ofisolating neoantigenspeciï¬c t cellsfront oncol 103389fonc202001347frontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsidentification and isolation ofneoantigenspecific t cells fromtilsfor most metastatic patients this time frame is unacceptable toaddress these issues additional attempts have been made usingeither surface markers or t cell receptor tcr redundancyresearchers have long attempted to isolate neoantigenspeciï¬csubpopulations from the of transferred tils in earlystudies an autologous tumor cell cdna library was constructedand used as a pool to screen for neoantigenspeciï¬c t cells in a study of a melanoma patient who experienced acomplete response going beyond years following adoptive tiltransfer one t cell clone speciï¬c for a mutated antigen ppp1r3bwas identiï¬ed and shown to be responsible for the antitumoreï¬ects advanceshowever the timeconsuming and laborious process requiredto identify neoepitoperesponsive t cells has hindered theirextensive functional assessmentin nextgeneration sequencing have enabled the rapid assessment of themutational landscape of human cancers and made it possibleto identify immunogenic mutated tumor antigens throughin silico analysis rosenbergs group ï¬rst employed predictedneopeptides obtained by wholeexome sequencing and humanleucocyte antigen hla class ibinding algorithms for tilscreening using this approach they identiï¬ed neoantigensrecognized by therapeutic bulk til cultures that mediatedobjective tumor regressions in three individuals with melanoma using a similar method neoantigenspeciï¬c cd8 tilscould also be identiï¬ed in hematological malignancies such asacute lymphoblastic leukemia all prickett and stevanovic also demonstrated that neoantigenspeciï¬c t cells could be identiï¬ed from therapeutic tils byscreening tandem minigene tmg libraries encoding cancermutations identiï¬ed from patients tumors by wholeexomesequencing this ï¬nding might further facilitate the recognitionof neoantigenspeciï¬c t cells because it circumvents the needfor prediction of hlapeptide binding and synthesis of a largenumber of peptideswith the advent of these techniques the ï¬eld of act took agreat leap from bulk tils to neoantigenspeciï¬c t cells a conciseï¬owchart showing the steps involved in identifying and isolatingneoantigenspeciï¬c t cells for act is summarized in figure tran successfully performed neoantigenspeciï¬c t celltherapy in a 43yearold woman with extensively metastatic andintensively treated cholangiocarcinoma after administration ofa bulk lymphocyte population containing a high percentage ofneoantigen erbb2ipspeciï¬c cd4t cells the patient showed alonglasting objective clinical response without obvious toxicitysubsequently neoantigenspeciï¬c t cells were identiï¬ed in onecolon cancer patient and another breast cancer patient andreinfusion of these speciï¬c t cells led to a partial response inone patient and a durable complete response in another currently act with neoantigenspeciï¬c t cells is beingtested in clinical trials in both solid and hematological tumorssupplementary table howeverthe extensive expansion of neoantigenspeciï¬ct cells during preparation compromises their proliferationpotential the method involved requiressophisticated equipment and a time period of several monthsin additionapproaches based on surfacemarkerscd137 belongs to the tumor necrosis factor receptor superfamily it functions as a costimulatory molecule to promote theproliferation and survival of activated t cells cd137expression is highly restricted to transiently activated cd8 tcells but almost undetectable in resting cells upregulated cd137can be detected on stimulated cd8 t cells of all phenotypeseg na¯ve t cells as well as early and late memory eï¬ector tcells naturally occurring tumorreactive t cells stimulatedby tumor antigens also express cd137 as proven by ye in a clinical trial trial registration id nct02111863 among patients with melanoma who underwent adoptive transfer withcd137selected tils only patient achieved partial responseand the remaining progressed the study was terminatedthis approach has its pitfalls because cd137 is an activationmarker cd137 t cells obtained by largescale productionare generally overactivated and highly diï¬erentiated withlimited proliferative potential a potential solution is to obtaintcrs from these cd137t cells instead this strategy wasreported by parkhurst brieï¬y cd8 t cells werestimulated overnight with immunogenic mutated tmg rnassubsequently the cd8 t cell population with the highestcd137 expression was sorted by ï¬uorescentactivated cell sortingfacs and expanded in vitro then dominant tcr α and chains were sequenced in the enriched populations twentyseven tcrs from patients that recognized neoantigensexpressed by autologous tumor cells were identiï¬ed howeverthis process was timeconsuming monthsa simpliï¬ed protocol was proposed by seliktaroï¬r here tils but not cd8 t cells were coculturedwith autologous tumor cells cd137 t cells were isolatedby magnetic bead separation and expanded no further tcrsequencing was performed the entire process took only dayst cells stimulated with neoantigens or other tumorassociatedantigens exhibit upregulated cd137 expression therefore a cd137based selection protocol was advocated forits broad antigen coverage including both neoantigen and sharedtumor antigens without prior knowledge of epitope speciï¬cityhowever the prerequisite of the establishment of autologoustumor cell lines poses a challengedirect and indirect evidence shows thatthe interactionbetween pd1 and pdl1 inhibits t lymphocyte functionleading to evasion of persistent ammatory or autoimmunereactions howeverthis protective mechanism ishijacked by tumors to escape immune surveillance pd1 hasbeen characterized as an inhibitory receptor on chronicallystimulated tcells in the tumor microenvironment atthe tumor site tils are exposed to tumor antigensthebinding of tcr and antigen upregulates either costimulatory orcoinhibitory receptors to promote or inhibit t cell activation andfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsfigure the general approach of identifying and isolating neoantigenspeciï¬c tils for act the tumor cells from excised tumor tissue and matched normal cellsunderwent wholeexome sequencing wes and rna sequencing to identify nonsynonymous mutations based on the information either tandem minigenes tmgsor peptides were then synthesized these tmgs or peptides were pulsed into autologous antigen presenting cells apcs such as dendritic cells dcs or b cells andthey were processed and presented in the context of major histocompatibility complex mhc on the other side the excised tumors were minced into ¼ mm3fragments and placed in 24well plates stimulated with il2 then the tils were cocultured with these pulsed apcs the identiï¬cation of the individualneoantigenspeciï¬c t subpopulation was dependent on the ifnγ enzymelinked immunospot elispot assay and the activation of the markers such ascd13741bb or cd134ox40 on the t cell surfaces when recognizing their cognate target antigen t cells with these activation surface markers would be puriï¬ed byï¬ow cytometry then the sorted t cells were subject to rapid expansion in vitro and reinfusion to the tumorbearing patientfunction respectively therefore pd1 t cell populationsamong tils may contain a large proportion of tumorspeciï¬ct cells the ï¬ndings of inozume and ahmadzadeh that tumorresponsive t cells are enriched amongcd8pd1 lymphocytes from fresh melanoma specimensprovide direct support for this notionin another study gros demonstrated that pd1expression on cd8 tils in fresh melanoma tumor specimensenabled identiï¬cation of a diverse patientspeciï¬c repertoireof clonally expanded tumorreactive cells including mutatedneoantigenspeciï¬c cd8 lymphocytes although pd1 is aninhibitory receptor expressed on t cells studies have shownthat il2 restored the antitumor function of t cells in vitro however on antigenexperienced terminally diï¬erentiatedeï¬ector memory temra cells pd1 is either not expressed orexpressed at very low levels therefore a pd1basedenrichment strategy may not be suitable for these cellsscreening strategies based on cd137 or pd1 expressionare suitable for cd8 t cells mainly in melanoma epithelialcancers which accountfor more than of all humanmalignancies harbor fewer mutations than melanoma theyexhibit compromised capability to induce mutationspeciï¬c tcell responses together with a limited number of ltratingneoantigenspeciï¬c tils in addition cd4t cells havebeen shown to play an important role in mediating tumorregression in animal models and patients however cd137 or pd1 is expressed on cd8 cells as a solemarker therefore it may not be reliably used to enrich activatedcd4 cells cd134 is transiently expressed on cd4 t cells stimulated by antigens and can be used as a marker forthe classiï¬cation of mutant reactive t cells recently yossef reported an approach in which thetils that expressed cd134 or cd137andor pd1 were isolatedby facs thus both cd4 t and temra cells were rescuedwhich would otherwise be missed if a single marker were usedsorted cells underwent limitingdilution in microwell plates toavoid the overgrowth of nonspeciï¬c t cells cultures were testedfor the ability to recognize a 25mer peptide pool encompassingpossible neoantigens notably the highly oligoclonal natureof these t cells makes possible the convenient applicationof single cell sequencing of their tcrs in patients withmetastatic epithelial cancer this highthroughput approach ledto the detection of cd4 and cd8 t cells targeting and neoantigens respectively whereas only and neoantigenswere identiï¬ed by using the til fragment screening approachin patients in which no neoantigen was found by traditionalfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsscreening the novel approach identiï¬ed distinct neoantigenspeciï¬c tcr clones for one patient and a highly potent mhcclass iirestricted krasg12vreactive tcr for the other in ametastatic tumor sample from a patient with serous ovariancancer mhc class iirestricted tcrs targeting the tp53g245shotspot mutation were identiï¬edtcr frequencytcr sequence analysis is used as a tool to monitor t cellresponses to speciï¬c antigens by measuring the abundance oft cell clonotypes the advent of nextgenerationsequencing has enabled identiï¬cation of the full tcr repertoireof tils this valuable data for tcrs from tumorreactive tils could be used to modify t cells tcrt howeverthe lengthy expansion process and excessive stimulation wouldresult in tcr repertoire switching to avoid this problempasetto directly performed tcr sequencing of thefresh enzymatically digested melanoma tissues prior to in vitroexpansion as described earlier tumorreactive clonotypes wereenriched in cd8pd1 til subsets in melanoma the authors analyzed the tcr repertoire of tils in cd8cd8 cd8pd1 or cd8pd1 subsets respectivelyand found that many of the most frequently occurring tcrclonotypes present in the cd8pd1 til subset recognizedthe autologous tumor and tumor antigens included neoantigensthis report provided a much more convenient approach toeï¬ciently identify tumorreactive t cells based solely on thefrequency of tcr and pd1 expression without prior knowledgeof the speciï¬c neoantigen however this strategy must be appliedwith caution because the isolated tcr clones may be selfreactiveand result in deleterious ontarget oï¬tumor toxicities isolation of neoantigenspecific tcells from peripheral bloodlymphocytes pblsin some situations neoantigenspeciï¬c t cells were undetectablein the til compartment possibly owing to the following factorspresentation of neoantigens in a nonammatory context impaired t cell ltration because of the sparse distributionof adhesion molecules on these cells and presence ofimmunosuppressive cytokines and cells eg regulatory t cellsin the tumor microenvironment furthermore the tissuefrom which tils may be obtained poses a challenge in thisregard peripheral blood is an alternative and reliable source forneoantigenspeciï¬c t cellsthe ï¬rst attempt is considered to have been made by agroup led by lennerz in this study a systemof mixed lymphocytetumor cells mltc was establishedwherein peripheral blood mononuclear cells pbmcs froma patient with metastatic melanoma were cocultured withautologous tumor cells the mtlc system could be viewed asa simpliï¬ed in vitro simulation of the tumor microenvironmentfurthermore cytotoxic t lymphocyte ctls clone derived bylimiting dilution from the mltc system or mltc were subjectto autologous tumor cell cdna library screening t cell clonesreactive to mutated epitopes were obtainedthe use of mhcpeptide tetramers is a canonical methodto identify and study a certain antigenspeciï¬c t cell subset for act tetramers were used to isolate and expandtumor antigenspeciï¬c t cells moreoverin immunecheckpoint inhibitor icitreated cancer patients mhcpeptidetetramers have been successfully used to monitor neoantigenspeciï¬c t cells cohen used this method tosort neoantigenspeciï¬c t cells from the pbls of patients withmetastatic melanoma in brief a panel of mhcpeptide tetramersconsisting of predicted neoepitopes was synthesized and usedto screen pbls neoantigenspeciï¬c t cells targeting of the mutated epitopes identiï¬ed from tils could be isolated fromautologous peripheral blood with frequencies ranging between and in cancers with intermediate mutational loadssuch as multiple myeloma the use of mhcpeptide tetramerscould also isolate neoantigenspeciï¬c t cells from the pbls however this method was only applied to cd8 t cellsand required hlabinding prediction algorithms to guide thesynthesis of hlapeptide tetramersa previous study has shown that pd1 expression couldguide the identiï¬cation of neoantigenspeciï¬c cd8 t cellsfrom the tumor microenvironment the same strategycould be adopted for isolation from pbls in one study patients with metastatic melanoma were enrolled cd8pbls were expanded in vitro and cocultured with autologousdcs which were electroporated with in vitro transcribed tmgrna for mutant epitopes in out of patients neoantigenspeciï¬c lymphocytes could be isolated from the cd8pd lymphocyte subset but not the cd8pd1 lymphocytesubset the isolation of neoantigenspeciï¬c cells from the pblsof patients with epithelial cancer is even more challengingpreexisting antigenspeciï¬c memory t cells may represent apotential solution memory t cells including central memoryt cells tcm eï¬ector memory t cells tem and temrafrom pbls were cocultured with dcs loaded with candidateneoantigens in the tmg or peptide form after coculturingmemory cells were restimulated with dcs loaded with all tmgsand then sorted by the expression of cd134 and cd137 to enrichfor neoantigenreactive t cells the resulting cells were thenexpanded and screened against all tmgs to test for neoantigenrecognition with this highly sensitive in vitro stimulationivs method t cells targeting krasg12d and krasg12vwere successfully isolated from out of epithelial cancerpatients this new method enabled identiï¬cation and isolationof neoantigenreactive t cells from the blood circulation at verylow frequenciesthe identiï¬cation of neoantigenspeciï¬c t cells from na¯vet cells is also of interest a previous report showed that bothna¯ve and activated neoantigenspeciï¬c t cells could be expandedfrom the peripheral blood of follicular lymphoma patients bypriming with peptidepulsed dcs using the same methodneoantigenspeciï¬c t cells were successfully expanded fromthe peripheral blood of hlamatched healthy donors these preliminary results support the use of na¯ve t cells asfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsfigure a strategies of identifying neoantigenspeciï¬c t cells the limitations of current methods of identifying neoantigenspeciï¬c t cells and strategies toimprove neoantigenspeciï¬c t cells identiï¬cation tils tumor ltrating lymphocytes pbls peripheral blood lymphocytes pd1 programmed cell death1 temracells terminally differentiated effector memory cells tcr t cell receptor tmg tandem minigene mhc major histocompatibility complex b the blueprint ofisolating neoantigenspeciï¬c t cells from peripheral blood after neoantigentargeting vaccine after several rounds of immunization with neoantigen vaccines t cellsare collected from the patients peripheral blood and neoantigenspeciï¬c t cells are identiï¬ed and isolated from these t cells then the neoantigenspeciï¬c t cellsundergo rapid expansion rep or their tcrs are exploited to modify autologous lymphocytes the expanded neoantigenspeciï¬c t cells or modiï¬ed tcrt cells arereinfused to the patientan alternative source for act however their exceptionally lowfrequencies in peripheral blood and requirement for repeatedstimulation pose hurdles recentlyalargelibrarybased minilinesscreeningapproach was proposed which aimed to identify na¯ve antigenreactive t cells from small volumes of blood this systembegan with a smallscale culture in 96well plates with initial t cells in each well the smallscale culture underwent arapid to 5000fold expansion miniline thousands ofsuch wellscaled cultures were conducted simultaneously eacht cell clone was maintained at a frequency of in butampliï¬ed to an absolute number of cells which isa suï¬cient number for routine detection applying this highthroughput parallel t cell culture system neoantigenspeciï¬ct cells were identiï¬ed and expanded months prior to theï¬rst tumor recurrence in a patient with highgrade serousovarian cancer however the long duration of culture possiblyrendered this method more suitable as a preemptive therapeuticstrategy discussionafter decades of eï¬orts the adoptive transfer of neoantigenspeciï¬c t cells is ï¬nally close to readiness for clinical applicationhigh eï¬cacy of this immunotherapeutic strategy has beenachieved in a number of cancer patients and the prospects arepromising however these approaches are also quite costly andhard to apply to large numbers of patients the current methodsof identifying neoantigenspeciï¬c t cells are summarized infigure 2a and supplementary table more convenient andfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellseï¬ective screening methods for neoantigenspeciï¬c t cellsremain necessary some strategies to improve neoantigenspeciï¬ct cells identiï¬cation are shown in figure 2ait is feasible to obtain neoantigentargeting t cells frompbls although their frequencies are generally much lower thantils however increasing the frequencies of thesevaluable neoantigenspeciï¬c t cells in peripheral blood remainsa challengevaccination with neopeptides has been shown to primecd4 and cd8 tcell responses in mouse models patients treated with vaccines generated neoantigenspeciï¬c tcells it could be reasonably inferred that the isolationof neoantigenreactive t cells from the peripheral bloodwould be more easily achieved following neoantigenspeciï¬cvaccination this neoantigenbased combo immunotherapy hasits advantages ï¬rst isolation and expansion of tils in vitro isnot necessary second cancer vaccines not only elicit neoantigenspeciï¬c t cell responses and amplify existing tumorspeciï¬c tcells responses but they also increase the breadth and diversityof the tumorspeciï¬c t cell response multiclonal t cellsmay thus be obtained third the relatively easy preparation ofcancer vaccines would buy time for the isolation of neoantigenspeciï¬c t cells in maintaining the performance of patients theblueprint is shown in figure 2bconclusionthe previous decade has witnessed theemergence ofimmunotherapy for cancer accumulating evidence suggests thatneoantigenspeciï¬c t cells underlie successful immunotherapytherefore the isolation of neoantigenspeciï¬c t lymphocytesrepresents the holy grail for cancer immunotherapy howevera fundamental challenge is to eï¬ectively identify and isolateneoantigenspeciï¬c t cells the developments summarizedin this review and future breakthroughs are anticipated totranslate the adoptive transfer of neoantigenspeciï¬c t cells intoa powerful weapon in our armamentarium against cancerauthor contributionsql prepared the manuscript draft zyd revised it critically forimportant intellectual content and approved the ï¬nal versionql and zyd contributed to the conception and design of thereview all authors contributed to the and approved thesubmitted versionfundingthis work was supported by the national clinical researchcentersichuanuniversity z2018b18for geriatrics west china hospitalsupplementary materialthe supplementary materialonline202001347fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatreferences hu z ott pa wu cj towards personalizedtherapeutic 101038nri2017131vaccinesforcancer natrevtumourspeciï¬cimmunolin an ipilimumabresponsive melanoma j clin oncol 31e439 101200jco2012477521 gubin mm zhang xl schuster h caron e ward jp noguchi t checkpoint blockade cancer immunotherapy targets tumourspeciï¬c mutantantigens nature 101038nature13988 chen fj zou zy du j su s shao j meng fy neoantigen identiï¬cationstrategies enable personalized immunotherapy in refractory solid tumors jclin invest 101172jci99538 le dt uram jn wang h bartlett br kemberling h eyring ad pd1blockade in tumors with mismatchrepair deï¬ciency new engl j med 101056nejmc1510353 keskin db anandappa aj sun j tirosh i mathewson nd li sq neoantigen vaccine generates intratumoral t cell responses in phase ibglioblastoma trial nature hilf n kuttruï¬coqui s frenzel k bukur v stevanovic s gouttefangeas c actively personalized vaccination trial for newly diagnosed glioblastomanature 101038s415860180810y ott pa hu zt keskin db shukla sa sun j bozym dj animmunogenic personal neoantigen vaccine for patients with melanomanature 101038nature22991 sahin u derhovanessian e miller m kloke bp simon p lower m personalized rna mutanome vaccines mobilize polyspeciï¬c therapeuticimmunity against cancer nature 101038nature23003 carreno bm magrini v beckerhapak m kaabinejadian s hundal jpetti aa a dendritic cell vaccine increases the breadth anddiversity of melanoma neoantigenspeciï¬c t cells science 101126scienceaaa3828 tanyial personalized cancerjl bobisse s ophir e tuyaerts s roberti a genolet rantitumorett cell10eaao5931 101126scitranslmedaao5931eï¬ectively mobilizessci transl medimmunityovarianvaccineincancer van rooij n van buuren mm philips d velds a toebes m heemskerkb tumor exome analysis reveals neoantigenspeciï¬c tcell reactivity rizvi na hellmann md snyder a kvistb p makarov v havel jjlandscape determines sensitivity to pd1 blockade in mutationalnonsmall cell lung cancer science 101126scienceaaa1348 van allen em miao d schilling b shukla sa blank c zimmer l genomic correlates of response to ctla4 blockade in metastatic melanomascience 101126scienceaad0095 dudley me wunderlich jr robbins pf yang jc hwu p schwartzentruberafterregression and autoimmunity in patientsal cancerrepopulation withdjetclonal 101126science1076514lymphocytesantitumorscience dudley me yang jc sherry r hughes ms royal r kammula u adoptive cell therapy for patients with metastatic melanoma evaluation ofintensive myeloablative chemoradiation preparative regimens j clin oncol 101200jco2008165449 rosenberg sa yang jc sherry rm kammula us hughes ms phan gq durable complete responses in heavily pretreated patients with metastaticmelanoma using tcell transfer immunotherapy clin cancer res 10115810780432ccr110116 besser mj shapirafrommer rlevy d adoptive transfer ofpatients with metastatic melanomaitzhaki o treves aj zippel dbtumorltrating lymphocytes inintenttotreat analysis and eï¬cacyfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspeciï¬c t cellsafter failure to prior immunotherapies clin cancer res 10115810780432ccr130380 andersen r donia m ellebaek e borch th kongsted piversentz longlasting complete responses in patients with metastaticmelanoma after adoptive cell therapy with tumorltrating lymphocytesand an attenuated il2 regimen clin cancer res 10115810780432ccr151879 stevanovic s draper lm langhan mm campbell te kwong mlwunderlich jr complete regression of metastatic cervical cancer aftertreatment with human papillomavirustargeted tumorltrating t cells jclin oncol 101200jco2014589093 huang j elgamil m dudley me li yf rosenberg sa robbins pf tcells associated with tumor regression recognize frameshifted products ofthe cdkn2a tumor suppressor gene locus and a mutated hla class i geneproduct j immunol 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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 | 2 |
"requires an intraoperative fluoroscopy to confirm an adequate excision as well as lead to increased radiation exposure (10-13). The use of mixture has been reported to make up for the weakness of marking materials. For example the problem of dye diffusion has led to attempts to use a mixture of dye with various materials such as cyanoacrylate adhesive or collagen or autologous blood (14-16). However they have not been widely used for localization due to difficulties in making and manipulation. Lipiodol and methylene blue are commonly used materials for localization (17-20). We hypothesized that lipiodol reduces the spread of methylene blue and provides additional localization opportunities by its radio-opacity. The use of a mixture of lipidol and methylene blue (MLM) for a percutaneous injection material requires a high success rate for appropriate localization and a low complication rate. To our knowledge there have been no reports that evaluate the availability of MLM as a percutaneous injection material in human lungs. This study compared MLM with methylene blue as a percutaneous injection material for pulmonary localization in rabbit lungs. MATERIALS AND METHODS Animal preparation This study was performed after approval by the Institutional Animal Care and Use Committee (IACUC) in Seoul National University Hospital biomedical research institute (IACUC approval No. 11-0356). Twenty-four adult New Zealand White rabbits were used. We recorded their weight before the procedures. The animals were randomly divided into two groups: Group A (n=12) and Group B (n=12) each sacrificed at about 6 hr and 24 hr after percutaneous injections respectively (Fig. 1). Six hours after percutaneous injections were same day operations of the preoperative localization; and 24 hr after percutaneous injections were next day operations of the preoperative localization. The injection of each material was done in all 24 subjects because we injected methylene blue and MLM at two different lung sites for each subject. Percutaneous injection materials: mixture of lipiodol and methylene blue versus methylene blue A pilot study was performed to decide the optimal amount of materials for percutaneous injections. Methylene blue (1% 100 mg/mL TERA Pharmaceuticals Buena Park CA USA) of 0.3 to 0.9 mL was used for human lung localization in previous studies by Wicky et al. (18) and Vandoni et al. (19). In the pilot study with rabbit lungs we injected 0.1 mL and 0.05 mL of methylene blue and MLM in four subjects. We found that staining was extensive (more than half height of one lobe) with 0.1 mL and localized (about 1 cm of staining diameter) with 0.05 mL for both methylene blue and MLM. Extensive dispersion made it difficult to find exact injecting sites; subsequently 0.05 mL of methylene blue was administered. We made variable mix ratios of lipiodol and methylene blue in vitro; 1:1 1:2 1:3 1:4 and 1:5 in order to find an appropriate mixing ratio of lipiodol (480 mg Iodine/mL Andre Guerbet Aulnay-sous-Bois France) and methylene blue. The separation of two materials occurred instantly after mechanical blending to the fat-soluble character of lipiodol and the water-soluble character of methylene blue. A higher concentration of lipiodol in MLM resulted in increased uneven blending and rapid separation. A mixture with a 1:6 (or lower) mixing ratio contained a minimal amount of lipiodol and it might make it difficult to be detected on the fluoroscopy; subsequently we decided that 1:5 was an appropriate mixing ratio for injection. A total of 0.06 mL of MLM (0.01 mL of lipiodol plus 0.05 mL of methylene blue) was administrated in each subject to avoid the effect of different volumes of methylene blue to the diffusion extent of the materials. CT guided percutaneous injections Percutaneous injection was performed with computed tomography (CT) guidance (Discovery CT750 HD; GE Healthcare Waukesha WI USA). We performed pre-procedural CT scans in order to determine an appropriate skin entry site for the successful placement of a needle in the desired location. The desired location was the basal portion of both caudal lobes around the mid-scapula line. We tried to situate the needle tip at 5 mm depth from the visceral pleura and avoid passing through the pulmonary vessels. We placed the needle of 20 gauze and 3.5 cm length in the lung parenchyma after marking the appropriate skin entry site. The parameters of CT used in our study were: tube voltage of 120 kV tube current of 25 mA slice thickness of 2 mm thickness and gantry rotation speed of 350 milliseconds. We connected 1 mL syringe to the needle hub and retracted the syringe piston to confirm that no blood was aspirated after the needle tip was accurately located within the desired location. We then injected the materials and immediately removed the needle. On the procedural CT scan we measured the distance from the skin-entry to the needle tip and the depth from visceral pleura to the needle tip. A post-procedural CT scan identified procedure-related complications that included the leakage of injecting materials and pneumothorax; in addition we recorded the extent shape and density of radio-opacity of MLM after injection. The extent of MLM was defined as a maximum diameter of the radio-opacities. The shape of radio-opacity was categorized into 3 groups (small faint nodular scattered nodular and discrete compact nodular). We recorded the injection time to measure the time interval between injection and sacrifice. Fluoroscopic examinations A successful localization of lipiodol was determined by fluoroscopic examination; subsequently we evaluated the radio-opacity of MLM using the fluoroscopy X-ray unit (BV Pulsera; Philips Medical Systems Best The Netherlands) at the immediate post-procedure session and the follow up session at 6 hr in Group A and 24 hr in Group B. The parameters of fluoroscopy were: tube voltage of 59 kV and tube current of 946 mA. We obtained anteroposterior fluoroscopic images of the thorax of the rabbit with a 17 cm of field of view. A radio-opaque ruler of 5 cm was located near the rabbit in order to estimate the exact size of lipiodol opacity. We recorded the time of the fluoroscopic examinations and the radiographic findings of MLM (size and shape of the radio-opacity). Evaluation of the staining and radio-opacity We assessed the directly visible staining on the freshly excised lung surface and radio-opacity of MLM on the fluoroscopic examinations using 4-point scoring in order to compare the localization ability of MLM and methylene blue as a percutaneous injection material. A blind reviewer who was unaware of the injection materials assessed the staining ability. In order to evaluate the staining ability the blind reader reviewed the photographic images of the freshly excised lung specimens obtained before formalin fixations and rated the staining by 4-point scores: 0=non-visualization of staining 1=inappropriate; extensive dispersion made it difficult to find accurate injecting locations 2=acceptable; available to estimate injecting locations in spite of the dispersion and 3=excellent definitely localized staining (Fig. 2). The maximum diameter of the staining extent on the lung surface was measured. We calculated and compared scores and extent of staining between two materials. For the fluoroscopic findings the radio-opacity of MLM was evaluated using 4-point scoring: 0=no detectable radio-opacity 1=inappropriate minimally increased opacity 2=acceptable low density of increased opacity 3=excellent compact nodular increased opacity (Fig. 3). We compared the average scores of initial and follow up fluoroscopic examinations. We considered a score of 0 or 1 as inappropriate and a score of 2 or 3 as appropriate for localization for both staining and radio-opacity. We compared the number of appropriate or excellent localization between MLM and methylene blue. Sacrifice and histopathologic examinations Both freshly excised entire lungs were used as final specimens. The lung tissues were fixed in 10% neutral formalin embedded in paraffin and cut into 5 µm thick slices after we took photographs to record staining on the lung surface. We made 4 axial slices that covered the center of the staining. The slices were subjected to hematoxylin-eosin (H-E) stain to the evaluate lung parenchymal change. We evaluated the presence or absence of neutrophil infiltration vasculitis necrosis hemorrhage and foam cell in alveolus. The extent of each histopathologic finding was estimated using visual grading scores as 0 (no) 1 (focal) or 2 (diffuse). Localized parenchymal change (<50% of total area) surrounded by normal lung was defined as focal. Extensive lung parenchymal change (?50% of total area) that replaced normal lung was defined as diffuse. An experienced pathologist with eight years of experience reviewed all slices. The overall severity of the lung parenchymal change was defined as a total score by adding visual grading scores for each histopathologic finding. We compared the overall severity score between MLM and methylene blue as well as between Group A and Group B. Statistical analysis All data are expressed as mean±standard deviation (SD) unless otherwise stated. Comparisons of the average scores were performed by two-tailed unpaired Student's t-test or Mann-Whitney test. We used a Fisher's exact test to compare the number of subjects in the subgroups. Linear by linear association evaluated the association of the extent of lung parenchymal change and materials or groups. Null hypotheses of no difference were rejected if the P values were less than 0.05. The statistical analysis was performed with commercially available statistical software IBM SPSS Statistics version 20.0 (IBM Corp. in Armonk NY USA). RESULTS Subject characteristics procedural records time interval of injection and examinations Among the 24 subjects included in our study successful CT-guided percutaneous injections into the desired location of the lung were achieved in 21 subjects (11 in Group A and 10 in Group B). Three subjects died during anesthesia. Mean weight was 3.2±0.2 kg for Group A and 3.3±0.2 kg for Group B. Injection depth from visceral pleura to needle tip was 0.4±0.1 cm (range: 0.3-0.6 cm) for MLM and 0.4±0.1 cm (range: 0.3-0.7 cm) for methylene blue (P=0.43). Distance from skin to needle tip was 2.8±0.6 cm (range: 2.1-5.0 cm) for MLM and 2.8±0.3 cm (range: 2.2-3.5 cm) for methylene blue (P=0.83). Of 42 CT-guided percutaneous injections total number of procedure related complications was 10 (24%) including 7 leakage (all in MLM) and 3 pneumothorax (2 in MLM 1 in methylene blue). The complication rate in MLM was significantly higher than methylene blue (43% vs 5%) (P=0.004). On post-procedural CT images the extent of the radio-opacity of MLM was 1.3±0.4 cm (range: 0.7-2.0 cm) for Group A and 0.6±0.3 cm (range: 0.3-1.1 cm) for Group B. Discrete compact nodular opacity was achieved in 15 subjects (72%) scattered nodular opacities in 3 (14%) and small faint opacity in 3 (14%) (Fig. 4). The average value of radio-opacity of MLM was 1415±856 HU (range: 307-2768 HU). The interval between injection and sacrifice was 7.9±0.1 hr (range: 7.8-8.0 hr) for Group A and 23.5±0.1 hr (range: 23.4-23.7 hr) for Group B. Time from injection to initial and follow up fluoroscopy was 3.4±0.5 hr (range: 2.5-4.2 hr) and 6.8±0.4 hr (range: 6.3-7.7 hr) for Group A and 1.5±0.4 hr (range: 0.9-2.1 hr) and 22.6±0.4 hr (range: 21.9-23.2 hr) for Group B respectively. Scores and extent of staining and radio-opacity Table 1 demonstrates the staining extent and localization ability of MLM and methylene blue. In total groups the staining extent of MLM was significant smaller than methylene blue (0.6 cm vs 1.0 cm P<0.001). MLM showed a significantly higher staining ability score than methylene blue (2.8 vs 2.2 P=0.010). Radio-opacity in the initial fluoroscopy was not significantly different from the follow up (2.0 vs 1.9 P=0.49). Table 2 showed the number of subjects in each score of localization ability of staining or radio-opacity. In Group A appropriate staining was 100% for both MLM and methylene blue. In Group B appropriate staining was 90% for MLM and 70% for methylene blue. Appropriate staining of MLM was not significantly different from that of methylene blue (95% vs 86% P=0.61); " | 1 |
previous studies have shown a strong coexistence of colorectal neoplasia crn and cardiovascular diseases cvd this study was aimed to summarize the available evidence on association of cvd risk with early crn detection in asymptomatic populations pubmed web of science and embase were systematically searched for eligible studies published until dec studies exploring the associations of recommended cvd risk assessment methods eg risk scores carotid artery plaque and coronary artery calcium score [cacs] with risk of crn were included metaanalyses were conducted to determine the overall association of cvd risk with the crn a total of studies were finally included the association of carotid artery plaque with the risk of colorectal adenoma ad was weakest pooled odds ratio [or] confidence interval [ci ] participants with cacs100 had about 2fold increased risk of ad than those with cacs0 the pooled ors were ci and ci for the risk of advanced colorectal neoplasia an and ad respectively in participants with framingham risk score frs20 when compared to participants at low risk frs10 frs might help identify subgroups at increased risk for an but further studies are needed keywords cardiovascular disease risk assessment colorectal neoplasiaintroductionboth colorectal cancer crc and cardiovascular diseases cvd are the leading causes of mortality and morbidity worldwide12 previous studies have shown a strong coexistence of colorectal neoplasia crn and cvd probably due to the shared risk factors eg smoking obesity and metabolic syndrome and pathophysiological mechanisms eg chronic inflammation and oxidative stress3current guidelines8 recommend assessing the cvd risk in healthy people using risk estimation scores such as framingham risk score frs1112 procam13 and the pooled cohort equation14 which are based on individuals medical history and easily available laboratory data in addition assessment of subclinical atherosclerosis by imaging modalities could be added as risk modifiers to help make clinical decisions for borderline or intermediaterisk adults8 routine use of imaging modalities is not recommended for cvd risk assessment in clinical practice due to the medical costs or invasiveness but incorporation of imaging data such as the anklebrachial index abi coronary artery calcium score cacs and carotid artery plaques cap could improve the prediction of cvd risk15clinical epidemiology chen 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 0cchen dovepressvarious risk scores have also been developed for predicting advanced colorectal neoplasia an18 although several studies2526 have reported that elevated blood lipids the well documented cvd risk factor and history of cvd were associated with increased risk of crc the majority of risk scores developed for an did not include them into the models27 recent studies have reported the associations between cvd risk assessment and risk of1 crn higher frs estimating the 10year risk of developing coronary heart disease chd1112 was significantly associated with the higher risk of an frs vs frs10 odds ratio [or] confidence interval [ci] abi was associated with 13fold increased risk of an in a recent study29 cap and cacs were also found to be positively related to the increased risk of adenoma ad and an in several studies30given a number of shared risk factors and mechanisms between cvd and crc and the emerging epidemiological evidence of association between cvd risk and crc there is a possibility that cvd risk assessment could help trigger crc screening therefore the aim of this review was to provide an overview of the cvd risk assessment methods and their associations with the risk of crn fully understanding of the current knowledge and existing gap might promote better prevention and treatment for cvd and crc circulating and urinary biomarkers have either no or only limited value when added to cvd risk estimation score systems834 thus only score models and imaging methods recommended as risk modifiers abi cacs and cap in the guidelines8 were included in this reviewmaterials and methodsthis systematic review was conducted following the procedure recommended by the cochrane collaboration35 and was reported according to the preferred reporting items for systematic reviews prisma checklist36 ethical approval and patient informed consent were not necessary since all the data included in the current study were obtained from previously published studiesand metaanalyses remaining publications and reference lists were scrutinized studies that fulfilled the predefined criteria were includedinclusion and exclusion criteriawe required that included studies meet the following criteria published as an original research in a peer reviewed cardiovascular risk has been assessed using either score models or imaging methods recommended as risk modifiers abi cacs and cap in the guidelines3 only included participants who were considered asymptomatic reported the association of cvd risk assessment results with the risk of crn studies were excluded if they were published as conference proceedings dissertations or s only or were not published in english pico eligibility criteria for this review were presented in the supplementary table s1data extractiontwo authors yc and xc independently performed data extraction of all included studies the following information was ed author publication year study period number of participants age number of males outcome ad an and so on data source medical records questionnaires or both cvd risk assessment and association indexdiscriminatory accuracy or hazard ratio [hr] specificity sensitivity or area under the receiver operator characteristic curve values] in case of any disagreement consensus was obtained by discussionquality assessment in eligible studiesrisk of bias and applicability were assessed according to quality assessment of diagnostic accuracy studies2 quadas237 quadas2 evaluates the risk level of bias composed of four basic components patient selection index test reference standard flow and timing clinical applicability is also assessed for the first three components the risk of bias and concerns regarding applicability for each study was then rated as high low or unclearliterature search strategiespubmed embase and web of science were searched up to december to identify the relevant papers the searched items were presented in the appendix which mainly covers expressions for cvd risk score models recommended imaging modalities crn and discriminatory accuracy or strength of association after removal of duplicates titles and s of records were screened according to the inclusion and exclusion criteria full texts of the statistical analysiswe pooled ors for the same cvd risk assessment index using r statistical software version and the r meta package version for frs and cacs ors were pooled separately for different levels of scores using the lowest level as reference two kinds of outcomes ad and an were reported in the studies using frs for cvd risk assessment and thus ors were pooled separately for different outcomes heterogeneity across studies was evaluated submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen using cochranes q statistic with p value and the i2 statistic if significant heterogeneity was observed i2 or pqstatistics a randomeffects model was used to calculate pooled estimates otherwise a fixedeffects model was used35 twosided p values of or lower were considered to be statistically significantresultsliterature search resultsa total of records were obtained in the initial search including citations from pubmed citations from embase and citations from web of science after removal of duplicates n1609 and exclusion due to our predefined criteria n5727 records were qualified for fulltext assessment fortyfour records were excluded due to the inclusion and exclusion criteria finally a total of studies28 including one study which was identified through crossreferences were included the detailed information of the selection process was presented in figure study characteristicstable summarized the basic characteristics of the included studies published between and of the included studies nine were from korea and the other three studies were from japan austria and turkey respectively the study periods stretched from to with sample sizes ranging from to only one was designed as a prospective study41 and the others were crosssectional studies most studies included participants aged older than years and only one study enrolled subjects aged years32 in addition most studies were predominantly in men with proportions of males among participants ranging from to four cvd risk assessment methods abi cap cacs and frs were used in the included studies all studies explored the role of cvd risk assessment method on the detection of ad and some of risk adenoma3032 and an2829384243focused on colorectal high them also figure flowchart of inclusions of studies about relation of cvd risk to crn note adapted from moher d liberati a tetzlaff j preferred reporting items for systematic reviews and metaanalyses the prisma statement plos med creative commons license and disclaimer available from httpcreativecommonslicensesby40legalcode36 abbreviations cvd cardiovascular disease crn colorectal neoplasiaclinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable basic characteristics of included studies about relation of cvd risk to colorectal neoplasiastudycountrystudy periodnumber of participantsyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj 2019a41lee jy niederseer d basyigit s japankoreakoreakoreakoreakoreakoreakoreakoreakoreaaustriaturkeyage years mean±sd ± ± 530b median± ± ± ± 526b± ± ±male n outcomec data sourcececum intubation ratedcvd risk assessment ad anad hraadad anad hraadadadadad anad anad anmrqmrqmrqmrmrqmrqmrmrmrqmrqmrqmrqnrnrnrnr¥nrabicapcapcapcacscacscacscacscacsfrsfrsfrsnotes ait is a retrospective followup study and all the other studies are crosssectional bsd was not reported cdetected by colonoscopies in all included studies d100 cecum intubation rate participants with failure of cecum intubation were excluded nr not reported studies mentioned that colonoscopies were extended to cecum in the methods section but did not reported the success rate of cecum intubation abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score hra high risk adenoma mr medical records nr not reported q questionnaires sd standard deviationquality assessment of studiesthe results for the quality of included studies using the quadas2 tool are presented in table regarding patient selection one study by kim did not provide detailed information about patient selection31 thus the risk of bias and applicability concerns were rated unclear for this domain in this study otherwise no major risk of bias or applicability concerns were identifiedassociation of cvd risk assessed by different methods with crc risktable described the details of the cvd risk assessment methods in the included studies abi was associated with 13fold ci increased risk of an29 three studies reported the weak association between cap and risk of ad303138 one of them also showed an increased risk of an in the participants with cap but the results were not statistically significant or table risk of bias and applicability judgements in quadas2studyrisk of biasapplicability concernstotalpatient selectionindex testreference standardflow and timingpatient selectionindex testreference standardyamaji y kim j kim h cha jm yun ke choi sh yang mh kim hb lee yj lee jy niederseer d basyigit s totalnotes _ high risk low risk unclear risksubmit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen table details of the cvd risk assessment methods in the included studies about relation of cvd risk to colorectal neoplasiastudycategoriesboutcome or[ ci]yamaji y kim j abnormal abiabnormal abicap yescap yeskim h cap yescha jm yun ke choi sh cap yescap yescacs cacs cacs cacs cacs cacs cacs cacs cacs yang mh201339cacs kim hb cacs cacs cacs ¥lee yj 2019a41cacs lee jy niederseer d basyigit s frs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highfrs intermediatefrs highadanadhraadadanadadadhrahrahraadadadadadadadadadadananadadananadadanan[ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]hr [ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ][ ]notes ain participants without adenoma cacs0 at baseline compared to cacs0 increased the risk of colorectal adenoma at followup colonoscopy hr ci bthe lowest level was defined as reference abbreviations abi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque frs framingham risk score hra high risk adenoma hr hazard ratio or odds ratio ci confidence interval ci in addition the presence of cap was associated with increased risk of colorectal high risk adenoma or ci four studies reported ors for different levels of cacs with cacs0 as reference32333940 highest cacs levels seemed to be associated with the increased risk of ad with or ranging from to the 10year chd risk estimated by frs was categorized as low risk intermediate risk and high risk ¥ participants with high risk of 10year chd had increased risk of either ad or an in the study by basyigit participants at high chd risk had about 4fold or ci increased risk of an28metaanalyses of available ors for different cvd risk assessment methodsmetaanalyses were performed in the studies that provided ors and their cis for the same cvd risk assessment index the association of cap with the risk of ad was weakest the pooled or ci a medium level of cacs cacs was associated with 134fold increased risk of ad when compared to the lowest category of cacs cacs0 participants with cacs100 had an increased risk of ad and the pooled or was ci the pooled ors were ci and ci for the risk of an and ad respectively in participants with high chd risk frs20 when compared to participants at low chd risk frs10 further details were presented in table and in the supplementary figures s1discussionthis systematic review summarized the associations of recommended cvd risk assessment methods with risk of crn in asymptomatic populations a total of studies including four different methods were identified among these methods frs was most strongly associated with risk of both an and ad participants with frs20 have about 34fold and 23fold increased risk of an and ad respectively when compared to participants at low chd risk frs10 only one study29 reported that abnormal abi greatly increased the risk of an thus it was not included in the metaanalysisboth crc and cvd are thought to develop via a process of insulin resistance inflammation and oxidative clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepresstable metaanalysis of odds ratios for different cvd risk assessment toolsstudycvd risk assessmentcategoriesaoutcomeor cicapcacscacscacsfrsfrsfrsfrsyes vs nocacs vs cacs0cacs vs cacs0cacs vs cacs0intermediate vs low riskhigh vs low riskintermediate vs low riskhigh vs low riskadadadadadadanan note athe lowest level was defined as reference abbreviations ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque cvd cardiovascular disease frs framingham risk score or odds ratio ci confidence intervalstress74547 which might partially explain why they share a number of risk factors eg alcohol consumption tobacco use physical activity use of antiinflammatory agents obesity and diabetes mellitus4548 in addition several cellular metabolismrelated pathways eg ampk and pparγ signaling pathways eg wnt signaling pathway and genetic pathways eg lrp6 mutation and tcf7l2 polymorphism are not only associated with accelerated atherosclerosis and an increased risk of cvd but also linked to cancer development and progression7 better understanding of these overlaps might promote shared management of prevention and treatment for both disordersrisk of an in this review the strength of associations between identified cvd risk assessment methods and the risk of crn was generally weak except frs which was modestly associated with frs20 vs frs10 frs was calculated based on age total cholesterol highdensity lipoprotein cholesterol smoking status systolic blood pressure and treatment of blood pressure which are typically available in the medical records44 compared to the more sophisticated risk calculators232449 for predicting an which need variables such as physical activity red meat intake and vegetable consumption frs has relatively higher generalizability and lower recall bias a recent study has recommended the combined preventive screening and research efforts in the prevention of both cvd and cancer50 if participants with highrisk of cvd predicted by frs could be recommended to have a screening for crn which will help increase compliance and uptake of crc screening as persons who are aware of their increased risk are more likely recommendations furthermore it also maximizes the medical values of the comply with to expert information participants obtain from a clinical examination or risk assessment and thus reduces the time and costs for health carehowever there are some issues that merit our attention firstly the included studies are all crosssectional which limits the comparisons between frs and the previously developed risk prediction models for crc secondly frs has its own limitations frs only estimates 10year chd risk for all individuals years or older but not the overall cvd risk in addition it is developed based on the american population while most of study participants are asians in the included studies studies have shown that frs overestimated cvd risk in the asian cohorts51 at last the included studies tended to yield results with wide ci probably due to the limited number of participants the wider the ci the less the precision in summary higher cvd risk might trigger concurrent crc screening which should be further validated on largescale studies and future studies could consider about using the overall cvd risk score models developed from data of local cohorts to predict the risk of crcas for imaging data the association of cap or cacs with risk of ad is not strong enough that imaging index alone might not be useful for informing early detection of crn similarly routine screening with imaging modalities to predict future cardiovascular events is generally not recommended in clinical practice but use of these imaging techniques has been shown to improve cvd risk assessment and serve as a guide for initiating preventive therapies8 a high cacs can help modify the predicted risk obtained from frs alone especially among patients in the intermediaterisk category16 up to now only one risk score developed in the multiethnic study of atherosclerosis mesa study used both cacs and submit your manuscript wwwdovepresscom dovepress clinical epidemiology 0cdovepress chen traditional risk factors to predict the 10year chd risk55 inclusion of cacs in the mesa risk score offered significant improvements in risk prediction cstatistic vs p factors in the risk models like smoking behaviors and blood lipids are closely related to the incidence and progression of cvd but they are not direct markers of current status of atherosclerosis this might help explain why the performance of risk models is improved by adding markers with anatomical delineation through imaging technology accounting for the higher performance of the combined use of risk scores and imaging tools on cvd risk assessment further studies could consider about exploring the association of combined form of them with the risk of crcwe also observed that less than half of included studies reported the associations of cvd risk with both risk of an and ad2829384243 colonoscopy is considered to as a valid primary screening tool for crc and is able to detect both ad and an the lower prevalence of an and the limited number of participants in several included studies might limit the power to explore the relation of an with cvd risk which could partly explain why most of studies did not include an as outcome therefore the findings should be carefully interpreted and further validated on largescale studiesour study has some strengths comprehensive search strategies along with welldefined eligibility criteria were used to help identify relevant s in addition two reviewers independently extracted data and assessed the risk of bias in the included studies however several limitations should also be addressed firstly the current meta analysis was based on observational studies there were the possibilities of potential effects of unknown or residual confounding factors on our results secondly as we only considered about established cvd risk models and recommended imaging modalities the potential of other cvd risk assessment index on the detection of crn was not summarized and compared in this study however it is also reasonable to just include these methods since their feasibility and performance for cvd risk prediction have been well approved in the clinical practice thirdly cut off values and group comparisons for the same cvd risk assessment method varied in the included studies which limits the synthesis of results for example the cut off values for cacs are the tertiles of cacs in the study by kim 40 however cacs was categorized into three groups with cut off values at and in the other studies3233 therefore less studies were included in the metaanalysis which might influence the accuracy of the pooled results lastly most of studies were conducted in asian populations which is an inherent limitation of the included studies thus our findings might not be applicable to other populations and needs to be externally validated in racially diverse populationsconclusionsto our knowledge this is the first review that applies metaanalyses to determining the overall association of recommended cv risk assessment methods with the risk of crn in the asymptomatic population frs calculated based on shared risk factors of cvd and crc shows potential to help identify subgroups at increased risk for an whether the combination of frs and imaging index is useful for the optimal evaluation of crn risk remains to be solved in the future studies cvd risk might inform crc screening which needs more research in the future to validate its feasibility and effectivenessabbreviationsabi anklebrachial index ad colorectal adenoma an advanced colorectal neoplasia cacs coronary artery calcium score cap carotid artery plaque chd coronary heart disease ci confidence interval crc colorectal cancer crn colorectal neoplasia cvd cardiovascular disease frs framingham risk score hr hazard ratio hra high risk adenoma mr medical records nr not reported or odds ratio prisma preferred reporting items for systematic reviews and metaanalyses quadas2 quality assessment of diagnostic accuracy studies2 q questionnaires sd standard deviationfundingthis research was funded by national natural science foundation of china grant number disclosurethe authors report no conflicts of interest in this workreferences 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 doidoi103322caac21492 joseph p leong d mckee m et al reducing the global burden of cardiovascular disease part the epidemiology and risk factors circ res doidoi101161circresaha117308903 chan aoo man hj kwok fl et al prevalence of colorectal neoplasm among patients with newly diagnosed coronary artery disease j am med assoc doidoi101001jama29812 clinical epidemiology submit your manuscript wwwdovepresscom dovepress 0cchen dovepress chan aoo lam kf tong t coexistence between colorectal canceradenoma and coronary artery disease results from patients aliment pharmacol ther doi doi101111j13652036200602958x wang sc schulmanmarcus j fantauzzi j et al colon cancer laterality is associated with atherosclerosis and coronary artery disease j gastrointest oncol doidoi1021037jgo20180918 kahr pc hammerl s huberschönauer u et al atrial fibrillation a new indicator for advanced colorectal neoplasia in screening colonoscopy j clin med doidoi103390jcm8071083 masoudkabir f sarrafzadegan n krahn a et al cardiovascular disease and cancer evidence for shared disease pathways and pharmacologic prevention cardiovascular disease and cancer evidence for shared disease pathways and pharmacologic prevention hhs public access atherosclerosis doidoi101016 jatherosclerosis201706001 piepoli mf hoes aw agewall s european guidelines on cardiovascular disease prevention in clinical practice the sixth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice constituted by representatives of societies and by invited experts developed with the special contribution of the european association for cardiovascular prevention rehabilitation eacpr atherosclerosis doidoi101016jatherosclerosis201605037 arnett dk blumenthal rs albert ma et al accaha guideline on the primary prevention of cardiovascular disease a report of the american college of cardiologyamerican heart association task force on clinical practice guidelines circulation 201914011e596e646 doidoi101161cir0000000000000678 mach f baigent c catapano al esceas guidelines for the management of dyslipidaemias lipid modification to reduce risk eur heart j doi cardiovascular doi101093eurheartjehz455 grundy sm becker d clark lt et al detection evaluation and treatment of high blood cholesterol in adults adult treatment panel iii circulation doidoi101161circ106 cleeman ji executive summary of the third report of the national cholesterol education program ncep expert panel on detection evaluation and treatment of high blood cholesterol in adults adult treatment panel iii j am med assoc doi doi101001jama285192486 assmann g cullen p schulte h simple scoring scheme for calculating the risk of acute coronary events based on the 10year follow up of the prospectiv | 0 |
Journal of International Medical Research The Authors reuse guidelinessagepubcomjournalspermissions journalssagepubcomhomeimrCase ReportNivolumab plus gemcitabinedexamethasone and cisplatinchemotherapy induce durablecomplete remission inrelapsedrefractory primarymediastinal Bcell lymphomaa case report andliterature reviewGang Huang1 Ju Huang2 Zhili Zhang2Chongchong Xue1 and Yuan Liu2AbstractPrimary mediastinal large Bcell lymphoma PMBCL is an uncommon but aggressive type ofBcell lymphoma Patients with relapsed refractory PMBCL rrPMBCL have limited therapeuticoptions and usually have a relatively poor outcome Immune checkpoint blockade has become apotential treatment for this disease We report here a case of a female patient with rrPMBCLwho was treated with nivolumab plus gemcitabine dexamethasone and cisplatin GDP chemotherapy Complete remission was achieved after four cycles of combined therapy With continuednivolumab maintenance monotherapy she has remained in complete remission for longer than months This is the first report of nivolumab plus GDP chemotherapy inducing completeremission in patient with rrPMBCL This case supplements the limited literature and providesimplications for clinical trial designs regarding the potential use of nivolumab in the treatment ofrrPMBCL1Department of Hematology Yuebei Peoples HospitalShantou University Medical College ShaoguanGuangdong Province China2Guangdong Women and Children Hospital GuangzhouGuangdong ChinaCorresponding authorYuan Liu Medical Genetic Centre Guangdong Womenand Children Hospital No Xingnan Rd PanyuDistrict Guangzhou Guangdong ChinaEmail yuanliu005163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical ResearchKeywordsRelapsed refractory primary mediastinal Bcelllymphoma nivolumab checkpoint blockadegemcitabine dexamethasone cisplatin chemotherapy programmed cell death completeremissionDate received February accepted July IntroductionPrimary mediastinal large Bcell lymphomaPMBCL is an uncommon but aggressivetumor that accounts for to of nonHodgkin lymphoma1 PMBCL is distinguished from diffuse large Bcell lymphomaby virtue of distinct clinical pathologicaland genetic features2 Recently PMBCLwas listed as a separate entity in the latestWorld Health anization classiï¬cation of hematopoieticand lymphoidtumors5 PMBCL has a similar clinical presentation as classical Hodgkin lymphomacHL and PMBCL also shares certain features at the molecular level particularly9p241 alterations and programmed celldeath protein ligand 1ligand PDL1PDL2 expression6 At present management and outcome of PMBCL are still critical and a more serious situation is faced bypeople who are diagnosed with relapsedand refractory PMBCL rrPMBCL19The optimal salvage chemotherapy andautologousforrrPMBCL are of limited efï¬cacy19stem celltransplantRecently agents targeting programmedcell death PD1 and PDL1 have beenimmunotherapy10developed in tumorAntiPD1 therapy with monoclonal antibodies has been approved for the treatmentof several types of solid tumor and cHLThe therapeutic potential of antiPD1 therapy in other malignancies is likely to beapproved soonIn a humanizedimmunoglobulin G1 recombinant monoclonal antibody for the PD1 receptor pidilizumab was approved by the US Food andDrug Administration FDA for treatingandpediatricpatientsthatwithadultrrPMBCL11 Another agenttargetsthe PD1 receptor called nivolumab isa fully humanized immunoglobulin G4monoclonal antibody that has been grantedapproval by the US FDA for treating several solid malignancies and cHL The therapeutic efï¬cacy of nivolumab in patientswith rrPMBCL remains unclearWe report here a patient with rrPMBCLwho received combined treatment with offlabel nivolumab and GDP chemotherapyComplete remission CR was achievedafter four cycles of such combined treatment At the time of this submission thepatient has remained in CR for longerthan months with continued nivolumabmaintenance monotherapyCase reportA 32yearold woman presented to YuebeiPeoples Hospital with intermittent dyspneaand chest pain A positron emission tomography PET scan showed a 10cm mass inthe anterior superior mediastinum with astandardized uptake value of Themass showed unclear margins and compressed the ascending aorta and pulmonarytrunk Small pericardial and left pleuraleffusions were also observedThe mass was diagnosed as PMBCL by asubsequent biopsy Immunohistochemicalstaining showed thatlarge lymphocyteswere positive for CD20 CD79a Pax5BCL6 CD23 CD30and multiplemyeloma1 and negative for CD10 CD3CD5chromogranin Asynaptophysin 0cHuang et alterminalencodingandincludingtwocycles positiveregion inendomysialdeoxynucleotidyltransferase cytokeratin CK CK19 andandS100 Ki67 wassituEpsteinBarrhybridization was negative She was initially treated with six cycles of frontline chemotherapyofrituximab cyclophosphamide doxorubicinvincristine and prednisolone RCHOPand four cycles of doseadjusted etoposidecyclophosphaprednisolonemiderituximabDAEPOCHR were administered Thetimeline of treatment is shown in aShe received tumor resection by thoracoscopic surgery after she continued twodoxorubicinvincristinecycles ofgemcitabine dexamethasonecisplatinumetoposide and rituximabtherapy Her ï¬rst CR was achieved inDecember However monthslater a PETcomputed tomography CT scan showedhypermetabolic lesions located at the leftlung and right adrenal gland but not inthe primary mediastinal site bThe patient reported no physical symptomsand received a repeat tissue biopsy whichconï¬rmed a relapse with PMBCL She wastreated with each cycle of a dexamethasoneifosfamide cisplatin and etoposide regimenand ibrutinib bendamustine and cytarabine therapy A chest CT scan showedFigure Summary of treatment and monitoring the tumor response a Patients timeline chart with thedates of treatment and monitoring the tumor response b Positron emission tomography images Upperpanel a scan of the relapsed hypermetabolic lesions located at the left lung and right adrenal gland beforecombined treatment Lower panel complete remission was achieved after four cycles of nivolumab plusGDP chemotherapyRCHOP rituximab cyclophosphamide doxorubicin vincristine and prednisolone DAEPOCHR doseadjusted etoposide prednisolone vincristine cyclophosphamide doxorubicin and rituximab GDPERgemcitabine dexamethasone cisplatinum etoposide and rituximab CR complete remission PMBCLprimary mediastinal large Bcell lymphoma DICE dexamethasone ifosfamide cisplatin and etoposideIBC ibrutinib bendamustine and cytarabine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical Researchthat the right adrenal gland lesion had partially responded while the lesions in the leftlung had progressed After those cycles ofchemotherapy the patient showed GradeIV myelosuppression and had to receiveblood transfusion treatment Moreover acerebrospinal ï¬uid examination showedthe presence of atypical lymphocytes andno symptoms of infection of the central nervous system were observed Intrathecal chemotherapy cytarabine mg methotrexate mg and dexamethasone mg was thenadministered and no atypical lymphocyteswere detected by repeated cerebrospinalï¬uid analysis These ï¬ndings highly suggested a potential risk of metastasis of thecentral nervous systemtreatmentBecause the disease had progressed withsevere myelosuppression and there were nostandard chemotherapy guidelines or alternative treatment options for the patientother salvage treatments of her refractorydisease needed to be considered Aftermuch discussion with the patient and herfamily she declined autologous hematopoietic stem cell transplantation and receivedcombinedgemcitabine mg dexamethasone mg and cisplatinum mg GDPchemotherapyand the offlabel antiPD1 antibody nivolumab mg After four cycles of combined treatment a repeated PETCT scanshowed thatshe had secondary CRb She received two more cyclesof combined treatment with nivolumab andGDP chemotherapy and then continuedsingle nivolumab maintenance treatment mg Since her ï¬rst dose in May she received doses of nivolumab Shereported moderate fatigue and pyrexia in to days after each administration ofnivolumab Blood tests indicated normalfunction of the liver kidney and thyroidFigure She also had normal bloodlevels of creatinine albumin globulin lactate dehydrogenase aspartate transaminasetotalaminotransferaseofalaninethey2b Neutrophilbilirubin and urea nitrogen during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the ï¬rst four combined therapies because of toxicity of GDPchemotherapy butrecovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2cFurthermore no adverse signs and symptoms were observed in the lungs brainand skin At the time of this submissionshe has remained in CR for longer than months with continued nivolumab maintenance therapyEthics approval was obtained from theethicalcommittee of Yuebei PeoplesHospital Written informed consent wasobtained from the patient for analysis ofthe samples and publicationDiscussionTreatment and outcome are critical in managing PMBCL Because there is no established standard approachthe ï¬rstlinetreatment of PMBCL is generally thesame as that for diffuse large Bcell lymphoma including RCHOP and DAEPOCHR Relapse of PMBCL usually occurs in theï¬rst to months after completion offrontline therapy with a lower incidenceapproximately than diffuselarge Bcell lymphoma19 There are varioussecondlineregimens for patients with rrPMBCL includingthe rituximab ifosfamide carboplatin andetoposide regimenthe rituximab dexamethasone cytarabine and cisplatin regimen and rituximabGDP12 Because of alack of standard guidelines or treatmentoptions for PMBCL the outcome greatlydepends on the patients response to theregimen Thisremains poordespite these secondline salvage chemotherapies and subsequent autologous hematopoietic stem cell transplantation912immunochemotherapyresponse 0cHuang et alFigure Blood test values during the whole treatment process since the first dose of nivolumab The firstfour cycles were nivolumab plus GDP chemotherapy and nivolumab maintenance monotherapy wasadministered since the fifth cycle a Thyroxine thyrotropin FT3 and FT4 levels b Levels of creatininealbumin globulin lactate dehydrogenase aspartate transaminase alanine aminotransferase total bilirubinand urea nitrogen c Neutrophil and platelet countsFT4 free thyroxine FT3 free triiodothyronine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical ResearchIn recent years strategies focusing on thecheckpoint blockade have been developedin tumor immunotherapy10 Therapeuticantibodies targeting the PD1PDL1 axispossess clinical activity and an acceptablesafety proï¬le in treating a growing list oflymphomas13solid tumors and BcellBased on a clinical study of patientswith rrPMBCL pidilizumab was approvedby the US FDA for treatment of adult andpediatric patients with rrPMBCL in Another antibody nivolumab has beengranted approval for treating several solidmalignancies and cHL However studiesregarding application of nivolumab forPMBCL are limited Only ï¬ve reportshave described using nivolumab for treatment of PMBCLrrPMBCL Table asfollows In a phase I study published intwo patients with PMBCL wererecruited and treated with nivolumab atdoses of or mgkg every weeks afterprevious systemic treatments14 No objective responses were observed in this previous study In another phase I study onewithrefractorypatient with PMBCL received combinedtherapy of nivolumab and ipilimumaband died during the therapeutic process15Recently two reports showed the potentialtherapeutic efï¬ciency of nivolumab forpatientsPMBCLrrPMBCL who showed failure with conventional immunochemotherapy1617 Both ofthese two cases had immunerelated adverseeffects during the antibody treatment process One patient with highgrade neutr ia had nivolumab stopped temporarilyand was treated with intravenous immunoglobulin16 The other patient with zosterreactivation was controlled by administration of valacyclovir17 Recently Zinzaniand colleagues showed that combined treatment of nivolumab and brentuximab vedotin had promising antitumor activity and amanageable safety proï¬le in patients withrrPMBCL18 In this phase II study patients were recruited and treated withnivolumab mgkg and brentuximab vedotin mgkg every weeks The objectiveresponse rate was and achievedTable Reports regarding application of nivolumab in primary mediastinal large Bcell lymphomarelapsedand refractory primary mediastinal large Bcell lymphomaNumberof cases DoseCombinedtreatmentAdverse events or mgkg mgkgIpilimumabResponseyesnoNoNo ofpatientsReportsLesokhin AMet al Ansell S et alWright Zet al Yassin R et alZinzani mgkgNoNoHighgrade neutr iaYesZoster reactivationYes mgBrentuximabNeutr iaPL et al vedotinthrombocyt iaand peripheralneuropathy of patientsPresent case mgkgGDPMild fatigue and pyrexiaYeschemotherapyNote means not indicated in the report 0cHuang et alofcyclescombinedCR and achieved partial remission Of patients of them had drugrelatedadverse events and the most common wereneutr ia thrombocyt ia and peripheral neuropathy18 In the present case weattempted several available approaches intreating the patients relapsed disease butfailed to control the progress of the massAfter much discussion with the patient andher family we considered an offlabel nivolumab and GDP chemotherapy as salvagetreatmentfor the patient In September her second CR was achieved afterfourtreatmentCurrently with continued nivolumab maintenance monotherapythe patient hasremained in CR for longer than monthsImmunerelated adverse events that areassociated with checkpoint blockade oftenstart within the ï¬rst few weeks to monthsafter treatment but can occur any time andin any an The most common immunerelated adverse events are hypothyroidismnausea diarrhea pyrexia and fatigue1920In the present case we were concernedabout immunerelated an damage sincethe ï¬rst dose of nivolumab The patientreported moderate fatigue and pyrexiaafter each administration of nivolumaband soon recovered within to daysBlood testing was performed during thewhole therapeutic process and the datawere reviewed and analyzed Blood levelsof thyroxine thyrotropin free triiodothyronine and free thyroxine indicated no thyroiditisFigure 2a Our patient alsoshowed normal metabolic data during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the ï¬rst four combined therapies because of toxicity of GDPchemotherapy but they then recovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2c2b NeutrophilUnlike otherarelymphomas prognosticbiomarkersinPMBCL12 Some serum molecules such aslackinglargelyCCL17 and CD163 are considered aspotential biomarkers for predicting andmonitoring responses and detection ofrelapses in patients with Hodgkin lymphoma1221 The role of serum biomarkers inPMBCL remainsinvestigatedRadiological imaging should only be usedin patients who have new clinical symptomsor signs suggestive of relapse but not inasymptomatic patients922betoTo the best of our knowledge this is theï¬rst reported case of nivolumab plus GDPchemotherapy that induced CR with nosevere immunerelated an damage in apatient with rrPMBCL We also reportthe longest followup observation of successful application of nivolumab in apatient with rrPMBCLThis report supplements the limited literature of nivolumab fortreatment ofPMBCLrrPMBCL and provides implications for clinical trial design regarding thepotential use of nivolumab in treatment ofrrPMBCL Further investigation needs to beperformed for potential application of singleor combined use of nivolumab for patientswith rrPMBCL who experience failure withconventional therapeutic approachesDeclaration of conflicting interestThe authors declare that there is no conï¬ict ofinterestFundingThis research received no speciï¬c grant from anyfunding agency in the public commercial ornotforproï¬t sectorsorcid000000034880ORCID iDYuan LiuReferences Martelli M Ferreri A Di Rocco A et alPrimary mediastinal large Bcell lymphomaCrit Rev Oncol Hematol 0cJournal of International Medical Research Savage KJ Monti S Kutok JL et al Themolecular signature of mediastinallargeBcell lymphoma differs from that of otherdiffuse large Bcell lymphomas and sharesfeatures with classical Hodgkin lymphomaBlood Rosenwald A Wright G Leroy K et alMolecular diagnosis of primary mediastinalB cell lymphoma identiï¬es a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma J ExpMed Mottok A Wright G Rosenwald A et alMolecular classiï¬cation of primary mediastinal large Bcell lymphoma using routinelyavailable tissue specimens Blood Swerdlow SH Campo E Pileri SA et alThe revision ofthe World Healthanization classiï¬cation of lymphoid neoplasms Blood XuMonette ZY Zhou J and Young KHPD1 expression and clinical PD1 blockadelymphomas Blood in Bcell Van Roosbroeck K Ferreiro JF TousseynT et al Genomic alterations of the JAK2and PDL loci occur in a broad spectrum oflymphoid malignancies Genes ChromosomesCancer rearrangements Twa DD Chan FC BenNeriah S et alGenomicinvolving programmed death ligands are recurrent in primary mediastinallymphomaBlood large Bcell Cwynarski K Marzolini MAV BarringtonSF et al The management of primary mediastinal Bcell lymphoma a British Societyfor Haematology Good Practice Paper BrJ Haematol Ribas A and Wolchok JD Cancer immunotherapy using checkpoint blockade Science HematologyOncology Cancer Approvals Safety Notiï¬cations Available online URL wwwfdagovdrugsinformationondrugsapproveddrugsucm610670htmdrugsapproveddrugsucm610670htmwwwfdagovdrugsinformationon Lees C Keane C Gandhi MK et al Biologyand therapy of primary mediastinal Bcelllymphoma current status and future directions Br J Haematol Goodman A Patel SP and Kurzrock RPD1PDL1 immunecheckpoint blockadein Bcell lymphomas Nat Rev Clin Oncol Lesokhin AM Ansell SM Armand P et alNivolumab in patients with relapsed orrefractory hematologic malignancy preliminary results of a phase Ib study J Clin Oncol Ansell S Gutierrez ME Shipp MA et alA phase study of nivolumab in combination with ipilimumab for relapsed or refractory hematologic malignancies CheckMate Blood Wright Z and Brown A Highgrade neutr ia in a patient successfully treated withnivolumab for refractory primary mediastilymphoma Blood Adv nal Bcell Yassin R Hajeer A Alshieban S et al HLAgenotype and response to nivolumab therapy in relapsed refractory primary mediastinal Bcell lymphoma Curr Res Transl Med Zinzani PL Santoro A Gritti G et alNivolumab combined with brentuximabvedotin forrelapsedrefractory primarymediastinal large Bcell lymphoma efï¬cacyand safety from the Phase II CheckMate Study J Clin Oncol Postow MA Sidlow R and Hellmann MDImmunerelated adverse events associatedwith immune checkpoint blockade N EnglJ Med Zinzani PL Ribrag V Moskowitz CH et alSafety and tolerability of pembrolizumab inpatients with relapsedrefractory primarylymphoma Bloodmediastinal large Bcell Jones K Vari F Keane C et al SerumCD163 and TARC as disease response biomarkers in classical Hodgkin lymphomaClin Cancer Res for Cheson BD Fisher RI Barrington SF et alinitial evaluationRecommendationsstagingofHodgkin and nonHodgkin lymphoma theLugano classiï¬cation J Clin Oncol assessmentandresponse 0c' | 2 |
" drug resistance leads to tumor relapse and further progression during chemotherapy in lung cancer close homolog of l1 chl1 has been identified as a tumor suppressor in most malignancies however to the best of our knowledge whether chl1 mediates chemoresistance remains unknown the present study observed that chl1 was significantly downregulated in cisplatin ddpresistant cells a549ddp and paclitaxel ptxresistant cells a549ptx compared with a549 cells when treated with or without ddp and ptx silencing of chl1 in a549 cells promoted the cell survival rate and clone formation and decreased apoptosis whereas overexpression of chl1 in a549ddp and a549ptx cells impeded the cell survival and clone formation and promoted apoptosis additionally chl1 overexpression enhanced the chemosensitivity of a549ddp cells to ddp in vivo notably the chemoresistance induced by chl1 depletion was reversed by the akt inhibitor sc66 in a549 cells the results of the present study demonstrated that chl1 enhanced sensitivity of lung cancer cells by suppressing the akt pathway which suggested that chl1 may be a potential target for overcoming chemoresistance in lung cancerintroductionlung cancer is the most common human malignancy accounting for of all cancerassociated deaths worldwide during in addition its morbidity and mortality rank the highest among all malignant tumor types worldwide according to the differentiation degree and morphological correspondence to dr rimao huang department of cardiothoracic surgery xiangya changde hospital moon avenue west of langzhou north road changde hunan pr chinaemail xyhuangrm163comkey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly classified into nonsmallcell lung cancer nsclc and smallcell lung cancer among patients with lung cancer nearly are diagnosed as nsclc which manifests with earlier diffusion and metastasis currently resection chemotherapy radiotherapy and targeted therapy are the primary treatments for lung cancer for patients with advanced nsclc or those who are clinically incapacitated for surgery chemotherapy is a remarkably important treatment cisplatin ddp is widely applied in the treatment of several malignancies and it exhibits a broad spectrum of antitumor effects by inducing dna damage and hindering dna damage repair paclitaxel ptx another commonly used chemotherapeutic agent in the clinic targets the microtubule cytoskeleton and impedes cell division the majority of patients have a good initial response to chemotherapy agents however subsequent relapse is common and largely due to the emergence of drug resistance thus chemoresistance is considered one of the main factors of poor prognosis in patients with advanced nsclc therefore there is an urgent need to investigate the target and mechanism of chemoresistance in nsclcclose homolog of l1 chl1 is a member of the l1 family of nerve cell adhesion molecules and is located on the 3q26 locus as a nerve cell adhesion molecule chl1 serves an important role in the development regeneration and plasticity of the nervous system the absence or mutation of chl1 can trigger 3p syndrome and schizophrenia the abnormal expression of chl1 may lead to reduced working memory and social behavior mental damage and abnormal behavior chl1 has been reported to be involved in carcinogenesis and progression in a variety of human cancers in esophageal squamous cell carcinoma escc chl1 downregulation is associated with invasion lymph node metastasis and poor overall survival functional studies revealed that chl1 has antiproliferation and antimetastasis abilities the expression of chl1 is downregulated by hypermethylation in human breast cancer and its negative expression contributes to breast tumorigenesis and progression in thyroid cancer and colonic adenocarcinoma chl1 impedes cell proliferation and invasion and acts as a tumor suppressor in lung cancer hÓ§tzel evaluated chl1 expression 0ccai chl1 enhances the chemosensitivity of lung cancer cellsin nsclc cases based on a tissue microarray and it was reported that chl1 expression is associated with t stage in adenocarcinomas as well as with metastatic lymph node status and improved survival additionally by analyzing the gene expression omnibus dataset gse21656 submitted by sun microarray results demonstrated that chl1 expression in ddpresistant h460 cells is significantly lower compared with that in parental cells suggesting that chl1 may be involved in nsclc chemoresistance however to the best of our knowledge the underlying mechanism remains unknownin the present study the expression of chl1 in ddp and ptxresistant a549 cells and the parental cells was assessed functional studies of chl1 were performed to investigate its potential role in chemoresistancematerials and methodsdata processing the human gse21656 microarray dataset was downloaded from the ncbi gene expression omnibus geo database wwwncbinlmnihgovgeo the available dataset gse21656 was based on the gpl6244 platform affymetrix human gene st array affymetrix thermo fisher scientific inc this data includes h460 cells and ddpresistant h460 cells sample and each cell has three repeats samples the online tool geo2r httpwwwncbinlmnihgovgeogeo2r was used to determine the differentially expressed genes in h460 and ddpresistant h460 cells p005 and log2foldchange¥ were set as cutoff standardscell culture the human nsclc cell line a549 the ptxresistant cell line a549ptx and the ddpresistant cells a549ddp were purchased from procell life science technology co ltd the cells were cultured in ham's f12k medium supplemented with fetal bovine serum both purchased from thermo fisher scientific inc uml penicillin and uml streptomycin cat no thermo fisher scientific inc in a Ëc humidified incubator with co2cell transfection the resistant cells a549ptx and a549ddp cells were transfected with µg chl1 recombinant expression plasmid cat no hg10143ny sino biological inc empty vector pcmv3spnha was used as the control a549 cells were transfected with pmol small interfering sirnas the sirna sequence for chl1 guangzhou ribobio co ltd were sirna 'gga gcu aau uug acc aua utt' sirna 'cag caa uau uag cga gua utt' and scrambled control 'uuc ucc gaa cgu guc acg utt' plasmids and sirnas were transfected into cells using lipofectamine® thermo fisher scientific inc following the manufacturer's instructions the time interval between transfection and subsequent experimentation was h for the rescue experiments the chl1 silenced a549 cells were treated with the akt inhibitor sc66 cat no s5313 selleck chemicals along with ddp µgml or ptx ngml both purchased from selleck chemicals for h at Ëcrna extraction and reverse transcriptionquantitative pcr rtqpcr assay total rnas were isolated using trizol reagent thermo fisher scientific inc according to the manufacturer's instructions and the mixed dnas were eliminated by dnase i new england biolabs inc firststrand cdna synthesis was conducted using the goscripttm kit promega corporation according to the manufacturer's instructions the reaction conditions for reverse transcription were Ëc for min Ëc for min and Ëc for min the sybr green realtime pcr master mix thermo fisher scientific inc was used to perform rtqpcr using a lightcycler480 system roche diagnostics gmbh the chl1 primer sequences were as follows forward 'ggc ttg gtc tct tgc ttt cc' and reverse 'atc ttc cct ccc ttt gca cg' and actin forward 'ttc ctt cct ggg cat gga gtc ' and reverse 'tct tca ttg tgc tgg gtg cc' the following thermocycling conditions were used for qpcr min at Ëc followed by cycles at Ëc for sec sec at Ëc and a final extension at Ëc for sec each reaction was conducted in triplicate relative expression levels were calculated using the δδcq method cell viability cell viability was detected by mtt assay a cell suspension µl was seeded into well plates at a density of 1x104 cellswell and incubated overnight at Ëc the concentrations of ddp used to treat a549 cells were and µgml while the concentrations of ptx used to treat a549 cells were and ngml the concentrations of ddp used to treat a549ddp cells were and µgml while the concentrations of ptx used to treat a549ptx cells were and ngml after treating with different concentrations of ddp or ptx for h at Ëc µl mtt mgml solution was added to each well and incubated for h at Ëc subsequently µl dmso was added to each well to dissolve the blue formazan crystals and the absorbance was measured using a microplate reader biotek instruments inc at nmclone formation assay a total of 1x103 cells were seeded into a mm dish in triplicate and maintained in f12k medium with or without ddp or ptx at Ëc for h a total of weeks later cells were fixed in paraformaldehyde for min at room temperature and stained with crystal violet dye at room temperature for min the rate of colony formation was calculated using the following equation number of coloniesnumber of seeded cells x100flow cytometry apoptosis was detected using a fitc annexin v apoptosis kit bd pharmingen bd biosciences according to the manufacturer's protocol cells 1x105 were collected and washed twice with pbs prior to being suspended in µl binding buffer subsequently cells were incubated with µl annexin vfitc and µl propidium iodide in the dark for min at room temperature and apoptosis was analyzed using a cytoflex flow cytometer beckman coulter inc data were analyzed using cytexpert software beckman coulter inc the ratio between early and late apoptosis was calculatedwestern blotting cells were collected washed twice with pbs and lysed with ripa lysis buffer thermo fisher scientific inc proteins were isolated from the cell lysis buffer and 0concology letters quantified using the piercetm¢ bca protein assay kit cat no thermo fisher scientific inc with bovine serum album as a standard equal amount of protein µg proteins were separated by sdspage gel next the proteins were transferred onto a polyvinylidene membrane thermo fisher scientific inc blocked with bsa thermo fisher scientific inc for h at Ëc and incubated overnight at Ëc with primary antibodies against chl1 cat no ap proteintech inc multidrug resistance gene mdr1 cat no ap proteintech inc multidrug resistanceassociated protein mrp cat no ig proteintech inc lowdensity lipoprotein receptorrelated protein lrp cat no ap proteintech inc phosphorylated pakt cat no ab38449 abcam and akt cat no ab227385 abcam after washing three times with pbs the membrane was incubated with horseradish peroxideconjugated goat antirabbit cat no ab6271 abcam_or rabbit antimouse cat no ab6728 abcam secondary antibodies for h at room temperature and the blots were detected with enhanced chemiluminescence reagent thermo fisher scientific inc protein expression was quantified using imagepro plus software media cybernetics incanimal experiments the animal experiments were approved by the medical ethics committee of xiangya changde hospital approval no and were performed in compliance with all regulatory institutional guidelines for animal welfare the national institutes of health publications no a total of male balbcnu mice weekold ± g hunan sja laboratory animals center of the chinese academy of sciences were used in this study all animals were kept at the spf level laboratory at Ëc a relative humidity of a h lightdark cycle and timesh of fresh air exchange all mice were given free access to food and water the bedding materials drinking water feeding cages and other items in contact with the animals were all autoclaved prior to use a549ddp cells 1x107 transfected with empty vector and chl1 overexpression vector using lipofectamine® reagent thermo fisher scientific inc were subcutaneously injected into the nude mice to establish xenograft models following anaesthesia with chloral hydrate mgkg xenografts were allowed to grow to mm3 over weeks and the mice were randomly divided into four groups n3group as follows i vector group a549ddp cells transfected with empty vector and treated with µl saline solution ii vectorddp group a549ddp cells transfected with empty vector and treated with mgkg ddp iii chl1 group a549ddp cells transfected with chl1 overexpression vector and treated with µl saline solution and iv chl1ddp group a549ddp cells transfected with chl1 overexpression vector and treated with mgkg ddp ddp was administered by intraperitoneal injection every days for weeks the mice were observed daily and the tumors were measured by a vernier caliper every days the tumor volumes were calculated as length x width22 a total of weeks postinjection mice were euthanized with co2 at volume displacement rate vdr per min using a programmable logic controller barrywehmiller design group inc mice were monitored continuously and once the mice were immobile except for breathing for min the vdr was provided at for min the animals remained in the euthanasia chamber for min and were then observed for an additional min breathing and heart rate were monitored to determine deathstatistical analysis all experiments were performed in triplicate and data are presented as the mean ± standard deviation all experiments were performed at least three times paired student's ttest was performed for comparisons between two groups and oneway analysis of variance followed by tukey's multiple comparison posthoc analysis was performed for comparisons between multiple groups spss ibm corp was used to perform the analysis p005 was considered to indicate a statistically significant differenceresultschl1 is downregulated in a549ddp and a549ptxresistant cells in order to investigate the mechanism of chemoresistance in lung cancer the lung adenocarcinoma cell line a549 the ddpresistant cells a549ddp and ptxresistant cells a549ptx were used in the present study cells were exposed to different concentrations of ddp µgml and ptx ngml and mtt assay was used to detect the cell survival rate a549ddp and a549ptx cells demonstrated higher resistance to ddp and ptx compared with a549 cells fig 1a the half maximal inhibitory concentration ic50 of ddp was significantly higher in a549ddp cells ± µgml compared with a549 cells ± µgml and the ic50 of ptx was significantly higher in a549ptx cells ± ngml compared with a549 cells ± ngml fig 1b in addition the expression levels of the drugresistant markers mdr1 mrp and lrp were significantly higher in a549ddp and a549ptx cells compared with a549 cells fig 1c additionally the mrna and protein expression levels of chl1 were significantly lower in a549ddp and a549ptx cells compared with those in a549 cells fig 1d and e and this was also observed in h460 ddpresistant cells obtained from the geo dataset gse21656 fig 1f these results suggested that chl1 may be involved in regulating ddp and ptx resistance in nsclcknockdown of chl1 enhances resistance to ddp and ptx in a549 cells as chl1 was upregulated in a549 cells chl1 was silenced in a549 cells using sirnas chl1 expression was significantly reduced in the chl1 sirna groups compared with that of the scrambled control group fig 2a as sirna demonstrated the greatest interference efficiency it was selected for use in the following experiments notably chl1knockdown enhanced the resistance to ddp and ptx in a549 cells fig 2b and c colony formation assay revealed that compared with the control group chl1knockdown significantly increased the rate of colony formation in the absence of chemotherapeutics and enhanced the resistance to ddp and ptx fig 2d flow cytometry results demonstrated significantly reduced apoptosis in chl1knockdown cells after ddp and ptx treatment compared with that of the control group fig 2e 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure chl1 is downregulated in ddp and ptxresistant a549 cells a cell survival of a549 and a549resistant cells a549ddp and a549ptx treated with increasing concentrations of ddp and ptx as assessed by mtt assay b the ic50 values of ddp in a549ddp and a549 cells and the ic50 values of ptx in a549ptx and a549 cells p005 vs a549 cells c western blotting demonstrated the expression of drug resistancerelated proteins mdr1 mrp and lrp in a549 cells and a549resistant cells a549ddp and a549ptx p005 vs a549 cells the protein and mrna expression levels of chl1 in a549 cells and a549resistant cells a549ddp and a549ptx were analysed by d western blotting and e reverse transcriptionquantitative pcr respectively p005 vs a549 cells f the mrna expression of chl1 in h460 and h460ddp cells in the gse21656 dataset p005 vs h460 cells chl1 close homolog of l1 ddp cisplatin ptx paclitaxel mdr1 multidrug resistance gene mrp multidrug resistanceassociated protein lrp lowdensity lipoprotein receptorrelated protein ic50 half maximal inhibitory concentration chl1 overexpression enhances the sensitivity of a549 resistant cells to ddp and ptx as chl1 is downregulated in a549ddp and a549ptx cells the present study successfully overexpressed chl1 in these cells using chl1 recombinant expression plasmids fig 3a the results demonstrated that chl1 overexpression alleviated the resistance to ddp and ptx compared with that of the control group fig 3b and c in addition chl1 overexpression inhibited colony formation in the absence or presence of ddp and ptx fig 3d additionally flow cytometry results demonstrated that restoration of chl1 expression promoted apoptosis in resistant cells following ddp and ptx treatment fig 3eto further validate the effects of chl1 overexpression on ddp or ptx sensitivity xenograft mice model experiments were performed the results demonstrated that chl1 overexpression or ddp treatment significantly impeded the tumor growth fig 3f and decreased the tumor weight fig 3g in addition chl1 overexpression further aggravated ddpmediated repression on tumor growth fig 3f and g these data suggested that chl1 overexpression suppressed tumor growth and enhanced the chemosensitivity in nsclcchl1 mediates chemosensitivity by inhibiting akt activity recently studies have confirmed that chl1 inhibits akt activity in escc and neuroblastoma cell lines thus the present study investigated whether chl1 mediates chemoresistance via the akt pathway in nsclc in a549 cells compared with the scrambled group chl1knockdown elevated the expression of paktser473 fig 4a by contrast restoring chl1 expression in a549ddp and a549ptx cells inhibited the akt phosphorylation compared with the control group fig 4a suggesting chl1 mediates chemosensitivity via the akt pathway subsequently chl1silenced a549 cells were treated with the akt inhibitor sc66 and it was demonstrated that inhibiting akt activity significantly reduced the promotive effects on cell survival fig 4b and clone formation fig 4c and the inhibitory effects on apoptosis fig 4d induced by chl1depletion these results confirmed that chl1 mediates chemosensitivity in nsclc by inhibiting the akt pathway 0concology letters figure chl1knockdown increases a549 cell resistance to ddp and ptx a western blotting was performed to validate the efficiency of transfection with chl1 sirnas p005 vs scramble mtt assays were performed to determine the survival rate of chl1knockdown a549 cells treated with b µgml ddp or c ngml ddp d colony formation assay of a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was used to detect apoptosis in a549 cells transfected with chl1 sirna in the presence or absence of µgml ddp or ngml ptx p005 p0001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering discussionthe results of the present study demonstrated that chl1 was significantly downregulated in a549ddp and a549ptx cells compared with a549 cells the knockdown of chl1 in a549 cells facilitated the cell survival and clone formation and decreased apoptosis when treated with or without ddp and ptx whereas chl1 overexpression in a549ddp and a549ptx cells inhibited cell survival and clone formation and increased apoptosis the results of the present study also demonstrated that chl1 enhances nsclc chemosensitivity through inhibition of the akt pathway these data suggested that chl1 may be a promising target to improve the efficacy of chemosensitivity in nsclcchl1 belongs to the l1 family of nerve cell adhesion molecules it was initially cloned in mice and its expression in mouse development was analyzed by senchenko through cellcell interactions and mediating cellcell and cellmatrix interactions chl1 has an important effect on the development regeneration and plasticity of the nervous system previous reports have demonstrated that chl1 also participates in carcinogenesis chl1 was observed to be significantly downregulated in up to types of tumor tissues compared with their adjacent normal tissues in most tumors chl1 is a potential tumor suppressor gene whose silencing is associated with tumor growth invasion and metastasis for example knockdown of chl1 expression results in enhanced cervical cancer cell invasion and migration a low expression of chl1 in patients with neuroblastoma predicts a poor prognosis and enhancing chl1 expression suppresses tumor progression in contrast chl1 has been reported to promote cell proliferation metastasis and migration in human gliomas however to the best of our knowledge research on chl1 and tumor chemoresistance has rarely been reported 0ccai chl1 enhances the chemosensitivity of lung cancer cellsfigure overexpression of chl1 increases the sensitivity of resistant a549 cells to ddp and ptx a western blotting was performed to detect chl1 expression in a549ddp and a549ptx cells transfected with chl1 expression plasmids p005 vs vector effect of chl1 overexpression on resistant a549 cell survival rate when treated with b µgml ddp or c ngml ptx as determined by mtt assay d colony formation assays demonstrated the number of colonies of resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx e flow cytometry analysis was performed to assess apoptosis in resistant a549 cells transfected with chl1 expression plasmids in the presence or absence of µgml ddp or ngml ptx chl1 overexpression enhanced chemosensitivity of a549ddp cells to ddp in vivo which was demonstrated by the effect of ddp treatment or chl1 overexpression on the f growth and g weight of xenografts derived from a549ddp cells p005 p001 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel the present study examined the differentially expressed genes in nsclc ddpresistant cells in a geo dataset chl1 was demonstrated to be upregulated in ddpresistant cells compared with parental cells suggesting that chl1 may be involved in nsclc chemotherapy resistance similarly a study that compared and analyzed the differentially expressed genes in chemosensitive tumors and chemoresistant ovarian adenocarcinomas tissues reported that the expression of chl1 in chemotherapysensitive tumor tissues is higher compared with that in drugresistant tissues suggesting that chl1 may help to predict the efficacy of chemotherapy for ovarian cancer in addition aberrant methylation of chl1 may be associated with the recurrence of colorectal cancer crc following chemotherapy azadc treatment restores flurouracil sensitivity in vitro which also suggests that chl1 may be involved in crc chemotherapy resistance the results of the present study demonstrated that chl1 was downregulated in a549ddp cells additionally as multiple drug resistance is a common characteristic another type of resistant cells a549tax cells were also used in the current study the results also demonstrated that chl1 was downregulated in a549ptx cells compared with control cells overexpression of chl1 significantly increased the sensitivity of cells resistant to ddp and ptx whereas knockdown of chl1 expression in 0concology letters figure chl1 mediates ddp and ptx sensitivity by inhibiting akt activity a western blotting was performed to detect the expression of pakt and total akt in chlsilenced and restored cell models p005 vs scramble or vector b mtt assays were performed to detect cell survival rates of a549 cells treated with chl1 sirna and akt inhibitor sc66 p005 c colony formation assays were performed in a549 cells treated with chl1 sirna and the akt inhibitor sc66 in the presence of ddp µgml or ptx ngml p005 vs sichl1 d apoptosis were measured in a549 cells treated with chl1 sirna and akt inhibitor sc66 in the presence of ddp µgml and ptx ngml p005 vs sichl1 chl1 close homolog of l1 ddp cisplatin ptx paclitaxel si small interfering p phosphorylated parent a549 cells displayed the opposite results to the best of our knowledge this study is the first study to suggest that chl1 may be involved in chemosensitivity in lung cancer the concentration of ddp used in vivo is mgkg however this may not be in line with the concentrations that would be used in a clinical setting in a clinical trial the human initial dose was calculated from the no observed adverse effect levels noaels verified in animal experiments noael is the maximum dose level without significant adverse reactions the noael verified in animal experiments can be converted to a human equivalent dose according to the body surface area conversion which is based on the area standardization mgm2 proportional among different species in the present study the concentration of ddp used in vivo was not the noael thus it was not consistent with the concentrations used in clinical settingsakt is a serinethreonine protein kinase that is activated by phosphorylation as a key molecule of the pi3kakt signaling pathway pakt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis additionally akt activation is associated with tumor chemoresistance the results of the present study demonstrated that compared with the control groups the expression of pakt was increased in chl1knockdown a549 cells and its expression was reduced in chl1 overexpressed a549ddp and a549ptx cells when akt activity was inhibited by the akt inhibitor the sensitivity to ddp and ptx in chl1knockdown a549 cells was restored this finding suggested that chl1 enhanced the chemosensitivity of nsclc by inhibiting the akt pathway considering numerous studies have confirmed that the akt pathway mediates chemoresistance via regulation of atp binding cassette abc members the present study didn't further investigate the specific abc members and mechanisms which was a of the limitation to the present study thus this research should be further investigated in vivoin summary the present study demonstrated that chl1 was downregulated in resistant cells a549ddp and a549ptx and upregulation of chl1 enhanced the chemosensitivity of nsclc via inhibiting the akt pathway to the best of our knowledge this was the first study to confirm the function and 0ccai chl1 enhances the chemosensitivity of lung cancer cellsmechanism of chl1 in mediating chemosensitivity in cancer thus the development of chl1based therapeutic strategies may improve the efficacy of chemosensitivity in nsclcacknowledgementsthe authors of the present study would like to thank mr dingliang li xiangya hospital changsha china for his guidance and assistance in flow cytometric analysisfundingno funding was receivedavailability of data and materialsthe datasets used andor analyzed during the present study are available from the corresponding author upon reasonable requestauthors' contributionsrh conceived and designed the present study xc bh yh and pl performed experiments and collected the data sl zz and zh analyzed and interpreted the data ml and lz analyzed the data and prepared the figure xc ml and lz drafted the initial manuscript and revised it for intellectual content all authors read and approved the final manuscriptethics approval and consent to participatethe animal experiments were approved by the medical ethics committee of xiangya changde hospital changde china approval no patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreferences parascandola m and xiao l tobacco and the lung cancer epidemic in china transl lung cancer res suppl s21s30 chen w cancer statistics updated cancer burden in china chin j cancer res oser mg niederst mj sequist lv and engelman ja transformation from nonsmallcell lung cancer to smallcell lung cancer molecular drivers and cells of origin lancet oncol e165e172 thatcher n faivrefinn c blackhall f anderson h and lorigan p sequential platinumbased chemotherapythoracic radiotherapy in early stage nonsmall cell lung cancer clin cancer res suppl s5051s5056 yano t okamoto t fukuyama s and maehara y therapeutic strategy for postoperative recurrence in patients with nonsmall cell lung cancer world j clin oncol fang z chen w yuan z liu x and jiang h lncrnamalat1 contributes to the cisplatinresistance of lung cancer by upregulating mrp1 and mdr1 via stat3 activation biomed pharmacother cai y dong zy and wang jy lncrna nntas1 is a major mediator of cisplatin chemoresistance in nonsmall cell lung cancer through mapkslug pathway eur rev med pharmacol sci han ml zhao yf tan ch xiong yj wang wj wu f fei y wang l and liang zq cathepsin l upregulationinduced emt phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in a549 cells acta pharmacol sin liu j meisner d kwong e wu xy and johnston mr translymphatic chemotherapy by intrapleural placement of gelatin sponge containing biodegradable paclitaxel colloids controls lymphatic metastasis in lung cancer cancer res hassan wa yoshida r kudoh s kameyama h hasegawa k niimorikita k and ito t notch1 controls cell chemoresistance in small cell lung carcinoma cells thorac cancer tang h jiang l zhu c liu r wu y yan q liu m jia y chen j qin y loss of cell adhesion molecule l1 like promotes tumor growth and metastasis in esophageal squamous cell carcinoma oncogene liu h focia pj and he x homophilic adhesion mechanism of neurofascin a member of the l1 family of neural cell adhesion molecules j biol chem tassano e biancheri r denegri l porta s novara f zuffardi o gimelli g and cuoco c heterozygous deletion of chl1 gene detailed arraycgh and clinical characterization of a new case and review of the literature eur j med genet morellini f lepsveridze e kahler b dityatev a and schachner m reduced reactivity to novelty impaired social behavior and enhanced basal synaptic excitatory activity in perforant path projections to the dentate gyrus in young adult mice deficient in the neural cell adhesion molecule chl1 mol cell neurosci he lh ma q shi yh ge j zhao hm li sf and tong zs chl1 is involved in human breast tumorigenesis and progression biochem biophys res commun martÃnsánchez e mendaza s ulaziagarmendia a monrealsantesteban i blancoluquin i córdoba a vicentegarcÃa f pérezjanices n escors d megÃas d chl1 hypermethylation as a potential biomarker of poor prognosis in breast cancer oncotarget zhu h fang j zhang j zhao z liu l wang j xi q and gu m mir targets chl1 and controls tumor growth and invasion in papillary thyroid carcinoma biochem biophys res commun yu w zhu k wang y yu h and guo j overexpression of mir5p promotes proliferation and invasion of colon adenocarcinoma cells through targeting chl1 mol med hötzel j melling n müller j polonski a wolterseisfeld g izbicki jr karstens kf and tachezy m protein expression of close homologue of l1 chl1 is a marker for overall survival in nonsmall cell lung cancer nsclc j cancer res clin oncol sun y zheng s torossian a speirs ck sc | 0 |
" 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 | 0 |
Breast cancer is a common malignancy in women Among breast cancer types triplenegative breast cancer TNBC tends to affect younger women is prone to axillary lymph node lung and bone metastases and has a high recurrence rate Due to a lack of classic biomarkers the currently available treatments are surgery and chemotherapy no targeted standard treatment options are available Therefore it is urgent to find a novel and effective therapeutic target As alteration of ion channels and transporters in normal mammary cells may affect cell growth resulting in the development and progression of TNBC ion channels and transporters may be promising new therapeutic targets for TNBC This review summarizes ion channels and transporters related to TNBC and may provide new tumor biomarkers and help in the development of novel targeted therapiesKeywords Triplenegative breast cancer Ion channels Ion transporters Pathological roles Targeted therapyBackgroundBreast cancer BC is the common malignancy in women its incidence is increased each year [] and it has become a significant threat to womens health [] BC is a heterogeneous disease that can be divided into multiple molecular subtypes based on estrogen receptor ER progesterone receptor PR and human epidermal growth factor receptor HER2 expression providing important prognostic and predictive information [] There are four BC subtypes depending on receptor status luminal A luminal B HER2overexpressing and triplenegative breast cancer TNBC Among them TNBC is defined as ER PR and HER2 negative and it tends to affect younger women a0years of age it is Correspondence onlyoneliuxuemei163com 0078029sinacom Chengli Lu and Zhiyuan Ma contributed equally and share first authorship Department of Thyroid and Breast Surgery Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province China Department of Gastroenterology Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province ChinaFull list of author information is available at the end of the prone to axillary lymph node lung bone metastases and has a high recurrence rate [ ] Lehmann et a0al classified TNBC into six subtypes based on gene cluster sequence expression basallike basallike immunomodulatory mesenchymal mesenchymal stemlike and luminal androgen receptor subtypes [] After analyzing the RNA and DNA profile of TNBC tumors Matthew et a0 al classified TNBC into four subtypes including luminal androgen receptor mesenchymal basallike immunesuppressed and basallike immuneactivated subtypes [] The two classification methods have similarities and both provide theoretical bases for exploring targeted therapies for TNBCAlthough TNBC is the BC subtype that responds best to chemotherapy its recurrence and metastasis rates are higher than those of other BC subtypes [] Furthermore due to the lack of classic biomarkers TNBC lacks standard treatments guided by tumor biology and only surgery and chemotherapy are currently available as treatments [] Previous studies have shown that ion channels and transporters play important regulatory roles in mammary physiology and the initiation and progression of BC 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 0cLu a0et a0al Cancer Cell Int Page of [] However the detailed functional role of ion channels and transporters in TNBC has not been clarified and summarized In recent studies upregulation of NaH exchanger has been shown to promote the proliferation migration and invasion of the TNBC cell line MDAMB231 [ ] In addition Ca2 channels such as mitochondrial calcium uniporter MCU can promote TNBC cell migration invasion and lung metastasis [] and Alvarez et a0al [] reported that the twopore domain potassium channel KCNK5 is associated with a poor prognosis in TNBC Therefore ion channels and transporters play important regulatory roles in the pathophysiology of TNBC but there is currently no relevant review on this topic Here we review the pathological roles of ion channels and transporters including AQPs Cl channels Ca2 channels K channels and acidbase transporters in the initiation and progression of TNBCAQP channelsAQPs which compose a family of transmembrane water channel proteins modulate the movement of water and small solutes into and out of cells and maintain suitable concentrations of water and solutes for cell survival [] At least AQP subtypes AQP012 have been identified in mammals and are divided into two families based on transfer specificity namely the classic watertransporting AQP family and the solute water and glyceroltransporting glycoprotein family [] AQP02 AQP4 to AQP68 are mainly waterselective AQP3 AQP7 AQP9 AQP10 and AQP12 also transport glycerol and possibly other small solutes AQPs also play roles in the transport of ammonia urea carbon dioxide metalloids nitric oxide and certain ions [] Expression of AQP1 AQP35 and AQP1012 has been detected in normal human mammary tissue and is closely related to milk secretion [ ] In addition deletion of CCAATenhancer binding protein a family of transcription factors isoforms results in changes in mammary ductal morphogenesis and changes in expression of transport proteins such as AQP5 suggesting that AQP5 may be involved in mammary development [] Recent studies have shown that AQPs play carcinogenic roles by promoting angiogenesis enhancing invasive and metastatic potential and enhancing the transport of reactive oxygen species ROS [ ] In femalespecific cancers such as BC AQP1 and are the most important AQPs and they are been reported to be upregulated []AQP1 the membrane protein was the first reported mammalian AQP and plays a significant role in tumor cell migration proliferation and angiogenesis [] Clinical studies have shown that patients with TNBC have higher levels of AQP1 expression and that upregulation correlates with a poor prognosis [ ] AQP1 expression is induced by hypoxia through the EBoxChoRE transcription element which is affected by increased glucose consumption and metabolism [] AQP1 expression has been detected only in a subgroup of CK14positive basallike breast cancer BLBC cases [] CK14 has been used as a marker of basal mammary epithelial cells with in vivo regenerative ability in studies on mammary gland progenitor and stem cells [] Therefore it is speculated that expression of AQP1 is related to the stem cell characteristics of BLBC cells Hu et a0al demonstrated that AQP1 upregulation promotes extravasation and increases migration in a0vivo and in a0vitro in the mouse TNBC cell line 4T1 suggesting that this aquaporin enhances the rate of cell migration by promoting water permeability in cell protrusions [] Thus upregulation of AQP1 can promote the proliferation migration and invasion in TNBC cells Moreover in a0 vivo experiments have shown that AQP1 deficiency can reduce tumor mass volume vessel density and lung metastases in MMTVPyVT mouse mammary tumor virusdriven polyoma virus middle T oncogene mice and inhibition of AQP1 function andor expression is predicted to attenuate angiogenesis via reduced migration and invasion of endothelial cells [] Recently Irene AbreuRodriguez et a0al [] revealed that AQP1 expression is also responsive to hypoxiainducible factor HIF which may play a role in the VEGFindependent signaling mechanism inducing angiogenesis in a hypoxic environment Helen et a0al [] also reported that the triterpenoid saponins bacopaside I and bacopaside II can synergistically reduce the transcriptional expression of AQP1 and inhibit proliferation migration and invasion in MDAMB231 cells Similarly ginsenoside Rg3 a compound with anticancer activity isolated from ginseng inhibits AQP1 to attenuate cell proliferation through a mechanism that involves downregulation of AQP1 to induce cell cycle arrest in G0G1 phase by inhibiting cyclin D and E and inhibition of chemoattractantinduced cell migration and invasion by blocking AQP1mediated water flux in MDAMB231 cells [] These findings indicate that AQP1 plays an important role in the development and progression of TNBCOverexpression of AQP3 has been detected in the membranes and cytoplasm of TNBC tumor cells and is significantly associated with poor prognosis [] XuChen Cao et a0 al [] found that the presence of fibroblast growth factor2 FGF2 induced cell migration and metastasis in MDAMB231 cells by increasing AQP3 expression Moreover FGF receptor kinase FGFRK inhibitors PI3K inhibitors and MEK12 inhibitors all inhibit AQP3 expression suggesting that FGF receptor kinases increase AQP3 expression and promote FGF2induced cell migration by initiating downstream PI3K and ERK pathways In addition CuSO4 a water transport 0cLu a0et a0al Cancer Cell Int Page of inhibitor of AQP3 inhibits migration in MDAMB231 cells AQP3 downregulation reduces the proliferation invasion and migration of MDAMB231 cells while increasing sensitivity to 5fluorouracil chemotherapy The mechanism may be related to a decrease in glycerol permeability caused by AQP3 downregulation [] Overall these findings demonstrate that AQP3 plays a pivotal role in the initiation and progression of TNBC and specific inhibitors of AQP3 in clinical applications may improve the therapeutic effect of TNBC patientsSimilarly overexpression of AQP5 in the membrane and cytoplasm of TNBC cells has been detected and is significantly associated with poor prognosis [] Moreover patients with higher Ki67 expression are more likely to have abnormal AQP5 protein expression than patients with lower Ki67 expression [] Ki67 is a widely accepted proliferation marker [ ] and it is speculated that upregulation of AQP5 may promote proliferation in TNBC cellsIn summary AQP1 AQP3 and AQP5 are significantly upregulated in TNBC this upregulation is related to a poor prognosis and can promote the proliferation migration and invasion of TNBC cells These AQPs are promising new targets for the diagnosis and treatment of TNBCCl channelsCFTRCFTR is a member of the ATPbinding cassette transporter family that localizes at the apical membranes of normal epithelial cells CFTR is mainly responsible for conducting HCO3 and Cl and promoting HCO3 secretion in many tissues including the airway intestines and pancreas [] However when the extracellular concentration of Cl is higher than a0mmolL the permeability of CFTR to Cl is much greater than that of CFTR to HCO3 thus CFTR mainly conducts Cl under physiological conditions [] CFTR can also transport two other anions glutathione and thiocyanate which are involved in airway inflammation and oxidative stress [ ] Interestingly Pierre et a0al [] reported that CFTR is required for the tightly connected functions of normal epithelial tissues loss of CFTR reduces epithelial resistance and epithelial integrity and this effect is not related to the anion channel function of CFTRCFTR has been reported to be associated with several cancers such as cervical cancer [] colorectal cancer [] prostate cancer [] and BC [] Significant downregulation of CFTR expression is observed in BC tissue compared to normal mammary tissue [] Zhang et a0al demonstrated that overexpression of CFTR inhibits EMT invasion and migration in MDAMB231 cells via a mechanism that involves CFTR inhibition of NFκB targeting of urokinasetype plasminogen activator [] In addition CFTR overexpression inhibits the EMT and the invasiveness of MDAMB231 cells and reduces lung metastasis in xenograft models Increasing evidence reveals that downregulation of CFTR occurs after treatment with EMTinducing factors such as TGF suggesting that as a downstream effector CFTR plays important roles in mediating various EMT effects [ ] Moreover hypermethylation of the cancer genome leads to activation of oncogenes or suppression of tumorsuppressor genes thereby resulting in tumorigenesis [] It has also been observed that the methylation level of CFTR in BC tissues is much higher than that in normal tissues and treatment with DNA methylation inhibitors in TNBC cell lines MDAMB231 and MDAMB435 can rescue CFTR mRNA indicating that CFTR methylation plays an important role in TNBC [] ÎF508 is the most common mutation in CFTR causing the protein to be retained and degraded in the endoplasmic reticulum due to misfolding [] It is worth noting that although there is no difference in the incidence of BC between ÎF508 carriers and noncarriers patients with ÎF508 CFTR mutations all have grade III cancer indicating that CFTR defects are associated with BC progression [] Therefore CFTR methylation or mutation need to be further investigated in the future which may provide novel therapeutic intervention for TNBCChloride channel The chloride channel ClC family also plays an indispensable role in the transport of Cl [] There are nine family members in humans which are divided into two categories based on their distribution and physiological function Cl channel proteins ClC1 ClC2 ClCKa and ClCKb which mainly exist in the plasma membrane and play roles in stabilizing membrane electric potential or mediating epithelial transport and ClH reverse transporter proteins ClC37 which mainly exist in the vascular intima of the endosomelysosomal pathway and are localized at the plasma membrane only to a limited extent due to protein degradation and hydrolase activity [ ] In recent years it has been discovered that ClC3 can transport one hydrogen ion in exchange for two chloride ions [] with important roles in cancers such as nasopharyngeal carcinoma [] and BCClC3 overexpression is observed in tissues and the TNBC cell line MDAMB231 [ ] Studies by Zhou et a0 al revealed that knockdown of ClC3 downregulates expression of cyclin D1 and cyclin E and increases levels of p21 indicating that knockdown of ClC3 can block the cell cycle of MDAMB231 cells at G0G1 phase inhibiting cell proliferation Moreover knockdown of ClC3 suppresses tumor growth in xenograft models and significantly reduces levels of pERK12 in MDAMB231 cells 0cLu a0et a0al Cancer Cell Int Page of This indicates that ClC3 can promote the progression of TNBC by acting on the ERK12 signaling pathway [] Nevertheless relative research on ClC3 in TNBC is still very limited and extensive work is needed in the furtherCa2 channelsCa2 is a key nutrient in milk that plays a vital role in the mineralization of bones and teeth and as a second messenger ionized Ca2 is a key regulator of proliferation migration cell cycle progression and apoptosis [] The level of Ca2 is very low in the cytoplasm a0molL whereas it is somewhat higher in anelles a0 molL and highest in the extracellular level milieu a0 molL Hence a small amount of Ca2 can significantly change intracellular levels to activate downstream signaling molecules including calmodulin nuclear factor of activated Tcells NFAT NFκB calmodulindependent protein kinase II calpain and others [ ] In nonexcitatory mammary cells calcium channels play important roles in lactation and the maintenance of normal physiological functions [ ]Continuous increases in intracellular Ca2 levels will drive expression of oncogenes resulting in tumor growth and development especially the metastatic behavior of cancer cells and conferring tumor cells with resistance to apoptosis [] Abnormal expression of several Ca2 transporters and ion channels such as calcium releaseactivated calcium modulator Orai1 has been observed in TNBC and may lead to oncogenic Ca2 signaling [] Interestingly specific changes in the expression and function of Ca2 channels are related to hormone receptor status and differ significantly among BC subtypes []Calcium modulator Ca2 influx mainly depends on storeoperated calcium channels SOCCs When the Ca2 concentration in the endoplasmic reticulum declines to a certain level the STIM stromal interaction molecule which is located on the endoplasmic reticulum membrane moves to a position close to the highly selective calcium channel protein Orai on the cell membrane Subsequently Orai is activated to cause Ca2 influx and storeoperated calcium entry SOCE is initiated thereby replenishing the calcium store Some researchers have proposed that the canonical transient receptor potential TRPC also participates in the above process though the mechanism remains controversial There are two different claims that both Orai and TRPC form independent channels activated by the STIM protein and that Orai and TRPC subunits form heterochannels triggered by STIM [] There are three Orai1 isomers Orai1 to Orai3 and two STIM homologs STIM1 and STIM2 SOCE has been found to be primarily mediated by Orai1 and STIM1 in TNBC [] Compared with that in nonmalignant breast epithelial cells expression of Orai1 and STIM1 is significantly higher in TNBC cell lines and is associated with a poor prognosis [ ] Liu et a0 al [] reported that hypoxia can induce expression of Orai1 Notch1 and Jagged1 and Orai1 is significantly downregulated after blockade of Notch signaling suggesting that hypoxia can increase Orai1 expression in TNBC by activating Notch signaling Notch1Orai1SOCENFAT4 axis Similarly Mognol et a0al [] found that Orai1 promotes the invasion and angiogenesis of TNBC cell lines and activates NFAT4 which can regulate genes involved in the cell cycle apoptosis angiogenesis and metastasis In addition Yang et a0 al [] demonstrated that Orai1 and STIM1 promote the migration and invasion of MDAMB231 cells both in a0vivo and in a0vitro and the authors proposed that these proteins may at least partially control cell migration by regulating focal adhesion turnover Furthermore treatment with TGF can reduce expression of STIM1 whereas blockade of SOCE can impair TGFinduced G0G1 cell cycle arrest and inhibit the proliferation of MDAMB231 cells [] Based on the above research Orai1 and STIM1 may be new therapeutic targets for TNBC Indeed some selective SOCE inhibitors have shown encouraging inhibitory effects on TNBC but they are still only in the preclinical trial stage For example phemindole a diindole derivative reduces SOCE by downregulating STIM1 expression significantly inhibits the proliferation and migration of MDAMB231 cells reduces the growth of solid tumors in mouse models and produces a targeted antitumor effect in TNBC [] In addition Miroslava Didiasova et a0al [] revealed that elevated cell surfaceassociated enolase1 ENO1 expression correlates with augmented MDAMB231 cell migratory and invasive properties Pharmacological blockade a selective SOCC inhibitor NS1643 or knockdown of STIM1 or Orai1 reduces ENO1dependent migration of MDAMB231 cells These results demonstrate the pivotal role of SOCE in the regulation of ENO1 exteriorization and thus in the modulation of TNBC cell migratory and invasive properties indicated that Orai1 and STIM1 might be promising threptic targets for TNBCSecretory pathway Ca2ATPaseThe secretory pathway Ca2ATPase SPCA can direct Ca2 and Mn2 from the cytoplasm to the Golgi and postGolgi vesicles Two isotypes SPCA1 and SPCA2 are known and the distribution and function of the two differ SPCA1 is commonly expressed in mammalian tissues expression of SPCA2 is limited to highly absorptive and secretory epithelial cells including mammary 0cLu a0et a0al Cancer Cell Int Page of and salivary gland cells [] SPCA1 is highly expressed in TNBC cell lines and SPCA2 is highly expressed in cell lines of other subtypes [] Interestingly based on clinical samples Desma et a0al reported SPCA1 levels to be significantly elevated in the basal subtype of BC compared with all other subtypes and it is worth noting that changes in its expression affect posttranslational modification and transport of certain proteins important for tumor progression without significantly changing cytosolic calcium signaling SPCA1 inhibition also decreased MDAMB231 cell proliferation [] Moreover SPCA1 is a key regulator of insulinlike growth factor receptor IGF1R processing in TNBC cells and promotes the production of functional IGF1R IGF1R activity is associated with poor prognosis suggesting that targeting SPCA1 is an alternative IGF1Rinhibiting strategy [ ] Overall upregulation of SPCA1 may promote the initiation and progression of TNBC The main mechanism reported to date involves SPCA1mediated increase in functional IGF1R expressionMitochondrial calcium uniporterUpregulation of MCU expression on the mitochondrial membrane is closely related to a poor prognosis in BC [] miR340 correlates negatively with the metastatic potential of TNBC cells [] it may directly inhibit MCU expression to reduce glycolysis and exercise capacity and knockdown or inhibition of MCU inhibits the growth invasion and metastasis of MDAMB231 cells [] Interestingly Anna et a0 al [] demonstrated that mitochondrial Ca2 uptake is required for TNBC progression in a0vivo and that absorption of Ca2 by mitochondria promotes the production of sustained mitochondrial reactive oxygen species activating the HIF1α signaling pathway and promoting tumor growth and metastasis In addition inhibiting or silencing MCU also block seruminduced migration of MDAMB231 cells and reduce serum or thapsigargininduced SOCE suggesting that MCU promotes TNBC cell migration by regulating SOCE [] The above results indicate that overexpression of MCU may play an important oncogenic role in the growth invasion and metastasis of TNBC cells However the precise mechanism is unclearOther promising calcium channel targets in TNBC include TRPV6 [] Overall calcium channels are promising targets for TNBC treatment but most compounds targeting these channels are only in the preclinical trial stage Thus further research is neededK channelsThrough the action of NaKATPase two K molecules are transported into a cell in exchange for three sodium molecules which increases the intracellular K concentration K channels on the cell membrane are numerous and in humans more than genes encode major K channel subunits [] K channels play key roles in maintaining acidbase balance by functioning in concert with the NaH exchanger and NaKATPase [] controlling electrical excitability of nerves and muscles and participating in energy metabolism and other physiological processes In addition K channels can help regulate cell proliferation and cell cycle progression and are involved in tumorigenesis [] Many studies have reported dysregulated K channel expression in human cancers including BC astrocytictype brain cancer and prostate cancer [ ] Tumorrelated K channels can be divided into four main categories according to their domain structures and activation mechanisms voltagegated potassium channels which are controlled by changes in membrane potential calciumactivated potassium channels which are activated by intracellular calcium inwardly rectifying potassium channels and twoporedomain potassium channels K2P KCNK [] However the carcinogenic mechanism of K channels remains rather clear Nuria et a0 al [] proposed that K channels may participate in and regulate tumor progression through permeationrelated mechanisms including changes in membrane potential Ca2 driving forces and cell volume regulation and nonconductive mechanisms dependent on proteinprotein interactionsThe Kv111 channel also known as human etheragogorelated gene hERG1 is not expressed in normal breast cells but is expressed in BC with a relationship with subtype Indeed TNBC exhibits lower expression of Kv111 compared with other subtypes [] Olivia Crociani et a0al [] showed that the mRNA levels of Kv111 change throughout the cell cycle peaking in G0G1 phase Moreover Lansu et a0al [] reported that stimulation of Kv111 led to inhibition of proliferation in MDAMB231 cells and that an agonist the diphenylurea derivative NS1643 caused a significant inhibition of cell proliferation This phenomenon can be linked to a rapid decrease in the cyclin E2 protein level which causes accumulation of cells in G0G1 phase and an increase in tumor suppressor proteins and markers for cellular senescence including p21 p16INK4a and galactosidase activity Therefore Kv111 inhibits TNBC cell proliferation by activating a cellular senescence program [] Breuer et a0 al confirmed that NS1643 reprograms the EMT by attenuating the Wntcatenin signaling pathway inhibits cancer cell stemness and significantly reduces the metastatic spread of breast tumors in a MDAMB231 mouse model [] Regardless cardiotoxicity is an important limiting factor for potential therapeutic molecules acting on Kv111 Although the activator is well tolerated 0cLu a0et a0al Cancer Cell Int Page of in BC potential effects include tachycardia [] Overall the potential benefits of Kv111 activators as anticancer drugs outweigh their side effectsIn addition many other channels are altered in TNBC For example some K2P channels with differential expression may serve as novel molecular markers associated with TNBC RNASeq analysis of K2P channels has shown that overexpression of KCNK5 KCNK9 and KCNK12 and low expression of KCNK6 and KCNK15 are related to TNBC [] The above findings indicate that K channels play an important role in TNBC and are expected to be diagnosis markersAcidbase transportersThe pH of milk is significantly lower than that of plasma indicating that there may be some acidbase transporters in the mammary gland that regulate the pH between the extracellular fluid and milk [] A uniform feature among solid tumors with high metabolic and proliferative rates is a significantly different pH from that of normal tissue [] Cancer cells can maintain a weakly acidic intracellular pH that is even more alkaline than that of normal cells suggesting that tumor cells have a powerful pH regulation system a0[]The NaH exchanger NHE a membrane transporter mainly catalyzes the exchange of intracellular H for extracellular Na in mammals thereby maintaining the pH balance inside and outside the cell [ ] There are subtypes of NHE with tissue and membranespecific expression patterns NHE15 are located on the plasma membrane and NHE69 are on intracellular anelle membranes NHE10 is only expressed in osteoclasts [] In addition NHE plays indispensable roles in maintaining normal mammary structure and physiological functions [] NHE1 SLC9A1 is universally expressed in epithelial cells and upregulated in BC tissues compared to normal tissues [] Studies have shown that hypoxia various growth factors and hormones among others can activate NHE1 and enhanced NHE1 activity can reduce extracellular pH and promote metastasis of MDAMB231 cells [] Furthermore it has been proposed that NHE1 promotes metastasis and remodeling of the extracellular matrix by acidifying the extracellular microenvironment [] In addition NHE1 knockdown reduces the migration invasion and growth of xenograft tumors of MDAMB231 cells increasing the susceptibility of these cells to paclitaxel [ ] Moreover knockdown of NHE1 or NBCn1 SLC4A7 in the MDAMB231 cell line significantly reduced the steadystate intracellular pH value after acid load the ability to restore pHi and the primary tumor growth of xenografts in a0 vivo but NBCn1 knockdown prolonged tumorfree survival and reduced cell proliferation [ ] It has been confirmed that NHE1 and NBCn1 promote the development of TNBC through different mechanisms There are two main NHE1 inhibitors amiloride and cariporide which are more effective than amiloride and highly selective [] Amiloride is a potassiumsparing diuretic and has blocking effects on a variety of ion channels such as NHE and the NaCa2 exchanger Cariporide is a highly specific and powerful NHE1 inhibitor that is relatively well tolerated in humans with heart disease [] Moreover a study has suggested that KR33028 a novel small molecule inhibitor of NHE1 produces a cellular phenotype comparable to that of NHE1 knockout cells and significantly decreases rates of migration invasion and colony growth in TNBC cell lines MDAMB231 MDAMB468 and Hs578T [] The above findings suggest that NHE1 may play an important role in the progression TNBCAdditionally other acidbase transporters are also altered in TNBC and are expected to emerge as new targets for TNBC treatment For instance NBCe1 SLC4A4 knockdown reduces cell proliferation invasion and migration in TNBC cells expressing high levels of NBCe1 [] The above findings all suggest that the acidbase transporters have essential functions in the occurrence and development of TNBC but further research is neededConclusionsDysfunction of ion channels and transporters in the mammary resulted in development and progression of TNBC Despite extensive work has been performed to investigate the expression pattern functional diversity regulatory mechanism and pathophysiology of different ion transporters in TNBC the systematic review is rare in this field Therefore this review focuses on different pathological function of multiple families in the development and progression of TBNC including the AQPs Cl channels Ca2 channels K channels and acidbase transporters Fig a0 Table a0 We hope that we can provide a basic systemics and summarised knowledge to this field advocating researchers play more attention on the pathophysiological role of ion channels and transporters in the development and progression of TNBC which may provide novel targets for the clinical diagnosis and treatment of TNBC 0cLu a0et a0al Cancer Cell Int Page of Fig Pathological roles of ion channels and transporters in triplenegative breast cancer cells Alteration and dysfunction of AQPs Cl channels Ca2 channels K channels NaHCO3 transporter and NaH exchanger results in abnormality of ion transport and disorder of multiple signaling pathway including WNT PI3K TGF Notch and VEGF etc eventually promoting TNBC cell proliferation migration and invasion but inhibiting apoptosis | 2 |
" 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 | 0 |
emergence of promising targeted therapies for the treatment of hepatocellular carcinoma HCCSorafenib has been the mainstay of treatment for a decade and newer modalities were ineffective and did not confer any increasedtherapeutic beneï¬t until the introduction of lenvatinib which was approved based on its noninferiority to sorafenib Thesubsequent success of regorafenib in HCC patients who progress on sorafenib treatment heralded a new era of secondlinetreatment and was quickly followed by ramucirumab cabozantinib and the most uential immune checkpoint inhibitors ICIsOver the same period combination therapies including antiangiogenesis agents with ICIs dual ICIs and targeted agents inconjunction with surgery or other locoregional therapies have been extensively investigated and have shown promise andprovided the basis for exciting clinical trials Work continues to develop additional novel therapeutic agents which could potentiallyaugment the presently available options and understand the underlying mechanisms responsible for drug resistance with the goalof improving the survival of patients with HCCSignal Transduction and Targeted Therapy 101038s4139202000264xINTRODUCTIONPrimary liver cancer remains a major problem for all health caresystems worldwide and is associated with a significant clinicaleconomic and psychological burden Hepatocellular carcinomaHCC accounts for of cases of nonmetastatic tumors of theliver1 During the past decades research has shed light on theepidemiology risk factors and molecular and genetic proï¬les ofHCC contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufï¬ciently early stage Unfortunately asignificant proportion of HCC patients present with intermediateand advanced stage disease often despite diligent surveillanceand curative treatments are frequently not possible4 In thesepatients systemic therapy remains essential and its pivotal roleand potential have stimulated considerable research over the pastdecade In this review we examine recent advances in targetedtherapy and discuss the impact this has had on the managementof HCC We also provide an overview of the most important areasof HCC research including novel clinical trials and technicalplatforms which promise to facilitate substantial progress withinthe next decadeAPPROVED FIRSTLINE AGENTS FOR HCCSorafenibThe success of SHARP and AsiaPaciï¬c trial promoted the approvalof sorafenib as ï¬rstline targeted therapy for advanced HCC5ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel ï¬rstline agents were compared andevaluated in an attempt to improve the prognosis of patients withHCC Unfortunately despite a number of trials which comparedthese novel agents including sunitinib10 brivanib11 cediranib12linifanib13 dovotinib14 and immunecheckpoint inhibitors ICIs tosorafenib none achieved the predeï¬ned primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asï¬rstline treatment for advanced HCC increased from monthsSHARP to months CheckMate459 further consolidating itsposition Meanwhile the antitumor activity and safety of sorafenibhave been validated in realworld setting Subanalyses of the SHARPand AsiaPaciï¬c trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15The safety of sorafenib was consistent across ChildPugh A and Bpatients in clinical practice18 and the occurrence of side effects suchas handfoot syndrome and diarrhea were associated with animproved OS19 Baseline hepatic function clinicopathologicalfactors and etiology also affect the prognosis in HCC patientstreated with sorafenib20 In addition sorafenib exerts antitumoreffects with recurrenttransplantationconferring a survival advantage when compared with bestsupportive care BSC21 Noticeably the application of sorafenibin clinical practice displays significantregional variations andincompliance with guidelines besides its usage as ï¬rstline therapyIt is common that initially unresectable HCCs got downstaged aftersorafenib treatment and underwent curativeintent surgery24 andlocoregional therapies before sorafenib were commonly encountered in realworld settings2930tumors following liver1Department of Liver Surgery and Transplantation Liver Cancer Institute Zhongshan Hospital Fudan University Shanghai China 2Key Laboratory of Carcinogenesis and CancerInvasion Fudan University Ministry of Education Shanghai China 3Shanghai Key Laboratory of an Transplantation Zhongshan Hospital Fudan University Shanghai China4Department of Hepatobiliary and Pancreatic Surgery University Hospitals of Leicester NHS Trust Leicester UK 5Institute of Biomedical Sciences Fudan University ShanghaiChina and 6State Key Laboratory of Genetic Engineering Fudan University Shanghai ChinaCorrespondence Jian Zhou zhoujianzshospitalshcnThese authors contributed equally Ao Huang XinRong YangReceived April Revised July Accepted July The Authors 0cTargeted therapy for hepatocellular carcinomaHuang et alFirstlineSorafenibSharpAsiaPacificCediranibNCT00427973SunitinibNCT0069937BrivanibBRISKFLLinifanibNCT01009593NintedanibNCT01004003DovitinibNCT01232296ATEZOBEVGO30140IMbrave150LenvatinibREFLECTPEMLENKEYNOTE524NivolumabCheckMate459DonofenibChina SecondlineBrivanibBRISKPSEverolimusEVOLVE1AxitinibNCT01210495RamucirumabREACHRegorafenibRESORCENivolumabCheckMate040PEMKEYNOTE224TivantinibMETIVHCCS1SCUBEPEMKEYNOTE240CabozantinibCELESTIALRamucirumabREACH2NIVIPICheckMate040CAMChinaApatinibChinaFig Overview of the targeted agents approved for HCC ATEZO atezolizumab BEV bevacizumab CAM camrelizumab LEN lenvatinib PEMpembrolizumab NIV nivolumab IPI ipilimumabThe clinical beneï¬t of sorafenib however remains modest andthe complex molecular pathogenesis of HCC stimulated theinvestigation of combinations of sorafenib with other moleculartargeting drugs Sorafenib has been combined with antiangiogenic agents MEKERK pathway inhibitors mTOR pathwayinhibitors histone deacetylase inhibitors EGFEGFR pathwayinhibitors and HGFcMet pathway inhibitors31 Other agentssuch as interferon32 selumetinib33 capecitabine34 tegafururacil35 gemcitabine and oxaliplatin GEMOX3637 and gemcitabinealone38 have also been evaluated but to date no treatmentsinvolving combinations containing sorafenib have succeeded inphase III trialsSince sorafenib and TACE are both recommended therapies foradvanced HCC it is reasonable to expect that their combined usewould confer beneï¬ts when compared with monotherapy Resultshowever highlighted regional differences and the heterogeneityof the trial protocolsIn TACE the multicenter randomizedplacebocontrolled phase European trial when compared withTACE alone the addition of concurrent sorafenib unlike the SPACEtrial39 did not improve progression free survival PFS40 It was alsoshown that the addition of sorafenib did not confer any survivalbeneï¬t in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these ï¬ndings retrospective studies from China have shown that combination therapywith sorafenib and TACE increased OS by more than compared with TACE alone42 which was supported by theï¬ndings from a number of other groups5455 RecentlytheTACTICS trial a randomized multicenter prospective trial fromJapan reported an improved PFS for TACE plus sorafenibcompared with TACE alone vs months p although this trial used a redeï¬ned PFS not conventional PFS buttime until unTACEable progression The TACTICS trial also usedtime to any cause of death plus OS as primary endpoints resultsnot reported and compared with sorafenib monotherapy TACEplus sorafenib was only superior in controlling tumor progressionand did not prolong OS5758The acceptance of sorafenib as the standard to which othernewer agents and nonsurgical interventions are compared hasresulted in studies comparing its use as monotherapy with TACEplus external beam radiotherapy59 and TACE plus intensitymodulated radiotherapy combined with sorafenib60 In the SARAHstudy selective internal radiotherapy with yttrium90 resin microspheres did not produce any survival beneï¬t compared withsorafenib in locally advanced and inoperable HCC median OS vs p and did not meet the primary endpoint of OS61Similarly the addition of selective internal radiation therapy tosorafenib did not result in a significant improvement in OScompared with sorafenib alone62 Bettinger et al63 however diddemonstrate that stereotactic body external beam radiotherapyemployed as monotherapy SBRT was able to improve OScompared with sorafenib and SBRT with TACE also providedimproved OS and PFS when compared with sorafenib and TACE incombination64 In a recentinfusionchemotherapy HAIC NCT02774187 He et al65 reported thatsorafenib plus HAIC with FOLFOX improved OS compared withsorafenib alone in advanced HCC when portal vein invasion waspresent which was supported by other studies6667 Although theSCOOP2 trial found sequential HAIC with cisplatin and sorafenibdid not improve the survival beneï¬t compared with sorafenibalone this is likely to have resulted from the study beingunderpowered for the primary and secondary endpoints68trial of hepatic arterialDue to the high recurrence rates following hepatectomy fortherapy has been extensivelyHCC approaches to adjuvantinvestigated although previous attemptsincluding the use ofantiviral agents have been largely unsuccessful Based on thepalliative use and success of sorafenib its potential in the adjuvantsetting was investigated and improved survivals following surgeryanticipated Unfortunately this has not been demonstrated and itfailed to reduce postoperative tumor recurrence in the STORMtrial69 and other western studies70 Explanations for the negativeoutcome in the STORM trial include highdose modiï¬cation ratesshort treatment durations and the enrollment of patients whowere not at high risk of tumor recurrence with no evidenceof tumor satellites with one lesion and with nomicroscopic vascular invasion71 Consistent with this viewpointWang et al72 reported no case of recurrence during the sorafenibdosing period whereas patients suffered recurrence of theirtumor within months of discontinuation of sorafenib72 andpersistent sorafenib intake following postoperative recurrenceimproved OS73 Considering that patients who respond tosorafenib may belong to limited clinical or biological subsetsthe effectiveness of sorafenib in an unselected population cohortsupports its use in the adjuvant setting A number of studies fromthe Far East including China Japan and Korea include patientswith HCCs who are treated with hepatectomy despite their tumorsbeing outside Barcelona Clinic Liver Cancer Classiï¬cation BCLCguidelines and although the results are difï¬cult to compare dueto heterogeneity ofthe protocols the results are positiveSorafenib significantly reduces tumor recurrence in BCLC stage Cpatients7475 and increases diseasefree survivalDFS76 andSignal Transduction and Targeted Therapy 0cZhuang et al77 demonstrated that adjuvant therapy increaseddisease free survival DFS and OS Sorafenib treatment followinghepatectomy significantly prolonged the OS of advanced HCCthan intermediate HCC78 In addition to BCLC stageratherpatients who underwent hepatectomy and were pathologicallydiagnosed with microvascular invasion MVI also beneï¬ted fromadjuvant sorafenib treatment79 In line with these results a largerecent study with propensity score matching analysis alsodemonstrated that sorafenib significantly improved overall andrecurrencefree survival following resection80 The results fromthese studies which include all eligible patients suggest that moreprecise stratiï¬cation would enable the identiï¬cation of thosepatients who will beneï¬t most from the use of adjuvant sorafeniband those in where additional treatment is not appropriateOngoing trials are attempting to evaluate the role of sorafenib inpatients with MVI following radical resection NCT02867280 andNCT02537158LenvatinibFollowing the approval of sorafenib for use in the treatment ofHCC it takes more than a decade before the second ï¬rstlinetargeted agent for HCC emerged Lenvatinib was approved for theï¬rstline therapy in advanced HCC following the results of theREFLECT trial a randomized phase III noninferiority trial publishedby Kudo et al81 Although not approved for long further multicenter data from realworld conditions conï¬rmed the efï¬cacy oflenvatinib regardless of previous tyrosine kinase inhibitor TKItherapies8283 and lenvatinib monotherapy demonstrated antitumor activity for more than years in unresectable HCC whenportal vein invasion was present84 In intermediatestage HCCpatients with tumors exceeding the uptoseven criteria for whomTACE is not helpful lenvatinib could provide significant longer OS vs months and PFS vs months85 Lenvatinibpharmacokinetics in HCC is affected by body weight8687 and asufï¬cient dose relative dose intensity RDI is required to achieve agood therapeutic effect and consequently improved outcomesand prognosis are associated with the preservation ofliverfunction which reduces the number of patients who need todiscontinue their treatment88 With lenvatinib unlike other TKIsthere are issues with thyroid toxicity and surveillance for thyroidabnormalities during treatment is important92 Hypothyroidism isnot unusual and there are also fewer common reports ofthyrotoxicosis and destructive thyroiditis93 From a healtheconomics standpoint however lenvatinib is more cost effectivethan sorafenib9495Secondline targeted agents for HCCStill sorafenib displays limited antitumor activity and someinitially sorafenibsensitive would eventually succumb to thedisease indicating the acquired resistance to sorafenib reducesits beneï¬cial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop ï¬rstline agentswhich were superior to sorafenib96 The RESORCE trial was arandomized double blind placebocontrolled and phase III trialdemonstrating the effectiveness of regorafenib in patients whohad progressed on sorafenib treatment Thisstudy ï¬nallyconï¬rmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival beneï¬tthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was conï¬rmed by Yoo et al99 in aretrospective study of safety and efï¬cacy in Korean patientswhere data were consistent with those from the RESORCE trialRegorafenib was even shown to be effective in patients with HCCrecurrence following liver transplantation with a median OS ofregardless ofSignal Transduction and Targeted Therapy Targeted therapy for hepatocellular carcinomaHuang et al months following regorafenib initiation and monthsfollowing sorafenib initiation CI for the sorafenibfollowed by regorafenib sequential therapy100However not all patients who progress on sorafenib arecandidates for secondline therapy101 In clinical practice only of patients are eligible forsecondline regorafenibtreatment102 Good liver functional reserve and ECOG performance status during sorafenib treatment contributed to theefï¬cacy and better outcomes of subsequent treatment103104including lenvatinib105 This may in part be due to the RDIrequired to achieve a clinically significantinprognosis106 This is supported by the demonstration that thenew liver reserve function biomarker albuminbilirubin gradeALBI107 successfully identiï¬ed regorafenib candidates and thatin the selected cohort a median OS of months was achievedcompared with months for noncandidates108 Even in patientsnot eligible for regorafenib the ones with an ECOGPS score of the absence of MVI and TTP time to progression ¥ monthscould still have acceptable postprogression survival109 Longtermtreatment with regorafenib has also been shown to reduceangiogenesis and improve portal hypertension PHT and acuteadministration ameliorates portal haemodynamics suggestingthat it may be especially suitable for patients with PHT andpreserved liver function110improvementCabozantinibCabozantinib is another small molecule inhibitor of the tyrosinekinases which are implicated in the progression of HCC and theacquired resistance to sorafenib Cabozantinib blocks the receptors involved in oncogenesis and angiogenesis including VEGFR hepatocyte growth factor receptor MET AXL and theangiopoietin receptors TIE2 RET cKit and FLT3 in vitro andin vivoIn CELESTIAL trial cabozantinib achieved the primaryendpoint with median OS of months compared with months for the placebo group111 and was consequentlyapproved in the EU and USA There remains a paucity of datahowever from realworld clinical practice examining the sequentialtreatment utilizing cabozantinib as the secondline agent it is acostly option associated with frequent highgrade adverse eventsConsequently several studies have addressed the costeffectivenessof cabozantinib using the cost and utility data extracted from theCELESTIAL trial The conclusion from these studies is consistent andconï¬rms that at its current cost point the gain of qualityadjustedlifeyears for cabozantinib QALYs and the incrementalcosteffectiveness ratio ICER mean that it isnot a costeffective treatment option for patients with sorafenibrefractory HCC112 compared with regorafenib QALY and ICER RamucirumabRamucirumab is a fully human recombinant IgG1 monoclonalantibody targeting the VEGF2 receptor Although ramucirumabfailed to meet its primary endpoint as secondline treatment in theREACH trial117 subgroup analysis found survival beneï¬t in patientswith AFP of ngml or higher118 This was later conï¬rmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the ï¬rstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent ï¬ndingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and conï¬rms the potential value of biomarkerdrivenclinical trials123Immune checkpoint therapy and TKI inhibitorsICIs stand as the mainstream of immunotherapy The CheckMate and KEYNOTE224125 studies evaluated the safety andefï¬cacy of nivolumab and pembrolizumab in patients with 0cTargeted therapy for hepatocellular carcinomaHuang et alphaseIIrandomizedparallelgrouptislelizumab sintilimabrealworld cohort studyadvanced HCC refractory to previous sorafenib treatment whichestablished the basis for accelerated approval by the FDA assecondline treatment Subanalysis of CheckMate040 data validated the safety and efï¬cacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efï¬cacy and safety in advanced HCCs as systemicï¬rstsecondthirdfourthline treatment with median OS andPFS of and months respectively127 and an excellentresponse to antiPD1 therapy has also been described in casereport128 Although the subsequent phase III KEYNOTE240 trialdid not meet its prespeciï¬ed statistical signiï¬cance in respect ofimproved PFS and OS the results were consistent with previousKEYNOTE224129 The KEYNOTE394 presently underway in Asianpatients may clarify the role of pembrolizumab in cases ofadvanced HCC with a viral background NCT03062358 RecentlyCheckMate459 the multicenter phase III randomized sorafenibcontrolled trial evaluating nivolumab as ï¬rstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbeneï¬t regardless of the level of PDL1 expression Furthermorenivolumab improved the survival of HCC patients whose etiologywas HBVHCV and did not reactivate hepatitis CamrelizumabSHR1210 Hengrui Pharmaceutical is an antiPD1 inhibitor fromChina investigated for the treatment of Hodgkin lymphoma andHCC It has been shown to have antitumor activity in previouslytreated Chinese patients with advanced HCC in a multicenteropenlabeltrialNCT02989922130 providing evidence for the effectiveness ofPD1 therapy for HBV related HCC in Chinese patients The resultsICIs including durvalumabfrom other trials investigating novelavelumabtremelimumabipilimumabspartalizumab and toripalimab will hopefully yield positive resultsand provide further options for the treatment of patients withHCC particularly those who have relapsed on ï¬rstline treatmentsFurther efforts to enhance the treatment effect of ICIs includedual ICIs treatment and combination therapy of ICIs with otherkinds of targeted agents For dual ICIs treatment the initial resultsfrom CheckMate 9DW were astonishing the objective responserate was higher than monotherapy of any ICIs alone FDA hasapproved nivolumab in combination with ipilimumab for patientswith HCC previously treated with sorafenib Treatment modalitiessuch as radiotherapy and antiangiogenesis agents which affectantigen release or modulate the tumor microenvironments havethe potential to increase the efï¬cacy of immunotherapy and thecombination oftargeted agents with ICIs are attracting theattention of a number of research groups and in vitro studies andearlyphase clinical trials assessing combination treatments haveshown promising antitumor effects in patients with advancedIn vitro evidence by Qui et al131 demonstrated thatHCClenvatinib and regorafenib could affect the expression of PDL1and realtime PCR results suggested that the mRNA expression ofPDL1 in the lenvatinib group was significantly higher than that inthe control group while its expression in the regorafenib groupwas significantly lower When combined with antiPD1 lenvatinibcan modulate cancer immunity in the tumor microenvironmentand enhance antitumor activity132133 In July the FDAannounced its approval of the ï¬rst combination therapy employing the TKI lenvatinib with the ICI pembrolizumab based on theresults from the KEYNOTE524Study NCT03006926 for thetreatment of HCC Recently results from Study Phase IbNCT03418922 showed marginally better results for lenvatinibwith nivolumab than lenvatinib with pembrolizumab METmediated phosphorylation leads to a decreased expression ofPDL1 using the combination of MET inhibitors tivantinib andcapmatinib antiPD1 and antiPDL1 produced an additive effectwhich slows the growth of HCCs in mice134 Clinically based onthe results from the experimental arm A of the GO studyNCT02715531 the FDA approved atezolizumab plus bevacizumab as breakthrough therapy for untreated advanced ormetastatic HCC135 Individual case studies also reported promisingresults for the use of combined TKI and antiPD1PDL1 agents foradvanced HCC136 Such results were conï¬rmed in the phase IIItrialIMbrave study NCT03434379 which reported thatatezolizumab combined with bevacizumab resulted in better OSand PFS than sorafenib in patients with unresectable HCC139Other combination therapies include Galunisertib with nivolumabNCT02423343 spartalizumab with and without capmatinibNCT02795429 FGF401 with spartalizumab NCT02325739regorafenib with pembrolizumab NCT03347292 cabozantinibwithaxitinibNCT03289533 ramucirumab with durvalumab NCT02572687and XL888 with pembrolizumab NCT03095781 Table avelumab withNCT03299946nivolumabImmunerelated adverse events IRAEs occur frequently duringtreatment with ICIs and the clinical consequences can besignificant140 Activation of the immune system leads to damageof normal healthy tissues and IRAEs can have myriad effects andinvolve a number of different ans and have been reported toproduce colitis hepatitis pneumonitis dermatitis myocarditisendocrine glands ammation and rheumatic and musculoskeletal phenotypesincluding ammatory arthritis arthralgiamyositis and sicca syndrome141 Although the precise pathophysiology underlying the IRAEs side effects during treatment withICIs remains unknown discontinuing administration and the useof steroids is generally effectiveIn severe cases howeveradditional immunosuppressants may be required but based oncurrent available evidence immunosuppression for IRAEs does notappearresponse to the ICItreatment142143to compromise the antitumorPromising agents and treatment regimensDespite abovementioned targeted drugs novel agents have beencontinuously under development Table Of note apatinib anovel inhibitor of VEGFR2 tyrosine kinase has attracted considerable attention and there is now a significant body of workdescribing clinical experience with its use Although less effectivethan sorafenib as a ï¬rstline treatment in a retrospective study144apatinib still displayed promising antitumor effects in sorafenibresistant HCC145 where portal vein invasion was present148when metastases have occured149150 and for unresectable andrelapsed HCCs151152 Combination therapy in studies utilisingapatinib with TACE have achieved better clinical effectivenessthan TACE alone with tolerable AEs153 Recentlythecombination of apatinib with the antiPD1 monoclonal antibodycamrelizumab achieved partial response rates of Theresults of other ongoing trials including the phase IIItrialcomparing TACE and apatinib with sorafenib as ï¬rstline treatmentfor locally advanced or metastatic and unresectable HCC NCT and the adjuvant apatinib after hepatectomy for theprevention of tumor recurrence NCT03722875 and NCT03261791will hopefully prove effective and add to the presently availabletherapeutic optionsThese promising results have stimulated the investigation ofother new agents the combinations of agents and regimenswhich have been thoroughly discussed in a recent review fromZhu and Sun154 The combination of bevacizumab and erlotinibhas been extensively evaluated as ï¬rst155 or secondline inadvanced HCCs156 but unfortunately the heterogeneousnature of the results precludes ï¬rm conclusions and recommendations Recently a singlearm metaanalysis of prospectivestudies found that combination therapy with bevacizumab anderlotinib used as secondline treatment was associated with afavorable PFS weeks P and OS months P suggesting that future welldesigned and sufï¬ciently poweredlargescale RCTsshould be able to identify the potentialcontribution of these agents163Signal Transduction and Targeted Therapy 0cTable Trials investigating the combination therapy of ICIs and TKIs in HCCTrial nameidentiï¬erPatient No Study type LineInterventionsPrimaryendpointStudy statusTargeted therapy for hepatocellular carcinomaHuang et alLEAP002NCT03713593Phase IIIFirstLenvatinib pembrolizumab vs lenvatinib PFSOSPhase IIIFirstPhase IIIPhase IIIPhase IIIFirstFirstFirstPhase IIIFirstFirstPhase IIFirstPhase IIPhase Ib II FirstFirstPhase IIPhase IbFirstNivolumab ipilimumab vs lenvatinib orsorafenibCabozantinib atezolizumab vs sorafenib PFSOSSintilimab IBI305 vs sorafenibOS ORRCamrelizumab apatinib vs sorafenibOSPFSOSCamrelizumab apatinib vs FOLFOX orsorafenibNivolumab lenvatinibNivolumab sorafenibSorafenib pembrolizumabAnlotinib sintilimabAvelumab axitinibOSORR AEMTD ORRORRORR AEAEActive notrecruitingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingOngoingCompletedCheckMate 9DWNCT04039607COSMIC312NCT03755791ORIENT32NCT03794440SHR1210III310NCT03764293SHR1210III305NCT03605706IMMUNIBNCT03841201NCT03439891NCT03211416KEEPG 04NCT04052152VEGF Liver NCT03289533KN743NCT03347292GOINGNCT04170556REGOMUNENCT03475953NCT02423343aaRegorafenib pembrolizumabFirstPhase ISecond Regorafenib nivolumabPhase IISecond Regorafenib avelumabPhase IIIPhase Ib II Second Galunisertib nivolumabAEAECR PRPhase Ib MTDOngoingOngoingOngoingOngoingPFS progressionfree survival OS overall survival ORR objective response rate AE adverse events MTD maximum tolerated dose CR complete response PRpartial responseaTrials enroll not only HCC patientsTable Trials investigating targeted therapy in advanced HCCTrial nameidentiï¬erPatient noStudy typeLineInterventionsPrimary endpointStudy statusIMbrave150 NCT03434379ZGDH3NCT02645981HIMALYYA NCT03298451RATIONALE301 NCT03412773PHOCUSNCT04344158ALTER0802 NCT02809534AHELPNCT02329860KEYNOTE394 NCT03062358NCT04080154Phase IIIPhase IIIIIPhase IIIPhase IIIPhase IIIPhase IIIPhase IIPhase IIIPhase IIIPhase IIFirstFirstFirstFirstFirstFirstFirstSecond Apatinib vs placeboSecond Pembrolizumab BSC vs placebo BSCSecond AnlotinibAtezolizumab bevacizumab vs Sorafenib OS PFSDonafenib vs sorafenibOSDurvalumab tremelimumab vs sorafenib OSOSTislelizumab vs sorafenibPexaVec sorafenib vs sorafenibOSAK105 anlotinib vs sorafenibOSPFS 12WAnlotinibOSOSPFSOS overall survival PFS progressionfree survival BSC best supportive careCompletedCompletedOngoingOngoingOngoingOngoingOngoingCompletedOngoingOngoingundergoingevaluationandPreclinical evidence for cyclindependent kinase CDK targetingtherapies in HCC has showed promise and supports theirinvestigation164 especially with the potential ability toabrogate the emergence of sorafenib resistance167 and sensitizeHCC to regorafenib treatment168 A number of CKD inhibitors arepalbociclibpresentlyNCT01356628 milciclibribociclibNCT02524119 The antiMET monoclonal antibody emibetuzumab exhibited the greatest antitumor activity in HCC whencombined with ramucirumab and had an excellentsafetyproï¬le169 and for HCC with high MET expression there was analmost 3fold increase in PFS vs months relative to thosewith low MET expression suggesting the potential for furtherbiomarkerdriven clinical trials Rigosertib is a synthetic benzylstyryl sulfone small molecule inhibitor which has been used in theNCT03109886includingtreatment of monomyelocytic leukemia and due to its activity as aRAS and PLK1signaling inhibitorit was investigated in HCCpatients who demonstrate upregulation of PLK1 during tumordevelopment and HRAS expression in advanced HCC Highexpression levels of PLK1 are also significantly correlated withpoor patient survival and the multiple effects of rigosertib couldbe beneï¬cially employed to produce a therapeutic dualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efï¬cacy when treating advanced HCC in phase1b trial and phase studies using sorafenib as the controlNCT02645981171 There are ongoing trials evaluating novelagents such as anlotinib another multikinase inhibitor which isorally administered and targets VEGFR ï¬broblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classiï¬cation of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassiï¬cationG1G6S1S3Msig iC1iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and lowMS1 SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell proï¬lingImmunomicroenvironmentTranscriptome and gonomeProteomicsCase no and ckit NCT02809534 Tivozanib is another oralinhibitor ofVEGFR123 with promising activity against HCC in vivoNCT01835223 and TRC105 which despite demonstrating clinicaltolerated in HCC patients followingactivity and being wellsorafenib has notto date met prespeciï¬ed criteria and itsdevelopment in HCC continues as combination therapy withsorafenib NCT02560779Biomarkerdriven targeted therapyDespite extensive research investigating potential biomarkers to aidthe development of protocols for the treatment of HCC none haveso far been identiï¬ed to be able to predict the effect of or responseto treatment with sorafenib172 Although the molecularclassiï¬cation of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183The continued belief that the future lies with personalizedtreatment which will be made possible through the rapiddevelopments in next generation sequencing and the precisionmedicine that it underpins have encouraged the development ofnovel trial designs191 These novel trials designs offer new hopethat biomarkerdriven targeted therapies can be modul | 2 |
"Twelve studies of various design reported statistically significant association between ICC and survival. General surgeons were surveyed to identify individual and anizational barriers to MCC adoption [22]. Surgeons said that MCCs were not well supported institutionally or widely accessible few had a designated coordinator and most reviewed only rare or select cases rather than all new cancer patients. Interviews and observation were used to explore MCC use in four hospitals [23]. Thirty-seven MCCs were observed at three hospitals and 48 clinicians and administrators were interviewed. Institutions lacked the capacity to fully implement MCCs as part of routine practice. MJD developed a measure of cancer services integration and conducted a population-based survey of Ontario health professionals to evaluate integration. The study identified 12 factors that accounted for the majority of variation in cancer services integration [24]. This work emphasized how leadership coordination resource allocation and communication influence overall integration of cancer services. Further analysis of this data revealed variability in access to electronic health records (EHRs) across different provider groups anization types and geographic locations which may limit ICC [25]. Another analysis focused specifically on the benefits of MCCs as a model of ICC [26]. Overall 74% of respondents were aware of MCCs within their region but only 58% were regular participants. Diagnostic assessment programs MCCs represent one model of ICC for treatment decision making. The time interval from suspicion to diagnosis of cancer involves numerous consultations and testing and is a confusing time for patients. Timely diagnosis can lead to improved access to MCCs or other consultation earlier treatment and a better prognosis [1-21]. Clinicians and managers suggested the need to improve ICC earlier in the cancer trajectory given barriers of access to and coordination of diagnosis and staging and recommended centralized diagnostic facilities [14-19]. An expert panel assembled by the provincial cancer agency issued anizational standards for Diagnostic Assessment Programs (DAPs) to coordinate diagnostic tests and integrate multidisciplinary expertise [2728]. A summary of recommended DAP features appears in . DAP standards Component Description Team composition Administrative ¢ Director/manager ¢ Reception clerical and bookings Health professionals ¢ Assessment coordinators (examples): ¢ Radiologists ¢ Pathologists ¢ Primary care ¢ Psychosocial support Specialists ¢ Surgeon specialists ¢ Respirologists (lung) ¢ Endoscopists (colorectal and other) Technicians ¢ Ultrasound technologists ¢ Mammographers (breast) Scope of diagnostic activity Examination (diagnostic activity differs depending on disease site) ¢ Physical exam ¢ Other disease site specific Imaging diagnostic and staging procedures ¢ Ultrasound ¢ MRI ¢ X-ray ¢ CT scan ¢ PET ¢ Upper endoscopy ¢ Colonoscopy ¢ Bronchoscopy ¢ Cystoscopy ¢ Bone scan ¢ Mammography ¢ Other disease site specific Surgical consultation and procedures ¢ Biopsy ¢ Fine needle aspiration cytology ¢ Biopsy Pathology and laboratory medicine ¢ Standardized surgical pathology requisition forms ¢ Routine analysis and pathology reporting ¢ Special pathological studies such as markers flow molecular etc ¢ Clinical lab testing of tumour markers hematology etc. Supportive care ¢ Education/psychosocial support ¢ Dietetics ¢ Genetic counselling ¢ Other supportive services ¢ Access ¢ Regionalized centralized ¢ One stop ¢ Virtual ¢ Entry point ¢ Primary care providers or specialist ¢ Screening program ¢ Self referral ¢ Operational features ¢ Entry ¢ Fast access booking ¢ Priority booking ¢ Open-access booking ¢ DAP core elements ¢ Assessment coordinator ¢ Multidisciplinary care conference (MCC) team/treatment team ¢ Cross-DAP collaboration ¢ Provincial indicators of quality for cancer DAPs ¢ Time intervals ¢ Clinical outcomes ¢ Quality of care ¢ Patient satisfaction ¢ Guidelines standards and services frameworks ¢ Guidelines and service frameworks for primary care providers ¢ Evidence-based investigative algorithms and guidance documents ¢ Wait-times benchmarks Study rationale ICC for cancer leads to multiple system anizational professional and patient benefits. However our analysis of conceptual literature did not reveal optimal ways to achieve ICC and our review of empirical literature revealed that no interventions apart from MCCs have been used to promote ICC for cancer care. Our research with health professionals identified limited support for use of and access to interventions that enable ICC for cancer particularly outside of designated cancer hospitals in community settings where the majority of cancer care takes place and for cancer diagnosis. DAPs appear to be a promising model by which to enable ICC. In 2007 the provincial cancer agency funded four pilot DAPs and all achieved reductions in wait times (http://www.cancercare.on.ca/pcs/diagnosis/diagprograms/). Hospital one reduced median time from suspicion of breast cancer to biopsy by 60% (38 to 15 days) and from suspicion to diagnosis by 53% (42 to 20 days); hospital two reduced time from referral to colonoscopy for patients with positive fecal occult blood test results by 78%; hospital three reduced lung cancer wait times from 113 to 69 days for referral to diagnosis; and hospital four reduced lung cancer wait times from 120 to 44 days for suspicion to diagnosis. Wait times are only one possible outcome of ICC. We require more information about how to design and implement DAPs to optimize ICC and achieve the range of associated beneficial outcomes. We conducted a systematic review of the cancer literature to describe clinical and economic evaluations of DAPs [29]. Most of the 20 eligible studies did not base their evaluations on guideline recommendations or quality indicators or include economic evaluations. Several DAPs were implemented across Ontario so more comprehensive evaluation was warranted and possible to better understand how various DAP models enabled ICC. The purpose of this study is to: 1. Describe DAP structure function and outcomes according to published DAP standards clinical guideline recommendations and a theoretical framework of ICC. 2. Conduct a pilot costing analysis of delivering diagnostic services with DAPs. 3. Explore challenges of DAP implementation and operation and associated ICC. 4. Issue recommendations that may optimize the implementation operation and outcomes of DAPs. This research will not evaluate DAPs from the perspective of patients. While crucial that objective warrants separate multi-year investigation to explore patient preferences for diagnostic care first through review of the literature then by interviewing patients with various characteristics who did and did not experience DAP care. This would establish patient-informed performance measures of diagnostic services and ICC which do not exist. We found that patient views about cancer care performance measures differed from those of health professionals thus development of patient-informed performance measures is necessary to fully evaluate the services provided by DAPs and the degree to which the DAP model enables ICC [30]. Instead this proposal responds to the expressed needs of our research partners and for multiple reasons including feasibility focuses first on evaluating DAPs according to evidence-based standards for DAPs and clinical care delivery and by soliciting the views of involved health professionals. This preliminary evaluation is needed to establish a baseline understanding of how DAPs were implemented and function. Only then will we have sufficient insight on factors influencing DAP outcomes that we could ask patients about and an established relationship with DAP collaborators to enable an expanded research study that would include patient recruitment. Methods Design A case study approach was chosen to explore multiple factors that influence ICC including DAP structure processes and outcomes [31]. These will be assessed according to DAP standards and guideline recommendations for staging and diagnosis of breast [32-34] and lung cancer [2728]. This will enable comparison between cases (DAPs) that vary by type of cancer academic status and geographic region. This may identify whether and how differences in DAP leadership staffing resources and referral patterns influence ICC or whether ICC must be enabled differently by condition. This approach is suitable for examining complex issues that require holistic interpretation (triangulation) of data collected in a variety of ways from different sources. Data will be collected from medical records and interviews. Four hospitals from different regions of Ontario agreed to participate. Two are considered academic teaching hospitals. Each site features a breast DAP and a lung DAP for a total of eight participating DAPs. This study was reviewed and approved by the University Health Network Research Ethics Board and research ethics boards at each of the four participating hospitals. Conceptual framework The overall goal of this study is to explore how DAPs enable ICC and associated outcomes. ICC was defined as interaction among various types of health professionals to plan or evaluate services or plan provide or review results or outcomes for individual patients. There is no single existing model or theory that describes factors influencing the quality of ICC so we compiled a conceptual framework from several sources. We had reviewed several bodies of knowledge describing models of health professional interaction for patient management according to the concepts of teamwork inter-professional collaboration continuity of care integrated service delivery inter-anizational collaboration and case management and extracted data on common domains and elements and associated outcomes [20]. This generated core components and enablers of ICC that were common across the models. anizational standards for DAPs that are described in [2728] were mapped onto this preliminary framework. The conceptual framework was expanded by adding challenges [10-26] and beneficial outcomes [9-21] identified in our background review of the literature. We also reviewed clinical guidelines for breast [32-34] and lung cancer [2728] to incorporate elements of desirable care delivery and outcomes. The resulting conceptual framework (Figure 1) will be used to inform the development of data collection instruments and data analysis. Findings will be used to validate and extend the conceptual framework to describe how DAP structure function and support enable ICC and associated outcomes. Figure 1 Conceptual framework describing factors that influence how DAPs achieve ICC and associated benefits. Medical record review to describe DAP services and outcomes Approach To describe DAP function and outcomes and gather information that will enable costing recommendations expressed in DAP standards and clinical guidelines will be assessed through retrospective observational study. Data will be acquired from databases maintained by participating DAPs and the provincial cancer agency and confirmed by and supplemented with review of medical records. Sampling Eligible patients include those 18 years of age or older with suspected lung or breast cancer who were referred to participating DAPs between 1 January 2012 and 31 December 2012. Based on input from collaborating sites we initially estimated that this includes a mean of 15 new patients per month by two types of cancer in four sites for an annual total of 360 patients per site and an overall total of 1440 patients. Assuming a type I error (alpha) rate of 0.05 power of 0.80 and equal sample sizes for two comparative groups (i.e. academic/community status or breast/lung cancer) 170 patients (85 in each group) in total would be required to detect a statistically significant difference in compliance with a given DAP or clinical guideline standard of 15%. Thus estimated patient sampling is more than sufficient to identify variations in process or outcome performance measures according to varying DAP features. However DAPs vary in case volume. To give equal weight to each DAP at each participating site based on patient volume 15% of patients will be randomly sampled from among those newly referred during the given time period. For all sites this is equal to or greater than minimal sample sizes estimated by traditional power calculation. Data collection and analysis Data reflecting DAP and guideline recommendations for diagnostic activity () will be extracted from medical records. A trained data abstractor will visit participating sites. Before this a data extraction form will be developed and independently pilot tested on five cases by two individuals. They will compare congruence of extracted data to assess how the form should be revised. This will be repeated until the format and content of the data extraction form is satisfactory and congruence of independently extracted data is high. During chart review 5% of charts will be re-abstracted for data quality monitoring. Summary statistics will be used to report compliance with DAP and guideline standards for patients overall and by DAP academic status and type of cancer. Statistical significance of differing outcomes will be reported with the Pearsons chi-square test. A generalized linear mixed model approach will be used to address binary and continuous process and outcome variables. Hierarchical modeling (patient level one hospital level two) will be used to allow for clustering by hospital. Pilot costing analysis of DAP diagnostic visits and services Approach Our ultimate goal is to conduct a cost-effectiveness analysis (CEA) but this is a complex undertaking requiring considerable data on the actual number and nature of services provided per patient and their cost. The purpose of CEA is not hypothesis testing but rather it is estimation [35]. To do a CEA one needs to compute estimates of the extra cost (?C) and the extra effect (?E) of an intervention. The ratio of ?C to ?E is called the incremental cost-effectiveness ratio (ICER) and is the main statistic in CEA. Such data are not readily available in administrative databases and often requires primary data collection from medical records. We currently do not know whether data required for a CEA of DAPs is available in medical records." | 1 |
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 | 0 |
ovarian cancer is a silent and largely asymptomatic cancer leading to late diagnosis and worseprognosis the latestage detection and low survival rates makes the study of the spacetime evolution of ovariancancer particularly relevant in addition research of this cancer in small areas like provinces or counties is still scarcemethods the study presented here covers all ovarian cancer deaths for women over years of age in the provincesof spain during the period spatiotemporal models have been fitted to smooth ovarian cancer mortalityrates in age groups [ [ [ and [ borrowing information from spatial and temporal neighboursmodel fitting and inference has been carried out using the integrated nested laplace approximation inla techniqueresults large differences in ovarian cancer mortality among the age groups have been found with higher mortalityrates in the older age groups striking differences are observed between northern and southern spain the globaltemporal trends by age group reveal that the evolution of ovarian cancer over the whole of spain has remainednearly constant since the early 2000s differences in ovarian cancer mortality exist among the spanish provinces years and age groups as theexact causes of ovarian cancer remain unknown spatiotemporal analyses by age groups are essential to discoverinequalities in ovarian cancer mortality women over years of age should be the focus of followup studies as themortality rates remain constant since highmortality provinces should also be monitored to look for specific riskfactorskeywords agespacetime models disease mapping inla ovarian cancer mortality smoothing the number and scientific impact of publications on ovarian cancer are continuously increasing not in vainovarian cancer is the eighth most common cancer inwomen and the 18th most frequent overall with nearly new cases worldwide in around of cases are concentrated in developed countries whereovarian cancer is the most lethal gynecological tumorcorrespondence lolaunavarraes1department of statistics computer science and mathematics publicuniversity of navarre campus de arrosadia pamplona spain2inamat public university of navarre campus de arrosadia pamplonaspainthe highest incidences are found in northern and easterneurope austria the czech republic germany irelandlatvia lithuania the nordic countries slovakia and theuk and the united states whereas in africa andasia this tumor is virtually nonexistent this pattern isattributed mostly to the low birth rates found in developedcountries in the past years the overall number of tumors hasundergone a constant increase in spain this is due notonly to the population growth but also to the increasedlife expectancy and the use of early detection techniquesin the estimated number of new cases of ovarian 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 0ctrandafir bmc public health page of cancer was representing of all female cancersbeing the fifth cause of cancer deaths in women afterlung breast colon and uterine tumors [ ] the agestandardized incidence rate calculated using the directmethod and the world standard population is per women which may be considered high as forits temporal evolution there was a slight decline between and but since then mortality rate starts toincrease slowly but constantly in addition there is a greatdeal of variability among the spanish provinces for example during the period one finds a rate of per women in the canary islands and a rate of per women in cuenca ovarian cancer is a disease affecting mostly older postmenopausal women with more than of cases beingdiagnosed in women over according to medicalexperts it is a silent and mostly asymptomatic cancer acircumstance that leads to late diagnosis and worse prognosis furthermore in the cases where symptoms doappear these may be confused with digestive problemsbloating early satiety abdominal andor pelvic pain orbenign gynecological alterations such as endometriosis orpolycystic ovary syndrome to date no method for earlydetection is available this is reflected in the fact that upto of cases are detected in the advanced stages of thedisease the etiology of ovarian cancer is poorly understoodhowever several factors associated with an increased riskof ovarian cancer have been identified age number ofovulations early menarche infertility low parity theuse of hormone replacement therapy hrt obesity physical inactivity a family history of breast andovarian cancer including brca1 and brca2 gene mutations and past and current smoking associations between exposure to asbestos in the workplace orat home and ovarian cancer have also been found further research is needed to corroborate this finding inspain as most jobs with a high exposure to asbestos arepredominantly maledominated eg mining milling orshipyard work nevertheless a study of asbestos exposure among italian women found that the main factorwas secondhand contact due to occupationally exposedrelatives for example from soiled work clothes broughthome as of this writing however there is no documented registry for asbestos exposure in the workplaceor at home in spain some epidemiological studies havedetected an increased risk of ovarian cancer in womenwith less exposure to sunlight and consequently with lowlevels of vitamin d in particular the higher the latitudethe less overall accumulated sunlight and the higher theincidence of ovarian cancer some protective factors against ovarian cancer have also been identified suchas multiparity oral contraceptives and tubal ligation orhysterectomy the 5year agestandardised relative survival in spain is estimated at less than similar tothe european average of as mentioned aboveovarian tumors are the eighth cause of cancer deathsamong women worldwide with deceases registered in overall among malign tumors in about women died yearly in spain from ovarian cancer representing of all cancer deaths and of all deaths among women the mean age of decease from ovarian cancer inspain is years cabanes analyzedthe ageadjusted mortality trends of ovarian cancer inspain for the period ovarian cancer caused deaths in this period with rates in women over showing a tenfold increase versus those in youngerwomen in women under rates increased peryear until and afterwards started to drop at a rateof per year in the age group mortalityrates significantly increased annually up to andbecame stable thereafter in women and older mortality rates increased annually up to the year anddecreased per year afterthe disproportionate impact on older women togetherwith the aforementioned concerns regarding latestagedetection and low survival rates makes the study of thespacetime evolution of ovarian cancer particularly relevant in addition it is important to mention that agegroups are not equally affected by ovarian cancer mortality and then it is necessary not just to standardize by agebut to analyze the different age groups hence the maingoal in this paper is to study the temporal evolution of thegeographical patterns of ovarian cancer mortality rates infour age groups of women aged years or moremethodsdata sourcethe study presented here covers all ovarian cancer deathscode c56 of the 10th edition of the international classification of diseases for women over years of agein the provinces of spain excluding the autonomouscities of ceuta and melilla recorded throughout theperiod by the spanish statistical officestatistical analysisa bayesian hierarchical spatiotemporal model is used toestimate rates the model is briefly described in whatfollows for better interpretation of resultsspain is divided into s provinces indexed by i s and data are available for t27 time periods corresponding to years labeled as t tfor each age group let nit represent the population at riskfor region i and time period t then conditional on themortality rates rit the number of ovarian cancer deathsoit is assumed to follow a poisson distribution with mean 0ctrandafir bmc public health page of table descriptive statistics of observed cases and crude mortality rates per women disaggregated by age groups provinceand year min minimum q1 first quartile q3 third quartile max maximumobserved casescrude ratesage group[ [ [ [ [ [ [ [ minq1medianmeanq3maxμit nitrit that isoitrit ¼ poissonμit nitritlog μit log nit log ritwhere the lograte is modelled aslog rit α ξi γt δithere α denotes the logarithm of the overall rate ξi andγt are the main spatial and temporal effects respectivelyand δit corresponds to the spacetime interaction effectssince each of these components are supposed to be gaussian markov random fields gmrf the integratednested laplace approximation inla technique hasbeen used for model fitting and inference specifically theleroux car prior distribution has been considered for the spatial random effects and a firstorderrandom walk prior distribution for the temporal randomeffects in addition the four different types of interactionintroduced by knorrheld have been considered forthe spatiotemporal random effects these interactionsallow the spacetime effects to be completely independenttype i interaction structured in time but not in spacetype ii interaction structured in space but not in timetype iii interaction or completely structured in spaceand time type iv interactionall the computations have been done using the interactive web application sstcdapp which can befound at httpsemisstcdappunavarraeslogin thisapplication provides a user interface for the analysis ofspatiotemporal areal count data allowing to fit a widevariety of spacetime models using the inla estimationtechnique in addition the application provides differentmodel selection criteria in this paper the model with the[[[[nemow rept shaed etar edurcyearfig temporal trends by age groups temporal trend of the ovarian cancer mortality crude rates by agegroups 0ctrandafir bmc public health page of lowest value of the deviance information criterion dic has been selected for further details about modelspecification prior distribution of the hyperparametersidentifiability constraints and additional model selectioncriteria see for example adin and the referencesthereinresultsa total of ovarian cancer deaths were registered inthe population of spanish women over years of age during the period since ovarian cancer is mainlyrelated to the onset of menopause the age groups we areconsidering in this paper are [ [ [ and [ a brief summary of observed cases and mortality rates per women by age groups provinceand year is shown in table clear differences areobserved in the mean and median mortality rates amongthe youngest and oldest age groups with values ranging from cases per women up to casesper women approximately respectively figure displays the global temporal trend of crude rates by agegroup here the different behaviour of the age groups iseven more evident a pronounced slope from the lastdecade of the twentieth century to the beginning of thetwentyfirst century is observed in the older age groupsmodel was fitted to smooth spatiotemporal rates ineach age group the interaction considered in the modelwas chosen on the basis of the dic values for each subgroup of ageclass the dic pointed toward a type ivinteraction for age groups [ [ and [ whereas a type ii interaction was selected for the agegroup [ to make the different terms in all themodels comparable a decomposition of the estimated logrates was computed by defining posterior spatial ξi t and spatiotemporal δtemporal γ it patterns see adini γ so that log rit α ξit note thatexpα represents the overall mortality rate for the wholeof spain during the period in order to facilitate interpretation of the results a map of spain showingits provinces is given in fig t δla coruñalugopontevedraorenseasturiasleoncantabriavizcayaguipuzcoaalavanavarrapalenciaburgosriojahuescaleridageronazamoravalladolidsoriazaragozabarcelonasegoviatarragonasalamancaguadalajaraavilamadridcacerestoledocuencabadajozciudad realalbaceteteruelcastellonvalenciaalicantebalearescordobajaenmurciahuelvasevillagranadaalmeriamalagacadizsanta cruz de tenerifepalmaslasfig administrative division of spain map with the administrative division of spain showing provinces source map was generated by the authorsusing the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of figure shows the map with the posterior meanieestimates of provincespecific mortality ratesexpα ξexceedance probabilitiesofrate being greaterthanthe overall spanish rate have also been computedsee fig i posteriorthis provincespecificthe estimated spatial pattern draws attention toasturias as a high ovarian cancer mortality rate provincefor all age groups in the age group [ the highest spatial rates are found in the northwestern provincesasturias lugo and la coruña but also in vizcaya andhuesca over cases per women in age group[ the regions with the highest estimated rates areasturias the balearic islands and valencia with an estimated rate of over cases per women in thethird age group [ the highest rates are located inthe centralnorthern areas with salamanca and asturiasleading the ranking and in the canary islands tenerifeprovince all of them with more than cases per women the oldest age group [ exhibits high rateareas in asturias barcelona gerona and guadalajarawith a rate of over cases per women all ofthese highrate provinces have a rate significantly higherthan the overall spanish rate in their respective age groupssee fig in general northern spain has greater ovarian cancermortality rates compared to the southern regions thelowest rates are found in guipúzcoa for age groups [and [ and in almerÃa for age groups [ and[ the northwestern province of la coruña showsage group [age group [ to to to to to to to to age group [age group [ to to to to to to to to fig provincespecific mortality rates estimates by agegroups posterior mean estimates of provincespecific mortality rates expα ξsource maps were generated by the authors using the library tmap from the r statistical software version no licenses are required touse or publishi 0ctrandafir bmc public health page of age group [age group [[][[[[[][[[[[][[[[age group [age group [[][[[[fig provincespecific posterior exceedance probabilities by agegroups posterior exceedance probabilities of each province in comparison withthe spanish overall rate pexpα ξstatistical software version no licenses are required to use or publish expαo source maps were generated by the authors using the library tmap from the ria surprising behaviour with high rates in the age group[ but one of the lowest rates in the age group [the estimated global temporal pattern expα γ t isvisualized in fig rates seem to have decreased duringthe last few years from to in age group [ but have remained nearly constant in the other threeage groups which experienced a sharp increase in ratesfrom up to the beginning of the twentyfirst centuryapproximatelyfigures and display maps showing the spatiotemporal evolution of ovarian cancer mortality rates foreach spanish province for the period dividedinto intervals of years for age groups [ [ [ and [ respectively specifically these mapsrepresent the posterior mean of rit expα ξi γ t δit the corresponding maps of probabilities showing theprobability that a particular rate in a given province andyear is greater than the spanish rate during that periodare not shown here to conserve space however we haveclassified a province as having a rate significantly greaterthan the spanish rate if this probability is greater than for age group [ we find that the mortality ratefor the period was highest in the northeasternregion with the province of huesca having a significantrate that lasted until in the latter years of the periodstudied only asturias showed a significant rate withinthis age group the percentage of the rates variabilityexplained by the spatiotemporal term was implying that the specific temporal evolution of each provinceis rather high in this age group 0ctrandafir bmc public health page of age group [age group [age group [age group [fig temporal trends estimates by agegroups for the whole of spain posterior mean estimates of yearspecific mortality rates expα γ and credible intervals dotted lines provide the estimated rate in each age group in the whole period in spain expαt to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publish 0ctrandafir bmc public health page of to to to to to to fig mortality rates estimates for age group [ posterior mean estimates of mortality rates rit for age group [ source maps weregenerated by the authors using the library tmap from the r statistical software version no licenses are required to use or publishin age group [ rates exhibit a greater variability among provinces between cases per women and about cases per women thehighest rates occurred in the late 1990s and early 2000smainly in the northern half of spain and in the balearicislands asturias lugo salamanca valladolid huescateruel gerona and the balearic islands show significantlyhigh rates at the end of the periodfor women between and years of age the lowest rates are found at the beginning of the study periodwith significantly low rates the highest significant ratesare located mainly in the northern and western parts ofspain and in the canary and balearic islands in the period the provinces with the lowest rates at the endof the period are gerona and madridwomen over years of age show significantly low ratesbetween again asturias shows the highestmortality rate at the end of the period some provinceslocated in southern spain mainly along the coastlineshow significantly low ratesthe temporal evolution of the rates for some selectedprovinces are plotted in fig the colors used in thebands are associated to the posterior exceedance probabilities of each province at year t in comparison withthe temporal pattern for the whole of spain in that yearnamely prit α γ t o in the case of barcelonathe probability that the mortality rate lies above the spanish rate reaches a maximum between for agegroups [ and [ and between forage groups [ and [ madrid behaves similar tospain in all age groups in asturias the probability is quitehigh for all age groups with a significant rising trend forage group [ from onwards la coruña showssignificantly low rates during the whole study period forthe age group [ whereas this provinces behaviourfor women over years of age is similar to the spanishratediscussionthe ovaries are one of the cancer sites where knownrisk factors are not enough to explain all the cases andthus spatiotemporal analyses provide additional important information allowing for the examination of thespatial temporal and spatiotemporal mortality patternsas the age groups are not equally affected by ovarian cancer mortality it is necessary not just to standardize by age but also to analyze the different agegroupsresults show large differences in mortality among theage groups with higher mortality rates in the older agegroups indeed the last age groups women of more than years of age double the rate of women between and more than cases in the last age groups vs casesper women on average in age group [ thiscan be explained by poor survival rates of gynaecologicalcancers in the elderly which in turn is influenced by late 0ctrandafir bmc public health page of barcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñabarcelonamadridasturiasla coruñafig temporal evolution of ovarian cancer mortality rates estimates for some selected provinces barcelona madrid asturias and la coruñatemporal evolutions of the posterior mean estimates of mortality rates rit for some selected provinces and twosided credible intervals for agegroups [ first row [ second row [ third row and [ fourth row the colors used in the bands are associated to theposterior exceedance probabilities of each province in time t in comparison with the temporal pattern of spain that is prit expα γ orepresented with a red line in the graphstdiagnoses and the failure of treatments due to comorbidity[ ]the global temporal trends by age group reveal thatthe evolution of ovarian cancer over the whole of spainhas remained nearly constant since the early 2000s particularly for women aged years or more after a sharpincrease during the period approximatelythe stabilization of the rates may be due to an increasedconcern among women regarding their personal health inparticular among older women that led them to be testedmore frequently access to better information via massmedia and the internet and the increasing effectiveness of 0ctrandafir bmc public health page of cancer treatments in the nineties access to informationvia the internet was more limited and health care was possibly less advanced which could explain in part the sharpincrease in mortality rates in the first half of the periodmortality in women between and years of age showsa slight decrease since until nearly althoughthis decrease does not seem to be significant with respectto the average mortality in this age group it seems thatin fact during the last two years of the study period ratesare starting to rise slightly but once again this trend is notsignificant as yetthe global geographical patterns show in general thatthe north has higher rates than the south a situation similar to that observed overall in europe the variabilityobserved in all age groups between northern and southernspain remains unknown although in general women inthe south are prone to marry earlier and have more children on average interestingly asturias the provincewith the highest rates in all age groups is one of thespanish provinces with the lowest average number of children the differences observed between the north and thesouth could also be explained taking into account the relationship between exposure to sunlight provitamin dand ovarian cancer although spain is in general a sunnycountry there is a great variability between the northand south in terms of average daily hours of sunlight forexample bilbao in the north receives about hoursof sunlight per year while sevilla in the south receives thus the observed increasing trend with latitude might be at least partly explained by a cumulativeexposure to sunlightheterogeneity among the provinces regarding ovariancancer mortality rates can be elucidated at least in partby a heterogeneous distribution of other risk factorsthere are differences among provinces in the age at whichwomen have their first period menarche the averageage of first childbirth and the total number of children[] the average fecundity rate in spain has beenmarkedly declining in particular the areas registering thelowest fertility rates were the basque country asturiasnavarre and aragón [ ] we should also point outthat the age of first birthing is closely related to socioeconomic development and is steadily increasing navarreand the basque country are the regions of spain wherewomen delayed childbearing the longest additionally hormonal replacement therapy has proven to have animportant influence on the appearance of ovarian cancerin postmenopausal women [] however its use inspain has been very limited another risk factor is the presence of a family history ofthe disease hereditary ovarian cancer syndrome presenting a mutation in brca genes is important between of ovarian cancer cases are linked to bcra mutation women who are carriers of the bcra1 mutationhave a chance of suffering ovarian cancer before age in spain the accumulated risk of developing ovarian cancer before age has been estimated at ci in carriers of a mutation in bcra1 and ci in carriers of a mutation in brca2 the prevalence of brca1 and brca2 mutation in spainis heterogeneous and varies according to geographical origin moreover blay showed that the brca1 andbrca2 spectrum of mutations in asturias was largely different from other areas of spain this could also explainin part the high mortality rates found in this provincediez studying a large group of spanish patientsshowed that there is only a slight difference betweenthe percentages of deleterious mutations in brca1 andbrca2 genes and respectively however somevariation due to geographic origin is present with a higherproportion of brca1 in families from the northwesternpart of spain according to vega the differencesfound in galicia could be due to founder effectsovarian cancer is also linked to lifestyle habits tobaccoand alcohol consumption the smoking habit is a riskfactor for epithelial ovarian cancer with an odds ratio of ci [ ] differences in alcoholand tobacco consumption can be found among spanishprovinces all in all to better understand the etiology of the disease and to better determine the effect of risk factors onthe spanish female population it would have been helpful to have the medical and workplace histories of all thewomen participating in this study this is the main limitation of the current work on the other hand as thereis a lack of scientific studies analyzing the associationbetween ovarian cancer mortality rates and risk factorsin the domains analyzed here age groups provinces andyears spatiotemporal analyses by age groups are essential to discover inequalities in ovarian cancer mortality todetect provinces with high risks in each age group and tokeep track of how the rates are evolving with timesdifferences in ovarian cancer mortality exist amongthe spanish provinces years and age groups as theexact causes of ovarian cancer remain unknown spatiotemporal analyses by age groups are very useful to look forpotential risk factors associated to the observed geographical patterns and to allocate funds among spanish regionsfor future clinical and epidemiological practice we recommend to followup women over years as the mortality rates remain constant since highmortalityprovinces should also be monitored to look more closelyfor specific risk factors some risk factors for ovarian cancer like getting old or having a family history cannot bechanged however women may slightly decrease their riskby avoiding other risk factors for example maintaining 0ctrandafir bmc public health page of a healthy weight avoiding tobacco and alcohol consumption or not receiving hormone replacement therapy aftermenopauseabbreviationsaei spanish research agency brca breast cancer dic deviance informationcriterion ue european union inla integrated nested laplaceapproximations gmrf gaussian random markov field sstcdapp spatialand spatiotemporal count data application uk united kingdomacknowledgementswe acknowledge the spanish statistical institute for providing the dataauthors contributionsstudy conception and design mdu pct acquisition of the data mdu aaanalysis of the data mdu pct aa interpretation of the data mdu aa pctwriting the pct mdu critical revision of the all authorsapproved the final manuscript and the decision to submit the manuscriptfundingthis research has been supported by the spanish ministry of science andinnovation project mtm 201782553r aeifeder ue the content of thispaper is solely the responsibility of the authors and does not represent theofficial views of the spanish ministry of science and innovationavailability of data and materialdata have been provided by the spanish statistical institute at municipalitylevel under a contract and aggregated later up on reasonable request thecorresponding author will make the datasets available mortality data fromcancer and other causes from by sex and province is available in theinteractive epidemiological information system ariadna httpariadnacneisciiiesevindexhtml of the spanish national center for epidemiologyethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesbrüggmann d pulch k klingelhöfer d pearce c groneberg d ovariancancer density equalizing mapping of the global research architectureint j health geogr bray f ferlay j soerjomataram i siegel r torre l jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin national institute for health and care excellence nice ovarian cancerthe recognition and initial management of ovarian cancer httpswwwniceukguidancecg122resourcesovariancancerrecognitionandinitialmanagementpdf35109446543557 accessed jan dos santos silva i beral v socioeconomic differences in reproductivebehaviour iarc sci publ ferlay j soerjomataram i ervik m dikshit r eser s mathers c rebelom parkin dm forman d bray f globocan v10 cancer incidenceand mortality worldwide iarc cancer base no [internet] lyoninternational agency for research on cancer available from httpglobocaniarcfr accessed jan ferlay j steliarovafoucher e lortettieulent j rosso s coebergh jcomber hea cancer incidence and mortality patterns in europeestimates for countries in eur j cancer forman d bray f brewster dh gombe mbalawa c kohler b piñerosm steliarovafoucher e swaminathan r ferlay j cancer incidence infive continents vol x iarc scientific publication no available from httppublicationsiarcfr_publicationsmediadownload3743e886b2754a75a0f70e190e9b56e5346047319c17pdfaccessed jan ledermann j raja f fotopoulou c gonzalezmartin a colombo n cs european society for medical oncology esmo guidelines workinggroup newly diagnosed and relapsed epithelial ovarian carcinomaesmo clinical practice guidelines for diagnosis treatment and followupann oncol coleman m gatta g verdecchia a esteve j sant m storm h allemanic ciccolallo l santaquilani m berrino f eurocare3 summary cancersurvival in europe at the end of the 20th century ann oncol bouchardy c rapiti e blagojevic s vlastos a vlastos g older femalecancer patients importance causes and consequences of undertreatment j clin oncol meindl a ditsch n kast k rhiem k schmutzler r hereditary breast andovarian cancer new genes new treatments new concepts deutschesärzteblatt | 0 |
" 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 | 0 |
"The patient had received two successive courses of first-line chemotherapy without tumor response. Tumor cells were negative for wild-type of epidermal growth factor receptor/K-RAS variants; thus she was not eligible for tyrosine kinase inhibitor therapy. Unfortunately increased levels of interleukin-6 and carcinoembryonic antigen and computed tomography scan results indicated cancer progression. Once crizotinib was approved by the China Food and Drug Administration and the ALK gene translocation was identified in tumor cells by fluorescent in situ hybridization the patient commenced treatment with crizotinib. Remarkably the tumor response to crizotinib was classified as partial response after only 26 days of treatment commencement. The partial response status has been maintained to date (23 weeks). Conclusion Considering this remarkable response to crizotinib we can safely conclude that patients with squamous cell lung cancer should have the option of undergoing ALK testing to determine if there is indication for crizotinib treatment even after they have failed chemotherapy. ALK Crizotinib Squamous cell lung cancer Chemotherapy Background Treatment of EML4-ALK fusion-positive non-small-cell lung cancer (NSCLC) with the anaplastic lymphoma kinase (ALK)-targeted agent crizotinib offers significant improvement of clinical outcomes [1]. Herein we report the successful case of a patient with squamous cell lung cancer and ALK gene translocation that experienced a remarkable response to crizotinib treatment after two courses of failed chemotherapy. Case presentation A 55-year-old woman presented in May 2013 with cough sputum and annihilation after activities of daily living for more than 20 days. She had no history of smoking or alcoholism but had undergone total hysterectomy because of hysteromyoma in 2010. She was diagnosed with hypertension three years earlier. On physical examination an enlarged right cervical lymph node was palpated. Chest computed tomography (CT) scan (Figure 1A) indicated the presence of a mass in the lower lobe of the right lung and mediastinal lymph node enlargement. The patient was then accepted and treated by the Department of Respiration for lung cancer stage IV with cervical lymph node metastasis (T4N3M1). Chest computed tomography (CT) scans. Before the first chemotherapy treatment (May 2013) (A). After the second course of chemotherapy (B). After 26 days (C) and 11 weeks (D) of crizotinib treatment. The whole enlarged right cervical lymph node was resected and followed by biopsy and histologic examination. Hematoxylin and eosin (H & E) staining showed the typical morphology of squamous cell carcinoma cells (Figure 2A and B). Immunohistochemistry (IHC) analysis demonstrated that tumor cells were positive for cytokeratin (CK) 5/6 (Figure 2C) and P63 (Figure 2D) and negative for CK7 CK20 TTF-1 and Napsin A. The positive rate of Ki-67 was 30%. Altogether these results confirmed the diagnosis of metastatic squamous cell carcinoma. The patient requested treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) because dozens of patients with squamous cell carcinoma and EGFR mutations responded well to TKIs at our institute. Amplification refractory mutation system (ARMS) was used to assess EGFR and K-RAS gene profiles to determine the presence of mutated variants. However evidence of a normal genotype excluded the patient from receiving EGFR-TKI treatment. Right cervical lymph node analysis. H & E staining (A and B). IHC staining of CK5/6 (C) P63 (D) and ALK (1A4) (E). ALK gene translocation (FISH arrows: split-apart signals for ALK gene translocation) (F). As first-line chemotherapy the patient was initially administered 135 mg/m2 (210 mg) of paclitaxel and 80 mg/m2 (120 mg) of nedaplatin. During treatment the patients condition did not seem to improve; thus after a 20-day treatment a second round of chemotherapy was administered with 75 mg/m2 (120 mg) of docetaxel and 80 mg/m2 (120 mg) of nedaplatin. Unfortunately increasing levels of interleukin-6 (IL-6) (25.41 vs 16.03 pg/mL) and carcinoembryonic antigen (CEA) (23.43 vs 7.13 ng/mL) indicated cancer progression which was confirmed by the presence of multiple metastases in both lungs on CT scan images (Figure 1B). Although not initially indicated the patient was then administered oral treatment with the EGFR-TKI erlotinib (1-month trial). However this treatment showed no efficacy. After the use of crizotinib was approved by the China Food and Drug Administration (CFDA) in July 2013 the patient underwent ALK testing. IHC staining (Clone 1A4 Origene 1:200) showed tumor cell positivity for ALK protein (Figure 2E). Then ALK break-apart fluorescent in situ hybridization (FISH) was performed on 4-?m formalin-fixed paraffin-embedded tissue sections. Slides were hybridized with the LSI ALK Break Apart Rearrangement Probe (Vysis Abbott Molecular Des Plaines IL USA) and read on an epifluorescence microscope (BX41 Olympus Tokyo Japan). The lung cancer cell line NCI-H2228 (American Type Culture Collection-ATCC) was used as positive control. At least 50 tumor cell nuclei were analyzed and at least 15% of interpretable tumor cells harboring break-apart signals were used as the cutoff value [2]. As a result the presence of ALK gene translocation was confirmed (Figure 2F). The patient underwent crizotinib treatment (250 mg/bid orally) for 26 days. After this period symptoms including neck constriction and cough were improved. Chest CT scan images demonstrated decrease in tumor size and metastases. According to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (version 1.1) such tumor response to crizotinib was classified as partial response (PR) (Figure 1C). Follow-up chest CT scan performed 11 weeks after the beginning of the treatment revealed a dramatic reduction in tumor size and mediastinal lymph node with nearly no presence of metastases in both lungs (Figure 1D). IL-6 (3.34 vs 25.41 pg/mL) and CEA (1.84 vs 23.43 ng/mL) levels were also reduced 23 weeks after the beginning of the therapy which demonstrated continuous partial response. Discussion According to the National Comprehensive Cancer Network guidelines ALK testing is not routinely performed in patients with squamous cell lung cancer. Therefore this patient was not tested for ALK until crizotinib was approved for marketing by the CFDA (June 23 2013). Unfortunately neither chemotherapy nor EGFR-TKI treatment produced effective tumor response in this patient. ALK gene translocations have been previously detected in patients with lung adenocarcinoma and light or non smoking history [34]. To date two studies have previously reported cases of patients with mixed carcinoma and squamous cell component harboring ALK rearrangements [56] but these studies did not describe specific therapy or diagnostic procedures. Another report showed that a patient with squamous cell lung cancer harboring c-MET amplification had partially responded to crizotinib [7]. Herein we report the case of a non-smoking woman with squamous cell lung cancer and ALK gene rearrangement who experienced remarkable response to crizotinib treatment after failed chemotherapy. In concordance with other studies on patients with lung adenocarcinoma treated with crizotinib [89] this patient has remained in remission (PR). Most importantly such remarkable response was obtained after two courses of failed chemotherapy. Conclusion Despite the low reconstruction rate of ALK gene if applicable patients with squamous cell lung cancer should have the option of undergoing ALK testing and receiving crizotinib treatment. ALK testing and subsequent targeted therapy could be an effective option for patients with non-small cell lung cancer who present progression following chemotherapy radiotherapy or any other treatment. Consent The patient provided written consent for publication of this case report. Abbreviations EML4: Echinodermmicro tubule associated protein like 4; ALK: Anaplastic lymphoma kinase; NSCLC: Non-small-cell lung cancer; CT" | 1 |
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 | 2 |
" Concentrations of HER2 and EGFR extracellular domains were determined by enzyme?linked immunosorbent assay. Patient?Reported Outcomes PROs of HRQoL disease symptoms specific to lung cancer and side effects of treatment were assessed using the 30?question European anisation for Research and Treatment of Cancer Quality of Life Questionnaire core module (EORTC QLQ?C30)13 which includes functional symptom side effects and global health status scales and the 13?item Lung Cancer symptom?specific module (QLQ?LC13).14 The impact of dacomitinib on patients' skin condition was assessed using the 10?item Dermatology Life Quality Index (DLQI) questionnaire. Statistical Design and Analyses The trial used a Fleming single?stage design for each patient population (adenocarcinoma and nonadenocarcinoma respectively). The primary objective of this study was to test the null hypothesis (H0) at the 0.05 significance level with 80% power that the objective response rate (ORR) in patients with adenocarcinoma did not exceed 5%. At the end of the study if there were at least 6 objective responders in 44 response?evaluable patients then the null hypothesis would be rejected demonstrating that treatment with dacomitinib is associated with a true response rate that exceeds 5%. A secondary objective was to test the H0 at the 0.05 significance level with 80% power that the ORR in patients with nonadenocarcinoma did not exceed 1%. At least 2 objective responders among 22 response?evaluable patients were required to reject the null hypothesis and conclude that treatment with dacomitinib demonstrates a true response rate that exceeds 1%. Target enrollment of 49 and 25 patients with adenocarcinoma and nonadenocarcinoma respectively was required and accounted for a rate of nonevaluability for response of up to 10%. Baseline characteristics PFS PFS6M OS OS6M and OS12M were evaluated in the intent?to?treat population safety in the as?treated population and response was assessed in response?evaluable patients. Results Patient Characteristics and Disposition Sixty?six patients were enrolled between April 2008 and November 2009 50 with adenocarcinoma and 16 with nonadenocarcinoma. Patient disposition is shown in Fig. 1. Enrollment of the nonadenocarcinoma arm was closed prior to reaching the planned target of 25 due to few nonadenocarcinoma patients identified with prior erlotinib treatment. Patient characteristics are summarized in . The majority of patients had received 2 or 3 prior treatment regimens (n?=?26 [39%] each). In addition to erlotinib prior EGFR?directed therapies comprised gefitinib (n?=?4) cetuximab (n?=?3) and neratinib (n?=?1). Fifty?five percent of the enrolled population were current or former smokers. Wild?type KRAS NSCLC was either directly confirmed (n?=?54) or assumed from a known EGFR mutation (n?=?12; EGFR mutation status was known for a total of 26 patients). Mutation and gene amplification data were collected from EGFR and HER2 according to availability of sufficient tissue for analysis (). Six patients had EGFR T790M resistance mutation identified after treatment with erlotinib (Supporting ; see online supporting information). T790M status was unknown in 54 patients who had biopsies taken prior to progression on erlotinib. Overall 74% of patients started dacomitinib within 3 months of discontinuing erlotinib. Of the 26 patients who had EGFR?mutant tumors at baseline the interval from discontinuing erlotinib to starting dacomitinib ranged from 15 to 544 days with 69% starting dacomitinib within 3 months of discontinuing erlotinib. Study flow diagram shows patient disposition and analysis populations. image Patient Baseline Characteristics Characteristic Adenocarcinoma (n?=?50) Nonadenocarcinoma (n?=?16)e Total (N?=?66) Median age years (range) 60 (37?79) 61 (50?84) 60 (37?84) Sex n (%) Male 15 (30.0) 14 (87.5) 29 (43.9) Female 35 (70.0) 2 (12.5) 37 (56.1) Race n (%) Caucasian 35 (70.0) 11 (68.8) 46 (69.7) Asian 12 (24.0) 2 (12.5) 14 (21.2) Other 3 (6.0) 3 (18.8) 6 (9.1) Smoking status n (%) Never?smoker 27 (54.0) 3 (18.8) 30 (45.5) Current smoker 1 (2.0) 2 (12.5) 3 (4.5) Exsmoker 22 (44.0) 11 (68.8) 33 (50.0) ECOG performance status n (%) 0 18 (36.0) 5 (31.3) 23 (34.8) 1 27 (54.0) 8 (50.0) 35 (53.0) 2 5 (10.0) 3 (18.8) 8 (12.1) Prior treatment regimens n (%) 1 regimena 4 (8.0) 1 (6.3) 5 (7.6) 2 regimens 18 (36.0) 8 (50.0) 26 (39.4) 3 regimens 19 (38.0) 7 (43.8) 26 (39.4) >3 regimensb 9 (18.0) 0 9 (13.6) Mutational status n (%) KRAS WT or EGFR sensitizing mutation 50 (100.0) 16 (100.0) 66 (100.0) KRAS WT 39 (78.0) 15 (93.8) 54 (81.8) KRAS unknown 11 (22.0) 1 (6.3) 12 (18.2) EGFR WT 10 (20.0) 13 (81.3) 23 (34.8) EGFR sensitizing mutation 24 (48.0) 2 (12.5) 26 (39.4) Exon 19 or 21 18 (75.0) 1 (50.0) 19 (73.1) Other 6 (25.0) 1 (50.0) 7 (26.9) EGFR unknown 16 (32.0) 1 (6.3) 17 (25.8) EGFR T790M secondary resistance mutation 6 (12.0)c 0 6 (9.1)c T790M unknown 39 (78.0) 15 (93.8) 54 (81.8) HER2 mutation 0 0 0 HER2 WT 29 (58.0) 13 (81.3) 42 (63.6) HER2 mutation unknown 21 (42.0) 3 (18.8) 24 (36.4) HER2 amplification positive 2 (4.0) 1 (6.3) 3 (4.5) HER2 amplification negative 22 (44.0) 11 (68.8) 33 (50.0) HER2 amplification unknown 26 (52.0) 4 (25.0) 30 (45.5) Prior EGFR?directed treatment n (%) Erlotinib 50 (100.0) 16 (100.0) 66 (100.0) Gefitinib 3 (6.0) 1 (6.3) 4 (6.1) Neratinibd 1 (2.0) 0 1 (1.5) Cetuximabd 2 (4.0) 1 (6.3) 3 (4.5) Response to immediately prior EGFR?directed treatment n (%) CR 1 (2.0) 0 1 (1.5) PR 13 (26.0) 0 13 (19.7) SD 21 (42.0) 5 (31.3) 26 (39.4) PD 9 (18.0) 10 (62.5) 19 (28.8) Unknown 6 (12.0) 1 (6.25) 7 (10.6) Abbreviations: CR complete response; ECOG Eastern Cooperative Oncology Group; EGFR epidermal growth factor receptor; PD progressive disease; PR partial response; SD stable disease. a Four patients in Arm A and 1 patient in Arm B had prior systemic treatment and prior erlotinib entered as 1 regimen. b Patients with >3 prior regimens includes patients with neoadjuvant and/or adjuvant therapies and/or investigational treatment regimen(s). c T790M status was derived from archival biopsies for 4 patients and fresh baseline biopsies for 2 patients. Three of the 4 patients with T790M status ascertained from an archival biopsy initiated dacomitinib more than 90 days after discontinuing from erlotinib. d Patients previously treated with investigational EGFR?directed therapies were eligible to participate in the study and did not represent protocol deviations provided the study sponsor provided written agreement. e Squamous n?=?12. Efficacy Best Overall Response In the overall population the ORR for response?evaluable patients was 5.2% (3 partial responses [PRs] of durations 1224 and 66 weeks). The ORR for patients with adenocarcinoma was 4.8% (2 PRs; 1?sided P?=?.372). For patients with nonadenocarcinoma the ORR was 6.3% (1 PR). The 25 response?evaluable patients with EGFR mutation?positive tumors (from both arms) achieved an ORR of 8% (2 PRs) and 17 (68%) achieved a BOR of stable disease (SD)??6 weeks (). Further details of the patients with PRs are presented in Supporting . Six patients had known EGFR T790M; of these 3 had SD??6 weeks (912 and 12 weeks respectively) and 3 had progressive disease (PD). Summary of Best Overall Response Per RECIST by Investigator Assessment PFS and OS Adenocarcinoma Arm A Nonadenocarcinoma Arm B Total Overall Patient Population No. of patients evaluable n 42 16 58 Objective response (CR?+?PR) n (%) [95% exact CI]a 2 (5) [1?16] 1 (6) [0?30] 3 (5) [1?14] P valueb 0.372 0.149 Clinical benefit (CR?+?PR?+?SD???24 weeks) n (%) [95% exact CI]a 10 (24) [12?40] 3 (19) [4?46] 13 (22.4) [13?35] Duration of response weeks 24c 66d 12e NA No. of patients enrolled n 50 16 66 No. of PFS eventsf n (%) 40 (80) 14 (88) 54 (82) PFS weeks [95% CI] 12 [8?20] 11 [6?18] 12 [9?19] PFS6M % [95% CI] 24 [12?38] 8 [1?30] 20 [11?32] No. of deaths n (%) 33 (66) 14 (88) 47 (71) OS weeks [95% CI] 45 [29?73] 27 [10?36] 37 [28?57] OS6M % [95% CI] 72 [57?82] 50 [25?71] 66 [53?76] OS12M% [95% CI] 46 [31?60] 22 [6?45] 40 [28?52] Patients With EGFR?Mutant Tumors No. of patients evaluable n 23 2 25 Objective response (CR?+?PR) n (%) [95% exact CI]a 2g (9) [1?28] 0 2g (8) [1?26] Clinical benefit (CR?+?PR?+?SD ?24 weeks) n (%) [95% exact CI]a 7 (30) [13?53] 0 7 (28) [12?49] No. of patients n 24 2 26 No. of PFS eventsf n (%) 19 (79) 2 (100) 21 (81) PFS weeks [95% CI] 18 [6?30] 21 [17?24] 18 [9?29] PFS6M % [95% CI] 36 [16?57] 32 [14?52] No. of deaths n (%) 17 (71) 1 (50) 18 (69) OS weeks [95% CI] 59 [42?76] [24 ] 57 [42?75] OS6M % [95% CI] 83 [62?93] 50 [1?91] 81 [60?92] OS12M % [95% CI] 61 [38?77] 59 [37?76] Patients With EGFR Wild?Type Tumors No. of patients evaluable n 7 13 20 Objective response (CR?+?PR) n (%) [95% exact CI]a 0 [0?41] 1h (8) [0?36] 1h (5) [0?25] Clinical benefit (CR?+?PR?+?SD ?24 weeks) n (%) [95% exact CI]a 2 (29) [4?71] 2 (15) [2?45] 4 (20) [6?44] No. of patients n 10 13 23 No. of PFS eventsf n (%) 8 (80) 11 (85) 19 (83) PFS weeks [95% CI] 8 [2?25] 9 [5?18] 8 [5?18] PFS probability at 6 months [95% CI] 14 [1?45] 6 [0?25] No. of deaths n (%) 6 (60) 12 (92) 18 (78) OS weeks 36 [2 ] 26 [8?36] 26 [10?47] Survival probability at 6 months [95% CI] 50 [18?75] 46 [19?70] 48 [27?66] Survival probability at 12 months [95% CI] 40 [12?67] 23 [6?48] 30 [14?49] A total of 12 patients were censored for PFS 10 in arm A and 2 in arm B (Arm A: 7 patients discontinued treatment before the first on?study assessment [4 because of AE and 3 because of global deterioration]; 1 had inadequate baseline assessment; 1 was still ongoing with study treatment; 1 was no longer willing to participate and had no PD documented. Arm B: 1 patient discontinued because of AE and had SD; 1 was no longer willing to participate and had no PD documented). a Using exact method based on binomial distribution. b For arm A: one?sided P?value for the hypothesis testing H0: ORR was ?5% using exact binomial test. For arm B: one?sided P?value for the hypothesis testing H0: ORR was ?1% using exact binomial test. c Confirmed EGFR mutation. d EGFR status unknown. e Confirmed EGFR wild type. f Objective progression or death. g One patient had E746_A750del5 exon 19; 1 patient had G719C exon 18 and S768I exon 20. h This patient had HER2 amplification and mutation. Abbreviations: CI confidence interval; CR complete response; H0 null hypothesis; NA not applicable; ORR objective response rate; PFS progression?free survival; PR partial response; SD stable disease. Of the 36 patients with SD as BOR (median duration 15 weeks) 10 patients (28%) had prolonged clinical benefit (SD??6 months); of these 5 patients had EGFR?mutant tumors 3 had EGFR wild?type tumors and 2 had tumors of unknown EGFR status. Of 56 patients with both baseline and??1 postbaseline tumor measurement 26 (46%) had some degree of tumor shrinkage (Fig. 2). Among patients with tumor shrinkage 6 (23%) and 12 (46%) were confirmed as having EGFR?WT tumors and EGFR?mutant tumors respectively; 8 (31%) had tumors of unknown EGFR status. Tumor shrinkage was noted in 1 patient with an EGFR T790M tumor; all other EGFR T790M tumors increased in size. Figure 2 Maximum percentage change is shown in target lesions per RECIST (Response Evaluation Criteria in Solid Tumors) in 56 patients with both a baseline and at least one on?study measurement reflected in the database. Six patients had no change in the size of their tumor; of these 1 had EGFR mutation 1 had EGFR of unknown status and 4 had EGFR wild?type tumors. image Progression?Free Survival Overall median PFS (n?=?66) was 12 weeks with 54 (82%) patients reaching PFS events and similar values in the adenocarcinoma and nonadenocarcinoma populations. Median PFS in the adenocarcinoma group was 12 weeks based on 50 patients. In the nonadenocarcinoma group median PFS was 11 weeks (Fig. 3A). Median PFS in patients with EGFR mutation?positive tumors was 18 weeks based on 26 patients with 21 (81%) achieving PFS events; this median was longer than that seen in the overall population. The 6 patients with documented T790M had a median PFS of 7 weeks which was similar to that of patients with EGFR wild?type tumors (8 weeks). Figure 3 Kaplan?Meier curves show (A) progression?free survival and (B) overall survival by arm (all patients). CI indicates confidence interval. image Overall Survival At the time of data cutoff 47 patients (71%) had died and median OS was 37 weeks in the overall population 45 weeks in patients with adenocarcinoma and 27 weeks in patients with nonadenocarcinoma (Fig. 3B). Of the 26 patients with EGFR mutation?positive tumors (both arms) median OS was 57 weeks OS6M was 81% and OS12M was 59%. Safety and Tolerability The majority of treatment?related AEs were of grade 1 or 2 severity (Table 3) and were manageable with standard supportive care. Common events included diarrhea (85%) dermatitis acneiform (68%) dry skin (38%) fatigue (38%) exfoliative rash (24%) stomatitis (24%) decreased appetite (23%) and pruritus (23%). One patient experienced treatment?related grade 4 AEs of dyspnea and pulmonary embolism considered by the investigator to be possibly related to study drug; 18 patients (27%) experienced treatment?related AEs with a maximum severity of grade 3. The majority of patients (n?=?44 67%) did not require a dose reduction and interruption of daily dosing was seen in 33% for evaluation and management of AEs. Of the 22 patients who did require dose reduction 17 patients had 1 dose reduction and 5 had 2 dose reductions. AEs resulting in dose modification were predominantly dermatologic or gastrointestinal. Six patients permanently discontinued dacomitinib due to treatment?related AEs which included grade 4 dyspnea (day 8) and grade 4 pulmonary embolism (day 9) (both in a single patient); grade 3 fatigue (day 14); grade 3 exfoliative rash (day 134); grade 2 allergic dermatitis (day 3); grade 2 fatigue (day 85); and grade 1 fatigue (day 43). Twelve deaths occurred within 28 days following the last dose of dacomitinib and were reported as serious AEs; none was considered to be treatment?related. Table 3 Treatment?Related Adverse Events Occurring in ?10% of Patients in the Overall Population (N?=?66) and Hematologic Laboratory Values by Maximum CTCAE Grade (All Cycles; N?=?66) Adverse Event Grade 1/2n (%) Grade 3n (%) Total n (%) Any adverse events 46 (69.7) 18 (27.3)a 65 (98.5) Diarrhea 48 (72.7) 8 (12.1) 56 (84.8) Dermatitis acneiform 41 (62.1) 4 (6.1) 45 (68.2) Dry skin 25 (37.9) 0 25 (37.9) Fatigue 23 (34.8) 2 (3.0) 25 (37.9) Exfoliative rash 14 (21.2) 2 (3.0) 16 (24.2) Stomatitis 15 (22.7) 1 (1.5) 16 (24.2) Decreased appetite 15 (22.7) 0 15 (22.7) Pruritus 12 (18.2) 3 (4.5) 15 (22.7) Nausea 13 (19.7) 0 13 (19.7) Vomiting 8 (12.1) 1 (1.5) 9 (13.6) Aspartate aminotransferase increased 8 (12.1) 0 8 (12.1) Mucosal inflammation 7 (10.6) 0 7 (10.6) Hematologic Laboratory Values Grade 1/2n (%) Grade 3n (%) Total n (%) Hemoglobin 36 (54.5) 1 (1.5) 50 (75.8) Lymphopenia 10 (15.2) 12 (18.2)b 40 (60.6) Neutropenia 2 (3.0) 1 (1.5) 4 (6.1) Thrombocytopenia 4 (6.1) 1 (1.5)c 5 (7.6) Leukopenia 10 (15.2) 0 11 (16.7) a Includes two grade 4 events (dyspnea and pulmonary embolism) both experienced by the same patient. b Includes 2 patients with grade 4 events. c Grade 4. Patient?Reported Outcomes Completion rates for the EORTC QLQ?C30/?LC13 and DLQI questionnaires were high throughout the study (generally?>90% of patients answered at least one question). Patients with radiographic disease control reported improvement in lung cancer symptoms of dyspnea cough pain in chest and pain in arm/shoulder relative to baseline scores first observed after 3 weeks on therapy (Supporting Fig. 1A). Diarrhea was the most commonly reported class?related AE; diarrhea peaked at cycle 3 day 1 (week 6) and remained stable over time (Supporting Fig. 1B). With a score of 0?=?no symptoms and 100?=?most symptoms patients on dacomitinib reported scores that were at the midpoint in the range at their worst. The impact of dacomitinib on PRO for NSCLC symptoms and dermatologic toxicity has been previously presented and will be subsequently reported in full (Campbell AK et al; unpublished data). Pharmacokinetics PK parameters (overall and by histology) following a single dose (cycle 1 day 1) and mean Ctrough values after multiple doses for dose?compliant patients (Supporting Table 3) were consistent with those previously reported.5 Pharmacodynamics Soluble HER2 and EGFR levels were slightly decreased on day 1 of most cycles compared with baseline for most patients. One patient with nonadenocarcinoma demonstrating HER2 amplification had elevated baseline soluble HER2 that significantly declined to population normal baseline levels upon treatment with dacomitinib. This patient's tumor also demonstrated a PR.16 Discussion In this phase 2 trial dacomitinib demonstrated an overall response rate of 5% but the primary endpoint of this study was not met. Three PRs were observed 2 in patients with EGFR mutation?positive tumors and 1 in a patient whose tumor was EGFR wild?type with HER2 amplification.16 In contrast patients with known EGFR T790M did not respond to dacomitinib therapy despite efficacy in preclinical models. These observations could be due to the presence of concurrent drug resistance mechanisms (such as MET amplification)18 or to the inability of dacomitinib to fully inhibit EGFR in tumors harboring EGFR T790M at the doses currently under clinical investigation.5 Strategies to improve EGFR inhibition in EGFR T790M cancers include the combination of irreversible EGFR inhibitors with the EGFR?directed antibody cetuximab (as reported for afatinib plus cetuximab)19; the development of more potent and specific inhibitors of EGFR T790M2021; and the use of intermittent but high doses of existing irreversible EGFR inhibitors.18 In contrast where resistance is mediated by compensatory signaling pathways or tumors harbor more than one concomitant drug resistance mechanism combination strategies with targeted agents in appropriately selected patients will be necessary to treat such cancers (eg inhibition of the MET pathway). In the absence of a known oncogene addiction patients with wild?type EGFR may still benefit from EGFR?directed therapy in the absence of a RECIST?defined radiographic response; endpoints such as PFS and patient report of HRQoL and symptom relief have become increasingly important in a noncurative setting.22 This is demonstrated in the BR21 trial of erlotinib versus placebo where the ORR was low and yet was associated with improvements versus placebo in OS and NSCLC symptoms.10 In the current study in refractory NSCLC 10 of 36 patients with SD as BOR derived prolonged clinical benefit (SD???6 months) with dacomitinib; patients also reported a rapid onset of improvement in key lung cancer symptoms with symptomatic improvements remaining durable over the course of therapy. Common AEs were typically gastrointestinal or dermatologic and consistent with targeting EGFR.24 By patient report both gastrointestinal and dermatologic symptoms peaked early in treatment and stabilized or improved over time (Campbell AK et al; unpublished data). The benefits seen in this study may reflect dacomitinib's broader mode of action in targeting all kinase?active HER family members irreversible binding to the tyrosine kinase domain retreatment in some of those patients with an EGFR?driven tumor following a period off treatment after a prior selective EGFR TKI or other as yet to be determined factors. Data from this and other phase 1 and 2 studies in post?EGFR TKI settings5 and from a head?to?head trial comparing dacomitinib with erlotinib in the second?line setting8 suggest that dacomitinib has clinically relevant activity in patients with NSCLC who do not harbor KRAS mutations. However in the absence of a control arm it remains unclear if this degree of benefit seen here could be due to patient selection or favorable prognostic factors. A phase 3 trial is underway to determine the efficacy and safety of dacomitinib compared with erlotinib in patients with KRAS wild?type NSCLC for whom first?line chemotherapy has failed (ARCHER 1009; ClinicalTrials.gov identifier NCT01360554). FUNDING SUPPORT This study was sponsored by Pfizer Inc. CONFLICT OF INTEREST DISCLOSURE Drs. Ruiz?Garcia Liang Taylor Gernhardt and O'Connell are employees of Pfizer and own Pfizer stock. Stephen Letrent and an immediate family member are employees of Pfizer and own Pfizer stock. Dr. Reckamp received research funding from Pfizer. Dr. Camidge served Pfizer in an advisory role. Dr. Engelman received honoraria from Genentech/Roche and received research funding from Novartis. He also received remuneration from Pfizer for use of cell lines for which he is a coinventor and has an EGFR/MET patent that has been licensed by Ventana and owned by Roche (no compensation to date). Dr. Koczywas received honoraria from Pfizer and Genentech. Dr. Gadgeel received honoraria from Pfizer. Alicyn K. Campbell and an immediate family member were previously employed by Pfizer (neither hold current employment with Pfizer). Dr. Campbell is currently employed by Genentech a member of the Roche Group. Dr. Jänne has been a consultant for Boehringer?Ingelheim Genentech/Roche AstraZeneca and Pfizer. Drs. Giaccone Khuri and Rajan have no conflicts of interest to disclose. Supplementary Material Additional Supporting Information may be found in the online version of this article. Supplementary information Click here for additional data file. Supplementary information . Click here for additional data file. Supplementary information . Click here for additional data file. 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Target Oncol. 2009;4:107?119 19452131 Biomed Res Int Biomed Res Int BMRI BioMed Research International 2314-6133 2314-6141 Hindawi Publishing Corporation 24804226 3997135 10.1155/2014/617868 Research Article Silencing miR-21 Sensitizes Non-Small Cell Lung Cancer A549 Cells to Ionizing Radiation through Inhibition of PI3K/Akt Ma Yongfu 1 Xia Hui 2 http://orcid./0000-0002-4004-1865 Liu Yang 1 * Li Min 1 1Department of Thoracic Surgery Chinese PLA General Hospital No. 28 Fuxing Road Beijing 100853 China 2Department of Thoracic Surgery The First Affiliated Hospital of Chinese PLA General Hospital No. 51 Fucheng Road Beijing 100048 China *Yang Liu: [email protected] Academic Editor: Hushan Yang 2014 7 4 2014 2014 617868 21 12 2013 28 2 2014 2 3 2014 Copyright © 2014 Yongfu Ma et al. 2014 This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. We investigated the role of microRNA-21 (miR-21) in radiotherapy resistance of non-small cell lung cancers (NSCLC) and the underlying molecular mechanism. A549 cells were transfected with anti-miR-21 or the negative control oligonucleotides and real-time PCR was applied to detect miR-21 expression level. After ionizing radiation (IR) the survival fractions proliferation apoptosis and expression of phosphorylated-Akt of A549 cells were determined by clonogenic survival analysis MTT assay flow cytometry and Western blotting. Downregulation of miR-21 in radioresistant NSCLC A549 cells inhibited the colony-forming ability and proliferation of A549 cells after IR. Moreover silencing miR-21 enhanced apoptosis of A549 cells induced by IR accompanied by decreased phosphorylated-Akt protein level. However PI3K activator IGF-1 reversed suppression of phosphorylated-Akt protein level and promotion of apoptosis of A549 cells after IR caused by miR-21 knockdown. Silencing miR-21 in radioresistant NSCLC A549 cells sensitized them to IR by inhibiting cell proliferation and enhancing cell apoptosis through inhibition of PI3K/Akt signaling pathway. This might help in sensitization of NSCLC to radiotherapy. 1. Introduction Lung cancer is the leading cause of cancer-related deaths worldwide [1] whereas non-small cell lung cancer (NSCLC) represents the most frequent type of lung cancer [2]. NSCLC accounts for approximately 80% of all lung cancer cases and has a 5-year overall survival rate of less than 15% [3 4]. Approximately 40% of patients diagnosed with NSCLC have unresectable stage III disease or medically inoperable disease [5]. Radiation therapy has been regarded as the main treatment strategy for NSCLC for a long time. However radioresistance is the key issue limiting the effects of radiotherapy [2 6]. It is possibly due to tumor heterogeneity in terms of cell of origin pathology etiology and molecular/genetic pathogenesis [7]. NSCLC cells are often resistant to radiotherapy [8] which in turn induces the local recurrence of NSCLC [9 10]. " | 1 |
"incidence of thyroid carcinoma is increasing all over the world Some studies have suggestedthat the change of adipokines expression can induce thyroid carcinoma However other studies have come to theopposite Therefore we studied the relationship between adipokines and thyroid carcinomaMethods DatabasesPubMed Cochrane Library SinoMed CNKI Wanfang and clinical trial registries weresearched A metaanalysis was then performed through a fixed or randomeffects model to calculate I values forheterogeneity analysisResults Twentynine s were finally included for analysis The level of serum tumor necrosis factoralpha TNFα [standardized mean difference SMD confidence interval CI to I2 P ]and the ratio of TNFα immunoreactivity in tissues [odds ratios OR CI to I2 P ]in thyroid carcinoma are significantly higher than those in control The serum interleukin6 IL6 in patients withthyroid carcinoma is higher than that in control SMD CI to I2 P There is nosignificant difference of the ratio of IL6 immunoreactivity in tissues between carcinoma and control OR CI to I2 P The ratio of leptin immunoreactivity in tissues is significantly associated with therisk of thyroid carcinoma OR CI to I2 P However after analyzing theexpression level of serum adiponectin in three studies no significant difference is found between thyroidcarcinoma and the control P Conclusions Adipokines TNFα IL6 and leptin show a strong relationship between elevated concentrations inserum andor tissue and thyroid carcinoma However the association between adiponectin and thyroid carcinomaneeds further researchKeywords Thyroid carcinoma Adipokines TNFα IL6 Leptin Metaanalysis Correspondence liaolinsdueducn cwc_llsdueducn Junyu Zhao and Jing Wen contributed equally to this work1Department of Endocrinology and Metabology The First Affiliated Hospitalof Shandong First Medical University Shandong Provincial QianfoshanHospital Jinan China5Department of Endocrinology and Metabology Qilu Hospital of ShandongUniversity Cheeloo College of Medicine Shandong University Jinan 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 0cZhao BMC Cancer Page of BackgroundThyroid carcinoma is the most common endocrine malignancy but mostly has good prognosis During the pastdecades a rising incidence of thyroid carcinoma worldwide has aroused the widespread attention of researchers[ ] Someone supposed that the growing use of diagnostic imaging and fineneedle aspiration biopsy may bethe main reason [] But this may be only partial andcan not totally explain the increased incidence of microcarcinoma Changes in the incidence of a cancer are notonly associated with increased detection and other unknown risk factors need further explore Recently somescientists found that the incidence of thyroid carcinomahas increased along with a marked rise in obesity rateand accumulating evidence of an association betweenobesity and increased thyroid carcinoma risk has beenproposed [] Various hypotheses have been supposedto interpret the relaitonship between obesity and thyroidcarcinoma including hyperinsulinemia upregulation ofaromatase activity chronic low grade inflammation altered immune response and DNA damage caused byoxidative stress [] Furthermore recent data supportingthe notion that a changed expression of adipokinescaused by obesity can affect the cell proliferation andeven induce a thyroid tumorigenesis [] Adipose tissue is a specialized connective tissue composed of fatcells which releases a number of biologically active molecules called adipokines or adipocytokinesincludingleptin adiponectin resistin and many cytokines of theimmune system such as tumor necrosis factoralphaTNFα interleukin6 IL6 and complement factor Dalso known as adipsin Adipokines refer to various enzymes hormones cytokines growth factors proteinsand other biological active substances secreted by adipocytes including adiponectin leptin resistin and interleukin The concentration of adipokines such as TNFαIL6 and leptin were significantly higher in obese subjects and the elevated levels was linked to obesity andeven positively correlated with body mass index []It is reported that adipokines took part in the biologicalprocesses of insulin sensitivity inflammation and proliferation [ ] which the proliferation have been recognizedthetumorigenesis and development At present many kindsof adipokines have been reported to be associated withthyroid carcinoma Rehem RA [] suggested thatserum leptin levels were higher in welldeffierentiatedthyroid carcinoma patients and a significant drop aftersurgery Another envidence showed that adiponectin related with tumor size [] However the opposite resultswere also found in other studies [] Some researchesreported the expression of adipokines is lower in tumortissue than normal control [] It is clearly that certain confounders such as age sex ethnicity and alsoimportantfactorleadingtoasanheterogeneity in study size methodology and original ofsample should be considered when trying to analyze theassociation between adipokines and thyroid carcinomaThese confunding factors above may be the cause of inconsistency results from different researches Additionaly the association between adipokines and thyroidcarcinoma are still not well documented Therfore theaim of this metaanalysis was to investigate the association between adipokines and thyroid carcinoma andpropose that adipokine as a risk factor for thyroidcarcinomaMethodsSearching progressWe conducted a search of all studies published until27th July regarding the association between adipokine and thyroid carcinoma Eligible casecontrol studieswere found by searching the database of PubMedCochrane library Sinomed CNKI and Wanfang and restricted to published results Clinical trial register centers httpwwwclinicaltrialsgov were also searchedThe following search terms Adipokine or Leptin oradiponectin or resistin or tumor necrosis factoralpha or Interleukin6 or Complement factor D orAdipocytokines or tumor necrosis factorα or TNFα or IL6 or adipsin and thyroid cancer or thyroid neoplasm or thyroid tumor or thyroid carcinoma or differentiated thyroid carcinoma or DTC orPapillary thyroid carcinoma or Thyroid carcinomapapillary or PTC or Thyroid cancer follicular orFTC or Thyroid Carcinoma Anaplastic or ATC orThyroid cancer medullary or MTC Hand searchingwas used to identify appropriate studies including reference lists of eligible s and related previous reviews Eligible studies met the following criteria published in English or Chinese language studyassessed the association between adipokine and thyroidcarcinoma study designed as the casecontrol study study reported the expression of at least one adipokine either in blood or tissue Studies were excluded ifany of the followings were identified insufficient information concerning adipokine or thyroid carcinomaoutcome cannot directly extract or calculate OR and95CI the type of study was not a casecontrol designhave not fulltext animal trialsStudy selection and data extractionTwo reviewers screened the studies and extracted dataindependently Any disagreement was resolved by discussion or consensus with a third senior reviewer Dataincluded the followingfirst author publication yearcountry participant characteristics ie mean age sample size sex ration pathological type of thyroid carcinoma source of controls measured outcomes or the 0cZhao BMC Cancer Page of scores were considered to be of high quality Disagreements were resolved by reevaluating and discussing between two reviewersinSearchingthis metaanalysisResultsSearch results and characteristics of included studies s regarding the association between adipokine and thyroid carcinoma were searched in therelated database and clinicaltrial websites Afterscreening the title and abstracts s were selected for fulltext review Finally studies were eligibleprogressincluded and excluded details are all shown in Fig Eighteen of these studies are published in Chinese[ ] and the rest are published in English[] Nineteen studies were conducted in Chinatwo in India and two in Turkey Brazil Greece IranItaly Denmark and Serbia each had one study Totally there are patients with thyroid carcinomain the case group and controls including healthysubjects patients with benign thyroid diseases or normal thyroid tissue near carcinoma were included inthe control group The sample size ranges from to in the case group while to in the controlgroup All the thyroid carcinoma patients were confirmed by pathologically Among these studiesfourteen studies reported papillary thyroid carcinomaPTC eight studies reported differentiated thyroidcarcinoma DTCreported differentpathological types in one paper one study reportedmedullary thyroid carcinoma MTC and the restfour studies did not show the pathological detailsThe detailed characteristics ofincluded studies aresummarized in Table three studiespercentage of samples show immunoreactivity for adipokines antibody both in the case and control groups Thecalculation method is shown below take thyroid cancerfor example the number of samples obtained from thyroid carcinoma that show immunoreactivity for adipokines antibody divided by the total number of thyroidcarcinoma samplesStatistical analysisFor metaanalysis dichotomous outcomes were analyzedby using the odds ratios OR computed using the MantelHaenszel method fixed or random models Continuousvariables measured on the same scale expressed as a meanvalue and standard deviation were analyzed by usingweighted mean differences WMD Otherwise standardized mean difference SMD were used for different scaleAll results were reported with confidence interval CI I2 was used to assess heterogeneity between studies and I2 values of and representing no lowmoderate and high heterogeneity respectively Visual inspection of the funnel plot was done to assess publicationbias The analyses were performed by Review Manager Cochrane Collaboration United Kingdom httpwwwcochraneQuality assessment and risk of biasThe methodological quality of casecontrol study wasassessed by the NewcastleOttawa Scale NOS Supplement Table which consists of the three parameterseight questions with nine possible scores Selection Exposure and Comparability A study can be awarded amaximum of one score for each numbered item withinthe Selection an Exposure categories A maximum oftwo scores can be got for Comparability A higher scoremeans better quality in methodology and five or moreFig Flow chart of the systematic search process 0cZhao BMC Cancer Page of Zhao Jianqiang []ChinaPTC FTC ATCand MTCthyroid adenoma andnormal healthUnknownUnknownTable Characteristic of included studiesFirst authorYearCountryPathologicaltype of thyroidcancerSource of controlsL Kayser []Denmark PTC and FTCCao Guangyao []ChinaUnknownMTrovato []ItalyDTC andundifferentiatedcarcinomamultinodular goitersadenomas Hashimotosthyroiditis hyperplasticglandsthyroid adenoma andnodular goiternormal thyroid tissues andbenign nodulesMelih Akinci []Wang Jingxia []ZhuangXiaoming []Yu Xiao []Hou Sen []SnezanaZivancevicSimonovic []Xu Xiaocheng []XeniProvatopoulou []TurkeyPTChealthy volunteersChinaPTC and FTCnormal thyroid tissuesChinaPTC FTC andMTCthyroid adenoma andnormal healthChinaPTCthyroid adenoma andnormal thyroid tissue nearcarcinomaChinaPTCthyroid adenomaSerbiaWDTChealthy subjectsChinathyroidcarcinomaGreecePTCthyroid adenomabenign thyroid disease andhealthy controlsSun Qinnuan []ChinaPTCnormal thyroid tissue nearcarcinoma and healthycontrolsChinaPTCthyroid adenomaChinaPTCthyroid adenomaMean age yearFemale Outcome indexNumber ofparticipants ncases control casesUnknowncontrolcontrolcasesUnknownUnknownUnknownUnknownUnknownTNFα tissueTNFα tissueIL6 tissueIL6ãTNFαblood ± ±Unknown leptinblood Unknown TNFα tissue Unknown IL6ãTNFαUnknownUnknownbloodleptintissueUnknown Unknown leptin ± ± tissueTNFαblood ± ± ± ± ± ± ± IL6blood IL6blood TNFαbloodandtissue ±Unknown Unknown leptinUnknownUnknowntissueadiponectintissueUnknown Unknown adiponectintissue IL6ãTNFαblood ± ± Zhang Zijie []Zhong Xiuxiu []Zhang Bo []Hu Jinhua []SnezanaZivancevicSimonovic []YanLan Fan []ChinaDTCChinaDTCnormal thyroid tissue nearcarcinomathyroid adenoma andhealthy controls ±SerbiaPTCcontrol subjectsUnknownUnknownIL6bloodChinathyroidcarcinomanodular goitre Hashimotosthyroiditis follicular adenomaand adjacent nonneoplasticthyroid tissue samplesUnknownUnknownleptintissue 0cZhao BMC Cancer Page of Table Characteristic of included studies ContinuedFirst authorSource of controlsYearCountryPathologicaltype of thyroidcancerChinathyroidcarcinomabenign thyroid disease andnormal thyroid tissue nearbenign thyroid diseaseChinaPTCthyroid adenomaTurkeyPTChealthy volunteersIndiaPTCIndiaPTCbenign thyroid diseases andhealthy individualsbenign thyroid diseases andhealthy individualsNumber ofparticipants ncases control cases ±Mean age yearFemale Outcome indexcontrol ±casescontrol TNFαtissue ± ±TNFα tissue IL6bloodUnknown Unknown TNFαbloodUnknown Unknown IL6bloodWangXinzheng []Song Runbo []Kemal Beksac []Toral PKobawala []Toral PKobawala []RaziyehAbooshahab []Zhang Bo []ZhouXiaodong []Ma Xiaokai []MarianaBonjiornoMartins []IranMTChealthy subjects ± ± leptinãadiponectinbloodChinaDTCnormal thyroid tissue nearcarcinomaUnknown Unknown leptintissueChinaDTChealthy subjects ± ±IL6ãTNFαbloodChinaPTCthyroid adenomaUnknown Unknown leptinBrazilDTCbenign thyroid nodules andhealthy controls ±tissue IL6blood ± ±ChinaIL6 Sun Zhenhua []tissueTNFα tumor necrosis factora DTC differentiated thyroid carcinoma IL6 interleukin6 PTC papillary thyroid carcinoma FTC follicular thyroid carcinoma ATCanaplastic thyroid carcinoma MTC medullary thyroid carcinoma WDTC welldifferentiated thyroid carcinoma FNAC fine needle aspiration cytologynodular goiterPTCQuality of included studiesThe quality assessment of these studies is assessed bythe NOS and the resultis shown in SupplementalTable Five or more scores are determined as highquality Two studies conducted by Cao G in [] and L Kayser in [] only get two scoresshowing a poor quality in methodology The rest studies are assessed as high qualityTNFα and thyroid carcinomaTwelve studies reported the expression of TNFα bothin patients with thyroid carcinoma and control subjects[ ] Among these sevenstudies [ ] had tested the level ofserum TNFα two studies [ ] had tested the expression of TNFα in tissues and the ratio of TNFα immunoreactivity was tested in four studies [ ] Firstly fixedeffect model is used to merge the SMDvalues of serum TNFα level however a large heterogeneity is found by the heterogeneity analysis heterogeneity test Chi2 P I2 and itmay be due to the different units differenttestingmethods in different researches or other unknown factors Then randomeffect model to merge the SMD isused and pooled effect size in favor of control group is CI to P Fig 2a SMDvalues of the expression of TNFα in tissues is mergedby fixedeffected model and the heterogeneity analysisshow a considerable heterogeneity heterogeneity testChi2 P I2 The different unitsand limited numbers of research may be the original ofheterogeneity So the pooled SMD with randomeffectmodel of the expression of TNFα in tissues is CI to P Fig 2b The pooled ORwith fixedeffect model of the ratio of TNFα immunoreactivity in thyroid carcinoma tissues is CI to P However a significant heterogeneity is detected heterogeneity test Chi2 P I2 The published by L Kayser in with a poor quality in methodology may attributeto this high heterogeneity Then randomeffect model ofpooled OR is used and pooled effect size in favor of 0cZhao BMC Cancer Page of Fig Forest plot of the TNFα level and the ratio of TNFα immunoreactivity in tissues in patients with thyroid carcinoma a Level of serum TNFα b Expression of TNFα in tissue c Ratio of TNFα immunoreactivity in tissuecontrol group is CI to P Fig 2c In level of serum TNFα and theratio of TNFα immunoreactivity in tissues of thyroidcarcinoma patients are significantly higher than controlsubjects which are without thyroid carcinomaIL6 and thyroid carcinomaAmong the included studies reported the level ofserum IL6 in patients with thyroid carcinoma and control subjects [ ] Due to thelarge heterogeneity of the merged SMD values of serumIL6 level by the heterogeneity analysis heterogeneitytest Chi2 P I2 randomeffectmodel was used to pooled the SMD values and thepooled effect size in favor of control subjects is CI to P Fig 3a which meansthat patients with thyroid carcinoma have a significantlyhigher level of serum IL6 than control subjects Twostudies reported the ratio of IL6 immunoreactivity bothin thyroid carcinoma tissue and noncarcinoma tissue[ ] The pooled OR of the limited two studies donot show an increased ratio of IL6 immunoreactivity inthyroid carcinoma tissues OR CI to P and a large heterogeneity always existsheterogeneity test Chi2 P I2 Fig3b Thus the level of serum IL6 is higher in patientswith thyroid carcinoma However it needs more clinicaldata to verify the relationship between the expression ofIL6 and thyroid carcinoma tissueLeptin and thyroid carcinomaTwo studies reported the level of serum leptin [ ]and another five studies reported the ratio of leptin immunoreactivity in tissues [ ] Because ofthe considerable heterogeneity of the pooled WMD ofserum leptin level heterogeneity test Chi2 P I2 and pooled OR of the ratio of leptinimmunoreactivity in tissues heterogeneity test Chi2 P I2 by the heterogeneity analysis with fixedeffect model randomeffect model is further used to merge the values and analysis Howeverthere is no association of higher level of serum leptin 0cZhao BMC Cancer Page of Fig Forest plot of the IL6 level and ratio of IL6 immunoreactivity in tissue in patients with thyroid carcinoma a Level of serum IL6 b Ratio ofIL6 immunoreactivity in tissueFig Forest plot of the leptin level and ratio of leptin immunoreactivity in tissuein patients with thyroid carcinoma a Level of serum leptin bRatio of leptin immunoreactivity in tissue 0cZhao BMC Cancer Page of with risk of thyroid carcinoma WMD 95CI to Fig 4a Moreover the pooled OR of theratio ofleptin immunoreactivity in tissues from fivestudies is 95CI to Fig 4b whichmeans a high ratio of leptin immunoreactivity in tissueis significantly related to thyroid carcinomaAdiponectin and thyroid carcinomaThree studies reported the expression of adiponectin inthyroid carcinoma including serum and tissue [ ] and the result is summarized in Table It could befound that the level of serum adiponectin is not staticallydifferent comparing thyroid carcinoma patients withcontrol subjects P Interestinglyit was foundthat the expression of adiponectin in thyroid carcinomatissue is significantly lower than control tissue while theopposite result is found when comparing the ratio ofadiponectin immunoreactivity However there was onlyone study for each result and this may be the reasonwhy the two results are diametrically opposed Thus itneeds more clinical studies to confirm in the futurePublication biasThe funnel plot was applied for assessing publicationbias of studies included in the three results includingTNFα Fig 5a IL6 Fig 5b and leptin Fig 5c InFig 5a and Fig 5b almost all studies lies inside the95CIs with an even distribution around the verticalindicating no evident publication bias was obtainedthrough the visual distribution of funnel plot Howevera potential publication bias was found in Fig 5c whencomparing the ratio of leptin immunoreactivity in tissues and that might influence the result of this metaanalysisDiscussionCurrently obesity affects one third of population amongUS adults [] and China has become a big country ofobesity with the incidence ranking first worldwide in theyear of [] Nowadays increasing clinical and experimental studies and documented the closely relationship between malignancies including colon esophaguskidney liver breast endometrium pancreas and prostate as well as nonHodgkins lymphoma and multiplemyeloma and obesityoverweight which affect its occurrence development and prognosis [] Becauseof the increasing incidence of thyroid carcinoma duringthe past decades lots of scientists focus on studying therisk factors of thyroid carcinoma It was found that theincidence of thyroid carcinoma has increased along witha marked rising rate of obesity [] Furthermore obesity is an independent risk factor for thyroid carcinoma[] Increased insulin resistance elevated serum cholesterol level and upregulated COX2 expression may be thetarget of the correlation between obesity and thyroidcarcinoma [] It is reported that people with higherbody mass index have a higher concentration of adipokines [] Adipokines take part in the followingpathological and physiological processes such as insulinsensitivity inflammation and proliferation [ ] andthese are important in the process of tumorigenesis anddeveloping So adipokines may be one of the targetslinking obesity with thyroid cancer The metaanalysiswas based on previous published studies In previousstudies the analysis of adiponectin and thyroid cancermostly focused on TNF IL6 Leptin and AdiponectinWhile few studies focused on other molecules includingIL1 and IL8 and we failed to combine statisticsTherefore in this metaanalysis only TNF IL6 Leptinand Adiponectin which are the most published adiponectin were analyzedTNFα produced by adipose tissue and inflammatorycells can lead to inflammatory response necrocytosisand assist other cytokines to kill tumor cells and improve the antitumor ability Meanwhile TNFα plays animportant role in the process of inflammation insulinresistance diabetes and obesity A moderate amount ofTNFα has a protective effect while an excessive amountwill cause damage which may lead to a resistant oftumor cells to TNFassociated apoptosisinduced ligandswhen the microenvironment of apoptosis is maladjustedTNFα has the ability to promote the production ofgranulocytecolony stimulating factor by thyroid fibroblasts [] which may play an important role in thyroidcancer Moreover TNFα can stimulate the vasoactivemediators such as interleukin and prostaglandin []and these mediators can promote the proliferation oftumor cells and significantly reduce the immune function TNFα can also induce an increased expression ofvascular endothelial growth factor VEGF [] the laterof that can promote the proliferation of tumor cells andprovide conditions for tumors metastasisTable Summary of adiponectin expression in thyroid carcinomaserum adiponectin []ratio of adiponectin immunoreactivity []Effect sizeWMD OR adiponectin in tissue [] CI confidence interval WMD weighted mean differences OR odds ratiosWMD 95CI PI2Not applicable 0cZhao BMC Cancer Page of Fig Funnel plots of a TNFα b IL6 and c leptin revealed no significant publication bias SE SMD standard error of standardizedmean differenceIn surprisingly the results of clinical studies provide evidence for basic research Simonovic SZ [] evaluated cytokine profiles determined in supernatants obtained from whole blood cultures in patients with DTC before and days after radioactiveiodine 131Itherapy and control subjects andfound that the expression of TNFα in DTC patients ishigher than control subjects and it showed a decreasedlevel after 131I therapy than those before therapy However no statistical difference found for the limited sample size Another study conducted by Kobawala TP et al[] with more patients patients with benign thyroiddisease PTC patients and healthy individuals determined the circulating levels of TNFα and it wasfound that the serum level of TNFα was significantlyhigher in PTC patients than benign thyroid disease patients and the later was also significantly higher thanhealthy individuals Furthermore serum TNFα was reported to be a significant prognosticator for overall survival in PTC patients It is a pity thatopposite result wasreported in a casecontrol study that included DTCcases and matched cancerfree cohort participantswhich found that TNFa was not associated with thyroidrisk in either gender []Based on current evidence our metaanalysis suggeststhat TNFα exhibit a strong association with thyroid carcinoma It may because that elevated TNFα may involved in the tumorigenesis and development of thyroidcancer Another possible reason is that the TNFα decreased with tumor cells less resulted the activation ofthe immune system by thyroid carcinomaThereforemore clincal studies and basic reseaches should be conducted in the futureIL6 a multifunctional cytokine plays important rolesin different types of cells including tumor cells It is reported that elevated serum IL6 level is closely related tothe tumorigenesis and development of a variety of tumors [] A strong positive association between theserum IL6 and the progression and poor prognosis oftumors in patients with several types of tumor wasalready found [] Serum IL6 level in thyroid cancer has been evaluated in numerous studies including 0cZhao BMC Cancer Page of in vivo and in vitro studies Provatopoulou X []found that serum IL6 were significantly higher in malignant and benign thyroid diseases compared to healthycontrols However other studies show a different resultthat no significance different of IL6 was found betweenthyroid cancer and nonthyroid cancer [ ] A limited sample size different inclusion criteriadifferent population characteristics or different pathological type of thyroid cancer may explain such a difference For in vitro research IL6 was also found to beexpressed in thyroid cancer cell lines and a potential roleof IL6 in PTC was confirmed indirectly []The underlying mechanism may be the followingsbelow Tumor cells including esophageal cancerlungcancer colorectal cancer and melanoma were foundhave the function of autocrine IL6 which can affect thegrowth and proliferation of tumor cells and participatein the tumor growth and metastasis by acting on themembrane receptors [] Also IL6R was found associated with the characterization of thyroid nodules malignancy and tumor aggressivenessIn additionIliopoulos D [] found that Src nonsomatic tyrosine kinase family oncogene can induce the normal epithelial cell transformation by activating NFκB and thistransformation contributes to tumorigenesis IL6 is considered as an important regulatory factor in this processAnother possibility is that the activation of the immunesystem of patients with thyroid cancer leads to an increase in adikopines level[]In general the data above support that IL6 is important for thyroid cancer but the detail mechanism remainto be further studyLeptin a circulating hormone secreted by adipocytesexerts its biological effect by combing with its receptorwhich is mainly presented in the hypothalamus Meanwhile gene of leptin receptor is also expressed in manyother tissues such as lung liver and kidney It is reported that obesity and overweight can lead to a highlevel of serum leptin which may because that obesity always accompanies with insulin resistance and hyperinsulinemia and insulin further enhance the expression ofleptin Moreover leptin acts as a growth factor in a variety of human cellsincluding both normal cells andtumor cells which regulates the process of differentiation proliferation and apoptosis thus stimulate thetumorigenesis and development of tumors through mediatingpathway RhoALIMK1Cofilinpathway and MAPKERK pathway [] Kim WG et al[] evaluated the effect of dietinduced obesity on thyroid carcinogenesis in a mouse model that spontaneously develops thyroid cancer Thrb PVPV Pten mice and found that obesity increases the frequency of anaplasia of thyroid cancer and exacerbatesthyroid cancer progression that were mediated byJAKSTAT3increased activation of the JAK2 signaling transducerand activator of STAT3 signaling pathway and inductionof STAT3 target gene expression Leptin is always reported a high expression on solid tumors [] and it isconfirmed that serum leptin levelis significantly increased in thyroid cancer mainly PTC while otherstudies showed a same results in cancer tissues [ ] Yu Xiao [] conducted a clinical studycomparing the level of serum leptin in PTC patientsincluding patients with lymph node metastasis and thyroid adenoma patients in Dalian China and foundthat patients with lymph node metastasis have a higherlevel of leptin than those without lymph node metastasisLeptin can induce the expression of vascular endothelialgrowth factor and promote neovascularization in tumortissue [] In addition it can also inhibit the apoptosisthrough Bcl2 dependent mechanism Meanwhile leptinreceptor exists in all thyroid cancer cells It is overexpressed in PTC and is involved in tumor invasion andlymph node metastasis [ ] Thus leptin may be involved in the tumorigenesis and metastasis of thyroidcancer through a complex pathway and a monitoringmay have some significance Due to the absence of directevidence elevated leptin levels can also be caused bythyroid carcinoma The cause and effect relationship between leptin and thyroid carcinoma are unclear now andneed further studiesCompared to lean women overweightobese womenhad lower serum adiponectin levels and this differencehas statistical significance [] In addition adiponectinis negatively associated with a variety of benign and malignant tumors especially those associated with obesityand insulin resistance such as leukemia [] renal carcinoma [] gastric carcinoma [] and colon cancer[] Moreover the association of adiponectin with potential tumorlimiting functions has been widely proposed []Otvos L Jr [] tried in vitro experiments andproved that adiponectin can inhibit the metastasis ofcancer cells Mitsiades N [] measured circulatingadiponectin levels in ptaients with PTC and found thatit is independently and inversely associated with the riskof thyroid cancer As the receptor that binds to adiponectin for biological effects adiponectin receptor hadbeen reported closely correlated with the developmentof PTC Adiponectin receptor1 and are higher expression in PTC tissues than that in the surrounding normaltissues and this is thought to be associated with a betterprognosis []However other studies have shown different results[ ] and more studies should be done furtherly tosupport the antitumor effect of adiponectin and thepositive correlation between the increased level of adiponectin in circulating blood and the prognosis of thyroid 0cZhao BMC Cancer Page of neoplasms and provide new ideas for the prevention andtreatment of thyroid neoplasmsFrom the above a strong relationship between elevatedconcentrations of adipokines in serum andor tissueand thyroid cancer can be concluded And this may explain why increased incidence of obesity and thyroidcancer are consistent Thus targeted drugs for adipokinemay be useful for the treatment of thyroid cancer in thefutureHowever some limitations in our metaana | 2 |
patients who have diabetes suffer from Type DMT2DM Many studies suggest using the significant role of lncRNAs to improve thediagnosis of T2DM Machine learning and Data Mining techniques are tools that canimprove the analysis and interpretation or extraction of knowledge from the dataThese techniques may enhance the prognosis and diagnosis associated withreducing diseases such as T2DM We applied four classification models including Knearest neighbor KNN support vector machine SVM logistic regression andartificial neural networks ANN for diagnosing T2DM and we compared thediagnostic power of these algorithms with each other We performed the algorithmson six LncRNA variables LINC00523 LINC00995 HCG27_201 TPT1AS1 LY86AS1DKFZP and demographic dataResults To select the best performance we considered the AUC sensitivityspecificity plotted the ROC curve and showed the average curve and range Themean AUC for the KNN algorithm was with standard deviation SD themean sensitivity and specificity were and respectively After applying theSVM algorithm the mean AUC obtained after stratified 10fold crossvalidationand the SD obtained The mean sensitivity and specificity were and respectively The mean AUC for ANN and the SD were and also the meansensitivity and specificity were and At last for the logistic regressionalgorithm our results showed of mean AUC and the SD of the meansensitivity and specificity were and respectively According to the ROCs theLogistic Regression and SVM had a better area under the curve compared to theothersContinued on next page The Authors Access This is licensed under a Creative Commons Attribution International License whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit tothe original authors and the source provide a link to the Creative Commons licence and indicate if changes were made Theimages or other third party material in this are included in the 's Creative Commons licence unless indicated otherwisein a credit line to the material If material is not included in the 's Creative Commons licence and your intended use is notpermitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyrightholder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public DomainDedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unlessotherwise stated in a credit line to the data 0cKazerouni BMC Bioinformatics Page of Continued from previous pageConclusion We aimed to find the best data mining approach for the prediction ofT2DM using six lncRNA expression According to the finding the maximum AUCdedicated to SVM and logistic regression among others KNN and ANN also had thehigh mean AUC and small standard deviations of AUC scores among the approachesKNN had the highest mean sensitivity and the highest specificity belonged to SVMThis studys result could improve our knowledge about the early detection anddiagnosis of T2DM using the lncRNAs as biomarkersKeywords Data mining Gene expression Machine learning algorithms Type diabetes mellitusBackgroundDiabetes mellitus DM is one of the most prevalent chronic noncommunicable diseases NCD around the world about of the patients who have diabetes sufferfrom Type DM T2DM [] The risk of developing T2DM is strongly associated withmany predispositions behavioral and environmental risk factors and also genetic factors [] Besides the genetic factors strong evidence indicates that factors such asobesity and physical inactivity are the main nongenetic determinants of the disease [] T2DM can range from predominant insulin resistance with relative insulin deficiency to dominant defective secretion with insulin resistance [] It is often related tometabolic syndrome problems Individuals who have impaired glucose tolerance arehighrisk subjects of type diabetes []Studies demonstrate a drastic increase of the disease in recent decades The trendsestimate that by more than million people will be affected by the disease []People who suffer from T2DM are susceptible to many forms of complications leadingto morbidity and mortality in these patients Many studies emphasize the genetic factors in the pathogenesis of T2DM [ ] Long noncoding RNAs long ncRNAslncRNA are subsets of RNA specified as being transcripts with lengths exceeding nucleotides that could not be translated into protein [] Long noncoding RNAslncRNAs belong to a heterogeneous class of regulatory lncRNAs with transcriptlengths nucleotides which have a positive role in the development and growth ofseveral various diseases including T2DM supporting the hypothesis that abnormal expression of LncRNAs is related to various diseases [] Besides considering the significant role of lncRNAs in disease pathogenesis increasing researches suggest using themto improve diagnosis prognosis and clinical management of T2DM Genomewide association studies GWAS have recently introduced several particular diabetesrelatedloci in the human genome [] Also many studies discovered the relationship betweenmore than susceptible loci and T2DM at a genomewide significant level [ ]Deregulation of genes located in GWAS defined loci may be risk factors for human diseases concerning which we applied the GWAS catalog to select six lncRNAsLINC00523 LINC00995 CG27_201 TPT1AS1LY86AS1 DKFZP as our gene targets for the present study [] Knowledge Discovery in Databases KDD or data miningare techniques for the computational process of discovering patterns in large datasetscontaining various approaches such as artificial intelligence machine learning statisticsand database systems [] These methods are applied to recognize patterns in dataprediction association and classification problems [ ] Considering the 0cKazerouni BMC Bioinformatics Page of importance of early detection of T2DM machine learning and Data Mining techniquesare tools that can improve the analysis and interpretation or extraction of knowledgefrom the data [ ] These techniques may enhance the prognosis and diagnosis associated with life quality reducing diseases such as T2DM [ ]To date several other studies tried to predict diabetes mellitus using outstandingdata mining techniques [] Vijayan et al[] applied the expectationmaximization algorithm KNN algorithm Kmeans algorithm amalgam KNN algorithm and ANFIS algorithm to predict and diagnose Diabetes Mellitus They usedthe UCI dataset containing blood test and demographic variables and their resultsshowed that EM possessed the least classification accuracy and amalgam KNN andANFIS provided better classification accuracy of more than and respectively Another study conducted by Saravananathan [] used popular classifiincluding J48 Support Vector Machines SVM Classificationcation algorithmsand Regression Tree CART and kNearest Neighbor kNNfor diabetic dataTheir performance indicators were accuracy specificity sensitivity precision errorrate They found that the J48 techniques performance was remarkably superior tothe other three techniques for the classification of diabetes data Meng []compared three data mining models of logistic regression ANN and decision treefor predicting diabetes mellitus or prediabetes by risk factors They gathered information about demographic characteristics family diabetes history anthropometricmeasurements and lifestyle risk The decision tree model C50 had the best classification performance with an accuracy of with a sensitivity of andspecificity of Another study performed by Saeidi [] used logistic regression to assess the diagnostic value of LY86AS1 and HCG27_201 as biomarkersfor T2DM They obtained a sensitivity of and specificity of Anotherstudy [] used two other lncRNAs including LINC00523 and LINC00994 expressions for the evaluation of their potential diagnostic value for T2DM They appliedlogistic regression and achieved a sensitivity of and specificity of Inour study we combined six lncRNAs as variables for the first time and appliedfour classification models including classification algorithms like Knearest neighbor KNN support vector machine SVM logistic regression and artificial neuralnetworks ANN for diagnosing T2DM and we compared the diagnostic power ofthese algorithms with each other In the present study we aimed to find the bestdata mining approach for the prediction of T2DM using six lncRNA expressionThe result of this study could improve our knowledge about the early detectionand diagnosis of T2DM using the lncRNAs as biomarkers []MethodsThe primary aim of the present study was to implement four models to predict DT2Mapplying data mining techniques based on the lncRNA variables The research objectives of our study wereImplementing data mining techniques for prediction of the DT2M Comparing the applied methodsselecting the best model for the T2DM prediction 0cKazerouni BMC Bioinformatics Page of We used the variables for predicting T2DM and comparing the performance of thevarious data mining techniques For the implementation of the algorithms we usedANACONDA3 bit a free and source platform distribution of pythonprogramming language with a vast number of modules packages and rich libraries thatprovide various methods for classification problems For obtaining the best amount ofperformance in the models 10fold crossvalidation performed on the dataset In dealing with the small data sets crossvalidation is a prominent strategy for estimating theperformance CrossValidation is a performance evaluation technique commonly usedin practice Here the data set is repeatedly partitioned into two nonoverlapping partsa training set and a holdout set For each partitioning the holdout set is used fortesting while the remainder is used for training The two most popular variants aretenfold crossvalidation 10fold CV where the data is split into ten mutually disjointfolds []Since our samples were more than and to be sure that each fold contains thesame proportion of healthy and diabetic individuals we used the stratified 10foldcrossvalidation approach [] Therefore the results are reliable and more credibleWe applied four popular data mining approaches on the lncRNA variables regression knearest neighbors SVM and neural network classification algorithmsKNN algorithmThe knearest neighbors algorithm kNN is an algorithm for classifying variables regarding the closest training data in the feature space KNN uses an instancebasedlearning method which is one of the simplest algorithms among data mining techniques This method considers the nearest neighbors to each object and decides todedicate the object to classes [ ]SVM algorithmSupport Vector Machine SVM is a supervised algorithm which divides the featurespace called hyperplanes considering the target classes SVM computes classification bymaximizing the margin of the hyperplane that intercepts classes This algorithm plots amultidimensional hyperplane that divides classes and increases the margin betweenclasses to enhance the accuracy of classification We used different kernel functionsembedded in the SVM class of SVC library in python framework as a quadratic polynomial radial basis etc to classify the instance and to detect the best accuracy amongthem []Artificial neural networkArtificial Neural Network is a data processing algorithm that simulates the biologicalneural network in its computations A common problem in using ANN is that they actfundamentally as a black box and the parameters are set by the model so we cannotdemonstrate them [] we can just apply the model in our problems and obtain thehigh performance We used Multilayer Perceptron Neural Networks MLPNN Thestructure of a multilayer perceptron neural network has been demonstrated in Fig It maps a set of input data into a set of appropriate output classes It includes threelayers input layer hidden layer output layer The principal function of neurons of the 0cKazerouni BMC Bioinformatics Page of Fig Artificial Neural Network structureinput layer is to divide input Xi into neurons in the hidden layer The neuron of thehidden layer adds the appropriate weights of Wij to the input variables The output formula isYj ¼ fWji XiX 10 11Where f is a simple threshold function that we considered sigmoid and hyperbolictangent function []In the present study a Multilayer Perceptron Neural Networks MLPNN was performed The structure of MLPNN is as shown in Fig It makes a map of input dataonto a set of suitable output dataThe RBF networks are another type of neural network In MLP each neuron considers the weighted sum of its input values in which each input value is multiplied by acoefficient and the results are the sum of values RBF is a more intuitive approach toMLP An RBFN classifies the inputs by calculating the inputs similarity to examplesfrom the training set Each RBFN neuron stores one of the examples from the trainingset as a prototype for classification of new input in each neuron the Euclidean distance between the input and its prototype is calculated The input is dedicated to aclass when it has more similar to that class than the other classesLogistic regressionLogistic regression is a common approach for predictive modeling practices The functionpX provides probability output between and for all values of X where X1Xp are thepredictors The coefficients β0βp are estimated using maximum likelihood estimationþβp Xð à ¼ eβ þ eβ1X1 þ¯Ã¾Î²Ã¾Î²pXp1X1 þ¯Ã¾Î²pXpDatasetThis study was based on the data obtained from three previous research conducted bySaeidi and Mansoori [ ] and the research of Parvizi and colleagues which 0cKazerouni BMC Bioinformatics Page of is not published yet We integrated these three studies and our data mining analysiswas implemented in their studies The data were collected from unrelated Iraniansubjects T2DM patients and healthy individuals matched for age and sexT2DM patients were recruited from individuals who referred to the Diabetic Clinic atShohada Hospital Tehran Iran In the current study we applied six lncRNAs expression and also six demographic variables including sex age weight height BMI andFBS for analysis and inputs of algorithms For the preprocessing phase we normalizedthe data inputs for KNN SVM and ANN models We also had low missing variablesand we replaced them with zero Table lncRNA extraction and selectionIncreasing evidence has suggested several lncRNAs are implicated in T2DM pathogenesis Recently human βcell transcriptome analysis showed lncRNAs dynamic regulation and abnormal expression of lncRNAs in T2DM [] However the extent oflncRNA deregulation in T2DM has yet to be determined To date more than100 susceptibility loci have been identified as being associated with T2DM at a genomewidesignificant level [ ] Considering this into account and by querying the GWAS catalog we candidated lncRNAs LY86AS1 HCG27_201 LINC00523 LINC00994TPT1AS1and DKFZP as target genes for this studyThe large scale GWAS have recognized approximately SNPs that were susceptibleto T2DM [] From there we used the GWAS catalog access in June to create alist of SNPs associated with T2DM In the current study we selected six lncRNA forexpression analysis according to the scan carried out in the study of Mansoori []and Saeedi [] We selected variants that had associations with increased risk ofT2DM We applied a quantitative PCR analysis of lncRNA expression levels in the samples We calculated the respective amount of each lncRNAs applying the 2ÎÎct asmeans of duplicate measurementsTable The lncRNAs as inputs of algorithmsnumberLncRNA VariablesDemographic VariablesVariablesLINC00523LINC00995HCG27_201TPT1AS1LY86AS1DKFZPSexAgeWeightHeightBMIFBS 0cKazerouni BMC Bioinformatics Page of Analysis and evaluation criteriaTo select the best performance data mining algorithms in predicting diabetic patientswe considered AUC sensitivity specificity and plotted ROC curve for the folds we ranand showed the average curve and its range [ ]ResultsTable shows the significant downregulation of PBMC expressions of the variables inthe T2DM group compared with the control group The AUC of each classificationtechnique has been demonstrated in Table AUC stands for Area under the ROC Curve AUC computes the entire twodimensional area under the whole ROC curve According to the finding the maximumAUC dedicated to SVM and logistic regression among others knn also had the highestmean AUC and minimum standard deviation of AUC scores among the approachesThe mean and standard deviation for AUC sensitivity and specificity of each algorithmis given in Table Apart from classification AUC sensitivity and specificity the Receiver Operating Characteristic ROC with stratified crossvalidation is shown for eachapproach in Figs and ROC curves generally plot true positive rate on the Yaxis and false positive rate onthe Xaxis In other words a false positive rate of zero and a true positive rate of onein the top left corner of the plot is called the ideal point It means that a larger areaunder the curve AUC is usually better According to the demonstrated ROCs theKNN and SVM have a better area under the curve in comparison with the othersDiscussionFor a medical diagnosis optimized approaches to gain useful and accurate outcomesare essential Applying machine learning and data mining methods to automate theprocess of diagnosis may assist practitioners to enhance the quality of their clinical decisions [ ]Since T2DM is one of the prevalent diseases with severe consequences [] developing efficient methods for early detection of the disease was the primary purpose of ourresearchRegardless of high number of lncRNAs in the RNA profile of human a few numbersof them has been proved to be biologically active The role of the few lncRNAs hasbeen identified but several studies discussed the significant impact of lncRNAs in diabetic people which may represent the role of abnormal expression of lncRNAs in thelncRNAs in theincidence of T2DM [] According to the possible function ofTable Relative expression of the variablesVariablesLINC00523LINC00995HCG27_201TPT1AS1LY86AS1DKFZPDiabetesÎCT ± SEM ControlÎCT ± SEM pvalue 0cKazerouni BMC Bioinformatics Page of Table The AUC of algorithms for each iterationNumberof foldsAUCKNNSVMANNLogistic Regressiondevelopment of T2DM we considered the expression levels of six lncRNAs in additionto the demographic data in diabetic and healthy individuals for our study Tomeasure the expression of the lncRNAs we applied PBMCs which demonstrate an extensive proportion of the genes encoded in the human genome [] Several studies haveinvestigated different machine learning and data mining methods to predict differentdiseases [ ] such as heart diseases thyroid tumors and also diabetestype diabetes prediction In the present study we combined four commonly used datamining algorithms KNN SVM neural networks and regression to predict type diabetes using Long noncoding RNAs expression and the demographic variables for thefirst time because most of the previous studies used blood test variables or the demographic data for their analysis Receiver operating characteristic ROC analysis AUCsensitivity and specificity measure was used to assess the diagnostic value of the sixbiomarkers for T2DM The mean AUC for the KNN algorithm was obtained andwith standard deviation and we obtained the highest sensitivity with thestandard deviation of among other approaches After applying the SVM algorithm the mean AUC obtained after 10folds with the standard deviation of and the highest specificity among other approaches obtained with the standarddeviation of For the ANN we applied a multilayer perceptron with five hiddenlayers and the mean AUC of folds was and the standard deviation was Atlast for the logistic regression algorithm our results showed of mean AUC andthe standard deviation of The lower standard deviations in the AUC scores ofcomputed folds means the algorithm has worked with more performance [ ]Other studies investigated data mining algorithms for several diseases Saravananathanand Velmurugan [] applied several classification algorithms in their study to analyzeincluding KNN Sadri Sadi [] compared three data miningdiabetes dataTable The mean and standard deviation of AUC sensitivity and specificity of algorithmsAlgorithmKNNMean AUC std Mean sensitivity std Mean specificity std SVMANNLogistic Regression 0cKazerouni BMC Bioinformatics Page of Fig The ROC for KNNalgorithms to predict T2DM and gained precision for ANN Sidiq [] reported about accuracy for KNN and accuracy for SVM algorithms applyingfor the Diagnosis of Various Thyroid Ailments In another study for the heart diseasesThe data mining algorithms indicated more than accuracy The investigated studies are in line with the findings of our study that these algorithms have a strong powerfor prediction and early detection of many diseases including T2DM and we obtainedremarkably better accuracy for prediction for example the SVM and logistic regressionaccuracy were In our study we also obtained a better accuracy for logistic regression that was and in comparison with other studies is a strong point for exampleSaeidi [] conducted a study to review two Long noncoding RNA expressions intype diabetes mellitus and with applying regressions reported about accuracyAnother research [] used two different Long noncoding RNA expressions in type Fig The ROC for SVM 0cKazerouni BMC Bioinformatics Page of Fig The ROC for MLPdiabetes mellitus and found of accuracy with the regression algorithm In thepresent study for the first time we performed four data mining algorithms on six Longnoncoding RNAs and compared their power with each other We demonstrated thatLong noncoding RNAs are effective biomarkers for data mining algorithms and have afeasible power to be applied for prediction of T2DM Also in this research we optimized the parameters of every algorithm and used stratified 10fold crossvalidation togain the best performance To be mentioned in the nearest neighbors algorithm theparameter k was varied between one and nine to find the bestoptimized method andwe selected k to have the best performance and the lowest standard deviation in theaccuracy of the folds In addition in choosing the parameters of the artificial neuralnetwork the number of hidden layer neurons significantly affects the accuracy of thenetwork so we set the parameters with two hidden layers with five and three neuronsFig The ROC for logistic regression 0cKazerouni BMC Bioinformatics Page of respectively to yield the best accuracy Considering the standard deviation of scores foreach algorithm the KNN had the lowest std Moreover the highest accuracy amongthe algorithms was the SVM algorithm and Logistic regression which had the maximum accuracy in folds among others We should mention that the strong points ofour study are using demographic data and six Long noncoding RNAs and combiningthem to get the best detection power of T2DM and performing four outstanding datamining algorithms and comparing their performances As the limitations of this studywe should account for the limited number of samples which is due to the high costs ofmeasuring the Long noncoding RNAs No doubt the higher number of samples wouldlead to higher performance and more reliable resultsConclusionIn this paper the performance of conventional data mining classification techniqueshas been calculated and compared for a dataset of patients referred for the screeningof type diabetes to the Shohada Hospital Iran The biomarker applied in this studydemonstrated high diagnostic value and the diagnostic process is suitable which couldhelp in the diagnosis of prediabetes and T2DMThe classification techniques compared were support vector machine artificial neuralnetwork decision tree nearest neighbors and logistic regression In data mining it isnot possible to say one classification technique will always work best and it often depends on the number of samples their distribution and the choosing of the right algorithm In this research work SVM and Logistic Regression had the best Area UnderCurve among methods of classification with the mean AUC of KNN and ANNalso had the high mean AUC and small standard deviations of AUC scores among theapproaches KNN had the highest mean sensitivity and the highest mean specificitybelonged to SVMFor future works performing other data mining and machine learning methods andusing higher numbers of samples are recommended to enhance the performanceAcknowledgmentsNot applicableAuthors contributionsAB and FA designed the study FA and AB collected the data and performed the statistical analysis AB and NPinterpreted the data FK ZM and LS wrote and revised the paper All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author onreasonable requestEthics approval and consent to participateEthical approval was obtained from the Shahid Beheshti University of Medical Sciences Ethics CommitteeIRSBMURETECHREC13951036 We informed all participants that their participation was voluntary and the study didnot state any potential risk and their identities will be private Informed written consent forms were taken from allparticipants before participationConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests 0ccoding RNAs LINC00523 and LINC00994 in type diabetes in an Iranian cohort Mol Biol Rep Saeidi L Ghaedi H Sadatamini M Vahabpour R Rahimipour A Shanaki M Mansoori Z Kazerouni F Long noncodingRNA LY86AS1 and HCG27_201 expression in type diabetes mellitus Mol Biol Rep Petersmann A Nauck M MüllerWieland D Kerner W Müller UA Landgraf R Freckmann G Heinemann L Definitionclassification and diagnosis of diabetes mellitus Exp Clin Endocrinol Diabetes Armoon B Karimy M Epidemiology of childhood overweight obesity and their related factors in a sample of preschoolchildren from Central Iran BMC Pediatr Tuomilehto J Lindström J Eriksson JG Valle TT Hämäläinen H IlanneParikka P KeinänenKiukaanniemi S Laakso MLouheranta A Rastas M Prevention of type diabetes mellitus by changes in lifestyle among subjects with impairedglucose tolerance N Engl J Med Guariguata L Whiting DR Hambleton I Beagley J Linnenkamp U Shaw JE Global estimates of diabetes prevalence for and projections for Diabetes Res Clin Pract Leti F DiStefano J Long noncoding RNAs as diagnostic and therapeutic targets in type diabetes and relatedcomplications Genes Heydari M Teimouri M Heshmati Z Alavinia SM Comparison of various classification algorithms in the diagnosis of type diabetes in Iran International Journal of Diabetes in Developing Countries Kazerouni BMC Bioinformatics Page of Author details1Department of Laboratory Medicine School of Allied Medical Sciences Shahid Beheshti University of MedicalSciences Tehran Iran 2Department of Health Information Technology and Management School of Allied MedicalSciences Shahid Beheshti University of Medical Sciences Tehran Iran 3Department of Clinical Biochemistry School ofMedicine Tehran University of Medical Sciences Tehran Iran 4Department of Genetics Faculty of Medicine BabolUniversity of Medical Sciences Babol IranReceived December Accepted August ReferencesLi X Zhao Z Gao C Rao L Hao P Jian D Li W Tang H Li M The diagnostic value of whole blood lncRNAENST00000550337 for prediabetes and type diabetes mellitus Exp Clin Endocrinol Diabetes Mansoori Z Ghaedi H Sadatamini M Vahabpour R Rahimipour A Shanaki M Kazerouni F Downregulation of long non Perkel JM Visiting noncodarnia In Future Science Kapranov P Cheng J Dike S Nix DA Duttagupta R Willingham AT Stadler PF Hertel J Hackermüller J Hofacker IL RNAmaps reveal new RNA classes and a possible function for pervasive transcription Science Cornelis F Martin M Saut O Buy X Kind M Palussiere J Colin T Precision of manual twodimensional segmentations oflung and liver metastases and its impact on tumour response assessment using RECIST European radiologyexperimental Liao M Liu Q Li B Liao W Xie W Zhang Y A group of long noncoding RNAs identified by data mining can predict theprognosis of lung adenocarcinoma Cancer Sci Deshpande S Thakare V Data mining system and applications a review International Journal of Distributed and Parallelsystems IJDPS Umar Sidiq D Aaqib SM Khan RA Diagnosis of various thyroid ailments using data mining classification techniques IntJ Sci Res Coput Sci Inf Technol Zou Q Qu K Luo Y Yin D Ju Y Tang H Predicting diabetes mellitus with machine learning techniques Front Genet Daghistani T Alshammari R Diagnosis of diabetes by applying data mining classification techniques InternationalJournal of Advanced Computer Science and Applications IJACSA Meng XH Huang YX Rao DP Zhang Q Liu Q Comparison of three data mining models for predicting diabetes orprediabetes by risk factors Kaohsiung J Med Sci Wu H Yang S Huang Z He J Wang X Type diabetes mellitus prediction model based on data mining Informatics inMedicine Unlocked Vijayan V Ravikumar A Study of data mining algorithms for prediction and diagnosis of diabetes mellitus Internationaljournal of computer applications Saravananathan K Velmurugan T Analyzing diabetic data using classification algorithms in data mining Indian J SciTechnol Nahar N Ara F Liver disease prediction by using different decision tree techniques International Journal of Data Mining Knowledge Management Process IJDKP Vol Airola A Pahikkala T Waegeman W De Baets B Salakoski T An experimental comparison of crossvalidation techniquesfor estimating the area under the ROC curve Computational Statistics Data Analysis Purushotham S Tripathy B Evaluation of classifier models using stratified tenfold cross validation techniques InInternational Conference on Computing and Communication Systems Springer Abdar M Kalhori SRN Sutikno T Subroto IMI Arji G Comparing Performance of Data Mining Algorithms in PredictionHeart Diseases International Journal of Electrical Computer Engineering Sambyal RS Javid T Bansal A Performance analysis of data mining classification algorithms to predict diabetesInternational Journal of Scientific Research in Computer Science Engineering and Information Technology Pradhan M Kohale K Naikade P Pachore A Palwe E Design of classifier for detection of diabetes using neural networkand fuzzy knearest neighbor algorithm International Journal of Computational Engineering Research Tzeng FY Ma KL ing the black boxdata driven visualization of neural networks IEEE Morán I Akerman Ä° Van De Bunt M Xie R Benazra M Nammo T Arnes L NakiÄ N GarcÃaHurtado J RodrÃguezSeguà SHuman β cell transcriptome analysis uncovers lncRNAs that are tissuespecific dynamically regulated and abnormallyexpressed in type diabetes Cell Metab 0cKazerouni BMC Bioinformatics Page of Voight BF Scott LJ Steinthorsdottir V Morris AP Dina C Welch RP Zeggini E Huth C Aulchenko YS Thorleifsson GTwelve type diabetes susceptibility loci identified through largescale association analysis Nat Genet Imamura M Maeda S Genetics of type diabetes the GWAS era and future perspectives Endocr J Soni J Ansari U Sharma D Soni S Predictive data mining for medical diagnosis an overview of heart diseaseprediction International Journal of Computer Applications Asadi F Paydar S Presenting an evaluation model of the trauma registry software Int J Med Inform Dangare CS Apte SS Improved study of | 2 |
Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly citedCutaneous metastases are unusual presenting symptoms of lung cancer erefore they are prone to be misdiagnosed and missede report describes a case of a fortynineyearold female with painful zosteriform rashes showing multiple vesiclelike papuleslocalized on the left breast for days e patient had been diagnosed as lung adenocarcinoma at the department of oncology oneyear ago Skin biopsy revealed blue nodular lesions in the dermis composed of clustered heterogeneous tumor cells with glandularformation Immunohistochemical stains conï¬rmed the diagnosis of metastatic lung adenocarcinoma IntroductionLung cancer can metastasize to almost all ans butmore often invades the hilar nodes liver adrenal glandsbones and brain [] e incidence of lung cancer withmetastases to the skin varies between [] A lungcancer metastasis is usually classiï¬ed only as adenocarcinoma squamous cell carcinoma SCC or undiï¬erentiated carcinoma Until the 1980s SCC was reported asthe most common type of lung cancer However adenocarcinoma has replaced SCC as the most common lungcancer subtype especially in women and in neversmokers Sun reported that the type of adenocarcinoma was times more frequent than that of SCC []Skin metastases can appear on any cutaneous surface andthe most common sites are the chest abdomen head andneck [ ] Cutaneous metastases have various manifestations such as single papulesnodules or multiplelesions on anywhere of the skin while other rare formsmay show plaquelike lesions erysipelaslike papuleszosteriform lesions and scars [ ] Case PresentationA fortynineyearold nonsmoker female was admitted toour department with multiple painful papules localizedon the left breast ey appeared eruptively for about days and initially diagnosed as herpes zoster in anotherhospital e patient had been diagnosed as lung adenocarcinoma at the department of oncology one year agoShe was given oxitinib mesylate a targeted drug for thetreatment of nonsmallcell lung cancer In addition thepatient exhibited symptom of pain signs of weight lossanorexia and fatiguePhysical examination showed zosteriform vesiclelikepapules measuring cm on the left breast elesions were pink or red ï¬rm and tender Figure Excisional biopsy was performed revealing blue nodularlesions ltrating in the dermis composed of clusteredheterogeneous tumor cells with glandular formationSome tumor cells were detected within vessels or lymphatic vessels Some cells were transparent Mitosis wassignificant Figures 2a2c In immunohistochemistumor cells were positive for cytokeratin CKtrycytokeratin7 CK7thyroid transcription factor1TTF1 and EMA and negative for cytokeratin20CK20 carcinoembryonic antigen CEA and grosscysticGCDFP15Figures 3a3c Proliferative index as measured byKi67 was approximately oftumor cellsAccording to the clinical and pathological features cutaneous metastatic lung adenocarcinoma was madeprotein15diseaseï¬uid 0cCase Reports in Dermatological MedicineFigure Zosteriform vesiclelike papules measuring cm on the left breast Pink or red ï¬rm and tenderabcFigure Skin biopsy revealed a blue nodular lesions ltrating in the dermis composed of clustered heterogeneous tumor cells withglandular formation HE magniï¬cation b some tumor cells were detected within vessels or lymphatic vessels HE magniï¬cation c some cells were blue and transparent and mitosis was significant HE magniï¬cation abcFigure Immunohistochemical stain highlighting the tumor cells showing a CK7 b EMA and c TTF1 positive DiscussionSkin metastases suggest the progression of primary cancerand portend a poor clinical prognosis Skin metastases fromlung cancer are rare e percentage of patients with lungcancer that develops cutaneous metastases ranges from to percent [] It is seen more often in men than in women[] It does not show any speciï¬c presentation It is oftenpainless and less likely to be noticed making it more diï¬cultto be diagnosed correctly which may delay treatment Although described cases show that metastatic nodules arepainless our patient showed severe pain e presence ofzosteriform painful vesiclelike lesions really mimics herpeszoster clinically in our casee mechanisms determining the metastasis of lungcancer in skin remain unknown Pathogenesis is suggested tobe by lymphovascular invasion with poor diï¬erentiationand upper lobe tumors increasing the risk [] Usually skinmetastasis develops after initial diagnosis of the primarymalignancy and late in the course of the disease Occasionally skin lesions that arise from lung cancer may develop before the primary tumor is recognized In our case 0cCase Reports in Dermatological Medicine[] R Koca Y Ustundag E Kargi G Numanoglu andH C Altinyazar A case with widespread cutaneous metastases of unknown primary origin grave prognostic ï¬ndingin cancer Dermatology Online Journal vol no p [] N A Babacan S Kilickap S Sene A case of multifocalskin metastases from lung cancer presenting with vasculitictype cutaneous nodule Indian Journal of Dermatologyvol p skin metastases occurred during the immunotherapy Histology shows most commonly adenocarcinoma and thensquamoussmallcell followed by largecell carcinoma []Immunohistochemical markers are useful for the identiï¬cation of the primary cancer or when a shorter diï¬erential isdesired AntiTTF is both sensitive and speciï¬c for primaryadenocarcinomas bronchioalveolar carcinomas and smallcell carcinomas when thyroid primary is excluded []CK7and CK20 are sensitive but not speciï¬c for primaryadenocarcinomas and bronchioalveolar carcinomas eCK7CK20tumors usually include the lung breast endometrium ovary thyroid salivary gland and mesothelioma [ ]Treatment of a single solitary skin lesion usually includessurgery alone or combined with chemotherapy andor radiation If lesions are more disseminated chemotherapy isthe primary option but may elicit an inadequate response[] Radiation can also be used alone andor in combinationwith chemotherapy andor surgery However despite thecombination of radiotherapy and chemotherapy patientswith lung cancer developing cutaneous metastases have apoor outcome Mean survival is short usually to monthsafter diagnosis of cutaneous metastasis []Conflicts of Intereste authors declare they have no conï¬icts of interestAcknowledgmentsis work was supported by a grant from the NationalNatural Science Foundation of China References[] T W Mollet C A Garcia and G Koester Skin metastasesfrom lung cancer Dermatology Online Journal vol no [] S Sun J H Schiller and A F Gazdar Lung cancer in neversmokersa diï¬erent disease Nature Reviews Cancer vol no pp [] S Dreizen H M Dhingra D F Chiuten T Umsawasdi andM Valdivieso Cutaneous and subcutaneous metastases oflung cancer Postgraduate Medicine vol no pp [] M Khaja D Mundt R A Dudekula Lung cancerpresenting as skin metastasis of the back and hand a caseseries and literature review Case Reports in Oncology vol no pp [] W T McSweeney and K Tan Cutaneous metastases as apresenting sign of metastatic NSCLC Journal of Surgical CaseReports vol no [] M H Brownstein and E B Helwig Metastatic tumors of theskin Cancer vol no pp [] R B McGrath S P Flood and R Casey Cutaneous metastases in nonsmall cell lung cancer BMJ Case Reportsvol [] V Jerome Marson J Mazieres O Groussard Expression of TTF1 and cytokeratins in primary and secondaryepithelial lung tumours correlation with histological type andgrade Histopathology vol no pp 0c' | 2 |
thyroid carcinoma is presently the malignancy with the most rapidly increasing incidence in the worldand is the most widely recognized endocrine carcinoma in the western world thyroid cancers derivedfrom follicular thyroid cells can be sorted into papillary thyroid carcinoma ptc follicular thyroid carcinoma ftc and anaplastic thyroid carcinoma atc according to the histological subtype clinicalresults vary across these subtypesthe annual rate of thyroid cancer has more than doubled within the past two decades with the vast majority of this increase being ascribed to ptc which accounts for of all thyroid carcinomas inaddition patients with ptc suffer from cervical lymph nodes metastasis or remote metastasis which leadsto unfavorable results and approximately of cases may progress to a potentially fatal recurrentailment due to these reasons uncovering the causes of ptc and its fundamental mechanisms andfinding molecular biomarkers for early diagnosis and customized treatment are significant and important 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 bsr20201555101042bsr20201555taskswith the advancement and continuous improvement of gene sequencing and geneediting technology it is nowconvenient to recognize the hub biomarkers related to neoplasm metastasis and survival status using a large amountof information available by applying bioinformatics currently there are no effective sensitive biomarkers for earlydiagnosis treatment and prevention of lymph node metastasis of ptc an examination of differentially expressedgenes degs between tumor and paracarcinoma tissue may help identify critical biomarkers of papillary thyroidcarcinoma as a form of molecular marker mrna containing the most abundant genetic information is necessaryfor protein translation and it is separate from the pathological process of cancer at various stages some studiesutilized public databases such as the cancer genome atlas tcga and the gene expression omnibus geo toidentify significant biomarkers of papillary thyroid carcinoma however these investigations were only founded onsingle datasets with constrained sample sizes or just based on online databases used to screen out the degsin the present study we analyzed the degs in ptc tissues versus the matched adjacent tissues by rnaseq andbioinformatics methods to obtain the degs then we screened out the key modules and extracted the key genes inthose modules by constructing degs interaction network then the possible role of differentially expressed geneswas analyzed using go annotation and kegg pathway enrichment analysis the expression validation survivalanalysis and functional enrichment analysis of key genes were conducted by using relevant databases finally wefound that the three genes adora1 apoe and lpar5 were highly expressed in ptc and were associated withptc methylation tnm staging and immune infiltrationmethodstissue samplesthirty pairs of ptc and adjacent tissues were collected from january to july at the first affiliated hospitalof hebei north university this experiment was approved by the ethical committee of the first affiliated hospitaland all patients provided informed consent all tissues were frozen in liquid nitrogen after surgical resectionrna library construction and sequencingtotal rna was isolated from four adjacent normal and cancerous thyroid samples utilizing trizol reagent qiagenvalencia ca usa as indicated by the manufacturers guidelines rnas of ptc tissues and paracancerous tissuessample numbers 1c 1p 2c 2p 3c 3p 4c 4p the number represents different samples the c indicates a cancersample and the p represents a matched paracancerous tissue sample were used six libraries were built utilizingan illumina standard kit as indicated by the manufacturers protocol all sequencing was carried out on an illuminahiseq lc bio chinadifferentially expressed genes screeningthe level of expression of mrnas was evaluated using stringtie by calculating fpkm the degs between ptcand paracancerous tissue were screened with log2 fold change1 and p005 was regarded as statistically significant the analyses were conducted using the r package ballgown functional enrichment analysis and pathway analysisto reveal the functional roles of the degs the annotation visualization and integrated discovery function annotation tool david httpdavidabccncifcrfgovhomejsp was used to perform gene ontology go enrichmentanalysis and kyoto encyclopedia of genes and genomes kegg pathway enrichment analysis p values less than were considered as cutoff criteriappi network construction and identiï¬cation of hub genesppi networks were constructed successively using string database tringdb the interactions ofdegs with a combined score were set as significant and cytoscape software version was utilized tovisualize and analyze the results of the string database to find key hub genes in this ppi network the significantmodule was analyzed by using the plugin mcode of cytoscape software the criteria for selection were set to thedefault the key genes were chosen with degrees ¥ subsequently genes in that module were used to analyse theirfunctional roles with funrich software 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 bsr20201555101042bsr20201555table pcr primersgene symboladora1actinapoelpar5bp base pair f forward primer r reverse primerprimer sequencef5cid3ccacagacctacttccacacc3cid3r5cid3taccggagagggatcttgacc3cid3f5cid3cactcttccagccttccttcc3cid3r5cid3aggtctttgcggatgtccac3cid3f5cid3 gttgctggtcacattcctgg 3cid3r5cid3 gcaggtaatcccaaaagcgaccid3f5cid3 cacttggtggtctacagcttg3cid3r5cid3 gcgtagtaggagagacgaacg3cid3data validation and analysisto verify the accuracy of our rnaseq results we used the gene expression profiling interactive analysis databaseto verify the expression of key genes in ptc and adjacent tissues the overall survival and diseasefree survivalanalyses were performed by kaplanmeier plots for these ptcrelated hub genes genetic alterations of hub genesin ptc and their correlations with other genes were analyzed utilizing the cbioportal for cancer genomics hub genesrelated to clinicopathological features were analyzed using the online database ualcan httpualcanpathuabedu the correlation of adora1 apoe and lpar5 expression with the immune infiltration level in ptc and theexpression of these three genes in different kinds of cancers was performed using the tumor immune estimationresource database for qrtpcr analysis total rna was isolated from normal and cancerous papillary thyroid samples utilizingtrizol reagent qiagen valencia ca usa cdna was synthesized with rna reverse transcription kit tiangenbiotech beijing china qrtpcr was performed with an abi realtime pcr system applied biosystems life technologies usa the expression of the genes of interest was normalized to actin the primers foradora1 apoe lpar5 and actin are shown in table for western blot ripa buffer was used to extract protein from four pairs of tissue from ptc patients and theprotein concentrations were measured via bca methods briefly the sdspage gel was used for electrophoresis andpdvf membrane was used for transmembrane transfer pdvf membrane was blocked and then incubated with primary antiadora1 antibodies dilution bioss bs6649r apoe dilution bioss bs4892r lpar5antibodies dilution bioss bs15366r and actin dilution bioss bs0061r at ¦c overnight followed by incubation with secondary antibodies zhongshanjinqiao dilution at ¦c for h the signal wasdetected using ecl methodstatistical analysisall the data were analyzed by r and spss spss inc usa kaplanmeier method was used to estimate thesignificant difference in survival between the overexpression group and the lowexpression group of key genes inpapillary thyroid carcinoma the statistical difference was set at p resultsdifferentially expressed genes screening based on rnaseqto screen out the genes or modules that may play a role in promoting cancer in papillary thyroid carcinoma weperformed rnaseq experiments on four pairs of thyroid cancer tissues and their matched paracancerous tissues toobtain differentially expressed genes after rnaseq we acquire million reads for each sample the fold changesbetween ptc cancer tissues and matched paracancerous samples were calculated setting the cutoff criterion as pvalue and a fold change there were upregulated and downregulated genes these degswere considered to be candidate genes for subsequent study figure 1a showed the expression of the top genes inptc versus matched paracancerous tissuesfunctional enrichment analysis and pathway analysisconsidering that there were many falsepositive genes among these degs we verified our results one by onethrough the tcga database we found that only genes in our data were consistent with the gene expression of the 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 bsr20201555101042bsr20201555figure identiï¬cation of degs by rnaseqthe heat map a and ppi network of the degs b c the volcano plots of the degs d the most significant module was selectedby mcode in cytoscape red represents the upregulated genes and blue represents the downregulated genesfigure go and kegg pathway enrichment analysis of degs through rnaseqa bubble plot of gene ontology enrichment analysis of degs b bubble plot of kyoto encyclopaedia of genes and genomespathway enrichment analysis of degstcga database to investigate the potential function of these degs in ptc genes functional enrichment was conducted by using go and kegg pathway analyses for the biological process category the degs were significantly involved in the regulation of axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion and urogenital system development thecellular component category results showed ptcrelated degs were enriched in collagencontaining extracellularmatrix synaptic membrane cellcell junction glutamatergic synapse neurontoneuron synapse postsynaptic membrane basolateral plasma membrane degs in molecular function were mainly involved in cell adhesion moleculebinding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycanbinding growth factor binding transmembrane receptor protein kinase activity and transmembrane receptor proteintyrosine kinase activity figure 2aas figure 2b showed the kegg pathway results showed degs were enriched in cytokinecytokine receptorinteraction mapk signaling pathway proteoglycans in cancer rap1 signaling pathway axon guidance cushing 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 bsr20201555101042bsr20201555figure go enrichment analysis and kegg analysis for the key genesa top elements involved in biological processes b top elements involved in molecular function c top elementsinvolved in cellular components d top pathways related to the key genes through kegg analysissyndrome parathyroid hormone synthesis secretion and action agerage signaling pathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ecmreceptor interaction arrhythmogenic right ventricular cardiomyopathyarvc endocrine resistance renin secretion type ii diabetes mellitus bladder cancer nicotinate and nicotinamidemetabolism and apoptosismultiple speciesppi network construction and module analysisppi networks were constructed successively by the database by loading the ptc related dges into the stringdatabase figure 1bc using cytoscape we analyzed the most significant module in the ppi network figure 1dthe ppi network consisted of nodes and edges following the use of mcode in cytoscape the significantmodule was selected the top hub genes adcy8 adora1 adra2c apoe c5ar1 ccl13 ccl20 cdh2chgb cxcl12 eva1a fam20a fn1 gnai1 gpc3 grm4 lpar5 meltf or mfi2 mfge8 nmu oprm1serpina1 sstr3 timp1 and tnc were evaluated by degree in the ppi network figure 1d the resultsshowed that the functions of the key genes were mainly concentrated in signal transduction cell communicationgprotein coupled receptor activity cell adhesion molecule activity and gpcr ligand binding figure data analysis and validationafter the key genes were selected the expression of key genes in ptc and its adjacent tissues were verified by thegepia database figure adora1 apoe eva1a lpar5 mfge8 oprm1 serpina1 sstr3 and timp1were positively related to the overall survival analysis of ptc patients while c5ar1 and gnai1 were negativelyrelated figure adcy8 adora1 chgb fn1 lpar5 nmu and tnc showed positive associations withdiseasefree survival analysis of ptc patients but not apoe figure next we analyzed the alterations of the key genes by using the cbioportal database figure the key geneswere changed in of queried samples figure 7b figure 7a showed the frequency of alterations of eachptc related key gene sstr3 fn1 and adora1 were altered the most and respectively figure 7dshowed the network of the genes and their altered neighbouring genes in ptc patients out of a total of among these genes only adora1 apoe and lpar5 genes simultaneously showed statistical significance foroverall survival analysis and diseasefree survival analysis of ptc patients the qpcr experiments and western blotdata verified that these three survivalrelated genes were all overexpressed in ptc figure then based on ual 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 bsr20201555101042bsr20201555figure validation of the key degs in the gepia databaseadora1 apoe ccl13 cdh2 cxcl12 eva1a fam20a fn1 gnai1 lpar5 mfge8 nmu serpina1 timp1 and tnc areoverexpressed in ptc tissues compared with paracancerous tissue while gnai and gpc3 are downregulated 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 bsr20201555101042bsr20201555figure overall survival analysis of key genes in ptc using kaplanmeier plotsexpression levels of adora1 apoe c5ar1 eva1a fam20a gnai1 lpar5 mfge8 oprm1 serpina1 sstr3 and timp1are related to the overall survival of patients with ptccan the clinical features and degree of methylation of these three genes were analyzed the transcription levels ofadora1 apoe and lpar5 were significantly higher in ptc patients than normal tissues according to subgroupsof sample types individual stages and nodal metastasis status figure in addition ador1 and lpar5 exhibiteda hypomethylation state in the cancer group but apoe showed a hypermethylation state in ptc samples figure10ato further clarify the role of these genes we conducted an analysis of immune infiltration the ador1 expression level was positively corelated with infiltrating levels of b cells r0111 p151e2 cd8 t cells r0246p396e neutrophils r0162 p331e and dcs r0232 p232e the expression of apoe was 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 bsr20201555101042bsr20201555figure diseasefree survival analysis of key genes in ptc using kaplanmeier plotsexpression levels of adcy8 adora1 apoe chgb fn1 lpar5 nmu and tnc are significantly related to the diseasefreesurvival of patients with ptcpositively associated with b cells r0228 p439e cd8 t cells partialcor p930e neutrophils r0197 p114e and dcs partialcor p358e lpar5 expression level was positively related to b cells r0259 p815e cd4 t cells r0238 p103e macrophages r0175p105e neutrophils r027 p142e and dcs r0256 p104e and negatively related to purity r p294e and cd8 t cells r p618e figure 10b these findings stronglysuggested that lpar5 ador1 and apoe may play specific roles in immune infiltration in ptc especially those ofdcs finally we examined the expression of these three genes in common cancer tissues and adjacent tissues and wefound that these three genes were highly expressed in most cancer tissues figure 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 bsr20201555101042bsr20201555figure the key genes expression and mutation analysis in ptc by the cbioportal for cancer genomicsa the genetic alterations of key genes of ptc samples queried genes are altered in of queried patientssamplesb the expression heatmap of key genes c the alteration frequency of key genes in ptc d network of key genesmutations and their frequently altered neighboring genes in ptc 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 bsr20201555101042bsr20201555figure the mrna and protein expressions of adora1 apoe and lpar5 in ptc tissuesac validation of expression levels of adora1 apoe and lpar5 by rtqpcr in cases of ptc and matched adjacent tissuesd adora1 apoe and lpar5 protein levels are increased in four cases of ptc and matched adjacent tissues as measured bywestern blot p0001discussionptc is a common cancer with great heterogeneity in morphological features and prognosis although most papillary thyroid carcinomas exhibit low biological activity there are still a few patients with higher invasive and metastaticclinical features activation of oncogene expression and loss of function of tumor suppressor genes may lead tothe development or progression of ptc to better clarify the molecular mechanism of ptc occurrence development and metastasis we identified key genes related to ptc progression through comprehensive bioinformaticsmethods and we screened three of the ptc prognosisrelated genes for a comprehensive analysisin the present study we identified differentially expressed genes by rnaseq with go enrichment analysis showing that the degs were enriched in the regulation of the axonogenesis regulation of cell morphogenesis extracellular structure anization extracellular matrix anization synapse anization cellsubstrate adhesion urogenital system development collagencontaining extracellular matrix synaptic membrane cellcell junction glutamatergic synapse neuron to neuron synapse postsynaptic membrane basolateral plasma membranecell adhesion molecule binding passive transmembrane transporter activity extracellular matrix structural constituent glycosaminoglycan binding growth factor binding transmembrane receptor protein kinase activity andtransmembrane receptor protein tyrosine kinase activity and kegg pathway results showed degs were enrichedin cytokinecytokine receptor interaction mapk signaling pathway proteoglycans in cancer rap1 signaling pathway axon guidance cushing syndrome parathyroid hormone synthesis secretion and action agerage signalingpathway in diabetic complications growth hormone synthesis secretion and action salivary secretion circadian entrainment cholinergic synapse p53 signaling pathway ecmreceptor interaction arrhythmogenic right ventricularcardiomyopathy arvc endocrine resistance renin secretion type ii diabetes mellitus bladder cancer nicotinateand nicotinamide metabolism and apoptosismultiple speciesto further explore the interrelationship of differentially expressed genes in papillary thyroid carcinoma we constructed a ppi regulatory network a total of degs with nodes greater than were screened out in the networkthe key genes were adcy8 adora1 adra2c apoe c5ar1 ccl13 ccl20 cdh2 chgb cxcl12 eva1afam20a fn1 gnai1 gpc3 grm4 lpar5 meltf mfge8 nmu oprm1 serpina1 sstr3 timp1 andtnc biological process and molecular function analyses of these key degs indicated that they were significantly 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 bsr20201555101042bsr20201555figure relative expression of adora1 apoe and lpar5 in normal thyroid tissues and ptc tissues individual cancerstages and nodal metastasis status respectively ualcanp0001involved in cancer regulation processes such as adjustment of cell growth or maintenance cell immune response celladhesion molecular activity and extracellular matrix structural constituentto verify the credibility of the experiments and data the degs screened were verified by the gepia databaseamong the selected genes genes showed expression differences consistent with our rnaseq data among the 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 bsr20201555101042bsr20201555figure methylation level and immune inï¬ltration level of adora1 apoe and lpar5a relative methylation level of adora1 apoe and lpar5 based on normal thyroid tissues and ptc tissues individual cancerstages and nodal metastasis status respectively ualcan b the correlation between the three genes and tiics timer tiicstumor infiltrating immune cells genes adora1 apoe ccl13 cdh2 eva1a fam20a fn1 lpar5 mfge8 nmu serpina1 timp1and tnc levels were overexpressed in ptc tissues while gpc3 and gnai1 were downregulatedadora1 belongs to the gprotein coupled receptor family and protects human tissues and cells under physiological conditions lin et al suggested that adora1 may promote the proliferation of breast cancer cellsby positively regulating oestrogen receptoralpha in breast cancer cells similarly jayakar indicated that knockdown of apoe expression can reduce the level of mmps by regulating the ap1 signaling pathway and thus reducethe invasion and metastasis of oral squamous cell carcinoma bioinformatics predictions were that apoe mrnashows a significant increase in ptc ccl13 is a coding gene involved in immune regulation and inflammatoryresponses and it has been reported that ccl13 has a role in promoting the proliferation of tumorforming volumein nude mice cdh2 is overexpressed in various cancers some research results indicate that overexpression ofcdh2 can increase the invasive ability and induce emt in lung cancer cells qiu et al confirmed cdh2 actsas an oncogene in papillary thyroid carcinoma which is consistent with our findings eva1a acts as a regulatorof programmed cell death and shen et al indicates that eva1a can inhibit the proliferation of gbm cells by inducing autophagy and apoptosis via inactivating the mtorrps6kb1 signaling pathway fam20a may play a keyrole in haematopoiesis there are few reports on the relationship between fam20a and cancer and our experimentfound that fam20a is more highly expressed in papillary thyroid carcinoma than in other cancers fn1 is involvedin regulating cell adhesion cell movement wound healing and maintaining cell morphology some researchersindicated that fn1 participates in regulating many types of cancer progression such as gastric cancer skin squamous cell carcinoma and papillary thyroid carcinoma it has been shown that lpar5 is related tothe pathogenesis of pancreatic cancer consistent with our study zhang et al believes that lpar5 may be involved in the development of papillary thyroid carcinoma according to previous reports mfge8 is involvedin the progression of various malignancies such as breast cancer melanoma bladder tumors and ovarian cancer 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 bsr20201555101042bsr20201555figure the expression of adora1 apoe and lpar5 in thyroid cancer tissues and normal thyroid tissuesthe three genes expression were analyzed in different kind of cancer tissues and normal tissues via the timer database p005p001 p001 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 bsr20201555101042bsr20201555[] mfge8 is considered to be a potential therapeutic target for ovarian cancer owing to its carcinogenic effect consistent with our data zhang et al indicate that nmu is one of the degs of papillary thyroid carcinoma recently a researcher has shown that abnormal expression of nmu is associated with a variety of cancers for serpina1 there are currently six s pointing out that serpina1 may be a key gene for ptc consistentwith our results [] clinical studies have shown that high expression of timp1 is positively correlated witha poor prognosis of colon brain prostate breast lung and several other cancers tnc is a component of theextracellular matrix ecm and is closely related to the malignant biological behavior of cancer in particular tncoverexpression is positively associated with liver cancer oral squamous cell carcinoma and lymph node metastasisof breast cancer gpc3 belongs to the glypicans family it has been reported that overexpression of gpc3can promote the metastasis of hepatocellular carcinoma but we found that it is expressed at low levels in ptcsimilar to gpc3 some scholars believe that gnai1 is a tumorpromoting gene and reported upregulated gnai1mrna in human glioma which is inconsistent with our data only the adora1 apoe and lpar5 genes simultaneously showed statistical significance for overall survivaland diseasefree survival of ptc patients considering that the occurrence and metastasis of cancer is a complexand multiregulated process we further analyzed the regulatory mechanisms of these three genes we found thatthe mrna and methylation levels of these three genes were significantly correlated with tnm staging in additionadora1 apoe and lpar5 were all related to immune infiltration especially to dendritic cells finally we foundthat these three genes were more highly expressed in cancer tissues than matched adjacent tissueshowever our research has certain limitations first only four pairs of cancer and adjacent tissues were analyzedusing rnaseq in this experiment so further research requires a larger sample size second further experiments areneeded to validate the specific mechanisms of these key genesdata availabilitythe data used to support the findings of this study are available from the corresponding author upon requestcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis study was supported by grants from the hebei provincial department of finance specialist capacity building and specialistleadership program [grant number ] the hebei provincial natural science foundation project [grant number h201840505]and the hebei north university basic research business expenses project [grant number jyt2019015]author contributionxu lin conducted the bioinformatics analysis xu lin and gang xue contributed as first authors xu lin wrote the manuscriptjingfang wu and gang xue critically revised the gang xue and da pei obtained clinical specimens and the others contributed to verification of the rnaseq resultsabbreviationsatc anaplastic thyroid carcinoma ecm extracellular matrix ftc follicular thyroid carcinoma ptc papillary thyroid carcinomareferences kitahara cm and sosa ja the changing incidence of thyroid cancer nat rev endocrinol 101038nrendo2016110 aschebrookkilfoy b ward mh sabra mm and devesa ss thyroid cancer incidence patterns in the united states by histologic type thyroid 101089thy20100021 pourseiraï¬ s shishehgar m ashraf mj and faramarzi m papillary carcinoma of thyroid with nasal cavity metastases a case report iranj med sci ullmann tm gray kd moore md zarnegar r and fahey iii tj current controversies and future directions in the diagnosis andmanagement of differentiated thyroid cancers gland surg 1021037gs20170908 jin x deng b ye k et al comprehensive expression proï¬les and bioinformatics analysis reveal special circular rna expression and potentialpredictability in the peripheral blood of humans with idiopathic membranous nephropathy mol med rep 103892mmr201910671 rapisuwon s vietsch ee and wellstein a circulating biomarkers to monitor cancer progression and treatment comput struct biotechnolj 101016jcsbj201605004 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 bsr20201555101042bsr20201555 pertea m pertea gm antonescu cm et al stringtie enables improved reconstruction of a transcriptome from rnaseq reads natbiotechnol 101038nbt3122 frazee ac pertea g jaffe ae et al ballgown bridges the gap between transcriptome assembly and expression analysis nat biotechnol 101038nbt3172 von mering c huynen m jaeggi d et al string a database of predicted functional associations between proteins nucleic acids res 101093nargkg034 chandrashekar ds bashel b balasubramanya sah et al ualcan a portal for facilitating tumor subgroup gene expression and survivalanalyses neoplasia 101016jneo201705002 li t fan j wang b et al timer a web server for comprehensive analysis of tumorinï¬ltrating immune cells cancer res e108e11010115800085472can170307 nikiforov ye and nikiforova mn molecular genetics and diagnosis of thyroid cancer nat rev endocrinol 101038nrendo2011142 pusztaszeri m and auger m update on the cytologic features of papillary thyroid carcinoma variants diagn cytopathol 101002dc23703 borea pa gessi s merighi s and varani k adenosine as a multisignalling guardian angel in human diseases when where and howdoes it exert its protective effects trends pharmacol sci 101016jtips201602006 lin z yin p reierstad s et al adenosine a1 receptor a target and regulator of estrogen receptoralpha action mediates the proliferativeeffects of estradiol in breast cancer oncogene 101038onc2009409 jayakar sk loudig o brandweingensler m et al apolipoprotein e promotes invasion in oral squamous cell carcinoma am j pathol 101016jajpath201706016 tan j qian x song b et al integrated bioinformatics analysis reveals that the expression of cathepsin s is associated with lymph 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Journal of International Medical Research The Authors reuse guidelinessagepubcomjournalspermissions journalssagepubcomhomeimrCase ReportNivolumab plus gemcitabinedexamethasone and cisplatinchemotherapy induce durablecomplete remission inrelapsedrefractory primarymediastinal Bcell lymphomaa case report andliterature reviewGang Huang1 Ju Huang2 Zhili Zhang2Chongchong Xue1 and Yuan Liu2AbstractPrimary mediastinal large Bcell lymphoma PMBCL is an uncommon but aggressive type ofBcell lymphoma Patients with relapsed refractory PMBCL rrPMBCL have limited therapeuticoptions and usually have a relatively poor outcome Immune checkpoint blockade has become apotential treatment for this disease We report here a case of a female patient with rrPMBCLwho was treated with nivolumab plus gemcitabine dexamethasone and cisplatin GDP chemotherapy Complete remission was achieved after four cycles of combined therapy With continuednivolumab maintenance monotherapy she has remained in complete remission for longer than months This is the first report of nivolumab plus GDP chemotherapy inducing completeremission in patient with rrPMBCL This case supplements the limited literature and providesimplications for clinical trial designs regarding the potential use of nivolumab in the treatment ofrrPMBCL1Department of Hematology Yuebei Peoples HospitalShantou University Medical College ShaoguanGuangdong Province China2Guangdong Women and Children Hospital GuangzhouGuangdong ChinaCorresponding authorYuan Liu Medical Genetic Centre Guangdong Womenand Children Hospital No Xingnan Rd PanyuDistrict Guangzhou Guangdong ChinaEmail yuanliu005163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical ResearchKeywordsRelapsed refractory primary mediastinal Bcelllymphoma nivolumab checkpoint blockadegemcitabine dexamethasone cisplatin chemotherapy programmed cell death completeremissionDate received February accepted July IntroductionPrimary mediastinal large Bcell lymphomaPMBCL is an uncommon but aggressivetumor that accounts for to of nonHodgkin lymphoma1 PMBCL is distinguished from diffuse large Bcell lymphomaby virtue of distinct clinical pathologicaland genetic features2 Recently PMBCLwas listed as a separate entity in the latestWorld Health anization classiï¬cation of hematopoieticand lymphoidtumors5 PMBCL has a similar clinical presentation as classical Hodgkin lymphomacHL and PMBCL also shares certain features at the molecular level particularly9p241 alterations and programmed celldeath protein ligand 1ligand PDL1PDL2 expression6 At present management and outcome of PMBCL are still critical and a more serious situation is faced bypeople who are diagnosed with relapsedand refractory PMBCL rrPMBCL19The optimal salvage chemotherapy andautologousforrrPMBCL are of limited efï¬cacy19stem celltransplantRecently agents targeting programmedcell death PD1 and PDL1 have beenimmunotherapy10developed in tumorAntiPD1 therapy with monoclonal antibodies has been approved for the treatmentof several types of solid tumor and cHLThe therapeutic potential of antiPD1 therapy in other malignancies is likely to beapproved soonIn a humanizedimmunoglobulin G1 recombinant monoclonal antibody for the PD1 receptor pidilizumab was approved by the US Food andDrug Administration FDA for treatingandpediatricpatientsthatwithadultrrPMBCL11 Another agenttargetsthe PD1 receptor called nivolumab isa fully humanized immunoglobulin G4monoclonal antibody that has been grantedapproval by the US FDA for treating several solid malignancies and cHL The therapeutic efï¬cacy of nivolumab in patientswith rrPMBCL remains unclearWe report here a patient with rrPMBCLwho received combined treatment with offlabel nivolumab and GDP chemotherapyComplete remission CR was achievedafter four cycles of such combined treatment At the time of this submission thepatient has remained in CR for longerthan months with continued nivolumabmaintenance monotherapyCase reportA 32yearold woman presented to YuebeiPeoples Hospital with intermittent dyspneaand chest pain A positron emission tomography PET scan showed a 10cm mass inthe anterior superior mediastinum with astandardized uptake value of Themass showed unclear margins and compressed the ascending aorta and pulmonarytrunk Small pericardial and left pleuraleffusions were also observedThe mass was diagnosed as PMBCL by asubsequent biopsy Immunohistochemicalstaining showed thatlarge lymphocyteswere positive for CD20 CD79a Pax5BCL6 CD23 CD30and multiplemyeloma1 and negative for CD10 CD3CD5chromogranin Asynaptophysin 0cHuang et alterminalencodingandincludingtwocycles positiveregion inendomysialdeoxynucleotidyltransferase cytokeratin CK CK19 andandS100 Ki67 wassituEpsteinBarrhybridization was negative She was initially treated with six cycles of frontline chemotherapyofrituximab cyclophosphamide doxorubicinvincristine and prednisolone RCHOPand four cycles of doseadjusted etoposidecyclophosphaprednisolonemiderituximabDAEPOCHR were administered Thetimeline of treatment is shown in aShe received tumor resection by thoracoscopic surgery after she continued twodoxorubicinvincristinecycles ofgemcitabine dexamethasonecisplatinumetoposide and rituximabtherapy Her ï¬rst CR was achieved inDecember However monthslater a PETcomputed tomography CT scan showedhypermetabolic lesions located at the leftlung and right adrenal gland but not inthe primary mediastinal site bThe patient reported no physical symptomsand received a repeat tissue biopsy whichconï¬rmed a relapse with PMBCL She wastreated with each cycle of a dexamethasoneifosfamide cisplatin and etoposide regimenand ibrutinib bendamustine and cytarabine therapy A chest CT scan showedFigure Summary of treatment and monitoring the tumor response a Patients timeline chart with thedates of treatment and monitoring the tumor response b Positron emission tomography images Upperpanel a scan of the relapsed hypermetabolic lesions located at the left lung and right adrenal gland beforecombined treatment Lower panel complete remission was achieved after four cycles of nivolumab plusGDP chemotherapyRCHOP rituximab cyclophosphamide doxorubicin vincristine and prednisolone DAEPOCHR doseadjusted etoposide prednisolone vincristine cyclophosphamide doxorubicin and rituximab GDPERgemcitabine dexamethasone cisplatinum etoposide and rituximab CR complete remission PMBCLprimary mediastinal large Bcell lymphoma DICE dexamethasone ifosfamide cisplatin and etoposideIBC ibrutinib bendamustine and cytarabine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical Researchthat the right adrenal gland lesion had partially responded while the lesions in the leftlung had progressed After those cycles ofchemotherapy the patient showed GradeIV myelosuppression and had to receiveblood transfusion treatment Moreover acerebrospinal ï¬uid examination showedthe presence of atypical lymphocytes andno symptoms of infection of the central nervous system were observed Intrathecal chemotherapy cytarabine mg methotrexate mg and dexamethasone mg was thenadministered and no atypical lymphocyteswere detected by repeated cerebrospinalï¬uid analysis These ï¬ndings highly suggested a potential risk of metastasis of thecentral nervous systemtreatmentBecause the disease had progressed withsevere myelosuppression and there were nostandard chemotherapy guidelines or alternative treatment options for the patientother salvage treatments of her refractorydisease needed to be considered Aftermuch discussion with the patient and herfamily she declined autologous hematopoietic stem cell transplantation and receivedcombinedgemcitabine mg dexamethasone mg and cisplatinum mg GDPchemotherapyand the offlabel antiPD1 antibody nivolumab mg After four cycles of combined treatment a repeated PETCT scanshowed thatshe had secondary CRb She received two more cyclesof combined treatment with nivolumab andGDP chemotherapy and then continuedsingle nivolumab maintenance treatment mg Since her ï¬rst dose in May she received doses of nivolumab Shereported moderate fatigue and pyrexia in to days after each administration ofnivolumab Blood tests indicated normalfunction of the liver kidney and thyroidFigure She also had normal bloodlevels of creatinine albumin globulin lactate dehydrogenase aspartate transaminasetotalaminotransferaseofalaninethey2b Neutrophilbilirubin and urea nitrogen during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the ï¬rst four combined therapies because of toxicity of GDPchemotherapy butrecovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2cFurthermore no adverse signs and symptoms were observed in the lungs brainand skin At the time of this submissionshe has remained in CR for longer than months with continued nivolumab maintenance therapyEthics approval was obtained from theethicalcommittee of Yuebei PeoplesHospital Written informed consent wasobtained from the patient for analysis ofthe samples and publicationDiscussionTreatment and outcome are critical in managing PMBCL Because there is no established standard approachthe ï¬rstlinetreatment of PMBCL is generally thesame as that for diffuse large Bcell lymphoma including RCHOP and DAEPOCHR Relapse of PMBCL usually occurs in theï¬rst to months after completion offrontline therapy with a lower incidenceapproximately than diffuselarge Bcell lymphoma19 There are varioussecondlineregimens for patients with rrPMBCL includingthe rituximab ifosfamide carboplatin andetoposide regimenthe rituximab dexamethasone cytarabine and cisplatin regimen and rituximabGDP12 Because of alack of standard guidelines or treatmentoptions for PMBCL the outcome greatlydepends on the patients response to theregimen Thisremains poordespite these secondline salvage chemotherapies and subsequent autologous hematopoietic stem cell transplantation912immunochemotherapyresponse 0cHuang et alFigure Blood test values during the whole treatment process since the first dose of nivolumab The firstfour cycles were nivolumab plus GDP chemotherapy and nivolumab maintenance monotherapy wasadministered since the fifth cycle a Thyroxine thyrotropin FT3 and FT4 levels b Levels of creatininealbumin globulin lactate dehydrogenase aspartate transaminase alanine aminotransferase total bilirubinand urea nitrogen c Neutrophil and platelet countsFT4 free thyroxine FT3 free triiodothyronine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical ResearchIn recent years strategies focusing on thecheckpoint blockade have been developedin tumor immunotherapy10 Therapeuticantibodies targeting the PD1PDL1 axispossess clinical activity and an acceptablesafety proï¬le in treating a growing list oflymphomas13solid tumors and BcellBased on a clinical study of patientswith rrPMBCL pidilizumab was approvedby the US FDA for treatment of adult andpediatric patients with rrPMBCL in Another antibody nivolumab has beengranted approval for treating several solidmalignancies and cHL However studiesregarding application of nivolumab forPMBCL are limited Only ï¬ve reportshave described using nivolumab for treatment of PMBCLrrPMBCL Table asfollows In a phase I study published intwo patients with PMBCL wererecruited and treated with nivolumab atdoses of or mgkg every weeks afterprevious systemic treatments14 No objective responses were observed in this previous study In another phase I study onewithrefractorypatient with PMBCL received combinedtherapy of nivolumab and ipilimumaband died during the therapeutic process15Recently two reports showed the potentialtherapeutic efï¬ciency of nivolumab forpatientsPMBCLrrPMBCL who showed failure with conventional immunochemotherapy1617 Both ofthese two cases had immunerelated adverseeffects during the antibody treatment process One patient with highgrade neutr ia had nivolumab stopped temporarilyand was treated with intravenous immunoglobulin16 The other patient with zosterreactivation was controlled by administration of valacyclovir17 Recently Zinzaniand colleagues showed that combined treatment of nivolumab and brentuximab vedotin had promising antitumor activity and amanageable safety proï¬le in patients withrrPMBCL18 In this phase II study patients were recruited and treated withnivolumab mgkg and brentuximab vedotin mgkg every weeks The objectiveresponse rate was and achievedTable Reports regarding application of nivolumab in primary mediastinal large Bcell lymphomarelapsedand refractory primary mediastinal large Bcell lymphomaNumberof cases DoseCombinedtreatmentAdverse events or mgkg mgkgIpilimumabResponseyesnoNoNo ofpatientsReportsLesokhin AMet al Ansell S et alWright Zet al Yassin R et alZinzani mgkgNoNoHighgrade neutr iaYesZoster reactivationYes mgBrentuximabNeutr iaPL et al vedotinthrombocyt iaand peripheralneuropathy of patientsPresent case mgkgGDPMild fatigue and pyrexiaYeschemotherapyNote means not indicated in the report 0cHuang et alofcyclescombinedCR and achieved partial remission Of patients of them had drugrelatedadverse events and the most common wereneutr ia thrombocyt ia and peripheral neuropathy18 In the present case weattempted several available approaches intreating the patients relapsed disease butfailed to control the progress of the massAfter much discussion with the patient andher family we considered an offlabel nivolumab and GDP chemotherapy as salvagetreatmentfor the patient In September her second CR was achieved afterfourtreatmentCurrently with continued nivolumab maintenance monotherapythe patient hasremained in CR for longer than monthsImmunerelated adverse events that areassociated with checkpoint blockade oftenstart within the ï¬rst few weeks to monthsafter treatment but can occur any time andin any an The most common immunerelated adverse events are hypothyroidismnausea diarrhea pyrexia and fatigue1920In the present case we were concernedabout immunerelated an damage sincethe ï¬rst dose of nivolumab The patientreported moderate fatigue and pyrexiaafter each administration of nivolumaband soon recovered within to daysBlood testing was performed during thewhole therapeutic process and the datawere reviewed and analyzed Blood levelsof thyroxine thyrotropin free triiodothyronine and free thyroxine indicated no thyroiditisFigure 2a Our patient alsoshowed normal metabolic data during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the ï¬rst four combined therapies because of toxicity of GDPchemotherapy but they then recovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2c2b NeutrophilUnlike otherarelymphomas prognosticbiomarkersinPMBCL12 Some serum molecules such aslackinglargelyCCL17 and CD163 are considered aspotential biomarkers for predicting andmonitoring responses and detection ofrelapses in patients with Hodgkin lymphoma1221 The role of serum biomarkers inPMBCL remainsinvestigatedRadiological imaging should only be usedin patients who have new clinical symptomsor signs suggestive of relapse but not inasymptomatic patients922betoTo the best of our knowledge this is theï¬rst reported case of nivolumab plus GDPchemotherapy that induced CR with nosevere immunerelated an damage in apatient with rrPMBCL We also reportthe longest followup observation of successful application of nivolumab in apatient with rrPMBCLThis report supplements the limited literature of nivolumab fortreatment ofPMBCLrrPMBCL and provides implications for clinical trial design regarding thepotential use of nivolumab in treatment ofrrPMBCL Further investigation needs to beperformed for potential application of singleor combined use of nivolumab for patientswith rrPMBCL who experience failure withconventional therapeutic approachesDeclaration of conflicting interestThe authors declare that there is no conï¬ict ofinterestFundingThis research received no speciï¬c grant from anyfunding agency in the public commercial ornotforproï¬t sectorsorcid000000034880ORCID iDYuan LiuReferences Martelli M Ferreri A Di Rocco A et alPrimary mediastinal large Bcell lymphomaCrit Rev Oncol Hematol 0cJournal of International Medical Research Savage KJ Monti S Kutok JL et al Themolecular signature of mediastinallargeBcell lymphoma differs from that of otherdiffuse large Bcell lymphomas and sharesfeatures with classical Hodgkin lymphomaBlood Rosenwald A Wright G Leroy K et alMolecular diagnosis of primary mediastinalB cell lymphoma identiï¬es a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma J ExpMed Mottok A Wright G Rosenwald A et alMolecular classiï¬cation of primary mediastinal large Bcell lymphoma using routinelyavailable tissue specimens Blood Swerdlow SH Campo E Pileri SA et alThe revision ofthe World Healthanization classiï¬cation of lymphoid neoplasms Blood XuMonette ZY Zhou J and Young KHPD1 expression and clinical PD1 blockadelymphomas Blood in Bcell Van Roosbroeck K Ferreiro JF TousseynT et al Genomic alterations of the JAK2and PDL loci occur in a broad spectrum oflymphoid malignancies Genes ChromosomesCancer rearrangements Twa DD Chan FC BenNeriah S et alGenomicinvolving programmed death ligands are recurrent in primary mediastinallymphomaBlood large Bcell Cwynarski K Marzolini MAV BarringtonSF et al The management of primary mediastinal Bcell lymphoma a British Societyfor Haematology Good Practice Paper BrJ Haematol Ribas A and Wolchok JD Cancer immunotherapy using checkpoint blockade Science HematologyOncology Cancer Approvals Safety Notiï¬cations Available online URL wwwfdagovdrugsinformationondrugsapproveddrugsucm610670htmdrugsapproveddrugsucm610670htmwwwfdagovdrugsinformationon Lees C Keane C Gandhi MK et al Biologyand therapy of primary mediastinal Bcelllymphoma current status and future directions Br J Haematol Goodman A Patel SP and Kurzrock RPD1PDL1 immunecheckpoint blockadein Bcell lymphomas Nat Rev Clin Oncol Lesokhin AM Ansell SM Armand P et alNivolumab in patients with relapsed orrefractory hematologic malignancy preliminary results of a phase Ib study J Clin Oncol Ansell S Gutierrez ME Shipp MA et alA phase study of nivolumab in combination with ipilimumab for relapsed or refractory hematologic malignancies CheckMate Blood Wright Z and Brown A Highgrade neutr ia in a patient successfully treated withnivolumab for refractory primary mediastilymphoma Blood Adv nal Bcell Yassin R Hajeer A Alshieban S et al HLAgenotype and response to nivolumab therapy in relapsed refractory primary mediastinal Bcell lymphoma Curr Res Transl Med Zinzani PL Santoro A Gritti G et alNivolumab combined with brentuximabvedotin forrelapsedrefractory primarymediastinal large Bcell lymphoma efï¬cacyand safety from the Phase II CheckMate Study J Clin Oncol Postow MA Sidlow R and Hellmann MDImmunerelated adverse events associatedwith immune checkpoint blockade N EnglJ Med Zinzani PL Ribrag V Moskowitz CH et alSafety and tolerability of pembrolizumab inpatients with relapsedrefractory primarylymphoma Bloodmediastinal large Bcell Jones K Vari F Keane C et al SerumCD163 and TARC as disease response biomarkers in classical Hodgkin lymphomaClin Cancer Res for Cheson BD Fisher RI Barrington SF et alinitial evaluationRecommendationsstagingofHodgkin and nonHodgkin lymphoma theLugano classiï¬cation J Clin Oncol assessmentandresponse 0c' | 2 |
Inflammation plays a leading role in the pathogenesis of nephrolithiasis The association of the dietary inflammatory index DII with urinary lithogenic factors is unclear This study aimed to evaluate the relation of DII to urinary risk factors of kidney stones formationResults Of participants n n n n and n had hyperoxaluria hypercreatininuria hypercalciuria hyperuricosuria hypocitraturia respectively There was a significant increasing trajectory in urinary calcium uric acid and creatinine as well as a decreasing trend in urinary citrate across tertiles of DII score all P After multivariate adjustment for energy intake age physical activity and body mass index high DII scores were associated with elevated odds of having hypercreatininuria OR 95CI Ptrend hypercalciuria OR 95CI Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend No association was identified between DII and hyperoxaluriaKeywords Dietary inflammatory index Kidney stones Hypercalciuria Hypocitraturia Hyperoxaluria Hyperuricosuria HypercreatinuriaIntroductionNephrolithiasis is one of the most prevalent urologic disorders and impose a substantial burden on human health globally [] The high recurrence rate of kidney stones is yet unsolved [ ]Thus there is an urgent need to target modifiable risk factors to prevent the development and recurrence of renal stonesHigher urinary excretions of oxalate calcium creatinine and uric acid as well as lower excretions of citrate are potential modifiable a0 urinary lithogenic risk factors Correspondence mina_mirzaei101yahoocom Department of Community Nutrition School of Nutritional Sciences and Dietetics Tehran University of Medical Sciences TUMS Tehran PO Box IranFull list of author information is available at the end of the involved in the formation of kidney stones [] Inflammation is also another mechanism which plays a leading role in the pathogenesis of nephrolithiasis [] Dietary modifications toward decreasing inflammation may have a potential to prevent kidney stones or their recurrence Several micronutrients or foods such as magnesium vitamin E vitamin C carotenoids fruits and fish had an antiinflammatory impact [] In contrast simple sugars red meats highfat dairy products and refined grains are associated with elevated inflammatory markers [] Nevertheless nutrients or foods are not consumed separately but as part of the whole diet [] The Dietary Inflammatory Index DII is developed to measures the overall inflammatory potential of diets [] which has been recognized to be related to the biomarkers of inflammation [] A proinflammatory diet has 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 0cMaddahi a0et a0al BMC Res Notes Page of been found to be related to the reduced kidney function [] However there is no study investigating the relation of DII to urinary lithogenic factors Therefore this study aimed to assess the association of DII with hypercalciuria hypocitraturia hyperoxaluria hyperuricosuria and hypercreatinuria in patients with nephrolithiasisMain textMethodsSubjectsThis crosssectional study was performed on a total of stone former men aged a0years in Tehran Iran in Participants were recruited from the Urology Research Center of Sina Hospital Tehran Iran Inclusion criteria for this study were having a history of kind stone formation and age ¥ a0years People with a history of thyroid disease fatty liver disease malignancy stroke diabetes cardiovascular disease and hypertension were excluded Participants who were on medications such as corticosteroids diuretics anticancer drugs multivitamins potassium citrate calcium and vitamin D or C supplements were not eligible for this study Furthermore all alcohol drinkers and drugs abusers were excluded Patients were included in the study after signing written informed consentsDietary assessmentUsual food intake of patients during the previous year was measured by a validated semiquantitative 168item food frequency questionnaire FFQ[] DII was calculated using the method reported by Shivappa et a0al [] The DII is based on scientific papers scoring food parameters based on whether they elevated reduced or had no impact on six inflammatory biomarkers [Creactive protein interleukin IL1 beta IL10 IL4 IL6 and tumor necrosis factoralpha As mentioned Shivappa et a0 al calculated DII according to the food parameters As dietary patterns of different populations are different with each other some food parameters used in the study by Shivappa may not be available in different FFQs Hence researchers calculate DII according to available foods in the FFQ by modification of the method used by Shivappa et a0al [] In the current study the DII score was calculated using the corresponding food parameters available from the FFQ used in our study This approach has been used broadly in the previous studies [] The DII score was calculated with the use of the corresponding nutrients or food parameters available from the FFQ including energy protein total fat carbohydrate dietary fiber monounsaturated fatty acids n3 fatty acids n6 fatty acids polyunsaturated fatty acids saturated fatty acids cholesterol trans fatty acids vitamin A thiamin niacin riboflavin Vitamin B6 folate vitamin B12 vitamin E vitamin C Vitamin D bcarotene iron magnesium zinc selenium as well as caffeine onion greenblack tea paper and garlic The inflammatory effect scores for dietary components used for calculation of DII in this study are reported in Additional file a0 Table a0S1 To calculate the DII score for each participant the mean intake of each nutrient or food parameter was standardized by subtracting mean global intake of food items from the actual individuals intake and dividing it by the global SD to create a zscore Zscore is used to express an individuals exposure relative to the standard global exposure This approach both anchors the individuals exposure to a robust range of dietary patterns in a variety of cultural traditions and obviates completely the problem of noncomparability of units because the a0Zscores is independent of the units of measurement These zscores then were converted to proportions and centered by multiplying values by and subtracting to normalize the scoring system and to avoid skewness The centered percentile values for food items were then multiplied by the corresponding food itemspecific inflammatory effect scores to obtain the food itemspecific DII scores Calculation of DII for carbohydrate intake in a participant in our study as an example for DII calculation is presented in Additional file a0 Table a0S2 similar approach was followed for the calculation of DII for other nutrients Information about global daily mean intake standard deviation for global intake and overall inflammatory effect score of all nutrientsfood items used for DII calculation is reported in the study by Shivappa et a0al [] The overall DII score for each individual was calculated by summing food itemspecific DII scores [] Higher DII scores indicate a more proinflammatory diet while lower DII scores indicate a more antiinflammatory dietMeasurements of a0study outcomesThe 24h urine samples were collected from all participants and urine was analyzed using an AutoAnalyzer as described previously [] Hyperoxaluria a0 was a0 defined a0 as a0 the urinary oxalate Ë a0 mgday hypocitraturia as a0urinary citrate of a0mgday hyperuricosuria as urinary uric acid over a0 gday hypercreatininuria as urinary creatinine of Ë a0 mgday and hypercalciuria as a0a urinary calcium ¥ a0mgday []Measurement of a0other variablesGeneral Information was obtained using interview Physical activity was measured using of a0 the a0 International a0Physical a0Activity a0Questionnaires a0IPAQ [] Body weight was measured in minimal clothing after removal of shoes by a digital scale Seca Germany with a precision about a0kg Height of individuals was assessed in 0cMaddahi a0et a0al BMC Res Notes Page of standing position without shoes using a calibrated stadiometer Seca Germany to the nearest a0cm BMI was calculated as weight divided by the square of height kgm2Statistical analysesDII was categorized into tertiles T1 to T2 to T3 to Analysis of variance ANOVA and Chi square tests were used to compare continuous and nominalordinal variables across tertiles of DII respectively Continuous variables are reported as mean ± SE and nominalordinal variables as frequency Odds ratio OR and confidence interval CI for the relation of DII to study outcomes was calculated using the logistic regression analysis Statistical significance was set at p for all tests All analyses were undertaken using the statistical Package for Social Science Version SPSS Inc Chicago IL USAResultsParticipants in the highest tertile of the DII had significantly higher total daily energy intake P and lower physical activity P than those in the other tertiles There was a significant increasing trajectory in urinary calcium P uric acid P and creatinine P and a decreasing trend in urinary citrate P across tertiles of DII score Table a0DII score and a0urinary lithogenic factorsIn the crude model it was found that higher adherence to the DII was significantly related to the increased odds of hypercreatininuria OR 95CI Ptrend hypercalciuria OR 95CI Table Characteristics of a0the a0study participants across a0tertiles of a0the a0DII scoreAge yearHeight cmWeight kgBMI kgm2Physical activity scoreEnergy intake kcaldayUrinary creatininekg weight mgdaykgTotalN ± ± ± ± ± ± ± ± ± ± ± Dietary inflammatory index scoreTertile n ± ± ± ± ± ± ± ± ± ± ± Tertile n ± ± ± ± ± ± ± ± ± ± ± Tertile n ± ± ± ± ± ± ± ± ± ± ± P value Urinary citrate mgdayUrinary oxalate mgdayUrinary uric acid gdayUrinary calcium mgdayJob status Engineerphysician Clerk Student Teacher Selfemployed Retired Worker UnemployedMarital status Married SingleEducation level Illiterate Diploma University degreeCategorical variables are presented as frequency n and continuous variables as mean ± SE Oneway ANOVA was used for continuous variables and persons Chi square test for categorical variables 0cMaddahi a0et a0al BMC Res Notes Page of Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend After multivariate adjustment for energy intake age physical activity and BMI high DII scores were associated with elevated odds of having hypercreatininuria OR 95CI hypercalciuria OR 95CI Ptrend hyperuricosuria OR 95CI Ptrend and hypocitraturia OR 95CI Ptrend The relation of DII to hypercreatininuria hyperoxaluria and hyperuricosuria was not significant after adjustment for carbohydrate fiber and protein intake Table a0 Ptrend adjustment for covariates dietary intakes of protein and fiber were slightly related to the decreased odds of hypocitraturia Protein OR 95CI fiber OR 95CI and hypercalciuria Protein OR 95CI fiber OR 95CI while the intake of protein and fiber was not to associated with hypercreatininuria hyperoxaluria and hyperuricosuriaDiscussionWe revealed that in stone former men a diet with a high DII is significantly related to the increased odds of having hypercreatininuria hypercalciuria hyperuricosuria and hypocitraturia but not to hyperoxaluriaIt has been confirmed that kidney stone formers could be susceptible to recurrence in stones formation We also evaluated the association of dietary protein and fiber intake on urinary risk factors Table a0 After Table Univariate and a0 multivariate logistic regression models for a0 the a0 relation of a0 DII score to a0 urinary risk factors of a0kidney stone formationDietary inflammatory index scoreModel Crude modelModel Model Model Odds ratio CIOdds ratio CIOdds ratio CIOdds ratio CIHypercreatininuria T1 T2 T3 P value for trendHypocitraturia T1 T2 T3 P value for trendHyperoxaluria T1 T2 T3 P value for trendHyperuricosuria T1 T2 T3 P value for trendHypercalciuria T1 T2 T3 P value for trend Model adjusted for energy intakeModel additionally adjusted for age BMI and physical activityModel adjusted for carbohydrate fiber and protein intake 0cMaddahi a0et a0al BMC Res Notes Page of Table Multivariate logistic regression models for a0the a0relation of a0dietary fiber and a0protein intake as a0continues variable to a0urinary risk factors of a0kidney stone formationFiberOdds ratio CIProteinOdds ratio CIModel Model Model Model HypercreatininuriaHypocitraturiaHyperoxaluriaHyperuricosuriaHypercalciuriaModel adjusted for energy intakeModel additionally adjusted for age BMI and physical activitybecause of unhealthy dietary patterns [] Inconsistent with our finding a study did not report any significant difference in creatinine across tertiles of DII in subjects with chronic kidney disease [] A randomized controlled trial a0 study by Noori et a0 al [] on recurrent stone formers showed that a a0DASH diet which in contrast to a diet with a high DII is featured by a high intake of whole grains fruits lowfat a0 dairy products and vegetables and a low intake of total fat cholesterol saturated fat meat and refined grains is significantly associated with a decrease in calcium oxalate supersaturation and an increase in citrate excretion Moreover another study reported that greater adherence to the Mediterranean a0dietary pattern a0is related to the reduced risk for incident kidney stones [] The relationship between systemic inflammation and nephrolithiasis has been identified previously [] Since both DASH and Mediterranean diets attenuate a0 inflammation [ ] the protective effects of these dietary patterns on kidney stones formation may be mediated at least partly by reducing systemic inflammation A crosssectional study conducted on diabetic patients also reported that higher intake of vegetable and fish dietary pattern is related to a lower creatinine rates [] Vegetables and fish as components of DII are identified to have antiinflammatory effects [ ] The DII is a tool to assess the overall impact of a diet on inflammatory potential [] and is associated with markers of systemic inflammation including such as IL6 [] and CRP [] [] IL6 and CRP are two of the inflammatory biomarkers considered in the calculation of DII [] It has been revealed that the DII score is inversely related to the Dietary Approaches to Stop Hypertension Score DASH Mediterranean Diet Score and Healthy Eating Index2010 [ ] Taken together these findings support that a likely mechanism for the relation of DII scores to hypercreatininuria hypercalciuria hyperuricosuria and hypocitraturia could be explained by the higher systemic inflammation level among people following a proinflammatory dietSince DII positively depends on protein intake that is also a metabolic risk factor for hypercalciuria hyperuricosuria and hypocitraturia and on contrary dietary fiber has a negative impact on DII and is a factor that can reduce calcium absorption we also adjusted the analysis for protein carbohydrate and fiber intake to differentiate the metabolic impact of DII from its proinflammatory impact It was found that DII is related to hypercalciuria and hypocitraturia independent of dietary intake of protein carbohydrate and fiber however the relationship between DII and hyperuricosuria disappeared showing that this association may be resulted from metabolic effects of DII Nevertheless protein and fiber intake was inversely associated with hypercalciuria and hypercalciuriaConclusionIn conclusion this study found that a diet with high inflammatory property might be unfavorably associated with urinary risk factors of kidney stone formation in men with a history of nephrolithiasisLimitationFirst since the participants of the current study were limited to men our findings may not be generalizable to women therefore it is essential to conduct such a study on women too Third causation cannot be inferred the crosssectional design of the present investigation Finally the calculation of DII by FFQ has a potential recall bias for the evaluation of dietary intake 0cMaddahi a0et a0al BMC Res Notes Page of Supplementary informationSupplementary information accompanies this paper at https doi101186s1310 yAdditional file a0 Table a0S1 Inflammatory effect scores for dietary components used for calculation of DII Table a0S2 calculation of DII for carbohydrate intake in a participant in our study as an example for total DII calculationAbbreviationsIPAQ International physical activity questionnaires DII Dietary inflammatory index PUFAs Polyunsaturated fatty acids CRP C reactive protein FFQ Food frequency questionnaire ANOVA Analysis of variance OR Odds ratio CI Confidence interval BMI Body mass indexAcknowledgementsWe would like to thank the Tehran University of Medical Sciences This work was supported by Tehran University of Medical Sciences Grant ID13951046Authors contributionsSMKA designed the research and collected the samples NSM and SHA wrote the paper HY and MSY analyzed data KhM conducted research and had primary responsibility for final content All authors read and approved the final manuscriptFundingNoneAvailability of data and materialsThe data are not publicly available due to containing information that could compromise the privacy of research participantsEthics approval and consent to participateEthics approval for the study protocol was granted by The Human Ethics Committee of Tehran University of Medical Sciences Grant ID IRTUMSVCRREC13951046 All participants signed written informed consent formsConsent for publicationNot ApplicableCompeting interestsAll authors declared that they have no competing interestsAuthor details Department of Community Nutrition School of Nutritional Sciences and Dietetics Tehran University of Medical Sciences TUMS Tehran PO Box Iran Department of Urology Sina Hospital Tehran University of Medical Sciences Tehran Iran Department of Epidemiology and Biostatistics School of Public Health Tehran University of Medical Sciences Tehran Iran Received March Accepted July References Li Y Zhang J Liu H et al Curcumin ameliorates glyoxylateinduced calcium oxalate deposition and renal injuries in mice Phytomedicine Fink HA Akornor JW Garimella PS et al Diet fluid or supplements for secondary prevention of nephrolithiasis a systematic review and metaanalysis of randomized trials Eur Urol Littlejohns TJ Neal NL Bradbury KE Heers H Allen NE Turney BW Fluid intake and dietary factors and the risk of incident kidney stones in UK Biobank a populationbased prospective cohort study European urology focus Ong CN Minerals from drinking water Bioavailability for various world populations and health implications Nutrients in Drinking Water Yagisawa T Chandhoke PS Fan J Metabolic risk factors in patients with firsttime and recurrent stone formations as determined by comprehensive metabolic evaluation Urology Grases F Melero G CostaBauza A Prieto R March J Urolithiasis and phytotherapy Int Urol Nephrol Khan SR Reactive oxygen species inflammation and calcium oxalate nephrolithiasis Translational Androl Urol Bo S Durazzo M Guidi S et al Dietary magnesium and fiber intakes and inflammatory and metabolic indicators in middleaged subjects from a populationbased cohort Am J Clin Nutri Chrysohoou C Panagiotakos DB Pitsavos C Das UN Stefanadis C Adherence to the Mediterranean diet attenuates inflammation and coagulation process in healthy adults the ATTICA Study J Am Coll Cardiol Upritchard JE Sutherland W Mann JI Effect of supplementation with tomato juice vitamin E and vitamin C on LDL oxidation and products of inflammatory activity in type diabetes Diab Care Neale E Batterham M Tapsell LC Consumption of a healthy dietary pattern results in significant reductions in Creactive protein levels in adults a metaanalysis Nutri Res Esmaillzadeh A Kimiagar M Mehrabi Y Azadbakht L Hu FB Willett WC Dietary patterns and markers of systemic inflammation among Iranian women J Nutri Mohseni R Mohseni F Alizadeh S Abbasi S The Association of Dietary Approaches to Stop Hypertension DASH Diet with the risk of colorectal cancer a metaanalysis of observational studies Nutrition and cancer Alizadeh S Djafarian K Alizadeh M ShabBidar S The relation of healthy and Western dietary patterns to the risk of endometrial and ovarian cancers a systematic review and metaanalysis Int J Vitamin Nutrition Res Alizadeh S ShabBidar S Mohtavinejad N Djafarian K A posteriori dietary patterns and risk of pancreatic and renal cancers Nutrition Food Science Mohseni R Abbasi S Mohseni F Rahimi F Alizadeh S Association between dietary inflammatory index and the risk of prostate cancer a metaanalysis Nutr Cancer Shivappa N Steck SE Hurley TG et al A populationbased dietary inflammatory index predicts levels of Creactive protein in the seasonal variation of blood cholesterol study SEASONS Public Health Nutrition Xu H Sjögren P Ãrnlöv J et al A proinflammatory diet is associated with systemic inflammation and reduced kidney function in elderly adults J Nutri Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relative validity of food group intake in a food frequency questionnaire developed for the Tehran lipid and glucose study J Epidemiol Shivappa N Steck SE Hurley TG Hussey JR Hébert JR Designing and developing a literaturederived populationbased dietary inflammatory index Public Health Nutri Bondonno NP Blekkenhorst LC Bird AL et al Dietary inflammatory index and the aging kidney in older women a year prospective cohort study Eur J Nutri Maddahi NS Mirzaei K Aghamir SMK Modaresi SS Yekaninejad MS Major Dietary Patterns and kidney stone formation among Iranian men J Nutri Sci Dietetics Moghaddam MB Aghdam FB Jafarabadi MA Allahverdipour H Nikookheslat SD Safarpour S The Iranian version of international physical activity questionnaire IPAQ in Iran content and construct validity factor structure internal consistency and stability World Appl Sci J Trinchieri A Mandressi A Luongo P Longo G Pisani E The influence of diet on urinary risk factors for stones in healthy subjects and idiopathic renal calcium stone formers Br J Urol Rouhani MH Najafabadi MM Surkan PJ Esmaillzadeh A Feizi A Azadbakht L Dietary inflammatory index and its association with renal function and progression of chronic kidney disease Clin Nutri ESPEN 0cMaddahi a0et a0al BMC Res Notes Page of Noori N Honarkar E Goldfarb DS et al Urinary lithogenic risk profile in sgÃ¥rd R Rytter E Basu S AbramssonZetterberg L Möller L Vessby B recurrent stone formers with hyperoxaluria a randomized controlled trial comparing DASH Dietary Approaches to Stop Hypertensionstyle and lowoxalate diets Am J Kidney Dis Leone A FernándezMontero A de la FuenteArrillaga C et al Adherence to the Mediterranean dietary pattern and incidence of nephrolithiasis in the Seguimiento Universidad de Navarra Followup SUN cohort Am J Kidney Dis Saneei P Hashemipour M Kelishadi R Esmaillzadeh A The Dietary Approaches to Stop Hypertension DASH diet affects inflammation in childhood metabolic syndrome a randomized crossover clinical trial Ann Nutr Metab Hsu CC Jhang HR Chang WT et al Associations between dietary patterns and kidney function indicators in type diabetes Clin Nutr Duda MK OShea KM Tintinu A et al Fish oil but not flaxseed oil decreases inflammation and prevents pressure overloadinduced cardiac dysfunction Cardiovasc Res High intake of fruit and vegetables is related to low oxidative stress and inflammation in a group of patients with type diabetes Scand J Food Nutri Wood LG Shivappa N Berthon BS Gibson PG Hebert JR Dietary inflammatory index is related to asthma risk lung function and systemic inflammation in asthma Clin Exp Allergy Hodge A Bassett J Shivappa N et al Dietary inflammatory index Mediterranean diet score and lung cancer a prospective study Cancer Causes Control Wirth MD Hébert JR Shivappa N et al Antiinflammatory dietary inflammatory INDEX scores are associated with healthier scores on other dietary indices Nutri Res Publishers NoteSpringer Nature remains neutral with regard to jurisdictional claims 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Human transcription factor and protein kinase gene fusions in human cancerKari Salokas Rigbe G Weldatsadik Markku VarjosaloOncogenic gene fusions are estimated to account for upto of cancer morbidity Recently sequencelevel studies have established oncofusions throughout all tissue types However the functional implications of the identified oncofusions have often not been investigated In this study identified oncofusions from a fusion detection approach DEEPEST were analyzed in detail Of the oncofusions we found almost are expected to produce functional proteins with features from both parent genes Kinases and transcription factors were the main gene families of the protein producing fusions considering their role as initiators actors and termination points of cellular signaling pathways we focused our indepth analyses on them Domain architecture of the fusions and their wildtype interactors suggests that abnormal molecular context of protein domains caused by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins To understand overall oncofusion effects we performed differential expression analysis using TCGA cancer project samples Results indicated oncofusionspecific alterations in gene expression levels and lower expression levels of components of key cellular pathways in particular signal transduction and transcription regulation the sum of results suggests that kinase and transcription factor oncofusions deregulate cellular signaling possibly via acquiring novel functionsAt any given moment multitudes of molecular networks are activated in cells throughout the body An important feature of these networks is highly concerted regulation of key signaling and deviation from homeostasis can result in diseases such as cancer Cancer is a complex progressive multistep disorder which stems from mutations caused by genomic instability1 The accumulation of genetic and epigenetic abnormalities ultimately leads to the transformation of normal cells into malignant derivatives Two highly enriched gene groups being mutated in the majority of cancer types are protein kinases PKs and transcription factors TFs23 PKs mediate most signal transduction events in cells by phosphorylation of specific substrates thus modifying their activity cellular localization andor association with other proteins TFs are the transistors of the cellular signaling circuits controlling the transcriptional outcome of activated signaling by binding to regulative elements of their corresponding target genes and driving or suppressing their expression Therefore it is easy to understand why mutational deregulation of these two gene groups can have such an impact on tumorigenesisIn addition to harboring activating or inactivating somatic point mutations PKs and TFs account for a large fraction of all human fusion genes involved in cancer COSMIC Catalogue of Somatic Mutations in Cancer cancersangeruk4 and dbCRID Database of Chromosome Rearrangements in Disease5 Chromosomal translocations creating fusion genes are among the most common mutation class of known cancer genes and they have long been identified as driver mutations in certain types of cancer6 Recently oncogenic fusion genes hereafter oncofusions OFs have been found in many hematological and solid tumors demonstrating that translocations are a common cause of malignancy78 Fusion mutations occur when two different gene regions fuse together via translocation Examples of consequences of chromosomal fusion to protein structure range from missense mutations to expressionchange inducing promotergene combinations to fully functional fusion proteins with neomorphic properties A classic example of gained functions is the breakpoint cluster regionAbelson tyrosineprotein kinase BCRABL1 translocation in chronic myeloid leukemia9 Alternatively a protooncogene is fused to a strong promoter and thereby the oncogenic function is upregulated due to the strong promoter of the upstream fusion partner This is common in lymphomas where oncogenes are juxtaposed to the promoters of the immunoglobulin genes10 and also in prostate cancer where ETS TF ERG is fused with TMPRSS2 regulatory Systems PathologyBiology Research Group Institute of Biotechnology HiLIFE University of Helsinki Helsinki Finland email markkuvarjosalohelsinkifiScientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0csequences thus obtaining androgen receptor ARresponsive expression11 The current understanding favors the aberrant gene function model rather than promoterinduced overexpressionThe frequency of recurrent OFs varies depending on the specific type of cancer12 but identified translocations are estimated to account for up to of cancer morbidity8 Recent fusion prioritization study found that inframe transcripts were the most powerful predictor of driver fusions16 confirming the intuition that inframe transcripts are crucial to function Notably breakpoints were also observed to preferentially avoid splitting of domains Together with frameshift conservation such trends could reflect a selection on fusion proteins to maintain protein stability and evade degradation pathways17Nextgeneration sequencing NGS of genomes and transcriptomes from primary human cancer cells is constantly revealing new gene fusions that are involved in driving tumorigenesis including examples found in colorectal carcinoma bladder carcinoma breast cancer and acute lymphoblastic leukemia ALL1518 Furthermore NGS has provided enough detailed sequence information of the fusion breakpoints allowing a0us to initiate systemslevel research on human oncofusions As a result various algorithms have been developed to mine OFs from large cancer datasets such as TCGA However the concordance among the different algorithms is very low that metacaller approaches utilizing consensus calls have been employed21 which limit novel OF discoveries Recently a new statistical method DEEPEST22 was developed to overcome these limitations In this study oncofusions that involve PKs and TFs were selected from the data produced by DEEPEST applied to the TCGA datasetIn most cases it is not possible to draw definite s about the mechanisms or extent by which individual translocations contribute to cancer Predicting protein function from a sequence has proven an extremely difficult task With gene fusions the task is even more daunting However an unexpectedly large number of PKs and TFs have been found to be mutationally activated or have increased expression due to gene amplification or translocation in cancer6 The high number of PKs and TFs with relatively low individual mutational frequency suggests either that a large number of signaling pathways can contribute to cancer or that many PKs and TFs can regulate the same pathways when activated unphysiologically Some additional support for this hypothesis comes from the interconnectivity of the PKTFoncofusionsIn this study fusions predicted to produce inframe proteins were analyzed to understand the proteinlevel implications of fusion events The fusions were analyzed from the perspective of their domain architecture to understand likely modes of action of the novel proteins Furthermore known interactomes of the participating wild type proteins were used to determine possible mechanisms of action pathways of interest and possible treatment vectors for affecting as many different fusions as possible As a result multiple cellular signaling pathways were found to intersect with major subsets of these fusions and multiple individual key interactors such as NTRK1 with over and EGFR with over interacting fusions were identified as potential targets of interestMaterials and methodsfusion selection and annotation Fusions that involve protein kinase genes23 and transcription factors24 were selected from the fusions that were identified by applying DEEPEST to the whole TCGA dataset22 Of these were determined to be unique by considering Ensembl gene IDs biotypes chromosomal breakpoints AGFusion assigned fusion effects and resulting protein sequences AGFusion was used to annotate these gene fusions to the human genome assembly GRCh38 v89 from Ensembl For analysis involving gene pairs the pair entry was used in alphabetical order eg ERGTMPRSS2 instead of TMPRSS2ERG in all cases Fusions were considered protein coding if both genes contributed over amino acids to the productclinical data Clinical data for TCGA samples was obtained from the GDC data repository The data was matched to AGFusion output data based on TCGA barcode eg TCGAWBA80K using a custom inhouse python script Stage information from the clinical data was simplified where possible eg Stage IIA was changed to Stage II Entries such as Stage Stage X and III NOS were ignored Tissue entries were simplified from detailed ICDO topographical codes to more general eg C569 C56 and mapped to names accordingly Chromosomal sequence information from GRCh38 v89 was used to categorize breakpoints into chromosomal interval groupsInteractor analysis Interactors for wild type proteins of all fusion partners were obtained from IMEx consortium25 and any interactions that were not confirmed to be physical by experimental methods were discarded Interactors were added to the interactor set from each fusion while leaving out the fusion pair genes themselves Annotations for interactors were obtained using Uniprot and Reactome From Reactome mappings to all levels of pathway hierarchy were used Dijkstras algorithm26 implemented with a custom python script was then used to establish shortest paths to Reactome root nodes for each network node A weight of was used for all network edgesDomain annotation For the protein producing fusions sequences of the protein products were produced using the AGFusion tools Duplicate fusions based on fusion genes and protein sequence were discarded Domains were taken from AGFusion output and mapped to protein sequence in the wild type protein The intactness of domains was then determined by matching the WT domain sequence to the predicted fusion protein sequence and only full length intact domains were picked for further study A domain was classified as PK or TFspecific if ¥ of all its occurrences were in PK or TF proteins respectivelyData visualization Data illustrations were made with CorelDRAW Excel and inhouse python scripts using Matplotlib and Seaborn Cytoscape27 was used for creating network figuresScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cDifferential expression analysis Gene expression quantification HTSeqcounts files were downloaded from GDC data portal Samples where OFs with intact fulllength PK or TF domains were detected were grouped together based on fusion gene pairs The groups were then analyzed with DESeq228 using other samples with protein producing nonPKTF fusions as controls For each pair group differential expression analysis against an equal number of control samples picked from samples in which other proteinproducing fusions were found Analysis was repeated times for each fusion pair For the resulting significantly differentially expressed genes qvalue basemean and expected values were averaged across all runs and a fold change value calculated based on these GO annotations were then added from ensembl annotations and Reactome pathways from first mapping ensembl gene IDs to Uniprot via Ensembl BioMart and then to Reactome lowest level pathway terms via Reactome Zscore value for pathway level overunderexpression was calculated by a method used in GOplot29 ieby deducting the number of underexpressed genes from the number of overexpressed genes and dividing the result by the square root of the number of significantly changed genes FDR corrected p ¤ ResultsDetection of oncofusions from TCGA dataset reveals enrichment of PK and TF fusions In this study we focused on protein producing OF genes Translocation of chromosomal regions can result in either inframe or outofframe OFs Fig a01A To characterize the proteins produced by known OFs in the TCGA dataset which currently contains data from different cancer projects we launched an analysis to understand the potential functional space of the protein producing fusions Fig a01B and especially those that involve either a PK or a TF or both PKTF fusionsThe DEEPEST dataset included fusions detected from cancer samples Of these were unique fusions Fig a01C upper panel Among the unique OFs were predicted to retain frame and also produce potentially functional proteins where both genes contributed over inframe amino acids Fig a01B Supplementary table a0S1 The limit of amino acids was the length of the shortest nonrepeat domain present in the fused proteins Examining the resulting protein producing OF set we noticed an abundance of those involving PK or TF Indeed these fusions constituted protein producing OFs Fig a01C lower panel Generally the proportion of PKTF fusions a0was under except in the PKTFfusion prone cancers acute myeloid leukemia cholangiocarcinoma thyroid carcinoma and thymoma The number of OFs per sample varied across cancer types The types most prone to protein producing fusions were sarcoma SARC with an average of protein producing fusions per sample esophageal carcinoma ESCA fusions uterine corpus endometrial carcinoma UCEC stomach adenocarcinoma STAD breast invasive carcinoma BRCA uterine carcinosarcoma UCS and ovarian serous cystadenocarcinoma OV Due to the prevalence of PK and TF genes in the fusions we next investigated if they are enriched in particular cancers While in most cancers PKTF fusions made up around of all protein producing OFs the percentage reached in acute myeloid leukemia LAML samples in thymoma THYM in thyroid carcinoma THCA and and in kidney renal papillary cell carcinoma KIRP and cholangiocarcinoma CHOL respectively Fig a01C Acute myeloid leukemia is well known as an OFprone cancer30 However aside from the four fusions detected between ABL1 and BCR the high percentage was mostly TFdriven with KMT2A RUNX1 and RARA being found in and fusions respectively This is in contrast to the peak in THCA which is driven by BRAF fusions fusions of RET of NTRK1 and of NTRK3 among other protein kinasesReading frame retention is common in pK and tf oncofusions The fusions consisted of unique gene pairs and individual genes of the pairs did not have any protein producing fusions The top protein producing fusion was RPS6KB1VMP1 with unique protein producing fusions in the dataset all the others having less than Fig a02A There were fusion gene pairs that were predicted to produce protein in at least fusions in fusions in and in fusion Out of the fusion gene pairs that produced or more unique proteins were PKTF fusionsTo better understand the behavior of prolific gene pairs we next mapped tissue annotations from TCGA to fusions of each gene pair based on barcodes from samples where a fusion of the gene pair was present In contrast to RPS6KB1VMP1 and ITGB6RBMS1 which were seen in samples from different cancers pairs were seen in samples of only one cancer type Out of these cancerspecific fusions were predicted to produce or more unique proteins with ERGTMPRSS2 predicted to produce different unique proteins supplementary table a0S2 PKTF fusions featured different PK or TF genes ERG being the most common TF and ERRB2 the most common PK supplementary table a0S3 Between and percent of oncofusions in each cancer project were unique highest being sarcoma with unique gene pair combinations and thyroid carcinoma the lowest with supplementary table a0S4 Protein producing fusions followed a similar theme unique protein producing fusions making up between and of all oncofusions in each given cancer project supplementary table a0S4We next looked in more detail what cancer stages PK and TF fusions were detected in The most prominent group was stage II breast invasive carcinoma which also had the most samples in the data set Fig a02B In total of the PK and TF fusions were found in stage I samples in stage II in stage III and in stage IV On average samples had PKTF fusions per sample However in some cancers PK or TF fusions are enriched towards the more severe stages Discounting stage groups with less than samples groups had more than fusions per sample ESCA stage III samples in particular had PKTF fusions per sample while STAD and BRCA stage IV samples had and respectively and STAD stage I had Supplementary table a0S5 The distribution of protein producing OFs mirrored that of PKTF fusions quite closely supplementary figure S1A In terms of chromosomal breakpoint locations those in the PKTF fusions varied compared Scientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cFusion gene Fusion gene PromoterProtein coding regionetis noisuFTranslocationChr Chr Chr Chr Inframe fusionOutofframe fusionTCGA DEEPEST dataset gene fusionsAGFusionEnsemblGRCh38TCGAclinical dataUniprotKinases Manning et al TFs Lambert et al et al Gene expressionquantiï¬cation dataprotein producing uniqueoncofusions Protein codingProtein kinase or Transcription factoroncofusionsPfamdomainsIMEx consortium interactor databaseCOSMICcancer genecensusReactomepathwaysClinical overviewDomain analysisInteraction analysis Pathway analysisDifferential expressionanalysisSamplesOncofusions per sampleABCselpmas fo rebmuNCCAACLBACRBCSECLOHCDAOCCBLDACSEMBGCSNHHCKICRKIPRKILMALGGLCHILDAULCSULOSEMVODAAPGPCPDARPDAERCRASMCKSDATSTCGTACHTMYHTCECUSCUMVUnietorp ni FT ro KP fo noitroporPsnoisufocno gnicudorpelpmas rep snoisufocno fo rebmuNACCBLCABRCACESCCHOLCOADDLBCESCAGBMHNSC Adrenocortical carcinomaBladder urothelial carcinomaBreast invasive carcinomaCervical squamous cell carcinomaand endocervical adenocarcinomaCholangiocarcinomaColon adenocarcinomaLymphoid neoplasm diffuselarge Bcell lymphomaEsophageal carcinomaGlioblastoma multiformeHead and neck squamous cell carcinoma Kidney chromophobeKidney renal clear cell carcinomaKidney renal papillary cell carcinomaKICHKIRCKIRPLAML Acute myeloid leukemiaLGGBrain lower grade gliomaLIHCLiver hepatocellular carcinomaLUADLung adenocarcinomaLUSCLung squamous cellcarcinomaMESOMesotheliomaOVOvarian serous cystadenocarcinomaPAADPancreatic adenocarcinomaPCPGPheochromocytoma and paraganglioma PRADREADSARCSKCMSTADTGCTTHCATHYMUCEC Prostate adenocarcinomaRectum adenocarcinomaSarcomaSkin cutaneous melanomaStomach adenocarcinomaTesticular germ cell tumorsThyroid carcinomaThymomaUterine corpus endometrialcarcinoma Uterine carcinosarcomaUCSUVM Uveal melanomaScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Schematic illustration of the gene fusions workflow and the number of gene fusions in human cancer A Schematic description of gene fusions formation Fusions are formed mainly via balanced and unbalanced chromosomal rearrangements such as translocations deletions inversions and insertions This usually leads to formation of a fusion gene with the ² end of Gene and ² end of Gene If the fusion occurs between two protein coding genes depending on whether the reading frame is violated and where exactly the fusion occurs a fusion protein may be transcribed with features and domains from both partners Other possible outcomes include full or truncated ² gene under the control of the promoter of the ² gene B Workflow used in this study Analysis progressed from the total set of fusions discovered by the DEEPEST method22 and moved towards more specific kinase TF containing protein producing oncofusions We started with TCGA databased fusion set from Dehghannasiri et a0al for which we generated protein sequences with AGFusion Domains were added by matching sequence to Uniprot proteins annotated with Pfam domains after which nonunique entries were dropped Fusions were classified as protein producing if both gene fragments were predicted to produce AA of protein sequence From this set the two most prominent protein groups were protein kinases and transcription factors and thus we focused further analysis on the unique protein kinase or transcription factor containing fusions using the full protein producing fusion set for comparison Known interactions for wild type fusion proteins were obtained from IMEx consortium and used for estimating maximal foreseeable effect on signaling pathways from Reactome Finally TCGA gene expression quantification data was used to probe observable effects of kinaseTF fusions using other protein producing fusions as background C Top Breakdown of samples and fusion mutations by TCGA project Largest single contributor of samples with fusions was TCGA breast invasive carcinoma project BRCA which had the highest number of samples and identified fusion mutations Bottom Proportion of protein producing fusions that include PK or TF genesto all protein producing fusion mutations but the prominent role of PKTF fusions is illustrated by overlapping hotspots supplementary figure S2Intact inframe domains are commonly retained in OFs To understand the contribution of each OF to the overall development or survival of cancerous cells the functional consequences of any given mutation and its impact on the pathways the proteins are involved in must be understood To this end we analyzed all identified unique protein producing fusions and the fulllength inframe domains of the fusion proteinsWhile AGFusion does predict protein sequence for each fusion partner and corresponding conserved or lost domains a domain is counted as conserved already if only amino acids are included in the sequence To adapt this to the study of fulllength domains we first mapped the Pfam identifiers of the domains to sequences in the wild type proteins from Uniprot The domains were then defined as conserved only if the full sequence was present in the fusion protein This resulted in conserved domains in all protein producing fusions Over of these domains are in PKTF fusions which account for of all protein producing fusions supplementary tables S1 S6 suggesting overall domain count strongly favors PK and TF genes perhaps indicating that these fusions produce more functional proteins in comparison to all protein producing fusionsThe most conserved domain was the protein tyrosine kinase domain Fig a03A supplementary table a0S6 which was conserved in fusions This was followed by the PH domain a common domain in intracellular signaling proteins and proteins of the cytoskeleton and the protein kinase domain To assess retention of nonobvious PK or TF domains we classified domains as PK or TF specific if over of the copies were found in PK or TF halves of the fusion proteins This resulted in copies of different TFspecific domains predicted to exist in the fusions compared to copies of PKspecific domains Most common TF domains were zinc finger C2H2 type KRAB and HLH DNA binding domains present in and copies respectively Many TF domains such as KRAB are involved in both transcriptional activation and repression depending on the molecular contextOn average protein producing fusions in samples of most cancer projects tended to have close to intact full length domain per protein producing OF PKTF fusions on average had more intact domains in all except for projects Fig a03B On average fusions in all projects tended to have between and intact domains while PKTF fusions featured a slightly higher average Although some cancers do appear to have particularly many domains this is mostly due to low count of fusions detected in the project Exception seems to be acute myeloid leukemia with detected protein producing fusions of which contain either a PK or a TF Most striking differences being seen in mesothelioma thyroid carcinoma rectum adenocarcinoma and uveal melanoma with and more retained domains on average in PKTF fusions compared to protein producing fusions respectivelyOn the cancer project level thyroid carcinoma had the highest percentage of PK domains of all domains identified in the samples of the project supplementary table a0S7 supplementary Figure S3 which totaled to only exceeded by breast invasive carcinoma with PK specific domains of all BRCA domains and lung adenocarcinoma LUAD with Proportion of TF domains varied less Kidney renal papillary cell carcinoma had of its intact domains in the TFspecific set followed by acute myeloid leukemia with and rectum adenocarcinoma and prostate adenocarcinoma both at Aside from prostate adenocarcinoma these projects had samples in the TCGA datasetinteractors of fusion partners can point to impact of ofs To understand what kind of implications the functional changes of lost conserved PK or TF specific domains in new combinations could have for the Scientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cNonprotein producing fusionProtein producing fusionLMEKLASMBRBGTIPMVBKSPRTXNURXNURHPMRYLKNWCREATMKLLEPXOFDNCCNPTPCDCCMUPNAKNICCATRFGFSSRPMTGREPAGPBBREARARLMPGRPTPTRCBLBAKRTNVTEBTBZASARFNZPBPSLEHYTTLDAMIUCMCCSAHDTMBMTPALDNYMZAYEDHSABRLBRSMTONCSRAYMUPSGRHPCMProtein kinaseTranscription factorProtein kinase and transcription factorAsnoisufocno fo rebmuNBIJNNAMDKCCMNBDDZDPONEPIRTSLYCHALAGTSfroCIV IIIIIIACCBLCABRCACESC CHOLCOADDLBC ESCAGBMHNSC Adrenocortical carcinomaBladder urothelial carcinomaBreast invasive carcinomaCervical squamous cell carcinomaand endocervical adenocarcinomaCholangiocarcinomaColon adenocarcinomaLymphoid neoplasm diï¬uselarge Bcell lymphomaEsophageal carcinomaGlioblastoma multiformeHead and neck squamous cell carcinoma Kidney chromophobeKidney renal clear cell carcinomaKidney renal papillary cell carcinomaKICHKIRCKIRPLAML Acute myeloid leukemiaLGGBrain lower grade gliomaLIHCLiver hepatocellular carcinomaLUADLung adenocarcinomaLUSCLung squamous cellcarcinomaMESOMesotheliomaOVOvarian serous cystadenocarcinomaPAADPancreatic adenocarcinomaPCPGPheochromocytoma and paraganglioma PRADREADSARCSKCMSTADTGCTTHCATHYMUCEC Prostate adenocarcinomaRectum adenocarcinomaSarcomaSkin cutaneous melanomaStomach adenocarcinomaTesticular germ cell tumorsThyroid carcinomaThymomaUterine corpus endometrialcarcinomaUCS Uterine carcinosarcomaUVM Uveal melanomaFigure a0 Clinical characterization of protein producing oncofusions by cancer stage A The most common protein producing gene pairs in oncofusions In total protein producing fusions were comprised of unique gene pairs predicted to produce one or more unique protein products The most common pair was RPS6KB1VMP1 with over unique proteins followed by ITGB6RBMS2 and ALKEML4 with each and LYRM9P3H4 and RUNX1RUNX1T1 at Kinase and TF fusions were common in top protein producing gene pairs illustrated by blue shading for the presence of a protein kinase in gene pair red for TF and orange for both B Sunburst diagram of project and stage distribution of PKTF oncofusions The innermost layer represents the number of fusions in each project The layers radiating out are the proportion of fusions detected in Stage I II III and IV samples in order from in to out Total numbers of fusions from each stage is marked under the stage indicatorsScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cProtein kinase or TF containing oncofusionsProtein producing oncofusionsAsniamod fo rebmuNniamodHS staeper niryknAniamod III epyt nitcenorbiFniamodC taeper atebG niamodDW niamod HC ygolomohninoplaC fitomnoitingocer ANRregnï¬DHPniamodZDP epyt HC regnï¬ cniZniamod lanimret Cdevresnoc esacileHfitom ahpla eliretS niamodMAS xobBARKniamodHS niamodZOPBTBniamod HS tnairaV niamodnirehdaC niamodgnidnib lorecylglycaidsretse lobrohP niamodgnidnibANDxilehpoolxileH niamodFEGohR niamodPAGohRtaeper nirtcepSnietorp tropsnart noIniamod nilubolgonummIA ssalc niamod rotpecer LDLniamodomorBniamodAHF taeper hcir enicueL niamod tesVnilubolgonummI niamod epyt nidnopsobmorhTniamodMIL niamodWW niamod ekilFGEriap niamoddnahFEniamod lartnec MREF niamodFGEgnidnibmuiclaC esacilehxobHAEDDAEDniamodstEniamodoemoHfitom ahpla eliretS niamodMAS etis yrotibihni aX rotcaFnoitalugaoCrotpecer enomroh raelcun foDBLniamodYRPS niamod htaeDniamodXP niamod HMretropsnart CBAtaeper editpepocirtarteTniamod FASDGlaR noitaicossa saRylimaf nispodohr rotpecer enarbmemsnartniamodHP esanik enisoryt nietorPniamod esanik nietorPniamod tesI nilubolgonummIBnoisufocno gnidoc nietorp rep sniamoDProtein producing oncofusionsProtein kinase or TF containing oncofusionsCsnoisufocnoFT ro esanik nietorp gnitcaretni fo rebmuNLMALOSEMACHTLOHCDAERMVUPRKIMYHTMBGGGLGPCPTCGTDARPCSULDAOCMCKSCSECACLBDAULCRASCSNHCRKIACSECHILACRBVOCCADATSDAAPSCUCECUCBLDHCKIFound in COSMIC cancer gene censusPK oncofusionsTF oncofusionsPKTF oncofusionsOther protein producing oncofusions Percent of unique interactors in COSMIC canger gene census KRTNGAHWYOPXCBURGERFGEAPSHBAHWYRACADHAPSHPECHTRISALERMCMQAHWYFNRXDDNUJLCDCLBVUREAHWYCRSAPRCDCCDAMSMDMCADHPSLMPACPPPNFSAPRROTMBERCDAMSAPSHDNACCADHPCVCDKRPTATSACRBDNCCBDDABUIMBANCPARAROXBFUPNRNHFigure a0 Domain analysis of protein producing fusions A Intact fulllength domains identified in unique protein producing fusions In total intact domains were detected The protein tyrosine kinase domain was the most prevalent with identifications In addition protein kinase domain was detected with copies each Kinase or TF specific domains included and unique domains respectively copies of kinasespecific domains were seen and of TF specific Kinase domains focused more on the two top kinase domains whereas TF domains were a much more evenly distributed group the top TFspecific domain C2H2 type zinc finger having copies B The number of domains per protein coding oncofusion in the TCGA projects C Most common interactors of protein kinaseTF fusions Yaxis describes numbers of unique protein producing fusions where one or both of the fusion partner WT genes interact with the protein Top right inset Proportion of interactors found in COSMIC cancer gene census is higher in both protein kinase and transcription factor fusions and most common in fusions between protein kinase and transcription factor genesScientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cATranscriptional regulationpathwaysRNA Polymerase II transcriptionGeneric transcription pathwaySignaling by Rho GTPasesMAPK family signaling cascadeGPCR signalingSignaling by NotchTGFβ signalingSignal transductionTranscriptional regulationby TP53Hedgehog pathwaySignaling by Receptor tyrosine kinasesIntegrin signalingIntracellular signaling bysecond messengersWnt signalingDeath receptor signalingNode proteins interact withTF fusionsKinase fusionsBCytokine Signaling in Immune systemDevelopmental BiologyAxon guidanceMetabolismPIP3 activates AKT signalingIntracellular signaling by second messengersPTEN RegulationRegulation of PTEN gene transcriptionOxidative Stress Induced SenescenceHDMs demethylate histonesSUMOylation of transcription cofactorsTranscriptional Regulation by E2F6SUMOylation of chromatin anization proteinsTranscriptional regulation by RUNX1Chromatin anizationChromatin modifying enzymesCellular SenescenceSUMO E3 ligases SUMOylate target proteinsSUMOylationInterferon alphabeta signalingEPHEphrin signalingPhospholipid metabolismEPHephrin mediated repulsion of cellsEPHAmediated growth cone collapsePI MetabolismSynthesis of PIPs at the plasma membraneInterferon SignalingMetabolism of lipidsOncogenic MAPK signalingSignaling by moderate kinase activity BRAF mutantsParadoxical activation of RAF signaling by kinase inactive BRAFSignaling downstream of RAS mutantsrRNA modiï¬cation in the nucleus and cytosolRHO GTPases Activate ForminsMajor pathway of rRNA processing in the nucleolus and cytosolrRNA processing in the nucleus and cytosolrRNA processingProcessing of Capped IntronContaining PremRNAmRNA SplicingmRNA Splicing Major PathwayMacroautophagyAutophagyToll Like Receptor TLR3 CascadeMyD88independent TLR4 cascade TRIFTICAM1mediated TLR4 signaling Tolllike Receptor CascadesToll Like Receptor TLR4 CascadeSignaling by TGFbeta family membersSignaling by Nuclear ReceptorsTCF dependent signaling in response to WNTSignaling by WNTSignaling by | 2 |
expanding cancer predisposition genes with ultrarare cancerexclusive human variationsRoni Rasnic1 nathan Linial1 Michal Linial2It is estimated that up to of cancer incidents are attributed to inherited genetic alterations Despite extensive research there are still gaps in our understanding of genetic predisposition to cancer It was theorized that ultrarare variants partially account for the missing heritable component We harness the UK BioBank dataset of individuals of which were diagnosed with cancer to detect ultrarare possibly highpenetrance cancer predisposition variants We report on cancerexclusive ultrarare variations and nominate variants with additional independent evidence as cancer predisposition variants We conclude that population cohorts are valuable source for expanding the collection of novel cancer predisposition genesDiscovery of cancer predisposition genes CPGs has the potential to impact personalized diagnosis and advance genetic consulting Genetic analysis of family members with high occurrences of cancer has led to the identification of variants that increase the risk of developing cancer1 In addition to familybased studies efforts to identify CPGs focus on pediatric patients where the contribution of environmental factors is expected to be small Forty percent of pediatric cancer patients belong to families with a history of cancer2Tumorigenesis results from misregulation of a0one or more of the major cancer hallmarks3 Therefore it is anticipated that CPGs overlap with genes that are often mutated in cancerous tissues Indeed CPGs most prevalent in children TP53 APC BRCA2 NF1 PMS2 RB1 and RUNX12 are known cancer driver genes that function as tumor suppressors oncogenes or have a role in maintaining DNA stability4 Many of the predisposed cancer genes are associated with pathways of DNArepair and homologous recombination5 The inherited defects in cells ability to repair and cope with DNA damage are considered as major factors in predisposition to breast and colorectal cancers6Complementary approaches for seeking CPGs are largescale genomeexome wide association studies GWAS which are conducted solely based on statistical considerations without prior knowledge on cancer promoting genes7 Identifying CPGs from GWAS is a challenge for the following reasons limited contribution of genetic heritability in certain cancer types low effect sizerisk associated with each individual variant lowpenetrance in view of individuals background8 and low statistical power Large cohorts of breast cancer show that of cancer cases are associated with mutations in BRCA1 and BRCA2 which are also highrisk ovarian cancer susceptibility genes Additionally TP53 and PTEN are associated with earlyonset and highrisk familial breast cancer Mutations in ATM and HRAS1 mildly increase the risk for breast cancer but strongly increase the risk for other cancer types and a collection of DNA mismatch repair genes MLH1 MSH2 MSH6 PMS2 are associated with high risk of developing cancer9 A large cohort of Caucasian patients with pancreatic cancer reveal high risk CPGs that overlap with other cancer types CDKN2A TP53 MLH1 BRCA2 ATM and BRCA110Estimates for the heritable component of predisposition to cancer were extracted from GWAS familybased and twin studies11 These estimates vary greatly with maximal genetic contribution associated with thyroid and endocrine gland cancers and a minimal one with stomach cancer and leukemia14 Current estimates suggest that as many as of cancer incidents can be attributed to inherited genetic alterations eg single variants and structural variations1516 The actual contribution of CPGs varies according to gender age of onset cancer types and ethnicity17 It is evident that high risk variants with large effect sizes are very rare21 Actually based on the heritability as reflected in GWAS catalog it was estimated that only a fraction of existing CPGs is presently 1The Rachel and Selim Benin School of Computer Science and Engineering The Hebrew University of Jerusalem Jerusalem Israel 2Department of Biological Chemistry Institute of Life Sciences The Hebrew University of Jerusalem Jerusalem Israel email ronirasnicmailhujiacilScientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cFigure a0 UK Biobank CUVs collection The Caucasian filtered UK Biobank UKBB data set include individuals who had cancer and the nonCaucasian include such individuals a Cancer type distribution for the Caucasian data set b Cancer type distribution for the nonCaucasian data set c The data of UKBB participants was used for this study of which were confirmed Caucasian d Out of UKBB variants we curated heterozygous and homozygous CUVs total CUVs known22 Therefore instances of extremely rare mutations with high risk for developing cancer remain to be discoveredA catalog of CPGs was compiled from a0years of research1 with about half of the reported genes derived from family studies representing highpenetrance variants An extended catalog was reported with a total of CPGs that were tested against rare variants from TCGA germline data covering cancer patients from cancer types and included known pediatric CPGs23 The contribution of BRCA12 ATM TP53 and PALB2 to cancer predisposition was confirmedIn this study we report on known and novel cancer predisposition candidate genes We benefit from the UKBiobank UKBB an invaluable resource of germline genotyping data for individuals The UKBB reports on cancer patients and cancer free individuals considered as control group We challenge the possibility that CPGs can be identified from very rare events henceforth called cancerexclusive ultrarare variants CUVs These CUVs are expected to exhibit high penetrance Notably the presented CUVs were extracted from UKBB DNA array and therefore only cover the array preselected SNPs We report on exome variations of which are heterologous The majority of the matching genes are novel CPG a0candidates We provide indirect genomic support for some of the CUVs that occur within coding genes and discuss their contribution to tumorigenesisResultsThe primary UKBB data set used in the is comprised of Caucasian UKBB participants see Methods Fig a01c cancerfree and diagnosed with at least one malignant neoplasm Among participants with cancer were diagnosed with either skin or breast cancer The clinical ICD10 codes assembly is summarized in Supplementary Table a0S1 A total of of the cancerdiagnosed individuals had two or more distinct neoplasms diagnosed The validation UKBB data set includes nonCaucasian participants among them are cancerfree Figure a01ab provide further details on different cancer type prevalence in these setsNonmelanoma skin cancer is mostly attributed to environmental factors rather than genetic association24 However based on evidence for hereditary links for nonmelanoma skin cancer predisposition2526 we included these individuals in our analysis In addition focusing on extremely rare variations enables the identification of existing yet overlooked genetic associationsCompilation of cancerexclusive ultrarare variants CUVs We scanned genetic markers in our prime data set for cancerexclusive variations variations met our initial criteria appearing at least twice in individuals diagnosed with cancer and not appearing in cancerfree individuals Among them were heterozygous and were homozygous variations In order to target variations with additional supporting eviScientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cFigure a0 Exomic CUVs are mostly gene disruptive The partition of variant types for the compiled list of exomic CUVs The list is dominated by transcript disruptive variations that include missense frameshift stop gain and splicing sites a Distribution of variation types among the exomic CUVs b Dispersion of variant types among heterozygous and homozygous CUVsdence we considered only coding exome and spliceregion variants To assure the CUVs rarity in the general population we applied an additional filter based on the gnomAD data set see Methods The resulting final list is comprised of variants associated with genes heterozygous and homozygous Fig a01d The detailed list of all CUVs can be found in Supplementary Table a0S2Most of the CUVs are missense variants There is a strong enrichment for loss of function LoF variants ie frameshift splicing disruption and stop gains which account for of the CUVs Only a single homozygous CUV is synonymous Fig a02a The distribution of variation types varies greatly between homozygous and heterozygous CUVs Fig a02b Missense variants are of the homozygous variant set but only of the heterozygous CUVs The heterozygous CUVs are highly enriched for LoF variants which constitute the other Cancerexclusive ultrarare variants overlap with known cancer predisposition genes From the listed CUVs variants were previously defined as cancer inducing genes in genes Table a0 Specifically CUVs within genes appear in the updated list of CPG catalog23 and CUVs within genes are known cancer driver genes Fig a03a as determined by either COSMIC27 or the consensus gene catalog of driver genes listing genes coined C29928 More than half of the cancer associated variants result in LoF Many of the affected genes are tumor suppressor genes TSGs among which are prominent TSGs such as APC BRCA1 and BRCA2 Table a0 each identified by two distinct CUVs Notably of the variants had at least one appearance in nonmelanoma skin cancerThe heterozygous CUVs are enriched for known cancer predisposition genes Twentyfive of the cancer associated CUVs are heterozygous and one is homozygous However there is an inherent imbalance in the initial variant sampling performed by the UKBB As the UKBB use DNA arrays for obtaining genomic data the identifiability of ultrarare exome variants is restricted by the selection of SNP markers and the design of the DNA array There are heterozygous ultrarare exome variants from genes which pass our biobankethnic and the gnomAD allele frequency filtration A total of of the filtered ultrarare variants overlap with known CPGs as some genes are overrepresented among the ultrarare variants Supplemental Table a0S3 For example the exomic region of BRCA2 is covered by such SNP marker variants while most genes have noneIn order to account for the disproportional number of the ultrarare variant of some CPGs we calculated the expected number of cancer predisposed genes when gradually removing highlyrepresented genes from the collection of heterozygous ultrarare variants As shown in Fig a03b there is an enrichment towards CPGs and even more so as we remove variants of overrepresented genes eg BRCA2 The statistical significance estimates pvalues for each datapoint are available in Supplemental Table a0S3 see MethodsIndependent genetic validation Due to the extremely rare nature of the CUVs we require additional support for the collection of the CPG candidates We seek independent genetic validation of the noncancer related CUVs We apply three sources for validation the filtered Caucasian UKBB cohort the matched filtered nonCaucasian UKBB cohort the collection of germline variants from TCGA as reported in gnomAD The complete list of genetically validated novel CPG candidates is listed in Table a0 Ten out of the novel CPGs were identified based on appearances in individuals with nonmelanoma skin cancerWithin the Caucasian cohort we consider the following as additional genomic evidence a gene with CUVs or any CUV seen in more than two individuals diagnosed with cancer We found genes that have distinct CUVs of which are already known CPGs BRCA1 BRCA2 and APC The other genes are likely novel Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cRefEffecthg19TMissenseGMissenseMissenseTSplice region GSplice region AFrameshiftFrameshiftStop gainMissenseFrameshiftMissenseStop gainMissenseMissense MissenseFrameshiftMissenseFrameshiftFrameshiftMissenseMissenseFrameshiftStop gainFrameshiftSplice region CMissenseTAlt GeneGBACMSH6AVHLGTGFBR2AMLH1GAPCAAGGA APCGTCTGTCC CTG AG TCTTCCGCACAGGCGAACAAGAGCTGGGCCACCGTCTGFBR1SPTAN1RETBMPR1APTENEXT2NUMA1ATMBRCA2BRCA2RB1ERCC5TSC2NF1BRCA1BRCA1TGIF1RUNX1NF2COSMIC C299 CPG FunctionaYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYEnzymeDNA repairUbqcomplexKinaseTSGTSGTSGKinaseCytoskeletalKinaseKinaseTSG PhosphataseTSG EnzymeMT Spindle poleDDR KinaseTSG DNA repairTSG DNA repairTSGDNA repairTSGRAS regulatorTSG DNA repairTSG DNA repairTGF ligandTFCytoskeletalYYYYYYYYYYYYYYYYYYYYTable CUVs overlap with known cancer predisposition or driver genes a Function abbreviation DDR DNA damage response TSG tumor suppressor gene TF transcription factor MT microtubule Ubq ubiquitin Variants with at least one appearance in nonmelanoma skin cancerFigure a0 CUVs list is enriched with cancer predisposition genes Out of the genes in the CUVs list are known cancer genes a Venn diagram of the genes associated with CUVs known cancer driver genes as reported in COSMIC and the consensus CPGs b Expected number of known CPG CUV orange versus the actual number of known CPG in heterozygote CUVs blue An unbalanced representation of genes in ultrarare variants of UKBB results in overrepresentation of some genes We therefore ranked the genes based on number of ultrarare variants Supplementary Table a0S3 For each rank we present the expected number of CUVs from CPGs and the actual number observed for CUVs from CPGsScientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cGene SymbolZygote form People per CUV Distinct CUVs NonCaucasian cohortTCGA germlineAGR2AKR1C2DNAH3DSPEGFLAMENDOUHIST1H2BOHSPB2ICAM1ISLRKCNH2MAP3K15MRPL39MYBPC3MYO1ENAV3PCDHB16SARDHSCN5AWDFY4ZFC3H1HeteroHeteroHomoHeteroHeteroHomoHeteroHeteroHomoHomoHeteroHeteroHeteroBothHomoHeteroHomoHomoHeteroHeteroHomoYYYYYYYYYYYYYYYYYFunction in tumorigenesisAffects cell migration transformation and metastasis Wnt signaling tumor antigenExerts an inhibitory effect on oncogenesisCancer predisposed genes in Tunisian familyAffects cell adhesion Suppressed by TGFβPromotes matrix assemblyCancer biomarkerAffects major signaling pathwaysEpigenetically regulatedBiomarker under a clinical trialMarker for mesenchymal stem cells Deregulated gene in cancerAffects proliferation and migrationContributes to cell migrationTumor suppressor by targeting miR130Cytoskeletal modifierStimulates upregulation of motility and invasionActs as a suppressor of breast cancerActs as tumor suppressorPromotes breast cancer possess antipancreatic cancerPresentats viral tumor antigen on dendritic cellsIndirect activating DNA repairRefTable Novel validated CPG candidates Variants with at least one appearance in nonmelanoma skin cancerCPG candidates DSP KCNH2 MYBPC3 and SCN5A There are CUVs which we detected in three individuals with cancer Three of them are known predisposition or driver genes NF1 ATM and TGFBR2 The other genes are CPG candidates that were not previously assigned as such This set includes PCDHB16 DNAH3 ENDOU AGR2 HIST1H2BO and NAV3 Interestingly a certain homozygous CUV in the gene ICAM1 appeared in individuals with cancer in our filtered Caucasian cohortThe nonCaucasian UKBB cohort provides additional independent genomic evidence There are CUVs that appear at least once in an individual with cancer from the nonCaucasian cohort CUVs from the genes MYO1E SARDH and ISLR appeared in two distinct individuals with cancer from this nonCaucasian cohort while CUVs from PCDHB16 and known CPG BMPR1A appeared in a single individual with cancerTCGA germline variants were obtained using exome sequencing and thus offer an additional separate source for CUV validation Clearly the appearance of CUVs in TCGA germline data is not anticipated as we discuss variants that are ultrarare in both UKBB and gnomAD The TCGA collection within gnomAD includes only samples We identified CUVs that were also observed in TCGA gnomAD germline data one of a known cancer driver gene TGIF1 and novel CPG candidates PCDHB16 EGFLAM AKR1C2 MAP3K15 MRPL39 DNAH3 WDFY4 HSPB2 and ZFC3H1Based on the above support we compiled a list of validated CPGs which includes genes that are novel CPGs Among these genes CUVs are heterozygous are homozygous and MYBPC3 is supported by both heterozygous and homozygous CUVs Two of these genes have multiple validation evidence DNAH3 with a homozygous CUV which appears in individuals with cancer in the Caucasian cohort and within TCGA germline variant collection PCDHB16 with a homozygous CUV which appeared in individuals in the Caucasian cohort one individual in the nonCaucasian cohort and in the TCGA gnomAD resource In addition nonCPG cancerdriver genes with validated CUVs include TGFBR2 and TGIF1 that are also very likely CPG candidatesSome of the prominent genes in our list were signified by additional independent studies For example a novel oncolytic agent targeting ICAM1 against bladder cancer is now in phase of a clinical trial29 Additionally DNAH3 was identified as novel predisposition gene using exome sequencing in a Tunisian family with multiple nonBRCA breast cancer instances30Somatic mutations in novel CPGs significantly decrease survival rate There is substantial overlap between CPGs and known cancer driver genes Fig a03a This overlap suggests that somatic mutations in validated CPG candidates may have an impact on patients survival rate We tested this hypothesis for the novel CPG candidates Table a0 using a curated set of nonredundant TCGA studies compiled in cBioPortal3132 that cover patients By testing the impact of alteration in the novel CPGs in somatic data we expect to provide a functional link between the germline CPG findings and the matched mutated genes in somatic cancer samples Altogether of the patients had somatic mutations in one or more of the genes The median survival of patients with somatic mutations in these genes is a0months while the median for patients without Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Somatic mutations in CPG candidate effect cancer patient survival and disease progression The effect of somatic mutations in the novel CPG candidate Table a0 on the survival rate of TCGA cancer patients was tested via cBioPortal a MeierKaplan survival rate estimate b MeierKaplan diseaseprogressionfree estimatesomatic mutations in any of these genes is much longer a0months Applying the KaplanMeier survival estimate yields a p value of 178e in the Logrank test Fig a04a The KaplanMeier diseaseprogressionfree estimate was also worse for patients with somatic mutations in the novel CPGs with a p value of 603e Fig a04b Cancer types in this analysis are represented by varied number of patients and percentage of individuals with somatic mutations in any of the novel CPGs Supplemental Table a0S4 The trend in most cancer types match the presented pancancer analysis Survival and diseaseprogression estimate for each cancer type are available in Supplementary Figures a0S1S24 Hazard Ratios and confidence intervals were calculated see Materials and Methods and Supplemental Table a0S4We conclude that the CUVbased CPG candidate genes from UKBB carry a strong signature that is manifested in patients survival supporting the notion that these genes belong to an extended set of previously overlooked CPGsHomozygous variations are mainly recessive In order to ascertain whether the homozygous variations found are indicative of the heterozygous form of the variant as well we viewed the heterozygous prevalence within the UKBB Caucasian population In only a single variant in the gene MYO1E was the prevalence in healthy individuals significantly lower than in individuals with cancer p value As most of the variations have a strong cancer predisposition effect as homozygous variations it seems that their influence is explained by a recessive inheritance mode This phenomenon might explain the significant depletion of known CPGs within the homozygous variations in our listInspecting the heritability model of previously reported CPGs1 is in accord with our findings showing that while about twothirds of the genes comply with a dominant inheritance the rest are likely to be recessive Notably in the most updated CPG catalog of the genes were assigned with both inheritance patterns In our ultrarare list only MYBPC3 is associated with both heterozygous and homozygous variationsDiscussionWe present a list of CUVs from genes Among them variants from genes are associated with known cancer genes Most of these variants overlap with known cancer predisposition genes Expanding the number of currently identified CPGs is crucial for better understanding of tumorigenesis and identifying various processes causing high cancer penetrance Genetic consulting family planning and appropriate treatment is a direct outcome of an accurate and exhaustive list of CPGsKnown cancer predisposition variants only partially explain the cases of inherited cancer incidents CPGs identification has already impacted cancer diagnostics therapy and prognosis1 Genomic tests and gene panel for certain cancer predisposition markers are commonly used for early detection and in preventative medicine3334 It is likely that CPGs based on ultrarare variants are not saturated For example additional CPGs including CDKN2A and NF1 were associated with an increased risk for breast cancer35 Specifically CDKN2A has been also detected as a CPG in families of patients with pancreatic cancer36 Inspecting the function of genes associated with Scientific RepoRtS 101038s41598020704940Vol1234567890wwwnaturecomscientificreports 0cthe identified genes further supports the importance of protein modification eg kinases and phosphatase function chromatin epigenetic signatures37 membrane signaling DNA repair systems and moreNumerous CUVs are present in individuals with nonmelanoma skin cancer For the most part nonmelanoma skin cancers are attributed to environmental factors Nevertheless studies show that there are in fact genetic components associated with the majority of nonmelanoma skin cancers2526 Accordingly CUVs can unveil such rare genetic associationsWe chose to focus on cancerexclusive variants to shed light on mostly overlooked ultrarare cancer predisposition variants Naturally additional ultrarare variants in the dataset are presumably cancer inducing Detecting these variants requires developing a broader model expanding the scope to somewhat less rare possibly lowerpenetrance variants The impending availability of UKBB exome sequencing exomes will enable us to revisit the identified variants to further refine the list of candidate CPGs ie removing falsepositives and adding evidence to support true CPGs and to develop a less strict detection modelThe inheritably rare nature of CUVs raise concerns on the reliability of their initial identification38 We overcome this hurdle by only considering as candidate CPGs those genes that are supported by additional independent genomic evidence from either the UKBB or the TCGA cohort We nominate genes as CPG candidates two of which are known cancer drivers As we have shown Fig a0 somatic mutations in the nondriver validated CPG candidates resulted in a significant negative effect on the patients survival rateMaterials and methodsStudy population The UKBB has recruited people from the general population of the UK using National Health Service patient registers with no exclusion criteria39 Participants were between and a0years of age at the time of recruitment between and To avoid biases due to familial relationships we removed samples keeping only one representative of each kinship group of related individuals We derived the kinship group from the familial information provided by the UKBB fam files Additionally samples had mismatching sex between the selfreported and the geneticsderived and samples had only partial genotypingWe divided the remaining participants into two groups Caucasiansindividuals that were both genetically verified as Caucasians and declared themselves as white nonCaucasiansindividuals not matching the previous criterion The Caucasian cohort includes individuals of whom had cancer and the nonCaucasian cohort includes individuals had cancer We used the Caucasian cohort for our primary analysis and the nonCaucasian cohort for additional validation purposesVariant filtration pipeline We considered a heterozygous variation as cancerexclusive when there were at least cancer patients exhibiting the variation and no healthy individuals with the variation in the Caucasian cohort We considered a homozygous variation as cancerexclusive when there were at least cancer patients exhibiting the variation ie homozygous to the alternative SNP and no healthy individuals with the homozygous variation in the Caucasian cohort The ensemble Variant effect predictor40 was used to annotate the variantsWe applied two additional filtration steps for the exomesplicingregion variants The first filter was applied using the nonCaucasian data set we filtered heterozygous variations with MAF and homozygous variations with homozygous frequency in this set This filtration step is meant to diminish variations which are mostly ethnic artifacts The second filter was applied to assure the variations rarity We applied the same filter heterozygous variations with MAF and homozygous variations with homozygous frequency using gnomAD v21141 The used gnomAD threshold was based on the summation of gnomAD v211 exomes and genomes We also used gnomAD for the TCGAgermline validation by extracting TCGA appearances from the databaseStatistical analysis The UKBB ultrarare variants are enriched with CPGs variants We accounted for this imbalance by calculating the expected number of cancer predisposed genes when gradually removing highlyrepresented genes from the ultrarare variant collection for heterozygotes We calculated pvalues for each datapoint using a twoside binomial testWe downloaded survival data from cBioPortal The data only included survival months We used Cox regression without covariates to calculate Hazard Ratio and confidence intervals The results are listed in Supplementary Table a0S4Rare variants reliability Our CUV collection includes variants that appeared at least twice in the filtered Caucasian cohort thereby evading many SNPgenotyping inaccuracies38 We further ascertain the validity of prominent variants with additional genomic evidenceCancer type definition The UKBB provides an ICD10 code for each diagnosed condition We considered an individual diagnosed with malignant neoplasm ICD10 codes C00C97 as individuals with cancer and otherwise as cancerfree individuals The codes were aggregated to improve data readability using the assembly described in Supplementary Table a0S1Ethical approval All methods were performed in accordance with the relevant guidelines and regulations UKBB approval was obtained as part of the project Ethical approval for this study was obtained from the Scientific RepoRtS 101038s41598020704940Vol0123456789wwwnaturecomscientificreports 0ccommittee for ethics in research involving human subjects for the faculty of medicine The Hebrew University Jerusalem Israel Approval Number UKBB received ethical approval from the NHS National Research Ethics Service North West 11NW0382 UKBB participants provided informed consent forms upon recruitmentData availabilityMost of the data that support the findings of this study are available from the UKBB However restrictions apply to the availability of these data which were used under license for the current study and so are not publicly available Data are available from the authors upon a justified request and with permission of the UKBB Data extracted from gnomAD is available from the authors upon requestReceived February Accepted July a1508 References Rahman N Realizing the promise of cancer predisposition genes Nature 101038natur e1298 Zhang J et al Germline mutations in predisposition genes in pediatric cancer N Engl J Med 101056NEJMo Hanahan D Weinberg R A Hallmarks of cancer the next generation Cell 101016jcell201102013 Vogelstein B Kinzler K W Cancer genes and the pathways they control Nat Med 101038nm108 Bertelsen B et al High frequency of pathogenic germline variants within homologous recombination repair in patients with advanced cancer npj Genom Med 101038s4152 Easton D F How many more breast cancer predisposition genes are there Breast Cancer Res 101186bcr6 Hindorff L A et al Potential etiologic and functional implications of genomewide association loci for human diseases and traits Proc Natl Acad Sci U S A 101073pnas09031 Galvan A Ioannidis J P A Dragani T A Beyond genomewide association studies genetic heterogeneity and individual predisposition to cancer Trends Genet 101016jtig200912008 Baria K Warren C Roberts S A West C M Scott D Chromosomal radiosensitivity as a marker of predisposition to common cancers Br J Cancer 101054bjoc20001701 Hu C et al Association between inherited germline mutations in cancer predisposition genes and risk of pancreatic cancer J Am Med Assoc 101001jama20186228 Verkasalo P K Kaprio J Koskenvuo M Pukkala E Genetic predisposition environment and cancer incidence a nationwide twin study in Finland Int J Cancer 101002SICI1097021519991 210836743AIDIJC830CO2Q Frank S A Genetic predisposition to cancerinsights from population genetics Nat Rev Genet 101038nrg14 Law P J et al Association analyses identify new risk loci for colorectal cancer susceptibility Nat Commun 101038s4146 w Czene K Lichtenstein P Hemminki K Environmental and heritable causes of cancer among million individuals in the Swedish FamilyCancer Database Int J Cancer 101002ijc10332 Economopoulou P Dimitriadis G Psyrri A Beyond BRCA new hereditary breast cancer susceptibility genes Cancer Treat Grant R C et al Prevalence of germline mutations in cancer predisposition genes in patients with pancreatic cancer GastroenRev 101016jctrv201410008 terology 101053jgastr o201411042 Petersen G M et al A genomewide association study identifies pancreatic cancer susceptibility loci on chromosomes 13q221 1q321 and 5p1533 Nat Genet 101038ng522 Wolpin B M et al Genomewide association study identifies multiple susceptibility loci for pancreatic cancer Nat Genet Long J et al Genomewide association study in East Asians identifies novel susceptibility loci for breast cancer PLoS Genet 101038ng3052 101371journ alpgen10025 Thomas G et al Multiple loci identified in a genomewide association study of prostate cancer Nat Genet 101038ng91 Mancuso N et al The contribution of rare variation to prostate cancer heritability Nat Genet 101038ng3446 Jiao S et al Estimating the heritability of colorectal cancer Hum Mol Genet 101093hmgddu08 Huang KL et al Pathogenic germline variants in adult cancers Cell 101016jcell201803039 Griffin L L Ali F R Lear J T Nonmelanoma skin cancer Clin Med J R Coll Physicians Lond 107861 Nikolaou V Stratigos A J Tsao H Hereditary nonmelanoma skin cancer Semin Cutan Med Surg 101016jclinm edici ne16162 sder201208005 Roberts M R Asgari M M Toland A E Genomewide association studies and polygenic risk scores for skin cancer clinically useful yet Br J Dermatol 101111bjd17917 Forbes S A et al COSMIC exploring the worlds knowledge of somatic mutations in human cancer Nucleic Acids Res 101093nargku10 cell201802060 Bailey M H et al Comprehensive characterization of cancer driver genes and mutations Cell 101016j Annels N E et al Phase I trial of an ICAM1targeted immunotherapeuticcoxsackievirus A21 CVA21 as an oncolytic agent against non muscleinvasive bladder cancer Clin Cancer Res 10115810780432CCR184022 Hamdi Y et al Family specific ge | 2 |
" in head and neck cancer hnc the relationship between a delay in starting radiotherapy rt andthe outcome is unclear the aim of the present study was to determine the impact of the amount of time beforetreatment intervention tti and the growth kinetics of individual tumors on treatment outcomes and survivalmethods two hundred sixtytwo hnc patients with primary tumors treated with definitive chemo rt wereretrospectively analyzed the tti was defined as the time interval between the date of histopathologic diagnosisand the first day of the rt course volumetric data on tumors were obtained from diagnostic and rt planningcomputer tomography ct scans in order to calculate the tumor growth kinetic parametersresults no significant association between locoregional control or causespecific hazards and tti was found thelog hazard for locoregional recurrence linearly increased during the first days of waiting for rt although this wasnot significant the median tumor volume relative increase rate and tumor volume doubling time was 32dayand days respectively and neither had any impact on locoregional control or causespecific hazards the association between a delay in starting rt and the outcome is complex and does not harm allpatients waiting for rt further research into imagingderived kinetic data on individual tumors is warranted inorder to identify patients at an increased risk of adverse outcomes due to a delay in starting rtkeywords head and neck cancer radiotherapy waiting time treatment delay outcome with new cases and deaths reported in head and neck cancer hnc is the eighth mostcommon and most lethal cancer in men worldwide in addition to surgery and systemic therapy radiotherapy rt is one of the cornerstones for treatment of thiscancer owing to the rising cancer incidence rate in ageing populations and the widening list of indications forirradiation the demands for rt have increased dramatically over the past decades [ ] the increasing complexity of pretreatment diagnostics and rt technology correspondence pstrojanonkoisi1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia4chair of oncology faculty of medicine university of ljubljana ljubljanasloveniafull list of author information is available at the end of the has led to delays in treatment decisionmaking and thereduction in linear accelerator throughputthat hasresulted in a significant imbalance between the demandfor rt and the availability of rt capacities in manypublically funded health systems this is also the case inslovenia [ ]due to obvious ethical reservations the only way tostudy the impact of delays in starting rt on treatmentoutcomes are retrospective observational analyses of cohorts from different institutions or countries intuitively one would expect that the prolongation of thetime taken before treatment intervention tti is harmful to patients both the likelihood of tumor growth andthe acquisition of a metastatic phenotype increases as afunction of time furthermore advanced tumors aremore difficult to treat than smaller tumors 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 0cžumer radiation oncology page of indeed a systematic review of pertinent literature fromthe period by chen showed an increasein the risk of local hnc recurrence of for everymonth of delay in definitive rt however certainstudies included in chens metaanalysis and also somemore recent reports negated the association betweenthe delay in definitive rt and the increased risk of treatment failure [] several different biases inherent inretrospective analyses either related to the quality ofdiagnostic procedures and treatment or to the inhomogeneity of the studied population as well as a selectionbias ie patients with fasttumor progression or ahigher burden of symptoms receive priority in treatment and significant variability in the kinetics of individual hnc cases may abolish the effect of tti inoutcomes [] however if patients with advancedor fastgrowing tumors have to wait longer the magnitude of this effect may be overestimated furthermore no compelling relationship between treatmentdelay and prognosis was found in some other cancertypes []in order to determine what would be an acceptabletti in hnc patients treated with definitive rt or concurrent chemoradiation we aimed to analyze the impactof tti and growth kinetics of individual tumors on theoccurrence of localregional failure distant metastasisand survival in the present study of a cohort of slovenepatients with hncmethodsin a retrospective study patients with oral cavity oropharyngeal hypopharyngeal or laryngeal squamous cellcarcinoma scc who were treated with curativeintentdefinitive rt with or without concurrent chemotherapybetween january and december at the institute of oncology ljubljana slovenia were includedpatients with t1n0 or t2n0 glottic cancer were left outof this cohort the period was chosen because of fluctuations in the waiting time for irradiationas a result of intensive renovations and expansion in thedepartment of radiotherapy that took place over thistime span from patients medical and rt charts wecollected information on clinical gender age onset ofsymptoms date and type of disease recurrence anddeath tumor histology site of origin tnm stage andtreatment characteristics rt technique regimen anddose duration of rt type of concurrent chemotherapy[cct] and the number of cycles administered thetnm stage was determined according to the 7th editionof the uicc classification systemfor analysis of the impact of tumor growth kinetics ontreatment outcomes the volumes ml of primary tumors and neck nodal metastases as marked on diagnostic and rt planning computer tomography ct scanswere compared patients with the same basic clinicaldisease and treatment characteristics as indicated abovebut with both sets of ct scans available were selectedfor this part of the study diagnostic ct scans were performed through the acquisition of mm thin ct sections whereas planning ct scans had a slice thicknessof mm both with intravenous iodine contrast enhancements sets with extensive artefacts were excluded forthe purposes of rt planning patients were positionedsupine on the flat tabletop and a fivefixation pointthermoplastic mask was used lymph nodes were considered positive if the smallest diameter was more than cm andor the necrotic center or extracapsular extension was seen if available segmentation was guided bymagnetic resonance imaging mri sets and the resulting contours around the primary tumors and metastaticneck nodes represented a consensus between two radiation oncologists and a radiologist volumes of primarytumors and metastatic neck nodes were separately calculated by a computer software program used for rt planning xio computerized medical systems inc stlouis usa eclipse varian medical systems inc paloalto usa the end points in this part of the study werechanges in the tumornodal volume and tnm stage thecalculation of the primary tumor volume doubling timeand their impact on the treatment outcomestatisticsthe study protocol was approved by the republic ofslovenia national medical ethics committee no for retrospective studies a written consentis deemed unnecessary according to national regulationsbasic descriptive statistics were reported with meansstandard deviations and ranges for numerical variablesand as percentages for categorical variables in patientswith two simultaneous hncs some characteristics werereported in regards to patients while others were reported in regards to tumorsthe survival curves were computed using the kaplanmeier estimator and the aalenjohansen estimator wasused to estimate the cumulative probabilities of competing risks the effect of covariates was analyzed using amultiple cox regression analysis with all the analysesthe data were censored at a fiveyear followupwhen focused on the survival of patients the analyseswere completed with patients as the units and the timewas measured from the first day of therapy until deaththe overall survival os regardless of the cause ofdeath and the absolute risk cumulative probability ofdying due to index cancer were reported in the cox regression only the index cancer deaths causespecifichazard csh were considered to be events of interestin the analyses where locoregional control lrc wasof primary interest the calculations were performed in 0cžumer radiation oncology page of regards to tumors excluding the nonresponders to rtie those with residual local or regional tumors at weeks after rt completion for the latter group weconcluded that it is the radioresistance of tumor cellsthat are responsible for the persistence of the diseaseafter therapy and not that patients had to wait for rtthe followup time was calculated from the last day oftherapy the estimated cumulative probability of localandor regional recurrence distant metastases lrcprobability of being still alive and without local andorregional recurrence and diseasefree survivaldfsprobability of being still alive and without events locoregional and distant failure and deaths were the events ofinterest were reported all the analyses were conductedin regards to the tumors as independent units this assumption was checked in the sensitivity analysis andallowed for gamma frailtythe assumptions of the cox regression were checkeda nonlinear effect a spline with degrees of freedomwas allowed for the numerical variables and the proportional hazards assumption was tested using schoenfeldresidualsthe tti was defined as the time interval between thedate of histopathologic diagnosis and the first day of thert course tumor growth kinetics was expressed as thetumor volume relative increase rate per day and as thetumor volume doubling time in daysthe tumor volume relative increase rate per day was 13 calculated as 12v t 2ðþþv t 1ðt2 t1where vt1 gross tumor volume at time t1 ieon diagnostic ct scans and vt2 gross tumor volume at time t2 ie on planning ct scans it was reported as the percentage increase was reported as per day the tumor volume doubling time was calculated asðv t 2ðv t 1ðln 2ð þ t t þþlnþsince a onetoone relationship existed between thetwo only the tumor volume relative increase rate whichrequires no extrapolation was considered for modellingall analyses were performed using r statistical software version and a pvalue below was considered statistically significantresultsimpact of time to treatment initiationbetween and patients with oral cavity oropharyngeal hypopharyngeal or laryngeal primarysccs were treated with definitive chemo radiotherapywith curative intent there were men and women aged years mean the majority oftumors originated in the oropharynx tumors in patients and were tnm stage iv tumors in patients the tti ranges from to days with mean days the distribution of the tti is only slightlyasymmetric with median and interquartile rangeiqr fig patients were irradiated with dimensionaltechnique or 3dimensionalconformal isocentric technique to the median rt dose gy iqr delivered in gy dailyfractions iqr concurrently to rt patientsreceived chemotherapy consisted of platinbased monochemotherapy patients or mitomycinecbleomycin combination patients the characteristics of patients and their tumors are shown in table treatment outcome and survivalclinical andor radiologic assessment at weeksposttherapy confirmed disease persistence locally andor regionally in cases ie in patients and thesepatients were excluded from further analyses of lrcand dfs thus patients with tumors were analyzed and during followup a local andor regional relapse was recorded in cases with a mediantime from treatment completion of months range months the two and fiveyear probability of localrecurrence after the end of treatment was and respectively whereas the regional relapse was and respectively after threeyears posttherapy we only observed a small increase ie inlocal recurrence probability whereas the probability ofregional relapse was stable after the third year followupthe probability of occurrence of distant metastases at years was and at years it was at the second and fifthyear followup the lrc was confidence interval [ci] and ci respectively and the dfs was ci and ci respectivelyin the fifth year posttreatment out of patients had died the index cancer was the reason in of the cases during the first years posttherapythe probability of cancerrelated death steeply increasedfrom zero to but later the changes were less pronounced at the second and fifth year posttreatment theos independent of the cause of death was ci and ci respectivelyeffect of covariatesin the univariate analysis the following parameters weretested for lrc and csh age gender stage of diseasetype of treatment rt vs rt cct and tti for rtas continuous variable results are presented in table 0cžumer radiation oncology page of fig distribution of ttitable clinical characteristics of patients and their tumorscharacteristicsgenderno femalemaleage yearsatumor locationboral cavityoropharynxhypopharynxlarynxtumor stagebstage istage iistage iiistage ivtreatmentbradiotherapyconcurrent chemoradiotherapyrt duration daysatime to treatment interventiona ± ± ± the same parameters were also included in the multivariate model table the occurrence of locoregional recurrence was significantly related to the diseasestage p whereas the relationship with agewas only of marginal significance p higherage had a lower hazard no significant associationwith the type of treatment could be found p table the log hazard for locoregional recurrenceseemed to linearly increase during the first days oftable effect of covariates on locoregional control and indexcancerspecific hazard n patients with tumors anunivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherap cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt a mean ± sd rangeb eleven patients had two simultaneous primary tumorsci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapy 0cžumer radiation oncology page of table impact of tti on locoregional control and indexcancerspecific hazard n patients with tumors amultivariate analysisparameterlocoregional control cihrpvaluetnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rtcause specific hazardtnm iii vs iiitnm iv vs iiiagettigendertherapy cct vs rt ci confidence interval tti time to treatment interventioncct concurrent chemoradiotherapy rt radiotherapywaiting for rt although the association between thehazard and tti wasregardless ofwhether we made an allowance for nonlinearity ornot fig 2ainsignificantthe hazard of dying due to the index cancer ie cshwas found to be favorably associated with a lower disease stage p and the addition of cct to rtp whereas age gender and tti did not reachthe level of statistical significance table fig 2bassumptions and sensitivity analysisproportional hazards for all included variables and thelinearity assumption for age were analyzed and presented graphically some indication of nonproportionalhazards could be found in gender but allowing for nonproportional hazards did not change the interpretationof the other covariates as a part of the sensitivity analysis the assumption of the dependence of the tumorsbelonging to the same individual was relaxed but nochanges in the interpretation of the results could be observed as an additional confounder the timedependentcovariate duration of rt was considered but its effectdid not prove to be importantimpact of tumor growth kineticsdiagnostic and planning ct scans median interval days range days were available from patientsfive of these patients had two primary tumors the majority of patients were males with a meanage of years range and the patients hadprimary tumors located in the oropharynx table when two ct sets were compared the original tstage of the primary tumor was increased ie upgradedin two cases and the nodal stage was increased in six patients due to the limited number of npositive casesn only volumes of primary tumors werecompared between the two ct sets for the calculationof the tumor volume relative increase rate per dayand tumor doubling time the absolute increase intumor volume ranged from to cm3 per daymedian no increase in volume was observed infive patients ct scans in these five patients were takenfig trend of the hazard for locoregional recurrence a and for index cancerspecific death b 0cžumer radiation oncology page of table characteristics of patients with available diagnostic andplanning ct scanscharacteristicsgenderno femalemaleage yearsatumor locationboropharynxhypopharynxlarynxstagebstage istage iistage iiistage ivtherapybradiotherapyconcurrent chemoradiotherapytumor volume relative increase rate dayatumor volume doubling time daysaa mean ± sd rangeb five patients had two simultaneous primary tumors from days to days apart median days the median tumor volume relative increase rate was perday median range and the mediantumor volume doubling time was days range days no difference was observed when these two parameters were compared between different tumor stagestreatment outcome and survivalamong the tumors the time taken for local andorregional relapse and the occurrence of distant metastaseswere assessed for tumors in which treatment resultedin clinicalradiological eradication of the disease at weeks posttherapy local andor regional relapse wasrecorded in nine cases with a median time fromtreatment completion of months range monthsthe two and fiveyear probability of local recurrenceafter the end of the treatment was and respectively whereas for the regional relapse it was and respectively at years posttherapy only anincrease in the local recurrence probability of wasobserved whereas the probability of regional relapseremained stable the two and fiveyear probability ofdistant metastases was at two and years thelrc was ci and ci respectively and the dfs was ci and ci respectivelyin the yearperiod after the start of treatment outof patients died the index cancer was thereason in cases at two and years posttreatment the os rates were ci and ci respectivelyeventseffect of covariatesdue to the low number of events local andor regionalrecurrence ninecancerspecific deaths events only univariate regression models were fittedthe following covariates were tested in the modelsoverall disease stage initial tumor volume tumor doubling time and relative increase rate day of primarytumor volume measures of tumor kinetics did not showany impact on lrc or csh only an inverse relationshipbetween the initial primary tumor volume and csh wasobserved hr for index cancerspecific death perevery cm3 increase in the volume of primary tumorstable there was no difference in the value of dfsbetween patients with a primary tumor volume relativeincrease rate 1day and 1day p fig discussionwhile it is intuitively anticipated and confirmed by theresults of the metaanalysis that a delay in starting rtwould have a negative impact on the treatment of patients with hnc this association was not confirmed inour study we only found a statistically insignificantupward trend in the risk of locoregional recurrence forthe first days of rt delay in addition the differencesin the growth kinetics between individual tumors whichwere studied in a smaller group of patients were considerable but did not appear to be of significance for theprediction of treatment outcomesobviously the relationship between tti and diseaseprognosis in patients with hnc is more complex than itmight seem at first glance the first negative impact ofwaiting for treatment to begin is the risk that the tumorwill increase in size andor metastasize during this timemaking it harder to treat or resulting in it becoming untreatable [ ] however the time for a tumor to growis only one of the factors that determines prognosis andthe absence of a statistically significant association between tti and the treatment outcome in our study andis thus not surprising []many other reportsmoreover no obvious methodological differences couldbe found between these negative studies and the positive studies that confirmed the association between ttiand treatment outcomes [] in both groups thereare individual studies that have similar sample sizestumor sitestage mix and periods covered speaking infavor of comparable quality of diagnostics therapy andstatistics across the studies 0cžumer radiation oncology page of table effect of stage and tumor kinetics on locoregional control and overall survivalparameterstnm stageiv vs iiiivtuper cm3vtu relative increase rateper dayvtu relative increase rate¤ vs 1dayvtu volume of primary tumorlocoregional controlhr cipvaluecause specific hazardhr cipvalueon the contrary in more recently reported analyses ofthe national cancer registries data an adverse effect ofwaiting for radiotherapy was clearly established []however the results from this type of analysis should betaken with caution not only due to the limitations inherent in the tumor registry data unmeasured confounding selection biasincomplete data and codingerrors but also because the effect of delaying rt oncancerspecific outcomes was not evaluated in additionpatients irradiated in postoperative and definitive settings were not analyzed separately overall the researchmethodology and interpretation used in these studieswere criticized and the magnitude of the effect that theysupposedly demonstrated was questioned in the present study a linear increase in the log hazardfor locoregional recurrence was found during the first days of waiting for rt although it was not statisticallysignificant it is possible that unknown confounders thatwere not accounted in our analysis eg tumor growth kinetics reduced the statistical power of the tti the resultsfrom fortin and naghavi who reported on theincreased risk of locoregional failure with tti daysand days respectively [ ] are the most comparable to our own however optimal ttithresholdsfig impact of primary tumor volume relative increase rate to diseasefree survival in patients with no residual disease at weeks posttherapy 0cžumer radiation oncology page of identified in different studies showed considerable variations pointing to the uncertainty of such calculations andtheir dependence on the characteristics of the analyzedpopulation [ ] the possible role of classical prognostic factors such as location of primary tumor diseasestage and addition of cct is not expected to be relevant inthis respect as in our and other similar studies the statistical significance of tti was verified by multivariateanalysisthe effect of tti on treatment outcomes however isnot only conditioned by the duration of waiting for rtbut also on the rate of tumor growth in hnc a vast heterogeneity in tumor cell kinetics has been observed conditioned by the local milieu from which it arises whichdiffers from patient to patient [ ] historicallydifferent methods were explored to evaluate tumor cellkinetics but did not succeed in providing clinically relevant kinetic parameters [ ] more recently a comparison of two sets of imaging data acquired at twodifferent time points was successfully employed for thispurpose usually volumetric data are extracted from diagnostic and rtplanning ct scans for the calculation ofdifferent parameters reflecting the rate of tumor growtheg tumor volume doubling time or absolutepercentagetumor volume increase per day despite some differencesin the calculation methodology across studies a large variation in the individual values of kinetic parameters wasseen in all of them including ours indicating that all ofthe studied populations represent a unique mix of slowand fast growing tumors [] among our patientsfive had no measurable increase in primary tumor volumeeven when the interval between ct scans was up to days the inverse relationship between kinetic parametersdetermined by the comparison of two ct datasets andtreatment outcomes was implied or even confirmed inseveral smaller studies pointing to potential clinical utilityof imagingderived kinetic data [] in our grouphowever no such association was found a small numberof patients and in contrast to other studies the inclusion of different tumor sites could contribute to the negative result as well as differences in the changes in kineticproperties triggered by rtccr in individual tumors tumor radiosensitivity may also influence the effect oftti on outcome while to some extent it may be evaluated before rt begins eg by molecular profiling identification of hypoxic cells and the determination of hpvstatus in oropharyngeal primary tumors in daily clinicalpractice it is usually not considered when planning treatment [ ] in order to diminish the impact of intrinsic tumor radiosensitivity the patients in our study withresidual disease at weeks postrt were excludedfrom the analysis of lrc we hypothesized that the inability to achieve a complete tumor response to chemoradiation was due to the radiobiological characteristicsof the disease and not the delay in starting rt howeverthe exclusion of these patients from the analysis did notaffect the end resultsour study has weaknesses which are mostly due to itsretrospective nature however for obvious ethical reasons this is an inevitable feature of studying delays inthe initiation of cancer therapy we are aware that thereis always some doubt as to the accuracy of the diagnosticprocedures the staging the quality of planning and theimplementation of rt in the case of retrospective research nonetheless the same restraint exists in the caseof other similar studies and even more so in the case ofanalyses of cancer registry datasets an important feature of our data set is that no patients were lost duringthe followup and in our opinion is free from manyhidden biases that larger studies inevitably bring withsince almost half of our patients had oropharyngeal carcinoma missing information regarding the p16 or human papillomavirus hpv tumor status could be ofimportance howeverin the cohort of oropharyngealcancer patients treated at our institution between and only had a hpvrelated tumor thus we reasonably assume that the impact of p16hpv tumor status on the study results was negligible inaddition in hpvpositive cases perni found nosignificant association between tumor growth velocitycalculated from serial pretreatment ct scanslocaland distant control or os and the same was reportedby chu who measured metabolic growth velocityusing pretreatment petct scans [ ]other drawbacks to consider include technical limitationsin ct scan acquisition the accuracy of presentation of actual tumor volume on ct images and the precision of thedelineation of gross tumor volume to minimize errors inthe estimation of comparative tumor volumes only highquality pairs of ct images were selected for the analysis oftumor kinetics in addition physical findings documentedin clinical records and when available diagnostic mriswere used for this purpose and labelled tumor volumes represented a consensus between two experienced radiationoncologists and a radiologist all dedicated to hnc management like many other studies [ ] the comorbidity burden was not registered in our patientsalthough it should be taken into account when assessingsurvival outcomes finallylags in the prebiopsyperiod were not addressed in our study including patientdelay and delays in referral and diagnostics which may addsignificantly to the total tti these are also costly and potentially fatal but can be successfully reduced by effectivecoordination between providers []sthe relationship between tti and treatment outcomesis multifaceted so the controversy of published results is 0cžumer radiation oncology page of not surprising in this study we found that delays in theonset of rt do not harm all patients as tti is a problem in many public health systems further research iswarranted and should focus on two areas evaluatinglarge population surveys with highquality data andtreatmentrelated outcomes not just os and the prognostic relevance of imagingderived kinetic data of individualtumors which appeared promising in severalsmaller and statistically underpowered studies in orderto obtain a tool to identify patients at increased risk oftreatment failure due to delays in starting rt in a situation without clear knowledge to whom waiting for irradiation is harmful the only possible recommendationcould be that the waiting time for rt should be asshort as reasonably achievable asara abbreviationshnc head and neck cancer rt radiotherapy tti time to treatmentintervention scc squamous cell carcinoma cct concurrent chemotherapyct computer tomography mri magnetic resonance imaging os overallsurvival csh causespecific hazard lrc locoregional control dfms distantmetastasisfree survival dfs diseasefree survival iqr interquartile rangeci confidence interval hpv human papillomavirusacknowledgementsthis study was financially supported by the slovenian research agencyprogram no p30307authors contributionsstudy concepts žumer b strojan p study design žumer b pohar perme mstrojan p data acquisition all authors quality control of data strojan p dataanalysis and interpretation pohar perme m strojan p statistical analysispohar perme m manuscript preparation all authors manuscript editingstrojan p manuscript review žumer b pohar perme m strojan p the authors read and approved the final manuscriptfundingthis study was financially supported by the slovenian research agencyprogram no p3availability of data and materialsthe datasets analyzed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatethe study protocol was approved by the republic of slovenia national medicalethics committee no all patients gave consent for using theirdata for study purposes at the start of their treatment for retrospective studies awritten consent is deemed unnecessary according to national regulationsconsent for publicationthe republic of slovenia national medical ethics committee approved thestudy which was conducted in accordance with the ethical standards laiddown in an appropriate version of the declaration of helsinki theneed for consent was waived by the republic of slovenia national medicalethics committeecompeting intereststhe authors declare that they have no competing interestsauthor details1department of radiation oncology institute of oncology ljubljana zaloÅ¡ka si1000 ljubljana slovenia 2institute of biostatistics and medicalinformatics faculty of medicine university of ljubljana ljubljana slovenia3department of radiology institute of oncology ljubljana ljubljanaslovenia 4chair of oncology faculty of medicine university of ljubljanaljubljana sloveniareceived 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 world health anization who life expecta | 0 |
"unrestricted use distribution and reproduction in any medium provided the original work is properly citedPurpose The present study was aimed at determining the serum levels of actinin4 ACTN4 in cervical cancer CC andinvestigating the diagnostic and prognostic value of serum ACTN4 in CC Materials and Methods We included CC patients cervical intraepithelial neoplasia CIN patients and healthy women Serum ACTN4 levels were assessed using an ELISAmethod A receiver operating characteristic ROC curve was performed to evaluate the diagnostic value of serum ACTN4 Thesurvival curves were used to display the overall survival distributions Results Serum ACTN4 levels in CC patients were ± pgmL which is significantly higher than those in CIN patients ± pgmL P and those in healthycontrols ± pgmL P The ROC analysis demonstrated that the area under the curve AUC of ACTN4 was 95CI with sensitivity of and speciï¬city of Serum ACTN4 levels were associated with theFIGO stage lymph node metastasis and lymphovascular space invasion of CC all P The survival curve suggested thathigh serum ACTN4 levels were related to poor prognosis Conclusion Our ï¬ndings suggest that serum ACTN4 levels may bevaluable diagnostic and prognostic biomarkers for CC IntroductionCervical cancer CC is the second most common femalemalignancy globally and it is the most common femalemalignancy in developing countries which has high morbidity and mortality rates [] In recent years the incidence ofCC has increased greatly in young women under the age of [] Despite great advances in surgical and adjuvant therapy the overall survival of CC patients especially that ofadvanced patients is still very poor [] At present a Papsmear combined with an HPV test has been used for the earlyscreening of cervicalthe screeningmethods are invasive and costly leading to lower screeningcoverage in China [] Previous studies have reported thatthe human papillomavirus HPV screening results have arelatively high falsepositive rate and a relatively low speciï¬city [ ] In addition the results of TCT interpretation byï¬lmreading doctors are uneven which might cause somelesions Howevermisleadingness in the choices of prevention measures andtreatment for CC [] Noteworthily when applying the sametreatment plan to patients with similar pathological types theeï¬cacy and prognosis are quite diï¬erent Therefore it is necessary to identify new biomarkers directly related to the progression and prognosis of CCAlphaactinins ACTNs are actinbinding proteins inthe spectrin gene superfamily [] which are known to becrosslinked with ï¬lamentous actin Factin to maintainthe integrity of cytoskeleton and to control cell motility []The ACTN family has four members numbered ACTN1which are present in humans and other mammals []ACTN4 is encoded by the ACTN4 gene and is widelyexpressed in many tissues especially in glomerular podocytes[] ACTN4 has an actinbinding domain at the Nterminus and ACTN4 monomers can form a homodimer throughreverse binding forming a dumbbellshaped structure []As an actinbinding protein ACTN4 is closely related to 0cDisease Markersenhancing cell viability and tumor invasion and metastasis[] Recent researches have reported that the expression ofACTN4 is significantly elevated in multiple cancers including breast cancer [] pancreatic cancer [] ovarian cancer[] and lung cancer [] In addition the ACTN4 levels aremarkedly associated with the poor prognosis of lung cancer[ ] thyroid cancer [] and salivary gland carcinoma[] An [] have found that the expression level ofACTN4 in human cervical tumors is dramatically higherthan that in normal cervical tissues Their ï¬nding demonstratedepithelialtomesenchymal transition and tumorigenesis by regulatingSnail expression and the Akt pathway in CC [] Thereforethe expression of ACTN4 in cervical tissues may be used inthe clinical diagnosis and prognosis prediction of CCthat ACTN4promotestheHowever up to now the signiï¬cance of the serumACTN4 levels in CC has not been evaluated Hence in thecurrent study the serum levels of ACTN4 in patients withCC were measured In addition we estimated the potentialdiagnostic and prognostic value of serum ACTN4 expressionin CC Materials and Methods Study Population A retrospective study was designed toevaluate serum actinin4 as a biomarker for CC Between July and June newly diagnosed female CC patientsand newly diagnosed female cervical intraepithelial neoplasia CIN patients who received treatment at HuaianMaternal and Child Health Care Hospital Huaian JiangsuChina were recruited The diagnoses of all patients were veriï¬ed by the histopathological examination The patients withother types of tumor or autoimmune atherosclerotic andhematologic diseases were excluded The mean age of CCpatients was years with a range of years Meanwhile healthy women with no evidence of neoplasmsand other serious diseases were enrolled from the physicalexamination center in the same hospital There was no significant diï¬erence in age among the CC CIN and healthy control groups This study was consistent with the Helsinkideclaration and was authorized by the Ethics Committee ofHuaian Maternal and Child Health Care Hospital approvalnumber H20130504 All participantssigned writteninformed consent Clinicopathologic Feature Collection and FollowUp Byreviewing the medical records we collected the clinicopathologic characteristics of the patients including age at diagnosis pathological type FIGO stage tumor diï¬erentiationpelvic lymph node metastasis tumor size and lymphovascular space invasion The CC patients were classiï¬ed based onthe revised FIGO staging system for CC in The tumorsize was the maximum tumor diameter determined by agynecologic oncologist during pelvic examination Thepatients in stage 1A1 received hysterectomy the patients instages IB1 and IIB received radical hysterectomy and pelviclymph node dissection the patients with ¥stage IIB receivedradiotherapy or radiotherapy combined with chemotherapyA regular telephone followup was conducted after treatmentto obtain the overall survival OS time of CC patients andthe OS was deï¬ned as the time from diagnosis to death orthe last followup The followup was in accordance withthe FIGO guidelines Blood Sample Collection and Detection of Serum Actinin and SCCA A mL peripheral blood sample from eachpatient was collected before receiving any treatment Afterstanding at room temperature for minutes the blood samples were centrifugated at gmin for min and then°the supernatant was stored at C until further usageThe serum actinin4 concentration was measured by a quantitativeELISAmethod Uscn Life Science Inc Wuhan China The levelsof SCCA in serum were determined using an ELISA kitRD Systems Minneapolis MN The detection of all samples was strictly in accordance with the instructions providedby the manufacturer and was performed in duplicatesenzymelinked immunosorbentassay Statistical Analysis All statistical analyses were conducted by using SPSS and GraphPad Prism The continuous data following normal distribution were expressed asthe mean ± standard deviation°SD A ttest was used tocompare serum ACTN4 levels between the two subgroupsof each clinicopathological parameters and the serumACTN4 levels of CC patients CIN patients and healthy controls were compared by the SNKq test Receiver operatingcharacteristic ROC curves were performed to assess thediagnostic value of serum ACTN4 levels for diï¬erentiatingCC patients from CIN patients and healthy controls TheKaplanMeier method and logrank test were used to plotsurvival curves The Cox proportional hazards models in univariate and multivariate analyses were used for evaluating theprognostic value of serum ACTN4 expression A twotailed Pvalue was considered to be statistically significant Results Serum ACTN4 Levels Are Higher in Patients with CCSerum concentrations of ACTN4 were detected to rangefrom to pgmL with a mean ±SD of ± pgmL for CC patientsto range from to ngmL with a mean ±SD of ± pgmL forCIN patients and to range from to ngmL witha mean ±SD of ± pgmL for healthy controlsSerum ACTN4 levels in CC patients were significantly higherthan those in CIN patients and healthy controls P However no significant diï¬erence in serum ACTN4 wasfound between CIN patientscontrolsP as shown in Figure and healthy The Diagnostic Value of Serum ACTN4 Levels for CC Wenext used ROC curve analysis to estimate the diagnostic valueof serum ACTN4 expression for CC The ROC curve showedthat the serum levels of ACTN4 were robust for discriminating CC patients from benign and healthy control subjectswith an area under the curve AUC value of 95CI as demonstrated in Figure index we usedAccordingto maximum Youdens 0cDisease Markerslymph node metastasis were the independent prognostic factors for CC all P Table Lmgp NTCAnsCCCINCON DiscussionCervical cancer is a heterogeneous disease with complicatedetiology Genetic and environmental factors play a crucialrole in the pathogenesis of CC [] Although the diagnosisand prognosis of CC have improved greatly over the pastfew decades it is necessary to improve early detection andscreening methods to determine additional promising circulating biomarkers for better patient selection and more personalized treatments [] As far as we know this studyrepresented the ï¬rst eï¬ort to evaluate the serum expressionof ACTN4 as a new biomarker for CCAs an actinbinding protein ACTN4 can participate inregulating cell migration invasion and metastasis via regulating the actin ï¬lament ï¬exibility at the leading edge ofinvading cancer cells [ ] ACTN4overexpressing cancercells have the potential to metastasize because the overexpression of ACTN4 protein in cancer cells can stimulate thedynamic reconstruction of the actin cytoskeleton [] Upto now numerous studies have reported the associationbetween ACTN4 and multiple cancers Okamoto []observed that ACTN4 is expressed in smallcell lung cancerNSCLC and it had a significant correlation with invasionand distant metastasis Additionally ACTN4 was reportedto be a potential predictive biomarker for the eï¬cacy of adjuvant chemotherapy in patients with NSCLC [] Watabe [] revealed that the copy number increase of ACTN4is a novel indicator for poor overall survival of patients withsalivary gland carcinoma and the copy number variationwould aï¬ect the expression of protein A recent study demonstrated that serum ACTN4 levels were dramatically elevated in patients with breast cancer when compared tohealthy controls and serum ACTN4 may be an eï¬ective clinical indicator for diagnosing or predicting the clinical outcomes of breast cancer patients [] In addition ACTN4was proven to be associated with the pathogenesis of CCAn [] proposed a novel mechanism for epithelialtomesenchymal transition and tumorigenesis in CC whichcould be induced by ACTN4 through regulating Snail expression and βcatenin stabilization Hence it is significant toinvestigate the role of serum ACTN4 in CCIn the current study we observed that serum levels ofACTN4 in CC patients were statistically higher than thosein CIN patients and those in healthy controls Howeverserum ACTN4 levels were not significantly diï¬erent betweenthe CIN group and the control group It was shown thatserum ACTN4 expression could strongly diï¬erentiate CCpatients from CIN patients and healthy controls The ROCanalysis demonstrated that the AUC of ACTN4 was and at the optimal cutoï¬ of pgmL the sensitivity andspeciï¬city were respectively and suggestingthat serum ACTN4 might be a potential diagnostic biomarker for CC In a recent study which included Chinesewomen Hu [] reported that the sensitivity and speciï¬city of HPV screening in the diagnosis of CC were and The sensitivity of the HPV test was a litter higherFigure The serum ACTN4 levels in CC patients CIN patientsand healthy controls P pgmL as the cutoï¬ value and the sensitivity and speciï¬city were and respectively Association between Serum ACTN4 Levels andClinicopathological Parameters of CC Patients We furtherinvestigated the correlations between serum levels of ACTN4and clinical pathological data of CC patients and theresults are demonstrated in Table We observed that serumACTN4 levels were related to the FIGO stage lymph nodemetastasis and lymphovascular space invasion all P Nevertheless no significant association was found betweenserum ACTN4 levels and age pathological type diï¬erentiation degree and tumor size in CC patients all P Survival Analysis of Serum ACTN4 Levels in CC Duringthe followup period nine CC patients were lost and thefollowed up rate is Finally the prognostic value ofserum ACTN4 was assessed in patients The patients werefollowed up to December The range of followup timewas to months with the median time of months andmean time of months According to the median serumlevels of ACTN4 in CC patients pgmL the CCpatients were divided into the high ACTN4 level group pgmL N and low ACTN4 level group¥ pgmL N The estimated 5year OS of patientswith high serum ACTN4 levels and low serum ACTN4 levelswere and respectively The KaplanMeier survival curve and logrank test indicated that CC patients withhigh serum ACTN4 levels had a worse prognosis than thosewith low serum ACTN4 levels P Figure Univariate Cox regression analyses showed that theserum ACTN4 levels P FIGO stage P diï¬erentiation degree P lymph node metastasisP and lymphovascular space invasion P had significant prognostic value for OS Multivariate analysiswas further performed to evaluate the prognostic value ofserum ACTN4 as an independent factor for CC All the statistically significant factors from univariate analyses wereincluded and the results indicated that the FIGO stage and 0cDisease MarkersytivitisneS specificityFigure ROC curve analysis assessed the diagnostic performance of serum ACTN4 in CC The AUC was P Table Serum ACTN4 levels in CC patients according toclinicopathological parametersParametersAge years¤Pathological typeSquamous cell carcinomaAdenocarcinomaFIGO stageIA1IB1¥IB2Diï¬erentiationWell and moderatelydiï¬erentiatedPoorly diï¬erentiatedLymph node involvementNegativePositiveTumor size¤Lymphovascular space invasionNegativePositiveN ACTN4pgmLP ± ± ± ± ± ± ± ± ± ± ± ± ± ± than that of serum ACTN4 detection though the speciï¬cityof serum ACTN4 detection was well above that of the HPVtest Hence comparing with the HPV test in diagnosingCC detecting serum ACTN4 has some advantages Furthermore serum ACTN4 levels have been indicated to be a greatbiomarker for diagnosing multiple cancers Fang [] intheir study reported that serum ACTN4 was a promisingindicator for diagnosing breast cancer with the AUC of Wang [] used ACTN4 expression in peripheralblood to diï¬erentiate NSCLC patients from healthy individuals in two groups of participants and they obtained bothsatisfactory eï¬ects Furthermore we investigated the correlation between serum ACTN4 and clinical characteristics ofCC patients The serum ACTN4 levels were significantlyassociated with the FIGO stage lymph node metastasis andlymphovascular space invasion of CC which suggests thatACTN4 could contribute to the development invasion andmetastasis of CC In addition our results indicated that highACTN4 levels were associated with the poor survival of CCpatients In the multivariate analysis although ACTN4 levelsdid not reach the statistical signiï¬cance it still seems to beable to inï¬uence the OSHowever several limitations in the present study should betaken into consideration First the sample size was relativelysmall which was likely to reduce the statistical power of ourresults Second we only explored the relationship betweenserum ACTN4 and OS and other prognostic indicators werenot examined due to the incomplete data which needs to beimproved in the future Third this study was a primary studyto determine the clinical signiï¬cance of serum ACTN4 levelsfor the diagnosis and prognosis of CC but the speciï¬c molecular mechanisms remain unclear Hence further experimentsshould be conducted to elucidate the mechanismsIn conclusion our study showed that serum ACTN4levels were increased in CC patients and were related to the 0cDisease Markers lavivrus muCLog rank P Overall survival monthsLow ACTN4 groupHigh ACTN4 groupFigure KaplanMeier curve compared OS of CC patients with high serum ACTN4 levels versus those with low serum ACTN4 levelsTable Univariate and multivariate Cox regression analysis of OS in CC patientsUnivariate CIVariablesAge vs ¤ yearsPathological type squamous cell carcinoma vs adenocarcinomaFIGO stage ¥IB2 vs IA1IB1Diï¬erentiation poorly diï¬erentiated vs well and moderately diï¬erentiated Lymph node involvement positive vs negativeTumor size vs ¤ cmLymphovascular space invasion positive vs negativeSerum ACTN4 levels high vs low levelsHR PMultivariate CIPHRFIGO stage lymph node metastasis and lymphovascularspace invasion of CC patients In addition serum levels ofACTN4 have great diagnostic and prognostic value in CCNevertheless further studies with a larger sample size shouldbe carried out to conï¬rm our resultsAcknowledgmentsWe thank all the patients and blood donors who participatedin our study This study was funded by grants from the Science and Technology Project of Traditional Chinese Medicine Bureau of Jiangsu province China YB2015128Data AvailabilityReferencesThe datasets used andor analyzed during the present studyare available from the corresponding author on reasonablerequestConflicts of InterestAll authors declare that they have no conï¬icts of interestAuthors ContributionsXigui Ma and Huiying Xue contributed equally to this workand should be considered as coï¬rst authors[] M H Forouzanfar K J Foreman A M Delossantos et alBreast and cervical cancer in countries between and a systematic analysis The Lancet vol no pp [] E Pelkofski J Stine N A Wages P A Gehrig K H Kimand L A Cantrell Cervical cancer in women aged yearsand younger Clinical Therapeutics vol no pp [] Y Zhou W Wang R Wei Serum bradykinin levels as adiagnostic marker in cervical cancer with a potential mechanism to promote VEGF expression via BDKRB2 International Journal of Oncology vol pp [] Y J Hu H P Zhang B Zhu H Y Chen L H Ma andY Wang The role of FH detection combined with HPV 0cDisease Markers[] N Miura M Kamita T Kakuya Eï¬cacy of adjuvantchemotherapy for nonsmall cell lung cancer assessed by metastatic potential associated with ACTN4 Oncotarget vol no pp [] N Tanaka T Yamashita S Yamamoto Histologicalgrowth pattern of and alphaactinin4 expression in thyroidcancer Anticancer Research vol no pp [] Y Watabe T Mori S Yoshimoto Copy numberincrease of ACTN4 is a prognostic indicator in salivary glandcarcinoma Cancer Medicine vol no pp [] HT An S Yoo and J Ko αActinin4 induces theepithelialtomesenchymal transition and tumorigenesis viaregulation of Snail expression and βcatenin stabilization incervical cancer Oncogene vol no pp [] F Niu T Wang J Li The impact of genetic variants inIL1R2 on cervical cancer risk among Uygur females fromChina a casecontrol study Molecular Genetics GenomicMedicine vol no article e00516 [] W Li Y Zhao L Ren and X Wu Serum human kallikrein represents a new marker for cervical cancer Medical Oncology vol no p [] H Shao J HC Wang M R Pollak and A Wells αActinin4 is essential for maintaining the spreading motility andcontractility of ï¬broblasts PLoS One vol no articlee13921 [] K Honda T Yamada Y Hayashida Actinin4 increasescell motility and promotes lymph node metastasis of colorectalcancer Gastroenterology vol no pp [] D G Thomas and D N Robinson The ï¬fth sense mechanosensory regulation of alphaactinin4 and its relevance forcancer metastasis Seminars in Cell Developmental Biologyvol pp screening on the diagnostic signiï¬cance of cervical cancer andprecancerous lesions European Review for Medical and Pharmacological Sciences vol no pp [] KH Wang C J Lin C J Liu Global methylationsilencing of clustered protocadherin genes in cervical cancerserving as diagnostic markers comparable to HPV CancerMedicine vol no pp [] T Li Y Li G X Yang Diagnostic value of combination of HPV testing and cytology as compared to isolatedcytology in screening cervical cancer a metaanalysis Journal of Cancer Research and Therapeutics vol no pp [] K Honda T Yamada R Endo Actinin4 a novel actinbundling protein associated with cell motility and cancer invasion The Journal of Cell Biology vol no pp [] E de Almeida Ribeiro N Pinotsis A Ghisleni Thestructure and regulation of human muscle αactinin Cellvol no pp [] D Wang X W Li X Wang Alphaactinin4 is a possible target protein for aristolochic acid I in human kidneycellsin vitro The American Journal of Chinese Medicinevol no pp [] I V Ogneva N S Biryukov T A Leinsoo and I M Larina Possible role of nonmuscle alphaactinins in musclecell mechanosensitivity PLoS One vol no articlee96395 [] K Honda The biological role of actinin4 ACTN4 in malignant phenotypes of cancer Cell Bioscience vol no p [] X Zhao K S Hsu and J H Lim αActinin potentiatesnuclear factor κlightchainenhancer of activated BcellNFκB activity in podocytes independent of its cytoplasmic actin binding function The Journal of BiologicalChemistry vol no pp [] H Shams J Golji K Garakani and M R Mofrad DynamicRegulation of α Actinin's Calponin Homology Domains on FActin Biophysical Journal vol no pp [] C Fang J J Li T Deng B H Li P L Geng and X TZeng Actinin4 as a diagnostic biomarker in serum ofbreast cancer patients Medical Science Monitor vol pp [] T Watanabe H Ueno Y Watabe ACTN4 copynumber increase as a predictive biomarker for chemoradiotherapy of locally advanced pancreatic cancer British Journal of Cancer vol no pp [] S Yamamoto H Tsuda K Honda ACTN4 gene ampliï¬cation and actinin4 protein overexpression drive tumourdevelopment and histological progression in a highgrade subset of ovarian clearcell adenocarcinomas Histopathologyvol no pp [] M C Wang Y H Chang C C Wu Alphaactinin is associated with cancer cell motility and is a potential biomarker in nonsmall cell lung cancer Journal of ThoracicOncology vol no pp [] N Okamoto H Suzuki K Kawahara The alternativelyspliced actinin4 variant as a prognostic marker for metastasisin smallcell lung cancer Anticancer Research vol no pp 0c" | 2 |
"At present the relationship between hypothyroidism and the risk of breast cancer is still inconclusive Thismetaanalysis was used to systematically assess the relationship between hypothyroidism and breast cancer riskand to assess whether thyroid hormone replacement therapy can increase breast cancer riskMethods The relevant s about hypothyroidism and the risk of breast cancer were obtained on the electronicdatabase platform Relevant data were extracted and odd ratios OR with corresponding confidence intervalsCI were merged using Stata SE softwareResults A total of related studies were included in the metaanalysis including cohort studies and casecontrol studies The results show that hypothyroidism was not related to the risk of breast cancer odd ratios CI In the European subgroup we observed that patients with hypothyroidism have a lower risk ofbreast cancerodd ratios CI Furthermore no significant correlation was observed betweenthyroid hormone replacement therapy and the risk of breast cancer odd ratios CI Conclusion Hypothyroidism may reduce the risk of breast cancer in the European population and no significantcorrelation was observed between hypothyroidism and breast cancer risk in nonEuropean populations Due to thelimited number of studies included more largescale highquality longterm prospective cohort studies areneededKeywords Hypothyroidism Thyroid hormone replacement therapy Breast cancer MetaanalysisBackgroundAs a global public health problem breast cancer has anincreasing incidence on a global scale [] According tothe US cancer statistics breast cancer has becomethe most common malignant tumour in women withabout new cases each year accounting for of new malignant tumours in women [] Therefore theidentification of risk factors for breast cancer and the Correspondence Yanhuangdr163com Ruobaolidr163com2Department of Oncology Affiliated Hospital of Weifang Medical UniversityWeifang China3School of Basic Medicine Weifang Medical University Weifang ChinaFull list of author information is available at the end of the adoption of effective early prevention and interventionmeasures are of great significance for patients withbreast cancerThe physiology and pathology of the breast are closelyrelated to the endocrine of the body [] As the largestendocrine an in the human body the thyroid glandhas specific regulation effects on various hormone levelsand cell growth and development in the body whichbrings new enlightenment to the research of breast cancer [] In Kapdi et alfirst proposed thathypothyroidism maybe increase the risk of breast cancer[] Since then many scholars have studied the relationship between hypothyroidism and the risk of breast 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 0cWang BMC Cancer Page of []cancer However the relationship between the two diseases remains controversial [] Some studies haveshown that hypothyroidism increases the risk of breastcancerthathypothyroidism reduces the risk of breast cancer []Besides some studies have found no correlation betweenthyroid disease and breast cancer risk [] Thereforewhether hypothyroidism can increase the risk of breastcancer is worthy of further studystudiesshownSomehaveTwo metaanalyses have previously been studied forhypothyroidism and breast cancer risk [ ] Based onprevious research we have included more prospectivestudies and Asian population studies to assess the relationship between hypothyroidism and breast cancer risksystematically Besides the impact of thyroid hormonereplacement therapy on breast cancer risk was exploredin this metaanalysisMethodsSearch strategyRelevant clinical literature was extracted by systematicretrieval of PubMed Medline EMBASE Springer Webof Science and Cochrane Library electronic databasesup to date to October Our search strategy includedorhypothyroidism or HT and thyroid diseases orbreast cancer or BC or breast neoplasms or mammarmy cancer and risk orincidence At the sametime we manually screened out the relevant potentialliterature in the references extracteddysfunctionthyroidtermsforInclusion and exclusion criteria The inclusion criteria Types of studies Published studies exploring therelationship between hypothyroidism and breastcancer risk Subject Female Exposure factors Primary hypothyroidism thediagnosis needs to be based on the detection ofthyroid function Outcome indicators the occurrence of primarybreast cancerThe exclusion criteria Nonprimary hypothyroidism due to other causes Non observational studiesInsufficient information was provided or no fulltext Unable to obtain full text or quality assessment ofthe literature Studies were repeated or publications overlappedData extraction and quality assessmentTwo researchers separately conducted literature screening data extraction and literature quality evaluationand any differences could be resolved through discussionor a third inspector Information secured from the enrolled literature included first authors surname year ofpublication country ofthe population sample sizefollowup time and data on the relationship betweenhypothyroidism and the risk of breast cancerThe NewcastleOttawa Scale NOS was used to assessthe quality of the study from three aspects cohort selection cohort comparability and outcome evaluation []NOS scores of at least six were considered highqualityliterature Higher NOS scores showed higher literaturequalityStatistical analysisAll data analysis was performed using Stata120 softwareMetaanalysis was performed according to the PRISMAguidelines The OR and 95CI from included studieswere treated with the combined effect size After thatthe heterogeneity test was conducted When P ¥ orI2 was performed it mean that there was no apparent heterogeneity and the fixedeffect model shouldbe applied for a merger When P or I2 ¥ indicated high heterogeneity the randomeffect model wasapplied Combined effect size if OR indicates thathypothyroidism is an unfavorable factor for breast cancer If OR is the opposite Publication bias Begg funnel plot and Egger test linear regression test were usedto research publication bias detection of the literatureincluded If P indicates obvious publication biasResultsProcess of study selection and description of qualifiedstudiesA total of studies were identified on our online databases After exclusion of duplicate references129 s were considered After screening the andtitle s were excluded After careful review ofthe full texts studies have been excluded because ofthem did not provide relevant data and s didnot have fulltext Nineteen s published between and met the inclusion criteria Fig A total of samples from studies involvingwere enrolled in this metaanalysis [ ] Sixcohort studies and casecontrol studies were includedin the study Twelve s were studied in the European population five in the North American populationand two in the Asian population All s are of highquality because of NOS score no less than The chiefcharacteristics of the enrolled materials are detailed inTable 0cWang BMC Cancer Page of Fig Flow chart of search strategy and study selectionRelationship between hypothyroidism and breast cancerriskThere were studies reported the relationship betweenhypothyroidism and breast cancer risk With obviousheterogeneity I p among these studies so a random effect model was used for assessmentThe pooled analysis suggested that was not related tothe risk of breast cancer OR CI P 0001Fig explorethefurtherrelationshipSubgroup analysis of hypothyroidism and risk of breastcancerTobetweenhypothyroidism and breast cancer risk subgroup analysis was conducted from three aspects study typepopulation distribution and followup time The resultsof subgroup analysis were shown in Table In theEuropean subgroup we observed that patients withhypothyroidism have a lower risk of breast cancer OR CI P In the subgroup witha followup date of more than four years patients withhypothyroidism can reduce the risk of breast cancerwith borderline significance OR CI In otherP found thathypothyroidism was not related to the risk of breastcancersubgroups weRelationship between thyroid hormone replacementtherapy and breast cancer riskA total of studies reported the relationship betweenthe use of thyroid hormone replacement therapy and therisk of breast cancer [ ] Asobvious heterogeneity observed the fixedeffect modelwas usedI p The result suggestedthat patients who received thyroid hormone replacementtherapy was not related to the risk of breast cancerOR CI 109P Fig Publication biasFigure 4a shows the results of publication bias for the relationship between hypothyroidism and breast cancerrisk which were evaluated by funnel plots and Eggerstest The Begg test Pr and the Egger testP were used to detecting publication bias showedthat there was no possibility of publication bias Asshown in Fig 4b there were no publication biases in the 0cWang BMC Cancer Table Main characteristics of the included studies in ouranalysisStudySampleYearRegionAdamiKalacheHoffmanBrintonMosesonSmythSheringTalaminiSimonTurkenKuijpensCristofanilliSandhuHellevikDitschGraniSøgaardWengKimSwedenUKSwedenUSACanadaIrelandIrelandItalyUSAPragueNetherlandsUSACanadaNorwegianGermanyItalyDanishUSAKoreaMedianMean ageyearsNANA ± NANA ± ± ¥ ± ¥Page of NOSFollowupyearsNANANAStudydesignCasecontrolCasecontrolCohortCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCasecontrolCohortCasecontrolCohortCohortCasecontrolCasecontrolCohortCasecontrolCohortStudyIDAdami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Overall Isquared p ES CIES CI WeightWeightNOTE Weights are from random effects analysisFig Relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Table Stratiedanalysis of the relationship between hypothyroidism and breast cancer riskVariableOR95CINoofstudiesPHeterogeneityI2RegionEur orth AmericaAsiaStudy designCasecontrolCohortFollowup date ¤ Page of ModelusedFixedRandomedFixedRandomedFixedFixedRandomedPhStudyIDHoffman Kuijpens Sandhu Ditsch Cristofanilli Simon Moseson Brinton Adami Weng ES CIES CI WeightWeightOverall Isquared p NOTE Weights are from random effects analysisFig Relationship between thyroid hormone replacement therapy and breast cancer risk 0cWang BMC Cancer Page of A ]rr[golB ]rh[golBegg's funnel plot with pseudo confidence limitsEgger's publication bias plotse of log[rr]Begg's funnel plot with pseudo confidence limitstceffe idezdradnatstceffi edezdradnatsprecisionEgger's publication bias plotse of log[hr]precisionFig Publication bias assessment a hypothyroidism b thyroid hormone replacement therapy Metaanalysis estimates given named study is omitted Lower CI Limit Estimate Upper CI Limit Adami Kalache Hoffman Brinton Moseson Smyth Shering Talamini Simon Turken Kuijpens Cristofanilli Sandhu Hellevik Ditsch Grani Sogaard Weng Kim Fig Sensitivity analysis for relationship between hypothyroidism and breast cancer risk 0cWang BMC Cancer Page of s on the study of thyroid hormone replacementtherapy The Egger test was P and the Begg testwas Pr Sensitivity analysisThe results of sensitivity analysis are generally stableand the primary source of heterogeneity is in the research of Cristofanilli []Fig So we excludedthe literature of Cristofanilli and analyzed the otherstudies The results revealed that the hypothyroidismcould reduce the risk of breast cancer was borderlineOR096 95CI092 P andsignificantthere was no heterogeneityI2 P cohortstudy ofDiscussionMore than years ago Beatson used thyroid extracts to treat patients with metastatic advanced breastcancer The condition was significantly alleviated sparkinginterest in exploring the relationship between thyroid andbreast cancer [] Subsequently a prospective study enrolled women and women with earlier diagnosisof hypothyroidism observed the occurrence of breast cancer during followup showed that low serum free thyroxine levels increased the risk of breast cancer [] In aprospective women withhypothyroidism and hyperthyroidism found thathypothyroidism slightly reduced the risk of breast cancer[] However a prospective cohort study of women with autoimmune hypothyroidism and women with normal thyroid function indicated that autoimmune hypothyroidism was not associated with breastcancer risk [] Besides some animal experiments alsoreflect the relationship between the two [ ] Animalexperiments by López Fontanafound thathypothyroidism mice inhibit the development of breastcancer and promote the apoptosis of breast cancer cellsdue to the low expression of βchain protein and activation of the apoptotic pathway on the tumour cell membrane [] Due to the inconsistency ofthe aboveconclusions we performed a metaanalysis to evaluate therelationship between hypothyroidism and breast cancerrisketalA total of studies were included in this metaanalysis and the results showed that patients withhypothyroidism not related to the risk of breast cancerHowever there was significant heterogeneity among theincluded studies After subgroup analysis and sensitivityanalysis we found that Cristofanillis research may causeheterogeneity [] Cristofanillis research is a retrospective study and the diagnosis of hypothyroidism patientswas based on the information recorded in the medicalrecords which may lead to the bias risk of misclassification and have a positive impact on the positive results ofthis study [] After excluding Cristofanillis researchwe found that patients with hypothyroidism had a lowerrisk of breast cancer with borderline significance [] Theresults of the metaanalysis are inconsistent with the findings of Hardefeldt and Angelousi [ ] Perhaps because our study included more prospective studiesand Asian population cohort study In addition we evaluated the risk of breast cancer in thyroid hormone replacement therapy and show that patients who received thyroidhormone replacement therapy was not related to the riskof breast cancerIn the analysis of the European population the resultsshow that hypothyroidism may reduce the risk of breastcancer We also found that patients with hypothyroidismcan reduce the risk of breast cancer was borderline significance in the subgroup with more longer followupdate However the relationship between the two was notobserved in North American and Asian populationsThe possible reasons for these disparities may be as follows First followup time may be the main contributorsto this difference A longer followup is required to demonstrate the relationship between hypothyroidism andbreast cancer risk In the metaanalysis five studies provided North American population data and two reported Asian population data However only one ofseven nonEuropean studies followup time for morethan years Second the differences may be attributedto different ethnicities sharing different genegene andgeneenvironmental backgrounds Third social and environmental factors are another critical cause for thisdifference With these in mind our findings suggest thathypothyroidism may reduce the risk of breast canceronly in the European population and more largescalehighqualitylongterm prospective cohort studies arestill needed to study on different human populationsThe following may explain the potential relationshipbetween hypothyroidism and the risk of breast cancerHealthy mammary epithelial cells can express a largenumber of T3 receptors and breast cancer cells have asimilar ability to bind to T3 [] T3 has an estrogenlike effect that promotes the growth of mammary glandlobes and stimulates normal breast tissue differentiation[ ] Therefore T3 can mimic the effect of estrogenon the proliferation of breast cancer cells When theconcentration of T3 is low in vivo it may inhibit theproliferation of breast cancer cells Hypothyroidism mayreduce the risk of breast cancer by affecting T3concentrationSome basic experiments support this theory In GonzalezSancho studied the relationship betweenT3 and breast cancer [] It was found that there is anoverexpressed T1 gene in human breast cancer cellsand T3 inhibits the proliferation of mammary epithelialcells by inhibiting the expression of cyclin D1 and T1thereby inhibiting the proliferation of breast cancer cells 0cWang BMC Cancer Page of Author details1School of Clinical Medicine Weifang Medical University Weifang China 2Department of Oncology Affiliated Hospital of Weifang MedicalUniversity Weifang China 3School of Basic Medicine WeifangMedical University Weifang ChinaReceived December Accepted July foundthat MartinezIglesias[] Afterthathypothyroidism can inhibit the growth of breast cancercells [] In Tosovic conducted a prospectivestudy of T3 levels associated with breast cancer risk andfound that T3 levels in postmenopausal women werepositively correlated with breast cancer risk in a doseresponse mannerthathypothyroidism through lower levels of T3 could reducethe incidence of breast cancer Our metaanalysis resultsalso confirm the above conjecture[] Therefore we suspectHowever this conclusion needs to be taken with caution as this study has several limitations First the studies that have been included do not adjust for importantrisk factors for breast cancer Second in subgroup analysis for example there are only two s in Asianstudies and we should be cautious about the results ofAsian analysis Third the results of this metaanalysis indicate that there is a large heterogeneity between studiesFourth followup time at different endpoints cannot beuniform Finally publication bias cannot be avoidedentirelyConclusionHypothyroidism may reduce the risk of breast cancer inthe European population and no significant correlationwas observed between hypothyroidism and breast cancerrisk in nonEuropean populations Furthermore therewas no obvious correlation between thyroid hormone replacement therapy and breast cancer risk It is necessaryto conduct a large sample size strictly controlled prospective study of hypothyroidism patients further todemonstrate the relationship between hypothyroidismand breast cancer riskAbbreviationsOR Odd ratios CI Confidence intervals NOS NewcastleOttawa ScaleAcknowledgementsNot applicableAuthors contributionsStudy design BW ZL RLYH and TL Data extraction BW ZL TL and YH Dataanalysis BW ZL RLand YH Manuscript writing BW and RL Manuscriptedition RL and YH All authors have read and approved the manuscriptFundingNo sources of funding were used to conduct this study or prepare this letterAvailability of data and materialsAll the published s and data were available onlineEthics approval and consent to participateNot applicableConsent for publicationNot applicableCompeting interestsNoneReferencesSiegel RL Miller KD Jemal A Cancer statistics CA Cancer J Clin httpsdoi103322caac21442Praestegaard C Kjaer SK Andersson M StedingJensen M Frederiksen KMellemkjaer L Risk of skin cancer following tamoxifen treatment in morethan breast cancer patients a cohort study Breast cancer httpsdoi101007s1228201506605 Mittra I Hayward JL Hypothalamicpituitarythyroid axis in breast cancerLancet httpsdoi101016s0140673674903444Adami HO Rimsten A Thoren L Vegelius J Wide L Thyroid disease andfunction in breast cancer patients and nonhospitalized controls evaluatedby determination of TSH T3 rT3 and T4 levels in serum Acta Chir ScandDargent M Berger M Lahneche B Thyroid function in patients with Cancerof the breast Acta Mustacchi P Greenspan F Thyroid supplementation for hypothyroidism Anlatrogenic cause of breast cancer JAMA Kapdi CC Wolfe JN Breast cancer Relationship to thyroid supplements forhypothyroidism JAMA httpsdoi101001jamaKuijpens JL Nyklictek I Louwman MW Weetman TA Pop VJ Coebergh JWHypothyroidism might be related to breast cancer in postmenopausalwomen Thyroid httpsdoi101089thy200515 Weng CH Chen YH Lin CH Luo X Lin TH Thyroid disorders and breastcancer risk in Asian population a nationwide populationbased casecontrolstudy in Taiwan BMJ 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using thyroid supplements JAMA Brinton LA Hoffman DA Hoover R Fraumeni JF Jr Relationship of thyroiddisease and use of thyroid supplements to breast cancer risk J Chronic Dis httpsdoi1010160021968184900626 Moseson M Koenig KL Shore RE Pasternack BS The influence of medicalconditions associated with hormones on the risk of breast cancer Int JEpidemiol httpsdoi101093ije2261000Shering SG Zbar AP Moriarty M McDermott EW O'Higgins NJ Smyth PPThyroid disorders and breast cancer Eur J Cancer Prevent Smyth PP Smith DF McDermott EW Murray MJ Geraghty JG O'Higgins NJA direct relationship between thyroid enlargement and breast cancer J ClinEndocrinol Metab httpsdoi101210jcem813Talamini R Franceschi S Favero A Negri E Parazzini F La Vecchia CSelected medical conditions and risk of breast cancer Br J Cancer httpsdoi101038bjc1997289 0cWang BMC Cancer Page of Simon MS Tang MT Bernstein L Norman SA Weiss L Burkman RT DalingJR Deapen D Folger SG Malone K Marchbanks PA McDonald JA Strom BLWilson HG 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CGallwas J Toth B Thyroid function in breast cancer patients Anticancer Res Grani G Dicorato P Dainelli M Coletta I Calvanese A Del Sordo M DeCesare A Di Matteo FM D'Andrea V Fumarola A Thyroid diseases inwomen with breast cancer La Clin Terapeut 20121636e401Kim EY Chang Y Lee KH Yun JS Park YL Park CH Ahn J Shin H Ryu SSerum concentration of thyroid hormones in abnormal and euthyroidranges and breast cancer risk a cohort study Int J Cancer httpsdoi101002ijc32283 Beatson GT On The Treatment Of Inoperable Cases Of Carcinoma Of TheMamma Suggestions For A New Method Of Treatment With IllustrativeCases1 Lancet Lopez Fontana CM Zyla LE Santiano FE Sasso CV CuelloCarrion FDPistone Creydt V Fanelli MA Caron RW Hypothyroidism reduces mammarytumor progression via Betacateninactivated intrinsic apoptotic pathway inrats Histochem Cell Biol httpsdoi101007s004180171544x MartinezIglesias O GarciaSilva S Regadera J Aranda A Hypothyroidismenhances tumor invasiveness and metastasis development PLoS One 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"Methods: We updated a casecohort study nested within a cohort of 267?400 female textile workers in Shanghai China. We compared exposure histories of 1456 incident lung cancers cases diagnosed during 19892006 with those of a reference subcohort of 3022 workers who were free of lung cancer at the end of follow-up. We applied Cox proportional hazards modelling to estimate exposureresponse trends adjusted for age and smoking for cumulative exposures lagged by 010 and 20 years and separately for time windows of ?15 and >15 years since first exposure. Results: We observed no associations between cumulative exposure and lung cancer irrespective of lag interval. In contrast analyses by exposure time windows revealed modestly elevated but not statistically significant relative risks (?1.27) at the highest three exposure quintiles for exposures that occurred >15 years since first exposure. Conclusions: The findings do not support a protective effect of endotoxin but are suggestive of possible lung cancer promotion with increasing time since first exposure. endotoxin lipopolysaccharide lung cancer epidemiology textile industry occupational health Br J Cancer Br. J. Cancer British Journal of Cancer 0007-0920 1532-1827 Nature Publishing Group 24651386 3992504 bjc2014146 10.1038/bjc.2014.146 Clinical Study A multicentre randomised controlled trial of reciprocal lung cancer peer review and supported quality improvement: results from the improving lung cancer outcomes project Improving lung cancer outcomes project results Russell G K 1 Jimenez S 1 Martin L 1 Stanley R 2 Peake M D 1 3 Woolhouse I 1 4 * 1Clinical Standards Department Royal College of Physicians London NW14LE UK 2Clinical Audit Support Unit NHS Information Centre for Health and Social Care Leeds LS16AE UK 3Department of Respiratory Medicine Glenfield Hospital Leicester LE39QP UK 4Department of Respiratory Medicine Queen Elizabeth Hospital Birmingham Birmingham B152WB UK *E-mail: ian.woolhouseuhb.nhs.uk 15 04 2014 20 03 2014 110 8 1936 1942 19 12 2013 11 02 2014 24 02 2014 Copyright 2014 Cancer Research UK 2014 Cancer Research UK From twelve months after its original publication this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license visit http://creativecommons./licenses/by-nc-sa/3.0/ Background: Results from the National Lung Cancer Audit demonstrate unexplained variation in outcomes. Peer review with supported quality improvement has been shown to reduce variation in other areas of health care but has not been formally tested in cancer multidisciplinary teams. The aim of the current study is to assess the impact of reciprocal peer-to-peer review visits with supported quality improvement and collaborative working on lung cancer process and outcome measures. Methods: English lung cancer teams were randomised to usual care or facilitated reciprocal peer review visits followed by 12 months of supported quality improvement. The primary outcome was change in the following national audit indicators; mulitdisciplinary team discussion histological confirmation active treatment surgical resection small-cell chemotherapy and specialist nurse review. Patient experience was measured using a new lung cancer patient questionnaire in the intervention group. Results: Thirty teams (31 trusts) entered the intervention group and 29 of these submitted a total of 67 quality improvement plans. Active treatment increased in the intervention group (n=31) by 5.2% compared with 1.2% in the control group (n=48 mean difference 4.1% 95% CI ?0.1 to 8.2% P=0.055). The remaining audit indicators improved similarly in all groups. Mean patient experience scores in the intervention group did not change significantly during the study but a significant improvement was seen in the scores for the five teams with the worst baseline scores (0.86 to 0.22 P<0.001). Conclusions: Reciprocal peer review with supported quality improvement was feasible and effective in stimulating quality improvement activity but resulted in only modest improvements in lung cancer treatment rates and patient experience. lung cancer multidisciplinary quality improvement peer Lung cancer is the commonest cause of cancer death in England and Wales with around 38?000 cases diagnosed each year and ?35?000 deaths. Data from the National Lung Cancer Audit (NLCA) demonstrate significant variation in process and outcome measures across England. In 2009 there was a three-fold difference in survival and active treatment rates which persisted following case mix adjustment (Beckett et al 2012). Furthermore reported lung cancer outcomes in the UK are worse than other comparable European countries (Walters et al 2013) and have improved little in recent years (Khakwani et al 2013). It has been estimated that if survival rates were increased to that of the best in Europe around 1300 lives could be saved each year in the United Kingdom (Abdel-Rahman et al 2009). Variation in health care is not unique to lung cancer and addressing unwarranted variation is challenging (Wise 2010). Although external regulation may have a role in some areas this approach is more difficult to apply to the complex pathways involved in lung cancer diagnosis and treatment. Peer review with supported quality improvement offers a promising alternative but the evidence for its effectiveness is limited. The Washington State's Surgical Care and Outcomes Assessment Program utilised a peer support programme to share the best practice which led to a significant reduction in post-operative complications (Kwon et al 2012). Within the United Kingdom the national COPD resources and outcomes project demonstrated that reciprocal peer-to-peer review led to only limited quantitative differences in the quality of services offered (Roberts et al 2012). A qualitative analysis of this study identified a number of barriers to improvement including difficulties in establishing effective working relationships funding changes and service re-design. In 2003 the Institute for Healthcare Improvement described the collaborative model to achieve a breakthrough improvement (Institute for Healthcare Improvement 2003). Collaboratives allow teams working on the same issue to share good practice and innovation permitting others to take these ideas and implement them in the context of their own anisation resources and case mix. Pronovost et al (2006) successfully employed this collaborative approach together with supported quality improvement to implement five evidence-based interventions on the intensive care unit resulting in the reduction in catheter-related bloodstream infections to zero. These studies offer a persuasive proof of concept but the absence of a control group or of patient-specific outcomes measures limits their implementation in other disease areas such as cancer. The aim of the current study is to determine whether a programme of reciprocal peer-to-peer review visits with supported quality improvement and collaborative working can significantly improve lung cancer process and outcome measures and thus reduce unwarranted variation in outcomes. Materials and methods Study design We conducted a prospective randomised controlled trial. Study population One hundred and sixty-two English NHS trusts were identified from the 2008 NLCA annual report. Centres only providing treatment (not diagnostics) orthopaedic hospitals and ambulance trusts were excluded. Invitations to participate were sent to the remaining 152 trusts. Trusts who agreed to participate and who had 2008 NLCA case ascertainment rates of > 50% expected were paired before randomisation on the basis of contrasting results for four key indicators from the NLCA. The indicators were active treatment rates surgical resection rates median survival and the proportion of patients assessed by a clinical nurse specialist. Each trust was colour coded for each indicator red if below the national average and green if above. By placing each trust with its colour-coded indicators on a map we were able to pair trusts on the basis of a contrasting mixture of red and green indicators and a travel time between centres of around 2?h. On the basis of data from the national COPD resources and outcomes project we determined that we would be able to complete 30 peer review visits during the lifetime of the project thus allowing 30 lung cancer multidisciplinary teams (15 pairs) to be randomised into the intervention arm. Randomisation was performed in a blinded fashion by assigning a random number to each pair of trusts and then allocating pairs numbered 115 to the intervention group. The remaining trusts formed either the control group (if they had agreed to participate) or the non-participant group and had no further contact with the study team but continued to submit data to the NLCA as usual. Intervention The study timeline is shown in Figure 1. Following introductory workshops the multidisciplinary teams within each pair undertook facilitated reciprocal site visits. The visits consisted of observation of the host team's multidisciplinary team meeting three discussion sessions focusing on the functioning of the mulitdisciplinary team meeting the host team's NLCA data and patient experience questionnaire results. The final session aimed to identify the focus of improvement work to be undertaken by the host team. The quality improvement facilitator introduced a structured template for the quality improvement plans and provided a short introduction to using the model of improvement to guide implementation of the plans. Over the next 12 months the quality improvement facilitator provided support via electronic mail telephone and follow-up visits where required. Teams within the intervention group supported each other via mini-collaboratives in the form of web-based teleconferences and two face-to-face workshops. Outcomes Changes in process and outcome were assessed using data from local quality-improving plans and the following indicators from the NLCA: the proportion of patients discussed at a multidisciplinary team meeting histological confirmation rate active treatment rate surgical resection rate the proportion of patients with small-cell lung cancer receiving chemotherapy and the proportion of patients seen by a lung cancer nurse specialist. Patient experience was assessed in the intervention group using a new lung cancer-specific patient experience questionnaire designed in collaboration with the Roy Castle Lung Cancer Foundation. The questionnaire included 11 questions selected with permission from the previously validated 2004 national cancer patient survey. The questions covered the following domains: communication privacy respect and dignity and three free text questions (see Appendix I). Participating teams were asked to distribute 30 questionnaires to patients recently seen in their services. The clinical nurse specialists distributed the questionnaires to patients who anonymously returned them to the Royal College of Physicians. An independent qualitative ethnographic evaluation of the study was undertaken by the Social Science Applied to Healthcare Improvement Research Group at the University of Leicester. Statistical methods Data were tested for normality using the ShapiroWilk test. Baseline NLCA indicators were taken from the 2009 NLCA report and the intervention control and non-participant groups were compared using a ?2- test. The changes in NLCA indicators from 2009 to 2011 were compared using an independent t-test. Patient experience questionnaire responses for each question were labelled and re-coded to separate them into the worst patient experience category (score 1) vs all other responses (score 0). These scores were then summated to create a domain and a total patient experience score with a possible range of 011 whereby a higher score indicates a worse patient experience. Analyses were performed using the statistical software package SPSS (International Business Machines Corp. Armonk NY USA). Funding and ethics The study was funded by a Closing the Gap' grant from the Health Foundation. The National Research Ethics Service confirmed that the study was service evaluation and quality improvement and did not require ethical review. Results One hundred trusts (66%) replied to the invitation to participate and 91 (61%) agreed to participate in the study. Eighty-one trusts had 2008 NLCA data of sufficient quality to allow pairing. Two trusts provided a joint multidisciplinary team allowing 40 pairs of multidisciplinary teams to be created. One pair agreed to act as a pilot and was excluded from further analysis. Of the remaining 39 pairs 15 pairs (31 trusts) were randomised to the intervention group. The remaining 24 pairs formed the control group. During the study two trusts in the control group amalgamated to form one trust so the total number of trusts in the control group was 47 (Figure 2). Quality improvement plans Two hundred and thirty medical professionals from 31 trusts participated in the review visits. Twenty-nine teams submitted a total of 67 quality improvement plans. The issues identified in the quality improvement plans are shown in Table 1. Eighteen teams collected local data to measure impact. An example of such data is shown in Figure 3. This trust identified small-cell lung cancer chemotherapy as an area for improvement. They introduced a number of changes to their diagnostic and treatment pathways including prioritisation of small-cell pathology reporting faxing of the results to the multidisciplinary team coordinator and lung nurse specialist to allow early booking of oncology appointments. These changes were monitored using a run chart that demonstrated a reduction in the time from multidisciplinary team meeting to chemotherapy treatment and an increase in the proportion of small-cell lung cancer patients receiving chemotherapy from 60% in 2009 to 71% in 2011. National lung cancer audit indicators Baseline (2009) NLCA indicators for the intervention control and non-participant groups were similar (Table 2). The mean change for each NLCA indicator from baseline to 2011 in the intervention and control group is shown in Figure 4. The proportion of patients receiving active anti-cancer treatment in the intervention group increased by 5.2% compared with 1.2% in the controls (mean difference 4.1% 95% CI ?0.1 to 8.2% P=0.055). The remaining NLCA indicators improved similarly both in the intervention and control groups. Patient experience In the intervention group patient experience questionnaires were returned by 438 patients from 30 multidisciplinary teams at baseline (return rate 49%) and 372 patients from 27 trusts following the intervention (return rate 41%). Baseline total scores were low (01.31) indicating high levels of patient satisfaction with the care received although there was a statistically significant (P<0.001) variation in results by the multidisciplinary team (Figure 5). In particular the proportion of patients responding yes to the question did you find that the person who told you about your diagnosis did so with sufficient sensitivity/care?' varied significantly by 57%100% (P<0.001). The total questionnaire scores did not change significantly during the study (0.220.17 P=0.377) however the variation by the multidisciplinary team reduced (Figure 5). Given that the study aimed to bring the standard of the lower performing trusts to that of the best we performed a post hoc analysis for the five trusts with the worst baseline patient experience scores. This demonstrated that the mean total score improved significantly for these trusts from 0.86 to 0.22 P<0.001. The biggest improvement in this group was seen in the proportion of patients responding yes to the question did you find that the person who told you about your diagnosis did so with sufficient sensitivity/care?' which increased from 75% to 90% (P=0.05). One multidisciplinary team in this group achieved this improvement by using their baseline questionnaire results as a lever to encourage attendance at an advanced communications skills course. " | 1 |
"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 pr sity 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 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 pageConclusions 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 survivalBackgroundGastric 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 et al[] found that GNEC has shorter progressionfree survival PFS than gastric adenocarcinoma Huang et al[] 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 usedpr sity 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 egPr sity 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 pr sity 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 pr sity 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 procedure LaparoscopicT stageT1T2T3T4N stageN0N1N2N3M stageM0M1pTNM stageIIIIIIIV Gastricadenocarcinoman ± ± ± ± P value Gastricadenocarcinoman ± ± BMI Body Mass Index MiNEN Mixed neuroendocrinenonneuroendocrine neoplasm NEC neuroendocrine carcinoma PSM Pr sity 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 procedure LaparoscopicT 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 Pr sity 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 TNM 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 et alfound 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 wholeConclusionsOur 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 Pr sity 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 et alAdvanced 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 et alNeuroendocrine 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 neuroendocrineexocrinecarc | 2 |
Rapid repair of human diseasespecific singlenucleotide variants by OneSHOT genome editingYuji Yokouchi12 Shinichi Suzuki2 Noriko Ohtsuki12 Kei Yamamoto12 Satomi Noguchi12 Yumi Soejima3 Mizuki Goto34 Ken Ishioka5 Izumi Nakamura12 Satoru Suzuki6 Seiichi Takenoshita7 takumi era123Many human diseases ranging from cancer to hereditary disorders are caused by singlenucleotide mutations in critical genes Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases However current procedures for repairing deleterious singlenucleotide mutations are not straightforward requiring multiple steps and taking several months to complete In the current study we aimed to repair pathogenic allelespecific singlenucleotide mutations using a single round of genome editing Using highfidelity sitespecific nuclease AsCas12aCpf1 we attempted to repair pathogenic singlenucleotide variants SNVs in diseasespecific induced pluripotent stem cells As a result we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a singlenucleotide marker followed by absolute markerless scarless repair of the RET SNV with no detected offtarget effects The markerless method was then confirmed in human type VII collagenencoding gene COL7A1 Thus using this OneSHOT method we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one resulting in allelespecific repair that can be completed within weeks with or without a singlenucleotide marker Our findings suggest that OneSHOT can be used to repair other types of mutations with potential beyond human medicineThe human genome contains extensive variation including an estimated singlenucleotide variants SNVs that determine how we look and function as well as our specific disease tendencies1 Some SNVs are pathogenic and either directly or indirectly cause hereditary disorders4 such as multiple endocrine neoplasia type 2B MEN2B7 and dystrophic epidermolysis bullosa DEB8 MEN2B is an autosomal dominant syndrome characterised by thyroid adrenal gland and neuronal tumours and skeletal abnormalities The majority of MEN2B cases result from a single aminoacid substitution Met918Thr in the RET protooncogene which is caused by a pathogenic SNV RET c2753TC at the second base of the codon7 DEB is an inherited disease characterised by severe recurrent skin ulcers and blistering It is caused by genetic mutations in the human type VII collagenencoding gene COL7A1 the product of which is an anchoring fibril connecting the epidermis to the dermis8 To model diseases such as these in a0vitro diseasespecific induced pluripotent stem cells iPSCs carrying pathogenic SNVs or other genetic mutations can be obtained from patients9 Repairing these iPSCs to generate isogenic revertant cells is a promising strategy for genome repair and s up new avenues for drug 1Pluripotent Stem Cell Research Unit in Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Hikarigaoka Fukushima Japan 2Department of Thyroid and Endocrinology School of Medicine Fukushima Medical University Fukushima Japan 3Department of Cell Modulation Institute of Molecular Embryology and Genetics IMEG Kumamoto University Kumamoto Japan 4Department of Dermatology Faculty of Medicine Oita University Yufu Japan 5Department of Microbiology School of Medicine Fukushima Medical University Fukushima Japan 6Office of Thyroid Ultrasound Examination Promotion Radiation Medical Science Centre for the Fukushima Health Management Survey Fukushima Medical University Fukushima Japan 7Fukushima Medical University Fukushima Japan email yokouchyfmuacjpScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c discovery1314 However the repair process remains problematic and a precise and convenient genome editing procedure has not yet been developedArtificial genome repair andor modification generally starts from a targetspecific doublestrand break generated by sitespecific nucleases1516 Doublestrand breaks are then repaired by a cells own genome repair machineries1516 However most of the break sites are incorrectly repaired by nonhomologous end joining and can result in gene knockout through the generation of nonspecific insertions or deletions indels1718 If a repair template carrying a repair base is coadministrated however the cleavage sites can be accurately repaired via homologydirected repair HDR1516 Sitespecific nucleases such as transcription activatorlike effector nucleases or the Streptococcus pyogenes Sp Cas9 nuclease are typically used as genome editing tools for human iPSCs19SpCas9 is a type IIA endonuclease in the class clustered regularly interspaced short palindromic repeats CRISPRCas system that has been repurposed as a programmable sitespecific nuclease for genome engineering22 Indeed SpCas9 has become a popular genome editing tool for genetically modifying human pluripotent stem cells19 Despite its efficient cleavage activity wildtype SpCas9 has a low DNA repair rate using HDR following plasmidbased administration It recuts the repaired site because the guide RNA has a 2base mismatch tolerance during sequence recognition leading to incorrect repair by nonhomologous end joining1718 To prevent recutting a blocking mutation must be introduced into the seed sequence of the guide RNA or into the protospaceradjacent motif PAM26However for scarless genome editing repairs with wildtype SpCas92749 each method has its obvious strengths and weaknesses For example CORRECT which includes excellent tricks to prevent recutting of the edited target by the editing tool can be performed even if the target recognition ability of the genome editing tool is insufficient however the need for two consecutive editing steps27 In comparison MhAX has high genome editing efficiency but cannot achieve completely scarless editing because singlebase markers are required Further as with the CORRECT method MhAX editing requires two consecutive edits49 increasing cost and time requirementsAnother recentlyidentified bacterial programmable sitespecific nuclease CRISPRCas12aCpf1 is a type VA endonuclease belonging to the class CRISPRCas system32 Among identified Cas12a enzymes those from Acidaminococcus sp BV3L6 As and Lachnospiraceae bacterium ND2006 show strong cleavage activity in mammalian cells32 These Cas12a endonucleases have unique features that complement Cas9 and expand the genome editing range First Cas12a recognises a Trich PAM upstream of the protospacer whereas Cas9 recognises a Grich PAM downstream of the protospacer32 Second two PAMinteracting variants have been generated that expand the Cas12a target range3334 Third Cas12amediated cleavage generates a staggered cut on the PAMdistal region of the target sequence as opposed to the PAMproximal blunt ends generated by Cas932 Finally and perhaps most importantly the guide or CRISPR RNA crRNA exhibits highfidelity target recognition meaning that Cas12a can precisely distinguish the target sequence at a singlebase resolution3536 Consequently the resulting offtarget effects are kept to a background level These features suggest that Cas12a might be suitable for precise diseasespecific iPSC repair because its recut activity is lowCas12a has already been used to knock out pathogenic genes in cancer cells37 generate insertions or twonucleotide substitutions in iPSCs38 and to induce exonskipping in diseasespecific iPSCs39 Thus we investigated whether Cas12a could be used to carry out allelespecific singlenucleotide repair of iPSCs carrying the pathogenic SNVs found in MEN2B and DEB patients in a single round of genome editing To accomplish this we used an AsCas12a PAM variant and singlenucleotide mismatch detection polymerase chain reaction SNMDPCR analysis in diseasespecific iPSCs to develop a precise convenient genomeediting procedure we have called OneSHOT One allelespecific single HDR and singlestranded oligodeoxynucleotide ssODN transient drug selection with SNMDPCR screening OneSHOT provides scarless singlenucleotide substitution of a pathogenic SNV in diseasespecific iPSCs within a0weeks The final modification rate is within a practical range for handpicking cloning Our findings suggest that this simple low cost procedure could be used for genome editing in a single step drastically reducing the time currently needed for scarless SNV repairResultsPrinciples of OneSHOT repair of singlenucleotide mutations AsCas12a is a highfidelity RNAguided sitespecific nuclease that binds to the target genomic DNA site via a 20nt guide sequence in the crRNA allowing it to discriminate the target sequence at the single nucleotide level Fig a0 Following the addition of a crRNA designed for a specific target sequence containing a singlenucleotide mutation AsCas12a selectively binds to the target sequence on the mutant allele and induces a doublestrand break leaving the wildtype sequence on the alternative allele unaffected Fig a0 In the presence of a ssODN wildtype sequence template the mutant nucleotide in the target sequence can be repaired to the wildtype sequence via the cellular HDR machinery To mark the repaired allele we labelled the ssODN with a singlenucleotide marker in the vicinity of the mutant nucleotide This label allowed us to easily identify the generepaired clones by allelespecific amplification4042SNMDPCR detection of the singlenucleotide marker Fig a0 A complete outline of the OneSHOT workflow for SNV repair is provided in the Supplementary Information and in Supplementary Fig a0S1Allelespecific singlenucleotide substitution in MEN2Bspecific iPSCs Before carrying out allelespecific singlenucleotide repair of the pathogenic RET mutation we assessed whether the OneSHOT approach could be used to accomplish allelespecific singlenucleotide substitution of the wildtype alleleWe established FB414 human iPSCs from a patient with MEN2B using a Sendai viral vector protocol43 We then confirmed that the FB414 cells exhibited an embryonic stem celllike morphology and expressed pluripotent gene markers indicating that they were authentic iPSCs Supplementary Fig a0S2 and X7 To identify Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cguide seqcrRNAHigh Fidelity SiteSpeciï¬c Nuclease SSN AsCas12aCpf1target alleleDSBTYCValleleTYCVHDRTYCVTYCVby SNMDPCR ntssODN w markerFigure a0 OneSHOT principles AsCas12a pale yellow and crRNA orange and grey lines selectively bind to a target sequence containing a pathogenic SNV red triangle on the target allele Binding leads to a doublestrand break in the target sequence on the target allele left but not in the corresponding wildtype sequence containing the wildtype nucleotide blue triangle on the alternative nontarget wildtype allele right When the ssODN repair template bluegreen line with the wildtype nucleotide blue triangle and a singlenucleotide marker a silent mutation for SNMDPCR screening green triangle is cotransfected with AsCas12a into the cells the target site on the pathogenic allele is repaired using the template by the endogenous HDR machinery In this case the intended geneedited clones are easily identified by positive screening for the singlenucleotide marker because the repaired expathogenic allele now carries the singlenucleotide markerpossible target sites for AsCas12a around the SNV of interest we first searched for PAMs recognised by wildtype AsCas12a or the RR and RVR variants which recognise TYCV and TATV PAMs respectively3334 We identified two PAM sites for the RR variant TYCV Y CT V ACG TTCC located 12bp upstream of the target nucleotide on the sense strand and TTCA located 7bp upstream of the target nucleotide on the antisense strand Fig a02a magenta lines Based on this information we designed two pairs of crRNAs crRNA_RET1 and crRNA_RET1 a0m and crRNA_RET2 and crRNA_RET2 a0m which contain guide sequences that specifically recognise wildtype and mutant target sequences respectively Fig a02aTo test the cleavage activity and targetrecognition specificity of AsCas12a_RR using these crRNAs we performed a T7E1 assay using 409B2 human iPSCs carrying the wildtype RET sequence in the target site Fig a02a middle The crRNAs for the wildtype sequence crRNA_RET1 and crRNA_RET2 each exhibited significant cleavage activity towards the wildtype target sequence Fig a02b P and P respectively By contrast the crRNAs for the mutant sequence crRNA_RET1 a0m and crRNA_RET2 a0m showed extremely weak activity Fig a02b P for both A more accurate ICE analysis showed no significant activity of the crRNAs on the WT allele Supplementary Fig a0X2a These results indicate that the crRNAs for the mutant sequence do not have significant if any activity on the WT alleleHowever the observed cleavage activity of AsCas12a_RR in conjunction with crRNA_RET1 was significantly higher than that with crRNA_RET2 Fig a02b P Supplementary Fig a0X2a Puromycin treatment further promoted the cleavage activity of AsCas12a_RR with crRNA_RET1 Fig a02b P To test the applicability of the method to carry out allelespecific singlenucleotide substitution in human iPSCs we attempted to replace the wildtype nucleotide RET c2753T at the Met918 site in the wildtype allele in FB414 MEN2BiPSCs Fig a02c Following electroporation of the pY211puro vector which expresses AsCas12a_RR and crRNA_RET1 Fig a02c blue line and a ssODN modification template ssODN_RET_M918T_I913silentC carrying both a variant nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a02c red C and light green C respectively into FB414 cells we conducted SNMDPCR screening Overall clones were positive for the substitution Fig a02c d and GE1 in Table a0 Direct sequencing of the target sequence revealed that clones contained the wildtype allelespecific introduction of the mutant nucleotide at the target site T C substitution resulting in the Met918Thr substitution Fig a02e red arrow along with the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a02e blue arrow The HDR efficiency was Table a0We then searched for offtarget sequences corresponding to the target sequence using the web tool CHOPCHOP v244 and detected no indels in either of the predicted two offtarget sites by Sanger sequencing Table a0 GE1 and by AmpliSeq Supplementary Table a0X4 These results indicated that the OneSHOT method could be used to replace a single nucleotide in an allelespecific manner while minimising offtarget effects As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE1Scientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0c¸Figure a0 Singlenucleotide substitution of the RET wildtype sequence in MEN2B iPSCs a Human RET locus containing the MEN2B mutation and crRNA of AsCas12a_RR for the mutation Top exon of the RET locus Middle the wildtype WT allele sequence Blue letters indicate the wildtype nucleotide at Met918 underlined Bottom the mutant allele sequence Red letters indicate the single missense mutation caused by a TC substitution producing a Met918Thr substitution underlined Coloured lines indicate the guide sequence template for the crRNA The pink line indicates the AsCas12a_RR PAM Coloured dashed lines indicate the sites cleaved by AsCas12a_RR with the corresponding crRNA b T7E1 assay using human wildtype iPSCs 409B2 electroporated with AsCas12a_RR and the different crRNAs crRNA_RET1 crRNA_RET1 a0m crRNA_RET2 or crRNA_RET2 a0m targeting exon Left the cropped gel images Arrowheads indicate cleaved bands The fulllength gels are presented in Supplementary Figure a0S7 Right statistical analysis of the cleavage activity and specificity of AsCas12a_RR with the crRNAs following selection with different concentrations of puromycin c HDRmediated editing for generating artificial homozygous MEN2B using AsCas12a_RR with crRNA_RET1 selectively targeting RET_Met918 in the wildtype allele d SNMDPCR analysis of the first round of screening The cropped gel image is shown here The arrowhead indicates positive PCR amplicon a0bp The fulllength gel is presented in Supplementary Figure a0S8 e Sequencing of the original and modified MEN2B iPSCs FB414 Top original sequence of RET exon with a T C substitution in the MEN2B mutant allele Bottom the modified RET sequence The T C substitution resulting in a homozygous Met Thr substitution Red and blue arrows indicate the positions of the pathogenic SNV and the singlenucleotide marker respectively Underlining indicates the codons affected by the editing A more detailed explanation is provided in the Extended Figure Legends in the Supplementary InformationAllelespecific singlenucleotide repair of a pathogenic RET variant To repair the pathogenic SNV RET c2753TC in the mutant allele in FB414 cells we first tested the cleavage activity and target recognition specificity of AsCas12a_RR using crRNA_RET1 a0m and crRNA_RET2 a0m Fig a02a in a homozygous MEN2B iPSC line with mutations in RET exon in both alleles GE19 genotype RETMet918ThrMet918Thr RETIle913 silentC Fig a02e bottom The T7E1 assay confirmed that the MEN2B target sequence was selectively cleaved by AsCas12a_RR with either crRNA_RET1 a0m or crRNA_RET2 a0m but not with crRNA_RET1 or crRNA_RET2 Fig a03a The ICE analysis revealed that only the AsCas12a_RR with crRNA_RET1 a0m exhibited strong cleavage activity on the target sequence Supplementary Fig a0X2b therefore we selected the crRNA_RET1 a0m for use in subsequent experimentsWe then carried out OneSHOT repair in FB414 cells using AsCas12a_RR with crRNA1m and a ssODN repair template containing a repair nucleotide at Met918 and a singlenucleotide marker at Ile913 Fig a03b T in blue and C in light green respectively Subsequent SNMDPCR screening showed that clones were positive Fig a03c GE2 in Table a0 while direct sequencing confirmed that of the positive clones contained the introduced wildtype nucleotide at the target site C T substitution leading to a Thr918Met substitution repair Fig a03d red arrow These clones also contained the singlenucleotide marker T C substitution leading to a silent mutation at Ile913 Fig a03d blue arrow The overall HDR efficiency was Table a0 GE2 and we detected no offtarget effects by Sanger sequencing Table a0 GE2 and by AmpliSeq Supplementary Table a0X4 As in the preliminary experiment direct sequencing analysis around the target sites revealed no duplication events in the unintended geneedited clones suggesting that most of the intended geneedited clones had clonally proliferated Supplementary Fig a0S3 GE2Allelespecific single nucleotide repair of a pathogenic variants in RET and COL7A1 without a singlenucleotide marker We next investigated whether the OneSHOT method could be used to repair the pathogenic SNV in RET without including the singlenucleotide marker which would achieve true scarless repair We therefore performed OneSHOT repair in the FB414 cells using AsCas12a_RR crRNA_RET1 a0m and the ssODN repair template with only a wildtype nucleotide at Met918 In the subsequent SNMDPCR screening for the pathogenic SNV no amplicons were obtained from repaired clones because the pathogenic SNV was lost from the mutant allele Fig a04a Overall we identified negative clones by SNMDPCR screening for the pathogenic SNV and direct sequencing revealed that carried only the wildtype nucleotide at Met918 Fig a04cd and GE4 in Table a0 In this experiment the overall HDR efficiency was Table a0 GE4 and no indels were detected in the two predicted offtarget sites by Sanger sequencing Table a0 GE4 and by AmpliSeq Supplementary Table a0X4We next attempted to perform scarless repair of a pathogenic SNV in iPSCs derived from a patient with DEB to confirm the applicability of the approach for other hereditary diseases We generated iPSCs from a patient with DEB autosomal recessive compound mutation COL7A1pGly2138Ter COL7A1c3591del13insGG and aimed to substitute the pathogenic SNV c6412G T pGly2138Ter in exon Supplementary Fig a0S4ab Scarless OneSHOT using AsCas12a_RR with crRNA_COL7A11 a0m plus the repair template scarlessly repaired the pathogenic SNV in the mutant allele Supplementary Fig a0S4cde and GE5 in Table a0 with a substitution rate of No indels were detected in the seven predicted offtarget sites Supplementary Table a0S2 Supplementary Table a0X4 Unlike the scarless OneSHOT for RET_Met918Thr in FB414 cells Fig a0 GE4 in Table a0 identical sequences within the target site were observed among the unintended geneedited clones suggesting that these clones were likely duplicated Supplementary Fig a0S3 GE5Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0cabcRET locus on Chr 10q11exon M918PAM crRNA_ RET1 bpWT allelecrRNA_RET2 PAMPAM M918TcrRNA_RET1mMutant allelecrRNA_RET2m PAM M NCpuro crRNA_RET 1m 2m T7E1 assay in WT iPSC 409B2 nsns xedni ledniNCcr2mpuro cr2mpuro cr2mpuro cr2puro cr2puro cr1puro cr1puro cr1mpuro cr1mpuro cr2puro cr1puro cr1mpuro crRNApuro treatmentRETI913 C T PAMRET M918 C TcrRNA_RET1WT allele sequencessODN_RET_M918T_I913silentCModiï¬ed WT allele sequence Mutant allele sequencede1KM M1KIle913 A T T A T TCMet918 A TC G A C GIleIleMetThrThrScientific RepoRtS 101038s41598020704017Vol0123456789wwwnaturecomscientificreports 0cGene editing CellGE1GE2GE3GE4GE5FB414FB414FB414FB414B1173Genotype phenotypeOriginal DestinationRETM918T RETM918TM918T I913_silentCMEN2Ba MEN2B homo with SN MarkerRETM918T RET I913_silentCMEN2Ba MEN2B revertant with SN MarkerRETM918T RET I920_silentCMEN2Ba MEN2B revertant with SN markerRETM918T RETMEN2Ba MEN2B scarless revertantCOL7A1G2138X del13 ins GG COL7A1 del13 ins GGDEBb DEB scarless revertantNo of total picked clones TCNo of 1st screening passed clones SNMD PCRNo of 2nd screening passed clonessequencing1stTC 2nd1st 2ndTC Table OneSHOT and scarless OneSHOT gene editing GE experiments After electroporation of the AsCas12a_RR expression vector and the ssODN template into the cells the crude DNA samples from the singlecell derived colonies that expanded on the master plates were subjected to SNMDPCR in the first screening round For positive screening colonies with amplifiable 200bp fragments from the SNMDPCR primer pair were the intendedclone candidates GE13 For negative screening colonies lacking PCR amplification were the intendedclone candidates GE4 and In the second screening round we directly read the sequences around the target site of the DNA fragments amplified by Tks Gflex DNA polymerase in each sample silentC a silent mutation generated by replacement with a cytidine for SN marker a Multiple endocrine neoplasia type 2B B Dystrophic epidermolysis bullosa Positive screening results Negative screening resultsSampleSiteGenomic location No of mis matchesOriginalRET exon target1chr10 GE1GE1GE2GE2GE3GE3GE4GE4Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget Offtarget chr15 chr4 chr15 chr4 chr15 chr4 chr15 chr4 Sequencea including mismatchesTTCC AGT TAA ATG GAT GGC AAT TGTTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG TTCC cGTTAAtTGGtTGG CAA TTG TTCC AcTTA AAT GcATG GCA tTTG Indel ratio b Table Offtarget effects of AsCas12a_RR in gene editing experiments GE1GE4 After amplifying the offtarget candidates predicted by CHOPCHOP v2 from the intended geneedited iPSC clones we directly read the sequences around the candidate sites after Sanger sequencing with specific primers a Underline indicates the PAM of the AsCas12a_RR variant Lower letters indicate mismatched bases in the offtarget candidates as compared with the original target sequence b Number of indel clones relative to the number of analysed clonesDiscussionMany hereditary human diseases are caused by singlenucleotide mutations These singlebase alterations have the potential to drastically alter protein structure and function Although most singlenucleotide mutations are completely harmless silent repair of pathogenic SNVs would significantly improve the quality of life and life expectancy of patients with hereditary diseases Thus in the present study we investigated whether we could achieve scarless repair of pathogenic SNVs in pluripotent stem cells from patients with two different types of hereditary disease MEN2B and DEB More importantly we aimed to carry out the repairs in a single stepUsing the OneSHOT approach developed in this study we successfully repaired a RET gene SNV in MEN2B iPSCs with the addition of a singlenucleotide selective marker in a single step We then confirmed that the same technique could be used to carry out scarless repair in MEN2B and DEBspecific iPSCs without the need for the singlenucleotide marker Scarless repair where no trace of gene editing is left around the target sequence is the goal of any gene editing technique because it safely repairs mutations in noncoding genomic regions without any secondary effects In contrast the inclusion of marker sequences during gene editing can have downstream effects Such secondary effects include the introduction of noncoding SNVs to cryptic splice sites Scientific RepoRtS 101038s41598020704017Vol1234567890wwwnaturecomscientificreports 0ccausing abnormal RNA splicing4546 and mutations that introduce a premature termination codon resulting in unstable mRNA46 Noncoding mutations affecting regulatory elements can also interfere with gene regulation through loss of function resulting in reduced gene expression or gain of function resulting in gene mis or overexpression4748 Therefore scarless repair is crucial for maintaining genome integrity and preventing unknown secondary effects in the target geneSeveral other methods of pathogenic SNV repair have been developed including CORRECT2627 and MhAX49 However all currently available methods have inherent obstacles to achieving scarless SNV repair in a fast and errorfree manner To overcome some of these obstacles we used the AsCas12a nuclease which has highfidelity targetrecognition3536 circumventing the need for a blocking base to inhibit recutting as is required in other methods2627 We also performed SNMDPCRbased negative screening for the pathogenic SNV which easily detects candidate clones containing the intended alteration As a result of these modifications we achieved absolute scarless editing of the RET and COL7A1 SNVs see Fig a0 Supplementary Fig a0S4 and GE4 and GE5 in Table a0 Another advantage of the AsCas12a nuclease was the ability to carry out SNV repair in a single step because only one round of HDR is required for gene editing Fig a0 The OneSHOT method was used to repair the SNVs in RET and COL7A1 within a 3week period with sufficient efficiency for handpicking In contrast other methods can take up to a0months to generate the intended geneedited clone because two rounds of HDRMMEJ may be required262749 However similar to our approach the CORRECT method can achieve scarless singlenucleotide substitution thus ensuring high sequence fidelity around the target site in geneedited cells Fig a0 and Supplementary Fig a0S4 Conversely MhAX leaves a silent single nucleotide mutation around the target site for use in screening49 Another difference is that the dsDNA template in MhAX can be randomly integrated into the genome outside of the target site by nonhomologous end joining50 whereas the ssODN templates used for OneSHOTscarlessOneSHOT and CORRECT approaches are not randomly integrated51 Thus the OneSHOT method developed for SNV repair in the current study appears to have several advantages over currently available methods see Supplementary Table a0S3In the CORRECT procedure the cuttomutation distance the distance between the CRISPRSpCas9 cleavage site and the blocking mutation is a crucial factor for HDR efficiency and zygosity determination2627 We therefore searched for more appropriate sites for the singlenucleotide markers by first comparing the efficacies of three singlenucleotide markers set in different positions around the target site using a PCRrestriction fragment length polymorphism RFLP assay52 We found that two of the markers showed similar HDRspecific cleavage activity while no cleavage activity was detected for the third marker Supplementary Information and Supplementary Fig a0S5 suggesting that Ile920 could be used as an alternative singlenucleotide marker Testing of HDR efficiency in FB414 cells following OneSHOT repair using the alternative marker again confirmed that the singlenucleotide substitutions in the geneedited clones were effectively detected by positive screening using SNMDPCR for a singlenucleotide marker Supplementary Information and Supplementary Fig a0S5 We do note however that the efficiency of identification might depend on the position of the singlenucleotide marker and the primers used for SNMDPCRDespite our success in repairing the pathogenic SNVs in a single step the study has several limitations The OneSHOT method only requires one PCR run thereby reducing the time and cost compared with standard PCRRFLP screeningbased methods which require up to three steps5253 However we found that falsepositive clones are included in the population after the first SNMDPCR screen Supplementary Fig a0X8 Therefore we are currently designing a simple way to discriminate false clones from authentic clones using a PCRbased procedure We also noted that the geneedited cell lines generated by OneSHOT are not always clonal This situation arises because high cell densities occur in the culture during puromycin selection a0days and in the recovery culture a0days prior to clonal expansion However assessment of our data suggests that a 1day recovery culture and sufficient singlecell suspension at the reseeding stage can prevent duplication and ensure clonal establishment of the geneedited cells Using the current protocol we estimate that the HDR substitution rate is While this is sufficient to permit a handpicking cloning protocol it is lower than that achieved by Cas12a in fertilised eggs from model animals3954 We hoped to improve this rate by combining OneSHOT with other procedures based on alternative principles such as introducing a blocking base into the repair template2627 andor using HDRNHEJ modification compounds3863 We have examined whether the modification compounds can promote HDR however the compounds examined in this study had no HDRpromoting effects in our experimental system Supplementary Fig a0X4It is important to emphasise though that the procedure depends on highfidelity target recognition by the sitespecific nuclease Thus the only enzymes appropriate for the OneSHOT procedure include highfidelity variants of engineered SpCas955 or naturally highfidelity Cas9 orthologues59 Finally while we confirmed the expression of pluripotency markers in the geneedited clones data not shown we next aim to carry out functional analyses to confirm the differentiation potential of the repaired cells Therefore further work is needed to finetune the protocol and to confirm differentiation potential and functionality of the proteins in the corrected cell populationsTo increase the reliability of the OneSHOT method it is important to show the robustness of OneSHOT and the fidelity of the repair In order to demonstrate these issues we performed targeted NGSbased deep AmpliSeq analysis of the target sequence With regard to repair fidelity the AmpliSeq analysis showed that accurate singlenucleotide substitutions were achieved by HDR that were faithful to the ssODN template and occurred at sufficient frequency Supplementary Fig a0X3ac These results suggest that the method has good repair fidelity With regard to the robustne | 2 |
chrysoperla nipponensis okamoto which has the unique diapause phenotype distinguishable from nondiapause adult is an ideal model anism for studying the mechanism of reproductive diapause however there is no reliable and effective reference genes used for the reproductive diapause study of c a0nipponensis therefore in this study we evaluated the expression stability of candidate reference genes tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin αtub in adults under diapause and nondiapause induction conditions using four statistical algorithms including genorm normfinder bestkeeper and ct method results showed that arp3 and tub1 were the most stable reference genes in all samples and in the adult tissues group arp3 and rps5 were the most stable reference genes in the development degree group αtub and ef1a were unstable reference genes under the conditions of this study meanwhile to verify the reliability of the reference genes we evaluated the relative expression levels of vg and vgr in different treatments significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was respectively observed when using arp3 as the reference gene but not when using an unstable reference gene the reference genes identified in this work provided not only the basis for future functional genomics research in diapause of c a0nipponensis and will also identify reliable normalization factors for realtime quantitative realtime polymerase chain reaction data for other related insectskey words chrysoperla nipponensis okamoto reference genes qrtpcr reproduction diapausedue to the advantages of high sensitivity rapidity specificity and accuracy bustin et a0al valasek et a0al vanguilder et a0al shakeel et a0al quantitative realtime polymerase chain reaction qrtpcr has been widely used in the study of animals plants and microanisms roy et a0al jia et a0al zhang et a0 al ding et a0 al sun et a0 al qrtpcr is the most commonly used method for the expression analysis of target genes however the reliability of qrtpcr results in different samples is determined by a variety of factors among which the use of stably expressed reference genes is an important link for accurate detection of gene expression changes by qrtpcr bustin et a0 al at present several commonly used reference genes for data normalization include tubulin actin ribosomal protein elongation factor 1α glyceraldehyde3phosphate dehydrogenase 18s ribosomal rna and other genes bustin vanguilder et a0al however more and more studies have found that these reference genes do not show consistent expression patterns under different experimental conditions and even affect the reliability of experimental results therefore in order to obtain stable and reliable normalization factors reference genes with stable expression are usually used for correction and standardization to reduce errors between samples selection and evaluation of reference genes have become a necessary step before quantifying the expression of target genes accuratelynowadays there have been many studies on the selection of reference genes for insects such as sesamia inferens helicoverpa armigera aphis gossypii myzus persicae etc lu et a0 al shakeel et a0al zhang et a0al ma et a0al kang et a0al in these studies four statistical algorithms including genorm normfinder bestkeeper and ct method were mainly used to analyze the ct values obtained by qrtpcr of reference genes under various experimental conditions shakeel et a0 al finally the stability of the candidate reference genes was determined according to the geometric mean value of each gene ranked using different the authors published by oxford university press on behalf of entomological society of americathis 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 cited for commercial reuse please contact spermissionsoupcom 0c of insect science vol no algorithms and the most suitable reference gene was selected for the target gene expression analysis xiao et a0al kang et a0al chrysoperla nipponensis okamoto as one of the important predatory natural enemies of agricultural and forestry pests prefers to eat aphids thrips and other pests okamoto nie et a0al because of its characteristically wide geographical distribution and broad range of host prey niijima syed et a0al it has good prospects for widespread application in biological control mcewen et a0 al memon et a0 al reproductive diapause is an important way for c a0nipponensis adults to escape from adverse environments xu et a0al at present there have been many reports on the diapause of c a0nipponensis xu et a0al found that the body color of c a0nipponensis was green during the reproductive period but turned brown and yellow during the diapause period chrysoperla nipponensis belongs to the photoperiodic sensitive insect the adult diapause was induced by short photoperiods xu et a0al chen et a0al found that different photoperiods affected the material content eg protein and glycogen of c a0nipponensis diapause induced by the short photoperiod was beneficial to the storage of c a0nipponensis chen et a0al we expect an exponential increase of diapause research on c a0nipponensis at the molecular level in the near future thus stable and reliable reference genes are important for accurately quantifying gene expression of c a0nipponensisribosomal proteins and ribosomal rna have been used as reference genes in previous diapause studies for example williams et a0al used ribosomal protein rp49 as a reference gene to study the natural variation of drosophila melanogaster diapause williams et a0 al and sim and denlinger used ribosomal protein large subunit rpl19 as a reference gene in a study of ovarian development of culex pipiens during overwintering diapause sim and denlinger this indicates that under the same experimental conditions the selected reference genes in different species research are also differentin this research candidate reference genes were selected including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub whose expression profiles were measured by the qrtpcr the stability was analyzed by four statistical algorithms genorm normfinder bestkeeper and ct method in different developmental stages reproductive and diapause of adults and among different tissues the optimal reference genes under different conditions were determined which contributed to the accurate expression of target genes for future researchmaterials and a0methodsinsecta stable population of c a0 nipponensis was maintained in an artificial climate chamber rsz intelligent artificial climate chamber changzhou guohua jiangsu province under the following conditions a0± °c temperature a0± relative humidity rh and long photoperiod of l d h in our laboratory the eggs were collected by cutting the stalk and incubated in fingertip tubes a0cm in diameter and a0cm in height the primary hatching larvae were fed megoura japonica matsumura whose host plant was vicia faba l a0the adults were paired immediately after emergence in a bottle a0cm in diameter and a0cm in height fed a dry brewers yeast feed mixed with sucrose in a ratio of and then minced in a mortar and sifted through mesh and honey water the diapause adults used in this study were kept under the conditions of short photoperiod of ld h in all processes from eggs larvae pupae to adults whereas the nondiapause adults were kept under the conditions of long photoperiod of ld a0hsample collection development degree samples from individuals from varying developmental stages included female adults in the diapause induction period d under the short photoperiod the diapause maintenance period d under the short photoperiod the diapause termination period d under the short photoperiod and the reproduction period d under the long photoperiod each sample which included three to four females was independently replicated three times as three biological replicates adult tissues reproduction seven different tissues were collected from reproductive adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of reproductive adults under long photoperiod were collected on the 10th day after emergence each tissue required about a0mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues diapause seven different tissues were collected from diapause adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of diapause adults under short photoperiod were collected on the 20th day after emergence each tissue required about a0 mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues samples from reproductive and diapause adult tissues all samples samples from group and all treatments were immediately frozen with liquid nitrogen and stored in an ultralow temperature refrigerator at °c prior to rna extractiontotal rna extraction and cdna synthesisin this study total rna was extracted using minibest universal rna extraction kit takara japan and dnase i a0 was used for digestion of the membrane rna integrity was estimated by agarose gel electrophoresis rna concentration and purity were measured with a nanodrop one spectrophotometer thermo scientific then 1μg rna was reversetranscribed into the firststrand complementary dna cdna according to the hiscript ii q rt supermix for qpcr gdna wipers vazyme nanjing china instructions and stored at °c all cdna was diluted 10fold with dnasernasefree sterile water before usecandidate reference gene selection and primer a0designaccording to several commonly used reference genes candidate reference gene sequences were obtained by screening from the existing transcriptome of c a0 nipponensis in the laboratory namely tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub in order to ensure the predictive accuracy of the selected sequences we conducted blast alignment all primers were designed using primer premier based on the following criteria gc content annealing temperature °c and primers length a0 bp and the specificity of each pair of amplicons was determined by qrtpcr followed by agarose gel electrophoresis and melting curve analysis the amplification efficiency of the pcr was calculated by using the formula e a0 a0 slope the slope was obtained by the standard curve which was generated by qrtpcr of a series of continuously diluted cdna samples 0c of insect science vol no realtime qrtpcr analysisthe 20µl total reaction volume were configured according to the protocol of chamq sybr qpcr master mix vazyme contained 10µl a0à chamq sybr qpcr master mix a0µl a0μm of each gene specific primer a0µl of cdna and a0µl of ddh2o the amplification reaction program was set as follows predenaturation at °c for a0s followed by cycles of denaturation at °c for a0s annealing at °c for a0 s the melting curves were analyzed in the °c temperature range after amplification step the reaction was performed on a roche lightcycler96 instrument to obtain ct values amplification curves melting curves and standard curves all samples were carried out in four technical and three biological replicates and the negative control no template was performed in paralleldata analysisct values for all samples were exported into an excel spreadsheet and were used to analyze the stability of candidate reference gene expression by genorm normfinder bestkeeper and ct method the comprehensive ranking were performed following methods adapted from xiao et a0 al the optimal number of genes was determined by the pairwise variation vnn1 between the normalization factors calculated by genorm among the four algorithms genorm and normfinder need to convert the original ct value according to the corresponding requirements before analysis in genorm the stability ranking of genes was determined by the expression stability value m value in bestkeeper the stability ranking of genes was determined by the coefficient of variation cv and sd in normfinder the stability ranking of genes was determined by the gene expression stability value sv in ct method the stability of genes was ranked according to the sd of genes ct values in four statistical algorithms genes with the lowest value were the most stably expressedvalidation of reference a0genesin most insects vitellogenin vg and vitellogenin receptor vgr play important roles in the reproductive process of female insects vg is taken up by developing oocytes through receptormediated endocytosis rme thereby promoting the development of oocytes and the formation of eggs in this process vgr is the main receptor mediating endocytosis previous studies have shown that reproductive diapause arrests development of oogenesis and vitellogenesis tatar and yin and the expression levels of the vg and the vgr in nondiapause female were significantly higher than those in reproductive diapause female jiang et a0al in order to evaluate the effectiveness of the selected reference genes the expression levels of the target genes vg and vgr were respectively detected by qrtpcr in the different development degree and tissues of adults and the most unstable reference gene was used for comparison in parallel the reaction system and program were the same as for qrtpcr of reference genes and four technical and three biological replicates were performed for each treatment the relative expression levels of vg and vgr were respectively calculated in excel using the ct method the differences of target genes expression levels were analyzed by tukeys test using spss software spss inc under different experimental conditionsresultsselection and primer performance of candidate reference a0genesthe candidate reference genes including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub were selected to identify the normalization factors for qrtpcr analysis and the sequence information has been submitted to genbank and the accession numbers are shown in supp table online only to determine the amplification specificity of the primers agarose gel electrophoresis and melting curve analysis were performed all primer pairs showed a single band and a single peak fig a0 to obtain correlation coefficient r2 and amplification efficiency e of pcr a standard curve was generated with the 10fold dilution series of cdna the results showed that the amplification efficiency ranged from to and the correlation coefficient varied from to supp table [online a0only]expression profiling of candidate reference a0genesto evaluate expression levels of the candidate reference genes the cycle threshold ct values under different groups were obtained by qrtpcr and represented by boxplot fig a0 the results indicated that ct values of all candidate reference genes were different under different conditions and also varied under the same condition overall ct values ranged from to among them the genes with higher expression abundance were tub1 rps26e and actin followed by 128up arp3 arpc5 rps5 and ef1a the genes with lower expression abundance were αtub and gapdh according to the ct value range of each candidate reference gene the genes with relatively stable expression were tub1 and arp3 whereas the most unstable genes were ef1a actin and αtubexpression stability of candidate reference genes under different conditionsin this study four statistical algorithms were used to analyze the expression stability of the candidate reference genes under different conditions including genorm normfinder bestkeeper and ct method as different statistical algorithms would generate different ranking patterns the comprehensive ranking of genes was finally determined through the geometric mean of sequencing bestkeeper the original ct values were used for analysis evaluated the stabilities of the candidate reference genes according to the cv and sd of the ct values ct method the difference values of original ct values were used for analysis performed stability ordering according to the mean sd of ct value genorm and normfinder the original ct values were converted for analysis sequenced the candidate reference genes according to the stability values the lower the stability value the more stable the gene expressiondevelopment degree of a0adultsthe expression stability of the candidate reference genes at different periods of reproductive and diapause female showed that the top four ranked genes identified by the genorm bestkeeper normfinder and δct method were similar but the rank order was slightly different arp3 and rps5 were the first and second stably expressed genes in the four statistical algorithms supp table [online only] and comprehensive ranking analysis fig a0 as for the third and fourth ranked gene tub1 and actin identified by comprehensive ranking analysis were the same as those generated by genorm and normfinder while bestkeeper selected arpc5 and tub1 ct method selected actin and rps26e supp table [online only] αtub was ranked by genorm bestkeeper normfinder and δct method as the least stable gene among the candidate reference 0c of insect science vol no fig specificity a and product length b of qrtpcr amplification for ten candidate reference genesgenes during different development degrees of adults supp table [online a0only]adult a0tissuesthe expression stability ranking of candidate reference genes in different tissues of reproductive and diapause females was varied according to the four statistical algorithms in different tissues of reproductive females the top four genes were rps5 arp3 arpc5 and tub1 respectively fig a0 but the rank order of the four genes was significantly different among different statistical algorithms rps5 was ranked first by genorm and ct method and was ranked third and fourth by normfinder and bestkeeper respectively arp3 was ranked first by bestkeeper and was ranked second third and fifth by normfinder δct method and genorm respectively arpc5 was ranked first and second by genorm and δct method and was ranked fourth and fifth by normfinder and bestkeeper respectively tub1 was ranked first and second by normfinder and bestkeeper and was ranked fifth and sixth by δct method and genorm respectively supp table [online only] however the four statistical algorithms found that ef1a was ranked as the least stable gene in the tissues from reproductive females supp table [online only]in different tissues of diapause females the top four genes were different from those of different tissues of reproductive females tub1 was ranked first by the comprehensive ranking followed by rps26e arp3 and 128up fig a0 through analysis it was found that ct method and normfinder displayed the same rankings for expression stability of candidate reference genes under this condition supp table [online only] tub1 was identified as the most stably expressed gene by genorm ct method and normfinder although it was ranked fifth by bestkeeper rps26e ranked steadily among the four statistical algorithms was ranked second by ct method and normfinder whereas it was ranked first and third by genorm and bestkeeper respectively arp3 was ranked first by bestkeeper with the smallest coefficient of variation whereas it was ranked third by ct method and normfinder in addition to being ranked third by genorm 128up was ranked fourth by bestkeeper ct method and normfinder supp table [online only] however in tissue from diapause females actin was consistently identified as the gene with the most unstable expression by the four statistical algorithms supp table [online only]in the reproductive and diapause female tissues the expression stability of the candidate reference genes was different in order to accurately determine the expression of the target genes the expression stability of candidate reference genes under the two conditions was analyzed according to the comprehensive ranking 0c of insect science vol no fig expression profiles of candidate reference genes in c a0nipponensis expression data are displayed as ct values for each reference gene using a box and whisker plot in different experimental conditions the line across the box is the median the box indicates the 25th and 75th percentiles the whiskers represent the 10th and 90th percentilesfig expression stability and comprehensive ranking of reference gene measured by the geomean method a a0lower geomean value indicates more stable expressiontub1 and ef1a were the most stable and unstable genes respectively whereas arp3 128up and arpc5 were ranked second third and fourth respectively fig a0 tub1 was the best candidate reference gene identified by normfinder and ct method and was ranked third and sixth by bestkeeper and genorm respectively arp3 was the most suitable candidate reference gene selected by bestkeeper and was ranked second by normfinder and ct method and fifth by genorm 128up and arpc5 were the most stable candidate reference genes identified by genorm whereas they were ranked separately fifth and sixth by normfinder and ct method and seventh and fifth by bestkeeper respectively supp table [online only] ef1a was identified as the least stable gene by genorm normfinder and δct method although bestkeeper selected actin as the least stable gene supp table [online only]all a0samplesin order to determine the best reference gene suitable for the different conditions of adults the stability of the ten candidate reference genes was ranked for all samples arp3 was identified as the most stable gene by the comprehensive ranking followed by tub1 arpc5 and rps5 whereas ef1a was identified as the least stable gene fig a0 0c of insect science vol no but the most and least stable genes identified by different statistical algorithms were slightly different arp3 was selected as the most stable reference gene by bestkeeper and ct method although tub1 and arpc5 were selected as the most stable reference gene by normfinder and genorm respectively ef1a was selected as the least stable reference gene by genorm and ct method despite it being ranked ninth by bestkeeper and normfinder supp table [online only]the best combination of candidate reference genes under different conditionsaccording to the pairwise variation vnn1 between the normalization factors and cutoff value calculated by genorm the number of reference genes required for optimum normalization in each experimental condition was determined the cutoff value of vnn1 a0 suggested that n reference genes were enough to make gene expression normalization otherwise n reference genes were needed the analysis results showed that all v23 were indicated that the optimal number of reference genes under each condition was two fig a0 more specifically arp3 and rps5 were the most stable gene combinations under adult developmental stage and reproductive adult tissues conditions tub1 and rps26e were the most stable gene combinations under adult diapause tissues conditions and arp3 and tub1 were the most stable gene combinations under adult tissues and all samples groups table a0relative expression levels of target genes vg and vgr genbank mt308983 mt522179 in the whole adult and from tissues of reproductive and diapause adults respectively when the most stable reference genes arp3 andor rps5 were used as normalization factors at different periods of reproduction and diapause the expression patterns of the target genes vg and vgr were consistent with low expression in the diapause period and rich expression in the late diapause and reproduction period however when the most unstable reference gene αtub was used as a normalization factor neither the target gene vg nor vgr showed a consistent expression pattern fig a0 under different tissue conditions when the most stable reference genes tub1 andor arp3 were used as the normalization factors the expression level of the target gene vg in the fat body of the reproductive female was significantly higher than that in the ovary of the reproductive female the expression level of the target gene vgr in the fat body of the reproductive female was lower than the ovary of the diapause female while when the most unstable reference gene ef1a was used as the normalization factor the expression pattern differed with normalization by tub1 and tub1arp3 fig a0 in general when the most stable reference genes were used as the normalization factors the accurate expression pattern of the target gene could be obtainedvalidation of reference a0genesthe stability of reference gene is very important for the analysis of expression level of target gene vg and vgr which are important for insect reproduction were selected to verify the applicability of the selected reference gene we examined the discussionqrtpcr has become an important means to explore gene expression level due to its high sensitivity rapidity specificity and accuracy and was widely used in physiology studies that investigated insect diapause such as drosophila melanogaster williams fig pairwise variations vnn1 was calculated by genorm to determine the optimal number of reference genes for accurate normalization in different conditions the cut off values under indicate that no additional genes are required for the normalization 0c of insect science vol no et a0al culex pipiens sim and denlinger leptinotarsa decemlineata lehmann et a0 al chrysopa septempunctata liu et a0al and pieris melete wu et a0al however the selection of appropriate reference genes was the key to accurately analyze the gene expression level for example under conditions of injury heatstressed and experimentally varied diets table recommendation for the best combination of reference genes based on the genorm and comprehensive rankings under various experimental conditionsgroupreference genemostdevelopment degreeadult tissues reproductionadult tissues diapauseadult tissuesall samplesarp3rps5tub1tub1 arp3 rps5arp3rps26earp3tub1leastαtubef1aactinef1aef1athe best reference gene was different in drosophila melanogaster ponton et a0al under biotic factors and abiotic stress inappropriate selection of the reference genes in locusta migratoria resulted in significant differences in the expression level of the target gene chitin synthase chs1 yang et a0al diapause of most insects was mainly affected by photoperiod and temperature in past studies the screening of reference genes of drosophila melanogaster ponton et a0al leptinotarsa decemlineata shi et a0al helicoverpa armigera zhang et a0al bombyx mori guo et a0al and harmonia axyridis qu et a0al under main environmental factors was completed by genorm normfinder bestkeeper and ct method in this study the expression profiles of candidate reference genes of c a0nipponensis were analyzed under different conditions by the same four statistical algorithms genorm vandesompele et a0 al normfinder andersen et a0al bestkeeper pfaffl and ct method silver et a0al different algorithms produced different stability rankings in order to obtain statistically consistent and accurate results we finally ranked the gene based on their stabilities determined by fig validation of selected reference genes under different periods a and tissues b of reproductive and diapause female in c a0 nipponensis relative expression levels of the vg and vgr in different samples using different normalization factors the most and least stable genes asterisks indicate significant differences in the expression levels of the vg and vgr r reproduction period d1 the diapause induction period d2 the diapause maintenance period d3 the diapause termination period 0c of insect science vol no comprehensive analysis method xiao et a0 al and selected the most stable reference genes under each condition as far as we know actin which played an important role in cell contraction and cytoskeletal maintenance was found in virtually all eukaryotic cells and was considered as an ideal reference gene for many anisms sürencastillo et a0al shakeel et a0al for example actin was used as a reference gene for normalization in the determination of genes related to reproductive and nutritional signaling such as vitellogenin of chrysopa septempunctata liu et a0al however in this study three genes related to actin were selected for analysis among which actin was similar to actin of c a0septempunctata which was also a member of the neuroptera in our study actin was the most unstable gene in the diapause female tissues but the actinrelated protein arp3 which was structurally homologous with actin showed better stability arp3 was selected as the most stable reference gene in adults of different developmental levels and all samples while it was the second most stable gene in the reproductive adult tissues and all adult tissues although arp3 was ranked as the third most stable gene in the diapause adult tissues it showed relatively stable expression in the expression profile tubulin which played an essential role in maintaining cell shape movement and intracellular material transport was also often used as a reference gene but different types of tubulin have different stability for example in the study of helicoverpa armigera the expression of βtub was relatively stable compared with that of αtub under almost all conditions zhang et a0 al similarly in this study αtub showed unstable expression and was the least stably expressed gene in adults of different developmental stages whereas tub1 was considered to be the most stably expressed reference gene in diapausing adult tissues and all adult tissues and was the second most stable gene in all samples ribosomal protein rp widely distributed in various tissues played an important role in protein biosynthesis and was widely used as a reference gene in many insects lu et a0al koyama et a0 al sun et a0 al in this study rps5 was considered to be stable in the tissues of reproductive females and ranked second among different developmental stages of adults elongation factors ef was a protein factor which promoted polypeptide chain to extension during the translation of mrna and was recommended as the ideal reference gene under different conditions of a variety of insects chapuis et a0 al ponton et a0 al however some studies showed that ef1α was one of the most unstable genes under certain conditions fu et a0 al in our study ef1a was found to be the most unstable gene in the reproductive adult tissues all tissues and all samples and the second least stable gene in the adults of different developmental stages and diapause adult tissues therefore it was not suitable for the study of c a0nipponensisrecently an increasing number of studies have demonstrated the importance of using multiple stably expressed reference genes for the accuracy of qrtpcr analysis ling and salvaterra yuan et a0 al kang et a0 al however this does not mean that the more reference genes increase the reliability of the results the study has indicated that either too few o | 0 |
to machine learning with python a guide for data scientists oreilly media inc california demÅ¡ar j statistical comparisons of classifiers over multiple data sets j mach learn res yates a the ensembl rest api ensembl data for any language bioinformatics kim e r chang d k colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis world j gastroenterol diovasc dis schulte d small dense ldl cholesterol in human subjects with different chronic inflammatory diseases nutr metab car smedley d biomartbiological queries made easy bmc genom quinlan a r hall i m bedtools a flexible suite of utilities for comparing genomic features bioinformatics 101093bioin forma ticsbtq03 rom¡n j evaluation of responsive gene expression as a sensitive and specific biomarker in patients with ulcerative colitis inflamm bowel dis 101002ibd23020 song r identification and analysis of key genes associated with ulcerative colitis based on dna microarray data medicine baltimore e10658 101097md00000 schwegmann k detection of early murine colorectal cancer by mmp29guided fluorescence endoscopy inflamm bowel dis 101097mib00000 oliveira l g d positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis arq gastroenterol 101590s0004 shin js antiinflammatory effect of a standardized triterpenoidrich fraction isolated from rubus coreanus on dextran sodium sulfateinduced acute colitis in mice and lpsinduced macrophages j ethnopharmacol 158pt a 101016jjep201410044 owens d w lane e b keratin mutations and intestinal pathology j pathol 101002path1646 macfie t s duox2 and duoxa2 form the predominant enzyme system capable of producing the reactive oxygen species h2o2 in active ulcerative colitis and are modulated by 5aminosalicylic acid inflamm bowel dis 10109701mib00004 0e palmer n p concordance between gene expression in peripheral whole blood and colonic tissue in children with inflammatory bowel disease one e0222952 101371journ alpone02229 wei z large sample size wide variant spectrum and advanced machinelearning technique boost risk prediction for inflammatory bowel disease am j hum genet amrhein v greenland s mcshane b scientists rise up against statistical significance nature wasserstein r a0l schirm a a0l lazar n a0a moving to a world beyond p maeda y fully automated diagnostic system with artificial intelligence using endocytoscopy to identify the presence of histologic inflammation associated with ulcerative colitis with video gastrointest endosc 101016jgie201809024 acknowledgementsthis research was partially supported by grants from the natural sciences and engineering research council of canada nserc to hu grant number rgpin and to lpc grant number rgpin hmk was partially supported by funding from memorial universitys school of graduate studiesauthor a0contributionsconceptualization hu and lpc methodology hmk hu and lpc analysis hmk and lpc writing hmk hu and lpc experiments hmk supervision hu and lpccompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to hu a0or a0lpcreprints and permissions information is available at wwwnaturecomreprintspublishers note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020705830vol1234567890wwwnaturecomscientificreports 0copen access 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 license and indicate if changes 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 if material 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 obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020705830vol0123456789wwwnaturecomscientificreports 0c' | 0 |
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 | 2 |
" accurate detection of patients with minimal residual disease mrd after surgery for stage ii coloncancer cc remains an urgent unmet clinical need to improve selection of patients who might benefit formadjuvant chemotherapy act presence of circulating tumor dna ctdna is indicative for mrd and has highpredictive value for recurrent disease the medocccreate trial investigates how many stage ii cc patients withdetectable ctdna after surgery will accept act and whether act reduces the risk of recurrence in these patientsmethodsdesign medocccreate follows the trial within cohorts twics design patients with colorectal cancercrc are included in the prospective dutch colorectal cancer cohort plcrc and give informed consent forcollection of clinical data tissue and blood samples and consent for future randomization medocccreate is asubcohort within plcrc consisting of stage ii cc patients without indication for act according to currentguidelines who are randomized into an experimental and a control armin the experimental arm postsurgery blood samples and tissue are analyzed for tissueinformed detection ofplasma ctdna using the pgdx elio¢ platform patients with detectable ctdna will be offered act consisting of cycles of capecitabine plus oxaliplatin while patients without detectable ctdna and patients in the control groupwill standard followup according to guidelinethe primary endpoint is the proportion of patients receiving act when ctdna is detectable after resection themain secondary outcome is 2year recurrence rate rr but also includes 5year rr disease free survival overallsurvival time to recurrence quality of life and costeffectiveness data will be analyzed by intention to treatcontinued on next page correspondence mkoopman6umcutrechtnlsj schraa and kl van rooijen are shared first authorrja fijneman gr vink and m koopman are shared last author1department of medical oncology university medical center utrechtutrecht university heidelberglaan cx utrecht the netherlandsfull 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 0cschraa bmc cancer page of continued from previous pagediscussion the medocccreate trial will provide insight into the willingness of stage ii cc patients to be treatedwith act guided by ctdna biomarker testing and whether act will prevent recurrences in a highrisk populationuse of the twics design provides the opportunity to randomize patients before ctdna measurement avoidingethical dilemmas of ctdna status disclosure in the control grouptrial registration netherlands trial register nl6281ntr6455 registered may wwwtrialregisternltrial6281keywords colon cancer circulating tumor dna ctdna adjuvant chemotherapy twics in patients with stage ii colon cancer cc the recurrence rate rr after surgery is approximately disease management after surgical resection in stageii cc is still under debate because the overall survivalos benefit of adjuvant chemotherapy act in thisgroup of patients varies between and only [ ]moreover offering act in a lowrisk population induces an important amount of overtreatment with unnecessary but sometimes severe toxicity and costsseveral prognostic characteristics of stage ii cc havebeen identified to provide better selection of patientsthat might benefit from act patients with presence ofat least one of the following characteristics are classifiedas being at high risk of disease recurrence poorly differentiated histology pt4 lesions inadequately less than sampled lymph nodes lymphovascular or perineuralinvasion or tumor presentation with perforation or obstruction in contrast patients with a deficient mismatch repair dmmr status in stage ii cc have a low risk ofrecurrence and act is not considered beneficial irrespective of the presence of other risk factors [ ]other known prognostic factors in cc like gene expression profiles or braf v600e and ras mutations have been investigated but do not adequatelyidentify the patients that will benefit from act []despite the definition of high and low risk subgroupsof stage ii cc patients retrospective analyses demonstrated that improved survival after administration ofact was not observed in high risk patients or exclusivelyin patients with a pt4 tumor [] therefore in thenetherlands act is currently only recommended in stageii cc patients with a pt4 tumor without dmmrunfortunately also pt4 is not an absolute predictorfor disease recurrence in stage ii patients in a retrospective analysis of stage ii cc patients with pt4tumors the 3year diseasespecific survival rate aftersurgery was in patients who received act and in patients who did not receive act whichmeans that of these patients are exposed to actunnecessarily considering nonpt4 stage ii patients a population registry analysis of patientsshowed that in this group of patients sufferedfrom recurrences these data demonstrate thatusing pt4 as a prognostic factor results in significantunder and overtreatmentminimal residual disease mrd is defined as the presence of tumor cells in the blood bone marrow or lymphnodes not detected by conventional staging procedures patients who have mrd after surgery are not completely cured and therefore at high risk of developingdisease recurrence development of a highly specific andsensitive biomarker testindicative for mrd wouldallow identification of the subset of patients likely to experience recurrence of disease thereby improving the selection of patients who may benefitfrom adjuvanttreatment in adjuvant trials this would solve problemsof high numbers needed for inclusion and dilution of effectiveness of adjuvant treatment by inclusion of manyalready cured participants cellfree circulating tumor dna ctdna has a strongpotential for being this sensitive and yet specific biomarker ctdna consists of smallfragments usually bp of tumorderived dna containing tumorspecific mutations which can be detected in liquid biopsies such as blood samples [] because of the shorthalflife of ctdna estimates ranging from to min the presence of ctdna in blood samples taken several days after surgery presumably reflects a state ofmrd [] patients with mrd have the highest riskfor disease recurrencerecently the presence of ctdna after tumor resection demonstrated a very strong prognostic value fordisease recurrence in stage ii cc with a 2years rrof versus in patients with and without detectable ctdna after surgery respectively in thisstudy the univariate prognostic value of ctdna was muchhigher than that of pt4 status hazard ratio of versus respectively there are several ongoing trials that usectdna in prognostication nct03637686 nct03737539nct03416478 nct03312374 nct02842203 nct0361 and treatment nct03748680 actrn12615000381 nrggi of nonmetastatic cc but to date thereare no results available of randomized controlled trialsrcts that use ctdna for selection of act treatment 0cschraa bmc cancer page of the accumulating evidence for the strong prognosticvalue of ctdna raises an important ethical dilemma forrandomization of patients when designing a conventional rct in which patients with detectable ctdna arerandomized into act treatment or standard of carefollowup while disclosing ctdna status to the controlgroup indeed the knowledge of having a very highchance of disease recurrence will be a big burden for patients with detectable ctdna in the control group andtheir caregivers as they are not being offered any additional therapy this warrants an innovative trial designlike the trialdifferent from the conventional rctwithin cohorts twics design [] the twics design enables an experimental group in which ctdna status is disclosed and a control group that is unaware oftheir ctdna statusthe medocccreate trial is designed as a multicenter twics study with two parallel groups in whichwe will investigate whether stage ii cc patients with detectable ctdna after resection are willing to receiveact and whether act reduces the rr in these ctdnapositive patientsmethodsdesignaimthis study investigates the willingness of patients to receive act after detection of ctdna postsurgery andthe effect of ctdnaguided act on the rr in stage iicc patientsstudy designthe medocccreate trial follows the twics designand is performed within the prospective dutch colorectal cancer cohort plcrc wwwplcrcnl plcrc is set up by the dutch colorectal cancer groupdccg and collects clinical data and patient reportedoutcome measures proms at baseline and at multipletime points during followup fig at enrollment patients give informed consent for use of their clinical dataand optionally for receiving quality of life questionnairesfig schematic presentation of medocccreate using the trial within cohort twics design a plcrc is a nationwide cohort study in thenetherlands with inclusion of crc patients all stages by optional informed consent regarding collection of biomaterials and futurerandomization observational as well as interventional trials can be performed within the cohort b nonmetastatic crc patients are included inmedocc when the patient signs informed consent for plcrc including additional blood sampling blood samples are withdrawn beforeresection days after resection and every months during the first years of followup c eligible stage ii colon cancer patients arerandomized following the twics design in the experimental group informed consent is being asked for immediate ctdna analysis of theblood sample obtained after resection if ctdna is detectable patients are offered adjuvant chemotherapy the control group is not informedabout medocccreate and will receive standard of care 0cschraa bmc cancer page of collection of biomaterials for research additional sequential blood sampling and for being approached forfuture studies conducted within the infrastructure ofthe cohort either in accordance with the twics design or notpatients with pt4 tumors will be offered act therefore we will include eligible patients with pt4 tumors without a recommendation for act according totheir treating physician and use pt4 status as a stratification factorpatient selection and recruitmentpatients will be recruited in both academic and nonacademic hospitals in the netherlands that are participating in plcrc nonmetastatic colorectal cancercrc patients that give informed consent for plcrcincluding consent for additional blood sampling at enrollment will be included in the observational plcrcsubstudy medocc molecular early detection of coloncancer before surgery the participants are eligible forthe current medocccreate trial if they meet the following criteria after surgery histopathological confirmed and radically resected stage ii cc age ¥ years informed consent for plcrc and medoccincluding consent for randomization in future trials anduse of tissue physical condition allows treatmentwith combination chemotherapy consisting of a fluoropyrimidine and oxaliplatin and no indication foract according to the treating physician andor multidisciplinary board patients who are pregnant have hadanother malignancy in the previous years except forcarcinoma in situ or patients with contraindications forfluoropyrimidines andor oxaliplatin will be excludedcurrently the dutch guidelines recommend act forpatients with pt4 tumors however there is large ageand hospital dependent variation in administration ofact in this group and in clinical practice not all stage iiblood sample collectionblood samples are collected before and days aftersurgery for all patients included in the medocc clinicalstudy predominantly comprising stage i ii and iii ccpatients table blood samples two tubes of mlper timepoint are collected in cell free dna streckblood collection tubes for various research purposesamong which the medocccreate trialrandomizationabout week after surgery when the histopathologicalreport is finished medocc patients who are eligiblefor medocccreate will be randomized to theintervention or control arm using slim an onlineplatform to manage patientinclusion including arandomizationgeneratedcomputerservice therandomization schedule is stratified by tstage and usespermuted blocks of random sizes allocation concealment will be ensured as the service will not release therandomization code only patients randomized to theintervention arm will be informed about medocccreate according to the twics design experimental armafter randomization only patients randomized to theexperimental arm will be asked separate informedtable standard protocol items for intervention trials spirit schedule of enrollment interventions and repeated measurementsact adjuvant chemotherapy ctdna circulating tumor dna qol quality of life intervention group only intervention grouponly if ctdna is positive 0cschraa bmc cancer page of consent for the immediate analysis of ctdna status ofthe postsurgery sample a small proportion of patientsestimated approximately will have detectablectdna in their blood these patients will be offeredact patients decide whether they accept or refuse thistreatment patients without detectable ctdna will receive routine standard of careact will consist of months of capecitabine and oxaliplatin capox or months of fluorouracil leucovorinand oxaliplatin folfox treatment starts preferablywithin weeks and not beyond weeks after surgeryduring and after completing act routine followupwill consist of regular visits at the surgical outpatient department blood withdrawals for analysis of carcinoembryonic antigen cea and imaging standard ultrasoundofthe liver according to current guidelines in thenetherlands no additional imaging will be performed toprevent detection biascontrol armin the control arm patients will not be informed aboutthe medocccreate trial and receive routine followup care consisting of cea tests every months for thefirst years and abdominal ultrasound or ct every months in the first year and once a year afterwards oneyear after surgery a colonoscopy is performed postsurgery blood samples will not be tested for ctdna immediately but will be analyzed batchwise after severalmonths without result disclosure to patients and theirtreating physiciansfollowupblood samples will be collected at 6monthly intervalsfor the first years after surgery for both patients in theexperimental arm and the control arm conform themedocc study protocol these samples will not be analyzed for ctdna immediately and results will not bedisclosed to patients and treating physicianstumor tissueinformed ctdna analysisafter surgery the local pathologist will send a formalinfixed paraffinembedded ffpetissue block to thecentral laboratory where dna will be isolated for further analysisthe postsurgery blood sample is drawn between and days after surgery the sample is not withdrawnbefore day to reduce the risk of falsenegative ctdnatests due to the relatively large amount of cell free dnacfdna released due to cell damage after surgery theblood is taken no later than days after surgery to beable to start chemotherapy within weeks after surgerysamples are kept at room temperature and sent by regular mail to the central laboratory within days wherectdna will be isolated for further analysistumor tissue dna will be analyzed by targeted nextgeneration sequencing of a panel of more than genes using the pgdx elio¢ tissue complete assay frompersonal genome diagnostics pgdx baltimore mdusa plasma ctdna will be analyzed by targeted nextgeneration sequencing of a panel of more than genesusing the pgdx elio¢ plasma resolve assay from pgdxbaltimore md usa both panels include the mostcommonly mutated genes in cc including apc tp53kras and braf tumor tissue dna mutations are usedas input information for plasma ctdna mutation callingthereby increasing both sensitivity and specificity of thectdna testprimary endpointthe primary endpoint is the proportion of patients starting with act after detection of ctdna in the postsurgery samplesecondary endpointsthe most important secondary endpoint is 2year rr inpatients with detectable ctdna in their blood expressedas the proportion of patients that experience a recurrence within years after surgery detection of recurrences in months after surgery will occur by standardfollowup investigations including monthly bloodsampling of tumor marker cea and monthly imagingwith ultrasound liver or ct abdomen and when indicated by symptoms radiological andor histopathological evidence is used to confirm the recurrence thedate of the said investigation is considered the date ofrecurrencedata about followup recurrences and survival areroutinely collected within plcrc using the netherlandscancer registry ncr managed by the netherlandscomprehensive cancer anisation iknl to provideinsight in the characteristics and magnitude of cancer inthe netherlands other secondary endpoints include 2year rr in aperprotocol analysis 5year rr intentiontotreatand perprotocol analysis time to recurrence ttr and 5year disease free survival dfs rate and7year diseaserelated os rate and 5year rr inpatients with undetectable ctdna after surgery quality of life qol and costeffectiveness of the ctdnaguided strategytimetoevent outcomesos rate is expressed as proportion of patients that arealive and years after surgery dfs rate is expressed asproportion of patients that did not experience disease recurrence a second primary cc or death within and years after surgery ttr is expressed as time monthsbetween surgery and detection of disease recurrence 0cschraa bmc cancer page of patients will be censored at the last date of followup if adate of death is not recorded and at the date of death ifthe cause of death is not due to ccquality of lifeqol is measured within the cohort at regular intervalsin patients who gave consent to send questionnaires nationally and internationally validated questionnaires areused among which the european anisation for research and treatment of cancer quality of life questionnaire core and the colorectal cancer moduleeortcqlqc30 and cr29 the work ability indexwai the euro quality of life5 dimensions eq5dthe multidimensional fatigue inventory20 mfi20and the hospital anxiety and depression score hadscosteffectiveness of the ctdnaguided treatmentthe costeffectiveness analysis will be carried out from asocietal perspectiveincluding both direct health carecosts as well as indirect costs from productivity loss thehealth outcome measure in the costeffectiveness analysis will be the total quality adjusted life years qalyper group for analysis offactors related to qalysquestionnaires are used provided within plcrcsample size considerationsthe primary endpoint is the proportion of ctdna positive patients starting with act however 2year rr inthe ctdna positive patients after surgery is an importantsecondary endpoint and the power calculation is performed for this secondary endpoint we estimate thatsimilar to effectiveness in stage iii cc patients act inctdnabased highrisk stage ii cc patients will lead toa absolute reduction of recurrences within yearsafter surgery in the observational trial of patientswith detectable ctdna experienced disease recurrencewithin years after resection with a power of and an alpha of patientswith detectable ctdna need to be included in botharms assuming a prevalence of ctdna after surgery of and adjustment for loss to followup and rejection ofadjuvant therapy in the intervention arm of a totalsample size of patients is calculated in eacharm we expect few patients with detectable ctdna inthe intervention group to refuse act because patientsare selected upfront for being in a physical condition toreceive act and the established prognostic value of detectable ctdna is highwe assume that crossover from the control arm tothe intervention arm will not occur because only eligiblepatients randomly selected in the cohort and allocatedto the intervention arm will be informed about the trialand have the opportunity for immediate analysis ofctdna patients in the control group will not be informed about the trial or their ctdna statuswe assume that of patients in the interventionarm with detectable ctdna will be treated with actthe proportion of patients starting with chemotherapythe primary endpoint can in that instance be determined with a margin of error width of the confidence interval of we expect to complete recruitment of patients within years with more than participating dutchhospitalsdata analysisdata will be analyzed according to the intentiontotreatprinciple for the primary endpoint and the secondaryendpoint of 2year rr in patients with detectable ctdnaafter surgery in this analysis we expect to compare patients with detectable ctdna who received act inthe intervention arm with patients with detectablectdna in the control arm ie based on ctdna analysisperformed retrospectively at least months after surgery and not disclosed to patients and treating physicians the proportion of patients that experience arecurrence in both arms will be compared by means of achisquare test in addition for other secondary endpoints and exploratory analyses we will analyze timetoevent outcomes in patients in both arms with detectablectdna after surgery differences in timetoevent outcomes will be analyzed by standard survival methodseg kaplanmeier curves compared by logrank testscoxs proportional hazards models will be used for multivariable analysiscomparison of qol of the ctdna positive patients inboth study arms will be done using repeated measurements methods and including act as factor qol willalso be analyzed for the whole population in both armsof the study treatment differences at each qol assessment time point will be compared by means of thewilcoxon rank sum testa lifetime horizon will be applied forthe costeffectiveness analysis parametric survival functions willbe used to extrapolate dfs and os curves beyond yearsresponsibilitiesprotocol modifications will be submitted as amendmentto the medical ethical committee by the study coordinator the local principle investigator of each participating hospital is responsible for patient inclusion logisticsof biomaterials to the centrallaboratory and patientfollowup to ensure quality of data study integrity andcompliance with the protocol and the various applicableregulations and guidelines a data monitor of the iknlhas been appointed to conductto thesite visits 0cschraa bmc cancer page of participating centers and randomly check patient datathe study coordinator together with the principle investigator will have access to the final dataset and is responsible for publishing study results the results will besubmitted to a peerreviewed journaldiscussionmedocccreate is the first clinical trial using thetwics design to investigate ctdnaguided strategies instage ii cc taking an important step towards clinicalimplementation of ctdna in cancer diagnostics andcarewithctdnadetectablea few other trials with the aim to reduce recurrencesin cc by use of a ctdnaguided approach are in preparation or recently started the improveit trial adanish study started in october uses a classicalrct in stage i and ii crc patients randomizing between months of act or intensified followup for patientspostsurgerynct03748680 four hundred fifty stage ii crc patients are being included in the australian dynamicstudy and randomized to be treated according to thectdna result with to months of act or accordingto standard of care actrn12615000381583 thecobra study in the united states and canada has asimilar rct approach nrggi also several trialsin stage iii crc patients started recently dynamiciii actrn12617001566325 in the near future thesestudies will provide deeper understanding and lead toimplementation of ctdnaguided strategies in clinicalpracticein the current era of rapidly emerging new diagnosticand treatment strategies the classical rct is challengedbecause of inefficient and therefore timeconsuming recruitment of eligible patients main reasons for patientsto refrain from participation in rcts are preference forone ofthe treatment arms anxiety or aversion torandomization and difficulties understanding the concept of an rct resulting in a delay of availability ofpotential beneficial treatments modern trial designsare being adopted to avoid thistimeconsuming and costly way of conducting trials with highrates of unfinished studies therefore the medocccreate trial uses the modern twics design the twicsdesign has shown to have a positive impact on trialefficiency also by enrolling higher proportions of eligible patients generalizability to daily clinical practiceimproves inefficientthisseveralstudy design hasstrengths firstmedocccreate is nested within the large nationwide plcrc cohort study with currently almost included crc patients the infrastructure of this cohortin which clinical data and biomaterials are collected afterbroad informed consent of participating patients allowsinnovative and efficientcomprehensiveresearch incrc using this infrastructure the study can be quicklyimplemented in many participating hospitals savingcosts and complicated logistics several studies accordingto the twics design are performed within this or comparable cohorts therefore experience with this trialdesign has been gained and this will contribute to execution of the medocccreate study [ ]secondly a difficult ethical dilemma in an rct analyzing ctdna presence postsurgery is avoided by thetwics design with the current knowledge about thestrong association with recurrent disease disclosingctdna status to all participants would be a great burdenfor patients with detectable ctdna and their treatingphysicians in the control group because of disappointment bias in the control group we would expect highdropout and contamination due to crossover when aclassical rct design would be applied making accrualand interpretation of results unfeasible in thistwics study all participants already have blood withdrawn after surgery for research purposes and only theeligible patients allocated to the intervention arm willhave the opportunity to obtain a ctdna test result andact if ctdna is detected patients in the control armtreated according to current guidelines will not be informed about randomization and their blood sampleswill be analyzed at a later point in time beyond the window of act treatmentthis study has also potentiallimitations and challenges the twics design is potentially susceptible tolow statistical power and internal validity biases levelsof participants eligibility and consent should be substantial to achieve valid and reliable results and measurements taken in the control group should be sufficient foradequate comparisons to be made therefore thetwics design is not appropriate for every experimentalintervention in case of the medocccreate studywe argue that eligibility and also consent will be substantial because of the high incidence of cc the large cohortwith high inclusion rates and the assumption that eligible patients in the intervention group are willing toaccept act because of the very strong association of thepresence of ctdna with recurrent diseaseanother limitation is the small sample size for primaryoutcome analysis eventually only patients in botharms of the trial are expected to have detectable ctdnaafter surgery based on previous data relapses areexpected within years and with a high event rate smallnumbers are sufficient capecitabinewe recommend a 6month duration of act consistand oxaliplatin capox oring offluorouracilleucovorin and oxaliplatin folfox forpatients with detectable ctdna after surgery the firstadjuvant cc trials investigating the combination of a 0cschraa bmc cancer page of fluoropyrimidine and oxaliplatin reported results for month duration of act in the idea trialfound a large reduction in toxicity for months treatment compared to months treatment although thistrial could not confirm noninferiority for monthstreatment for all patients treated with capox or folfox in stage iii crc the small difference limits clinicalrelevance besidesit did show noninferiority of theshorter regimen in patients treated with capox consequently dutch guidelines recommend months of actfor cc since however among patients withhighest risk of recurrence t4 n2 or both superiorityof 6month duration of therapy was found additionalideafrance resultsthe esmocongress showed the worst prognosis for ctdnapositive patients who only received months of act therefore in this study we recommend 6monthsact for patients with a very high risk of disease recurrence due to the presence of ctdna after surgerypresentedatliquid biopsy ctdna detection has become a promising technology with multiple putative clinical applications including its potential use as a biomarker for earlydiagnosis prognosis prediction and monitoring of treatment response driven by the excitement of its possibilities the field of technology of ctdna detection andanalysis is rapidly evolving yet the clinical utility ofctdna testing still needs to be proven when to applywhat technology to address which unmet clinical need isa key question that remains to be addressed applying ctdna detection as a biomarker for mrd isa challenging task biologically only a very low amountof ctdna is present in postsurgery patients with mrdstochastically by looking at mutations in a panel ofgenes chances increase that in a given blood sample atleast in one of the genes a mutation can be reliably detected test sensitivity can be further increased by making use of dna mutation information from tumortissue because the stringency in the calling of plasmactdna mutations can be reduced once you know whatmutations to look for tissueinformed ctdna analysisalso increases the ctdna test specificity recent observations showed that ctdna mutation detection can beconfounded by mutations that are present in clonalhematopoiesisincluding mutations in genes that arecommonly affected in cc such as tp53 these confounding mutations can be filtered by applying tissueinformed ctdna analyses as suchtechnically themedocccreate trial makes use of a ctdna test thatis wellsuited for mrd detection clinically howeverthe medocccreate trial needs to resolvewhether a positive ctdna test also allows to select forpatients who truly benefit from act treatment a requirement for clinical implementation to further support clinical implementation of ctdna analyses in thenetherlands the dutch coin initiative aims to providea validation framework for clinicalimplementation ofctdna analyses in the netherlands zonmw projectnumber in conclusion the medocccreate study is thefirst study using the modern and innovative twics design to study ctdnaguided administration of act instage ii cc patients the study aims to answer the important clinical question whether ctdna has prognosticas well as predictive value if this study demonstrates asignificant and substantial difference in disease recurrence in the intervention group compared to the controlgroup ctdna analysis and ctdnaguided treatmentshould be implemented into clinical practice to improvethe prognosis of stage ii cc patientsabbreviationsact adjuvant chemotherapy cc colon cancer cea carcinoembryonicantigen crc colorectal cancer ctdna circulating tumor dnadfs disease free survival dmmr deficient mismatch repair eortcqlqc30 and cr29 european anisation for research and treatment ofcancer quality of life questionnaire c30 and colorectal cancer module eq5d euro quality of life5 dimensions ffpe formalinfixed para | 0 |
99mtclabeled nanocolloid drugs development methodsVladimir Sadkin1 Viktor Sкuridin1 Evgeny Nesterov1 Elena Stasyuk1 Alexander Rogov1 Natalya Varlamova1 Roman Zelchan2The work considers the problem of obtaining nanocolloid radiopharmaceuticals RPs and studying their functional suitability for diagnosing sentinel lymph nodes SLN in cancer patients Two principal approaches to the formation of technetium99mlabeled ps based on inanic and anic matrices were considered when carrying out research to develop methods for the production of nanocolloid RPs The composition of the reagents and the conditions for obtaining nanocolloid radiopharmaceuticals were determined The functional suitability of new RPs for scintigraphic diagnostics of sentinel lymph nodes has been studiedThe identification of sentinel lymph nodesthe first nodes that stand in the way of metastasizing of malignant neoplasms attracts increasing interest in modern oncological practice1 It is believed that if the SLN is not affected by the metastatic process then all other regional lymph nodes are intact so the results of biopsy of these nodes are an objective diagnostic criterion for the spread of malignant process Fig a0 The optimal method of detecting SLN is the use of colloid nanomaterials labeled with technetium99m for scintigraphic or radiometric determination of node localization6 Not so much the chemical nature of such ps but their size is the determining factor in the choice of the indicator in this case Thus according to Schauer14 a colloid with a p size of less than a0nm can accumulate not only in the SLN but also at nodes of and orders of magnitude Ps with the sizes of more than a0nm slowly migrate from the injection site The colloid with the p size from to a0nm was recognized as the optimal one for detecting SLNThe simplest method of obtaining colloids with given sizes and properties is immobilization of 99mTc on the surface of nanosized materialsTechnetium99m is by far the most popular radionuclide for conducting diagnostic studies practically in all fields of medicine15 This is primarily due to its nuclearphysical characteristics a relatively short halflife a0h and Îradiation energy of a0meV providing a low exposure dose and at the same time sufficient penetrating power for radiometric measurementsToday the Tc99m Tilmanocept radiopharmaceutical is widely used which has proven itself well and gives good results But its production is quite timeconsuming and requires expensive components We offer a less laborious method from the simple components1920Materials and methodsMaterials All the reagents were purchased from SigmaAldrich ACS grade and used without further purification Technetium99m was obtained from chromatographic 99Mo99mTc generator 99mTcGTTOM produced by Tomsk Polytechnic University TPUTomsk RussiaThree types of nanops were selected to obtain nanocolloids labeled with 99mTc The first type of colloids was created on the basis of metal chelates with chemically modified complexons of diethylenetriaminepentaacetic acid DTPA It should be noted that the DTPA molecule itself like its complexes with metals is hydrophilic and does not tend to form colloidal ps The introduction of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified DTPA complexes21 The original substance of the modified DTPA DTPAmod was synthesized in Tomsk Polytechnic University Preparation of colloid solution DTPAmod was produced using the following method A sample of modified DTPA with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of NaHCO3 solution by heating to a0°C After that the volume was adjusted with the same solvent up to the mark In order to reduce the p size the container with suspension was heated in water to a0°C and treated with ultrasound for a0min 1Tomsk Polytechnic University Lenina Avenue Tomsk Russia 2Tomsk National Research Medical Center Russian Academy of Sciences Kooperativny street Tomsk Russia email sadkintpuruScientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorFigure a0 The general scheme for the synthesis of 99mTcDTPAmodwhich reduced the average p radius up to a0nm The general scheme for the synthesis of 99mTcDTPAmod is shown in Fig a0The second type of colloids is iron nanops coated with a carbon shell of FeC Fig a03a These ps were obtained from the Institute of Metal Physics UrB RAS Ekaterinburg Russia In order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates ADT onto the surface of these ps has been developed An effective method for the synthesis of ADT followed by their application onto the carbon surface of ps was developed at the Tomsk Polytechnic University22 The general scheme for the synthesis of FeC ps and their subsequent interaction with 99mTc is shown in Fig a03bIn the third type of colloids technetium99m was adsorbed on aluminum oxide powder A powder of lowtemperature cubic modification of gammaoxide Al2O3 prepared from aluminum hydroxide powder AlOH3 by its calcination in a muffle furnace was used The substance was synthesized in Tomsk Polytechnic UniversityA reducing agenttin II chloride dihydrate was used in order to obtain complexes of 99mTc with colloidsGelatin powdered Ph Eur USPNF pure pharma grade CAS Number was purchased from AppliChem GmbH Darmstadt GermanyMethods Method for preparation of 99mTc labeled nanocells The introduction of the radioactive label 99mTc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent SnCl22H2O a0mgml in different ratios and then adding a a0ml of eluate of 99mTc a0MBqml to the mixtures The mixtures were incubated for a0min at a temperature of a0°C The preparation is ready for use after cooling at room temperature The reducing agent SnCl22H2O was used as a hydrochloric acid solution The amount of a0g of tin chloride II is added to the vial and a0ml of a0M hydrochloric acid HCl Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Carbon encapsulated iron nanop B the general scheme for the synthesis of FeC psis then added for its preparation After dissolution the volume is adjusted with distilled water to a0ml Dissolution was carried out in an inert gas argon mediumDetermination of the size of 99mTc labeled colloidal ps The determination of the size of the labeled nanocolloids was carried out by spectroscopy on a Nanophox p size analyzer Sympatec GmbH Germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm Three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity Calculations of the yield of products with different p sizes were determined according to the formulas given belowC220 Avc A1Avc C100 A1 A2A1 C50 A2 A3A2where Avc is the activity of the initial suspension prior to filtration A1 is the activity measured after filtration through a a0nm filter A2 is the activity after filtration through a0nm filter A3 is the activity measured after filtration through a0nm filterIn parallel determination of the radiochemical purity RCP of preparations by thin layer chromatography method was carried outThinlayer chromatography TLC procedure To determine radiochemical purity of 99mTcnanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip Sorbfil Russia a0 cm To determine Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cAmount of SnCl22H2O mgA[Sn99mTc]A ATcVIIA Table Change in relative activities of the complex [Sn99mTc] and 99mTc VIIpertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min In this system pertechnetate migrated with the front of the mobile phase Rf To determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min In this system the 99mTcnanocolloid migrated with the front of the mobile phase Rf Stability The stability of 99mTcnanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mTcnanocolloid following by incubation at a0°C for a0h At different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using TLC24Determination of the functional suitability of preparations for scintigraphic detection of SLN A study to assess the functional suitability of new nanocolloid RPs was performed in series of experiments involving white Wistar male rats weighing a0g Injection of RP in a dose of a0MBq was performed between the first and second fingers of the rats hind paw The animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study Since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame a0s in a pixel matrix Static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of with a set of pulses scintigraphy of animals was performed on an ECAM Signature gamma camera Siemens Germany The results of scintigraphic studies determined the percentage of accumulation of RP in the lymph node and the injection site The maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the European Convention for the Protection of Vertebrates Used for Experiments or Other Scientific Purposes Strasbourg The experimental protocols were approved by Cancer Research Institute Biomedical Ethics Committee Protocol number All invasive manipulations with animals were performed using inhalation or drug anesthesiaStatistical analysis All mean values are expressed as IDg ± SD Data were analyzed statistically using methods of general statistics with a commercially available software package Statistics for Windows StatSoft Inc Version Results and discussionTo carry out the labeling of colloids 99mTc extracted from a standard generator in the form of pertechnetate ions contained in a NaCl solution was used It has a higher degree of oxidation VII in this chemical form and is not prone to complex formation A reducing agenttin II chloride dihydrate widely used for the preparation of various compounds labeled with 99mTc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 As a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess SnCl2·2H2O or the additional formation of a complex of reduced 99mTc with tin26 All this required preliminary experimental studies to establish the necessary and sufficient amount of SnCl2·2H2O in the reaction mixtureDuring the experiments it was found that the optimal concentration of Sn II in the composition of the reaction mixture when it interacts with 99mTc should be in the range of a0mgml Table a0It was found that when the eluate with the preliminarily reduced 99mTc VII was added to the nanops the Sn II concentration introduced in the RP was CSn a0mgml almost the entire amount of 99mTc has time to enter the composition of the largesize complex with tin even before its mixing with nanops This means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the Sn II solution into the reaction mixture In this connection the reduction of 99mTc with tin II was carried out in the presence of the selected substance In this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex The technique of applying of the 99mTc label to the surface of nanosized ps is given in the previous sectionAs a result of the studies reagent compositions and conditions for obtaining three nanocolloid RPs were determined Table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of a0nmProceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mTc VII in the obtained preparations is Preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm Scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in Fig a0Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cComposition of the preparation per a0mlDTPAmod a0mg 99mTc a0MBq SnCl22H2O a0mg n FeC a0mg 99mTc a0MBq SnCl22H2O a0mg n Al2O3 a0mg 99mTc a0MBq SnCl22H2O a0mg n Colloid yield a0nm RCP ± ± ± Table Composition of reagents for production of technocium99 a0m nanocolloidsFigure a0 Distribution of the preparation in the rat when the preparation is administered [Al2O3 99mTc Sn II] A immediately after the administration of the drug B a0min after the administration C a0min after the administrationComposition of preparations per a0mlAl2O3 a0mg 99mTc a0MBq SnCl22H2O a0mg G a0mg n DTPAmod a0mg 99mTc a0MBq SnCl22H2O a0mg G a0mg n FeC a0mg 99mTc a0MBq SnCl22H2O a0mg G a0mg n Yield of colloid a0nm RCP ± ± ± Table Indicators of RCP and the yield of a colloid with a fraction of a0nm after the introduction of gelatin in the reagentsScintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mTc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid Along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed Gelatin G was introduced into the reaction mixture in this connection to increase the mobility of the labeled ps and increase the speed of their movement through the lymph system Matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mTc The results of the experiments showed that the addition of gelatin a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of a0nm Table a0In addition the size of these ps was determined on a Nanophox p analyzer The obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in Fig a0 A B C The average p size diameter is and a0nm respectivelyStability test showed that complex 99mTcnanocolloid was stable in normal serum at least for a0h Radiochemical purity of the tracer at the end of the experiment was ± A study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes Fig a0 Table a0 displays the Al2O3 99mTc DTPAmod 99mTc FeC 99mTc biodistribution data at different time points postinjectionThe level of accumulation of preparations in the lymph nodes is of the total injected activityconclusionAs a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid RPs were determined An experimental dependence of the change in the content of 99mTc VII impurities on the concentration of tin II was established and its minimum amount a0mgml was determined to reach a RHP greater than In this case the yield of the target colloid with p sizes of ± a0nm is Preliminary tests of the developed preparations on experimental animals showed that accumulation of RP in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range Increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition a0mgml In addition there was an increase in the yield of the colloid Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Change in the density of the distribution of the number of ps from their size in radiopharmaceuticals A 99mTcAl2O3 B 99mTcFeC C 99mTcDTPAmodwith p sizes of a0nm to with radiochemical purity of the preparations of Repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cStomachTime h99mTcAl2O399mTcDTPAmod99mTcFeC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Figure a0 Distribution of the preparation in the rat with injection of suspension [Al2O3 99mTc Sn II Gelatin] A immediately after the administration of the preparation B a0min after the administration C a0min after the administration D a0min after the administration The numbers indicate lymph node site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± BloodmlHeart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table Biodistribution data up to a0h after injection of a0MBq of 99mTc in healthy male rats Data represent the average value n accumulation in the SLN Thus the level of accumulation of RP 99mTcDTPAmod and RP 99mTcFeCADT in the SLN is and respectively At the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityReceived March Accepted July References Jakobsen J K Sentinel node biopsy in urooncology A history of the development of a promising concept Urol Oncol Weixler B et al Sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival A prospective singlecenter trial World J Surg 101007s0026 Beasley G M et al Sentinel Lymph node biopsy for recurrent elanoma A multicenter study Ann Surg Oncol Moser J et al Sentinel node biopsy in melanoma A singlecentre experience with consecutive patients Br J Dermatol 101245s1043 Buda A et al Optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer Comparison of realtime fluorescence with indocyanine green and methylene blue Int J Gynecol Cancer Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c Sahbai S et al Pericervical injection of 99mTcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in SPECTCT Clin Nucl Med Hoogendam J P et al 99mTcnanocolloid SPECTMRI fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer J Nucl Med Stoffels I Leyh J P¶ppel T Schadendorf D Klode J Evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies Prospective randomized clinical trial to compare ICG99mTcnanocolloid hybrid tracer versus 99mTcnanocolloid Eur J Nucl Med Mol Imaging Beisani M et al Initial experience in sentinel lymph node detection in pancreatic cancer Rev Esp Med Nucl Imagen Mol Schubert T Uphoff J Henke R P Wawroschek F Winter A Reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer Verification in consideration of the European guidelines BMC Urol Jaukovic L et al Lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions Hell J Nucl Med Subramanian S Pandey U Shah S Rangarajan V Samuel G An indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection Nucl Med Commun RuizDomnguez J M IbarzServio L Garcade Manuel G Calaf Peris© O Intraoperative injection of 99mTcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery Actas Urol Schauer A J Specific developments in sentinel node labling using 99mTccolloids In The Sentinel Lymph Node Concept Springer Berlin Wang Y et al Gasphase chemistry of technetium carbonyl complexes Chem Phys OConnor M K et al Comparison of Tc99m maraciclatide and Tc99m sestamibi molecular breast imaging in patients with Wang J Zhang R Evaluation of 99mTcMIBI in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer EJNMMI Res Br J Radiol Costa P et al Scintigraphic imaging with technetium99Mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats Acta Cir Bras Vera D R Wallace A M Hoh C K Mattrey R F A synthetic macromolecule for sentinel node detection 99mTcDTPAmannosyldextran J Nucl Med Hoh C K Wallace A M Vera D R Preclinical studies of [99mTc]DTPAmannosyldextran Nucl Med Biol Skuridin V et al Modified DTPA moleculebased nanocolloid radiopharmaceuticals J Radioanal Nucl Chem Filimonov V D et al Unusually stable versatile and pure arenediazonium tosylates Their preparation structures and synthetic applicability Lett Lukasz K Thin Layer Chromatography in Drug Analysis CRC Press London Skuridin V et al Radiopharmaceutical drug based on aluminum oxide Indian J Sci Technol 1017485 ijst2015v8i36 Sazonova S I et al Synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging Iran J Nucl Med Skuridin V S et al Synthesis and biological characterization of 99mTclabeled ciprofloxacin Pharm Chem J AcknowledgementsThis work was financially supported by Ministry of Education and Science of the Russian Federation RFMEFI57514X0034Author contributionsVS Conducting experimental research analysis and interpretation of the data Final approval for manuscript publication VS development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication EN development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication ES development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication AR conducting experimental research analysis and interpretation of the data Final approval for manuscript publication NV conducting experimental research analysis and interpretation of the data Final approval for manuscript publication RZ conducting tests of the functional suitability of drugs Preparation of the section Evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and Figures Final approval of the manuscript for publication of the manuscriptCompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to VSReprints and permissions information is available at wwwnaturecomreprintsPublishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsScientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0c Access 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 license and indicate if changes 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 If material 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 obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 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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 | 0 |
"Lung carcinoma is a prominent cause of mortality among patients with cancer Previous studies have reported the vital role of long noncoding RNAs lncRNAs in the malignant progression of lung cancer lncRNA RP11284F219 was originally identified to be expressed in lung carcinoma but its specific function remains unknown Therefore the present study aimed to elucidate the role of lncRNA RP11284F219 in lung carcinoma progression The expression of RP11284F219 in lung cell lines and tissues was measured using reverse transcriptionquantitative PCR The endogenous expression of RP11284F219 was silenced using RNA interference and cell viabilities were measured with a Cell Counting Kit assay The invasion and apoptosis of cells were determined via Transwell assays and flow cytometry respectively The protein expression levels were measured by western blotting An increased expression of RP11284F219 was identified in both lung carcinoma tissues and cells Knockdown of RP11284F219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis The present study identified the existence of a direct interaction between RP11284F219 and microRNA miRNAmiR6273p Mechanistically it was demonstrated that RP11284F219 promoted the proliferation and invasiveness of lung carcinoma cells in part via the regulation of miR6273p Furthermore cell division cycle and apoptosis regulator CCAR1 was identified as a target gene of miR6273p The in vivo tumor growth assay also demonstrated that the knockdown of RP11284F219 suppressed tumor growth upregulated miR6273p and downregulated Correspondence to Dr Yuan Wang Department of Medical Imaging The First Affiliated Hospital of Xi'an Jiaotong University West Yanta Road Xi'an Shaanxi PR ChinaEmail wangyuan8003126comAbbreviations CCAR1 cell division cycle and apoptosis regulator NSCLC nonsmall cell lung cancer SCLC small cell lung cancerKey words RP11284F219 lung carcinoma proliferation invasion microRNA6273p CCARCCAR1 in the xenograft model of nude mice Thus the present findings indicated the tumor promoting functions of RP11284F219 in the progression of lung carcinoma and provided a novel lncRNAmiRNA axis as a target for the management of lung cancerIntroductionPulmonary malignancies including lung and bronchus cancer rank first and second among different cancer types in terms of mortality and morbidity respectively in both men and women Furthermore of lung cancer cases are categorized as nonsmall cell lung cancer NSCLC while the remaining are classified as SCLC Although diagnostic methods and therapeutic strategies based on traditional surgical excision chemotherapy and chest radiotherapy have continuously improved the prognosis of lung carcinoma remains at for an overall 5year survival Therefore an increased understanding of the malignant progression and studies on novel therapeutic targets for the improved management of this disease are essentialLong noncoding RNAs lncRNAs are nucleotides in length and have little or no protein coding capacity The mechanisms via which lncRNAs regulate gene expression are diverse and include regulating the transcription of target genes functioning as transcriptional precursors of small RNAs generating different splice variants via regulating mRNA splicing patterns modulating protein activity and subcellular localization and scaffolding for the assembly of multiple component complexes In recent years previous studies have reported that various human cancer types exhibit lncRNAs dysfunction and these lncRNAs are involved in different aspects of pathogenesis such as the proliferation metastasis and apoptosis of tumor cells In lung cancer lncRNA metastasisassociated lung adenocarcinoma transcript is found to be upregulated in patients with advanced lung adenocarcinoma and may serve as a prognostic marker to predict the survival outcome of patients with cancer lncRNA HOX transcript antisense RNA is also highly expressed in lung cancer and it enhances the aggressiveness of lymph node metastasis and indicates a short diseasefree survival in patients with NSCLC Furthermore studies have shown that the expression of lncRNA Urothelial carcinomaassociated 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONis significantly upregulated in NSCLC and may induce resistance to treatment of EGFRtyrosine kinase inhibitors by activating the AKTmTOR pathway lncRNA RP11284F219 was primarily discovered in a Pancancer transcriptomic analysis lncRNA RP11284F219 exists as a cluster of three annotated lncRNAs RP11284F219107 antisense to brevican which is a proteoglycan linked to invasiveness in glioma but lacks expression in squamous cell lung carcinomas However the specific function and the underlying mechanism of RP11284F219 in lung carcinoma remain unknownTo the best of our knowledge the present study demonstrated for the first time that lncRNA RP11284F219 was significantly upregulated in lung carcinoma tissues and cell lines and was involved in the carcinogenesis of lung cancer Together with microRNA miRNAmiR6273p and cell division cycle and apoptosis regulator CCAR1 the regulatory axis of RP11284F219miR3pCCAR1 exists both in the lung carcinoma cells in vitro and in the tumor growth model in vivo The present study aimed to investigate RP11284F219 function in lung carcinoma and demonstrate the molecular mechanism underlying the regulation process via the RP11284F219miR3pCCAR1 axisMaterials and methodsTissue samples and cell lines Between May and Jan paired tumor and adjacent healthy tissues were isolated from patients with lung carcinoma age range years nine male patients four female patients who were diagnosed and treated in First Affiliated Hospital of Xi'an Jiaotong University The samples were dissected during the surgery and immediately flashfrozen in liquid nitrogen and transferred to ËC storage for further extraction of both RNA and protein All the tissue samples were obtained with written informed consent from the patients The protocol was approved by The First Affiliated Hospital of Xi'an Jiaotong University approval no A normal lung epithelial cell line BEAS2B and lung carcinoma cell lines NCIH460 NCIH1299 and A549 were purchased from American Type Culture Collection ATCC and cultured according to the ATCC guidelines 293T cells were purchased from Procell Life ScienceTechnology Co Ltd and cultured in DMEM supplemented with FBS cat no ATCC and 1X Penicillinstreptomycin Thermo Fisher Scientific Inc BEAS2B cells were cultured in bronchial epithelial growth medium BEGM cat no CC Clonetics Corporation according to the manufacturer's instructions NCIH460 and NCIH1299 cells were cultured in RPMI medium cat no ATCC and A549 cells in F12K medium cat no ATCC supplemented with FBS cat no ATCC and 1X Penicillinstreptomycin Thermo Fisher Scientific Inc All cells were culture at ËC with CO2RNA extraction and reverse transcriptionquantitative PCR RTqPCR Total RNA from both tissue samples and cell lines were extracted using TRIzol® reagent Invitrogen Thermo Fisher Scientific Inc For each sample ng total RNA was reverse transcribed to synthesize the firststrand cDNA using the PrimeScript RT reagent kit Takara Bio InccDNA samples were diluted times to perform the RTqPCR using SYBR Premix Ex Taq Takara Bio Inc on a CFX96 realtime PCR detection system BioRad Laboratories Inc Expression levels of mRNAs lncRNAs and miRNAs were normalized to GAPDH The primers used for RTqPCR analyses were as follows GAPDH forward 'AAC GAC CCC TTC ATT GAC C' and reverse 'TCC ACG ACA TAC TCA GCA CC' RP11284F219 forward 'AGG ATT GGC ACT CAC TTC GG' and reverse 'TCT CTC ACC ACG TCT GGT CT' and CCAR1 forward 'CTG ATG GCT AGC CCT AGT ATG GA' and reverse 'TGC CTT TCA TGC CCA CTA AAA ' The temperature protocol used to perform RT was ËC for h followed by ËC for min Thermal conditions of PCR reactions were Initial denaturation at ËC for min followed by cycles for sec at ËC and sec at ËC The mRNA expression levels were determined using the 2ÎÎCq method Oligonucleotides and cell transfection The small interfering RNA siRNA synthetic negative control siNC RP11284F219 siRNAs siRP11284F219 miRNC miR3p mimics and miR3p inhibitor were purchased from Shanghai GenePharma Co LtdAll primer sequence information is presented in Table I At a density of 2x105 cellswell the cells were plated in 6well plates h before transfection and were transfected at confluency All of the oligonucleotides were transfected at a final concentration of nM using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's instruction Cells were collected at h posttransfection for subsequent experimentsCell Counting Kit CCK assay and EdU labeling of proliferating cells A CCK was used for cell proliferation assay the cells were seeded into well plates 2x103 cellswell and observed for and days or indicated time points following the manufacturer's instructions Dojindo Molecular Technologies Inc The optical density was measured at nm using a spectrophotometer Thermo Fisher Scientific IncFor the EdU assay cells were incubated with µM EdU cat no ab219801 Abcam for h at ËC and fixed with formaldehyde at room temperature for min After a brief washing with PBS click reagent was added into each well and incubated in the dark for min at room temperature Followed by PBS washing the cells were stained with µgml DAPI at room temperature for min Images were captured using a fluorescence microscope Nikon Corporation and measured using Adobe Photoshop software Adobe Systems Inc The EdU labeled cells were analyzed with MoFlo Astrios BeckmanCoulter Inc Magnification x200Transwell assay and flow cytometry measurement of cell apoptosis Transwell assays were performed with a coating of Matrigel BD Biosciences mixed with culture medium mixed at ratio at ËC for h A total of 1x105 cells in µl serumfree medium were added to the upper layer of the Transwell chambers µm pore size Corning Inc and cultured for h The lower chamber contained the culture medium with FBS The migrated cells were fixed with 0cONCOLOGY REPORTS Table I Sequence of siRNAs and miRNA mimics and inhibitorsOligonucleotides siNC siRP11284F219 miRNC miR3p mimics miR3p inhibitor miR microRNA siRNA small interfering RNA NC negative controlSequence ''UUCUCCGAACGUGUCACGUTTUAUUGGCACCAAGGAUAGCUCGUUAAUCGGCUAUAAUACGCUCUUUUCUUUGAGACUCACUUCUUUUCUUUGAGACUCACU paraformaldehyde for min at room temperature stained with crystal violet for min at room temperature and images of six randomly selected fields in each well were captured under a light microscope Magnification x200Cellular apoptosis was detected using the Apoptosis Detection kit cat no KGF001 Nanjing KeyGen Biotech Co Ltd according to the manufacturer's instructions Cells were stained with fluorescein isothiocyanateconjugated annexin V and PI After incubated for min at ËC in the dark µl 1X Binding Buffer was added to each tube and stained cells were analyzed using BD FACS Canto II flow cytometry FACS Calibur BD Biosciences Data were analyzed using FlowJo software version Tree Star IncLuciferase reporter assay The RP11284F219 wildtype wt or mutant mut 'untranslated region 'UTR and CCAR1 wt or mut 'UTR sequences were cloned into the pmirGLO plasmid Youbio httpwwwyoubiocn cat no VT1439 The vectors µgml were cotransfected with miRNC or miR6273p mimic nM and Renilla plasmids ngwell used as an internal control into cells seeded in a 48well plate 1x104well using Lipofectamine® reagent Invitrogen Thermo Fisher Scientific Inc Cell lysates were collected at h after transfection and the luciferase activities were detected with the DualLuciferase Reporter Assay system Promega Corporation according to the manufacturer's instructionsWestern blotting Cell were lysed using RIPA lysis buffer SigmaAldrich Merck KGaA and protein concentrations were assessed with the BCA Protein Assay kit according to the manufacturer's instructions Beyotime Institute of Biotechnology Shanghai China Equal amounts µg of cell protein lysates were loaded and separated by SDSPAGE transferred to a PVDF membrane and blocked with nonfat milk at room temperature for h The membranes were then incubated with CCAR1 primary antibody cat no ab70243 Abcam overnight at ËC followed by incubation with goat antimouse or goat antirabbit IgGhorseradish peroxidase conjugate secondary antibodies cat no ab205718 Abcam at room temperature for h GAPDH cat no ab181602 Abcam was used as loading control The signals were detected using the ECL system Protein Simple according to the manufacturer's instructionsIn vivo tumorigenicity analysis in mice Male BALBc nude mice age weeks weight g were obtained from Beijing Vital River Laboratory Animal Technology Co Ltd and housed at a room temperature of ËC with a h lightdark cycle The mice were maintained in an individually ventilated cage system under specific pathogenfree conditions temperature ËC humidity and fed with sterile food and water free access To evaluate the effect of RP11284F219 knockdown on the growth of lung carcinoma in vivo 5x106 siNC or siRP11284F219 treated NCIH1299 cells in µl serumfree medium were subcutaneously injected into each mouse n5 per group under anesthesia which was induced by isoflurane and maintained by isoflurane flow rate 1lmin The animals were monitored daily and the following criteria for humane endpoint was used Severe tumor burden mm in diameter difficulty breathing significant bodyweight loss and clinical signs such as prostration hypothermia and significant abdominal distension Tumors were measured on days and and the volumes were calculated using the formula a x b22 [the largest diameter a and the smallest diameter b] Then weeks after inoculation the mice were euthanized by CO2 inhalation CO2 flow rate of cage volume and the death of animals were confirmed by cessation of heartbeat The xenografts were imaged and weighedThe total RNA was then extracted from the xenografts as aforementioned Animal care and study were approved by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Xi'an Jiaotong University approval no Target prediction Potential target miRNAs of RP11284F219 were predicted using LncBase V2 httpcarolinaimisathena innovationgrdiana_toolswebindexphprlncbasev2index The target genes of miR3p were predicted using three bioinformatics algorithms TargetScanV72 httpwwwtargetscanorgvert_72 and miRDB httpwwwmirdborgmininghtmlStatistics analysis Data were analyzed using the GraphPad Prism software GraphPad Software Inc and presented as the mean ± SD from ¥ independent experiments A twotailed unpaired Student's ttest or oneway ANOVA with Tukey's posthoc analysis were performed to evaluate the statistical significance P005 was considered to indicate a statistically significant difference\x0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11284F219 expression is upregulated in LC tissues and cell lines A Expression of RP11284F219 in LC tissues in comparison with adjacent healthy tissues was analyzed using RTqPCR P0001 vs adjacent tissues n13 B Expression of RP11284F219 in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 compared with normal human lung epithelial cell line BEAS2B was analyzed using RTqPCR P005 P0001 vs BEAS2B n3 LC lung carcinoma RTqPCR reverse transcriptionquantitative PCRResultsExpression of RP11284F219 is upregulated in lung carcinoma To investigate the potential role of RP11284F219 in lung carcinoma its expression was analyzed in tissue samples and matched adjacent healthy tissues from patients with lung carcinoma The results demonstrated that the expression of RP11284F219 was significantly upregulated in tumor tissues compared with healthy tissues Fig 1A The expression of RP11284F219 was also analyzed in human lung carcinoma cell lines NCIH460 NCIH1299 and A549 and normal human lung epithelial cell line BEAS2B Consistent with the findings in the tissue samples the expression of RP11284F219 was significantly increased in carcinoma cell lines compared with the normal epithelial cell line Fig 1B These results indicated that RP11284F219 may serve an oncogenic role in lung carcinomaKnockdown of RP11284F219 exerts antioncogenic effects in lung carcinoma cells To study the specific role of RP11284F219 in lung carcinoma cells RP11284F219 siRNA was transfected into NCIH1299 and NCIH460 cells Fig 2A After transfection the proliferation of these cells was measured using CCK and EdU assays Fig 2BD The results suggested that knocking down RP11284F219 significantly reduced the proliferation of lung carcinoma cells compared with the NC group Fig 2BD The invasiveness of siRP11284F219 transfected cells also significantly decreased as indicated by the data from the Transwell assay Fig 2F To further validate the invasive capability a RTqPCR assay was performed to detect the expression levels of invasionrelated genes and the results identified that both MMP2 and MMP9 were significantly decreased when RP11284F219 was downregulated Fig S1The results of flow cytometry measurement based apoptosis assay suggested that cells transfected with siRP11284F219 had a higher apoptotic rate compared with the siNC transfected group Fig 2E These data demonstrated the antitumor effects of RP11284F219 knockdown in lung carcinoma cells indicating an oncogenic role of RP11284F219RP11284F219 directly interacts with miR3p Based on the prediction of the online tool lncBase v2 from DIANA Prediction module httpcarolinaimisathenainnovationgrdiana_toolswebindexphprlncbasev2index which was used to identify the downstream miRNAs of RP11284F219 the first five miRNAs in the output list were tested Among the predicted potential targets it was found that miR6273p had the most significant upregulation in NCIH1299 cells transfected with siRP11284F219 Fig S2Using sequence alignment it was identified that miR3p was partially complementary with the 'UTR of RP11284F219 Fig 3A Subsequently 293T cells were transfected with the pmirGLORP11284F219wt or mut vector containing the wt or mut sequence of RP11284F219 'UTR with or without miR3p mimics Results from the luciferase reporter assay suggested that miR6273p mimics significantly decrease the signal of RP11284F219wt transfected cells but not the RP11284F219mut transfected cells indicating a direct interaction between the two noncoding RNAs Fig 3A Furthermore transfection of siRP11284F219 into NCIH1299 and NCIH460 cells resulted in the suppression of endogenous RP11284F219 leading to a significant increase in miR3p expression Fig 3B Thus these findings suggested an inhibitory effect of RP11284F219 on the expression of miR3p in lung carcinoma cellsThe expression of miR3p was detected in both lung carcinoma tissues and cell lines It was demonstrated that miR3p was significantly downregulated in carcinoma tissues Fig 3C and NCIH460 NCIH1299 and A549 cells Fig 3D compared with healthy tissues and cells Collectively these data suggested a direct interaction between RP11284F219 and miR6273p in which RP11284F219 suppresses the expression of miR3pRP11284F219 regulates the proliferation and invasiveness of lung carcinoma cells via miR3p To rescue the antitumor effects of siRP11284F219 in lung carcinoma cells the miR3p inhibitor which specifically downregulates the expression of miR3p was transfected into NCIH1299 and NCIH460 cells Fig 4A The results from the CCK and EdU assays demonstrated that treatment with siRP11284F219 0cONCOLOGY REPORTS Figure RP11284F219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis A RP11284F219 knockdown was achieved via RP11284F219 siRNA and the knockdown efficiency was verified using reverse transcriptionquantitative PCR n3 Cell Counting Kit assay was performed to measure the proliferation of B NCIH1299 and C NCIH460 cells after transfection with siRP11284F219 compared with the siNC group n5 D An EdU assay was performed to measure the proliferation of NCIH1299 and NCIH460 cells after transfection with siNC and siRP11284F219 Magnification x200 E Flow cytometry analysis was performed to determine the effects of RP11284F219 knockdown on apoptotic rates in NCIH1299 and NCIH460 cells n3 F Transwell assay was performed to determine the effects of RP11284F219 knockdown on NCIH1299 and NCIH460 cell invasion n3 Magnification x200 P005 P001 vs control group NC negative control siRNA small interfering RNA OD optical density and miRNC significantly decrease the proliferation of both NCIH1299 and NCIH460 cells Fig 4BD However the administration of miR3p inhibitor partially reversed the antiproliferative effect of siRP11284F219 indicating that RP11284F219 regulates the proliferation of lung carcinoma cells partially via miR6273p Fig 4BD In addition the 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11284F219 directly interacts with miR3p A Binding site between RP11284F219 and miR3p that was identified using the DIANA tools and a luciferase reporter assay was conducted in pmirGLORP11284F219wt or mut treated cells in the presence of miR6273p mimics or miRNC n3 P005 vs miRNC B Expression of miR3p in NCIH1299 and NCIH460 cells transfected with siRP11284F219 was analyzed using RTqPCR P001 vs siNC n3 miR3p expression in C LC tissues and D NCIH460 NCIH1299 and A549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using RTqPCR n3 P005 P001 vs adjacent tissue or BEAS2B cells NC negative control siRNA small interfering RNA wt wildtype mut mutant miR microRNA LC lung carcinoma miR3p inhibitor restored the reduction in the number of NCIH1299 and NCIH460 cells that migrated through the Transwell membrane induced by siRP11284F219 treatment Fig 4F These data indicated the participation of miR6273p in the RP11284F219mediated invasive effectThe qPCR assay results identified that both MMP2 and MMP9 expression levels were restored in RP11284F219downregulated cells when miR6273p was inhibited compared with the miRNC group Fig S3 In addition transfection with miR3p inhibitor also diminished the proapoptosis effect of siRP11284F219 in both NCIH1299 and NCIH460 cells Fig 4E Therefore it was suggested that RP11284F219 promoted the proliferation and invasion as well as suppressed the apoptosis of lung carcinoma cells by inhibiting the expression of miR3pRP11284F219 regulates CCAR1 via targeting miR3p To further evaluate how RP11284F219 exerts an oncogenic role via miR3p the publicly available algorithms of TargetScan httpwwwtargetscanorg and miRDB were used which identified CCAR1 as a potential target for miR6273p Fig 5A In order to validate this prediction miR6273p mimic was transfected into cells and the transfection efficiency was assessed The results demonstrated that transfection of miR6273p mimic increased the expression of miR3p by times compared with cells transfected with miRNC Fig S4After validating the upregulation of miR6273p mimic a CCAR1wt vector was constructed which contained the wt binding site between miR3p and the CCAR1 'UTR and CCAR1mut vector containing the mut sequence Fig 5A The results from luciferase reporter assays indicated that compared with the miRNC group the miR6273p mimic significantly decreased the luciferase activity of CCAR1wt treated cells but not the CCAR1mut treated cells suggesting a direct binding of miR3p to the 'UTR of CCAR1 Fig 5B Increased expression levels of CCAR1 were present in the lung carcinoma tissues compared with the adjacent healthy tissues Fig 5C Moreover a significant decrease in both mRNA and protein expression levels of CCAR1 was detected upon transfecting NCIH1299 and NCIH460 cells with miR6273p mimics Fig 5D and E Thus CCAR1 may be a direct target of miR3p in lung carcinoma cells and tissuesRP11284F219 knockdown inhibits tumor growth and the expression of CCAR1 in vivo In order to investigate the effect of RP11284F219 on in vivo tumorigenicity NCIH1299 cells were transfected with siNC or siRP11284F219 and injected into the nude mice After weeks a significantly 0cONCOLOGY REPORTS Figure RP11284F219 regulates proliferation and invasiveness in lung carcinoma cells via miR3p A Expression of miR3p in NCIH1299 and NCIH460 cells transfected with miRNC or miR3p inhibitor was detected using RTqPCR analysis n3 P005 vs miRNC Cell Counting Kit assay was performed in B NCIH1299 and C NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor n5 D EdU assay was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor Magnification x200 E Flow cytometry analysis was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor n3 F Transwell assay was performed in NCIH1299 and NCIH460 cells stably transfected with siRP11284F219 in the presence of miRNC or miR3p inhibitor Magnification x200 n3 P005 vs siNC NC negative control siRNA small interfering RNA OD optical density miR microRNA 0cLI RP11284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure miR3p directly targets CCAR1 A Bioinformatic analysis of the predicted binding sites between the CCAR1 'untranslated region and miR3p B Luciferase reporter assay in CCAR1wt or CCAR1mut treated cells in the presence of miRNC or miR3p mimic n3 P005 vs miRNC C CCAR1 expression in LC tissues compared with adjacent healthy tissues was analyzed using RTqPCR n13 P001 vs adjacent tissue Expression of CCAR1 in NCIH1299 and NCIH460 cells transfected with miRNC or miR3p mimics was detected using D RTqPCR and E western blotting n3 P005 vs miRNC miR microRNA NC negative control wt wildtype mut mutant RTqPCR reverse transcriptionquantitative PCR CCAR1 cell division cycle and apoptosis regulator LC lung carcinoma slower proliferative rate of the tumors was observed in the siRP11284F219 group compared with the siNC group Fig 6A and B Furthermore the tumor volume and weight were significantly decreased in the siRP11284F219 group compared with the control group Fig 6A and B RTqPCR analysis also demonstrated that compared with the siNC group the tumors in the siRP11284F219 group expressed higher levels of miR6273p Fig 6C and lower levels of CCAR1 Fig 6D providing further evidence to the existence of the RP11284F219miR3pCCAR1 regulatory axis in lung carcinoma tumor tissuesDiscussionThe present study investigated the function of RP11284F219 in lung carcinoma It was initially found that RP11284F219 was significantly upregulated in both lung cancer tissues and cell lines Following the deduction of a potential oncogenic role of this lncRNA siRP11284F219 was transfected into NCIH460 and NCIH1299 cells and it was demonstrated that knockdown of RP11284F219 inhibited the proliferation and invasion while promoting apoptosis of lung carcinoma cells In the mechanistic studies using online prediction tools and in vitro assays the results indicated that miR3p directly interacts with RP11284F219 by binding to its 'UTRThe function of miR627 was initially reported in colorectal cancer CRC Padi found that when upregulated by calcitriol miR627 targets the histone demethylase Jumonji domain containing 1A to increase methylation of histone H3K9 and suppresses the proliferative factors of CRC cells thus inhibiting the proliferation of CRC both in vitro and in vivo Moreover in CRC Sun discovered the role of miR in vitamin Denhanced efficacy of irinotecan via inhibition of the cytochrome P450 enzymemediated intratumoral drug metabolism miR is also reported to be a potential noninvasive diagnostic marker in gastric and breast cancer types In pulmonary diseases miR627 is downregulated in patients with chronic obstructive pulmonary disease and targets the highmobility group box protein to inhibit its expression thus improving transforming growth factorβ1induced pulmonary fibrosis The present results demonstrated the inhibitory effect of RP11284F219 on the expression of miR3p In addition it was identified that the miR3p inhibitor can neutralize the antitumor effects of RP11284F219 knockdown indicating that RP11284F219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating miR3p This antitumor role of miR6273p under the regulation of RP11284F219 in lung carcinoma tissues and cells is in accordance with the previous aforementioned findings on human CRC gastric and breast cancer types\x0cONCOLOGY REPORTS Figure RP11284F219 knockdown inhibits tumor growth in vivo A Macroscopic image of xenografted tumors B Tumor volume in nude mice injected with NCIH1299 cells transfected with siNC or siRP11284F219 measured over weeks n5 C Weight of tumors in nude mice at weeks after injection of NCIH1299 cells transfected with siNC or siRP11284F219 n5 Expression levels of D miR3p and E CCAR1 in the tumor tissues from nude mice injected with NCIH1299 cells transfected with siNC or siRP11284F219 for weeks were detected using reverse transcriptionquantitative PCR n5 P005 P001 P0001 vs siNC miR microRNA NC negative control sh short hairpin RNA CCAR1 cell division cycle and apoptosis regulator Using the publicly available RNA interaction prediction algorithms the current study identified that CCAR1 which was initially shown as the target gene of miR6273p is also regulated by RP11284F219 Furthermore the regulatory axis of RP11284F219miR3pCCAR1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model The interaction between RP11284F219 and miR3p and the interaction between miR3p and CCAR1 were demonstrated by the dualluciferase assay Although this method has been used to validate RNARNA interactions in previous studies other assays such as RNA pulldown and RNA binding protein immunoprecipitation that would provide more direct evidence for the RNARNA and RNAprotein interactions should be performedCCAR1 was initially reported as a protein essential for cancer cell apoptosis induced by retinoids or chemotherapeutics such as Adriamycin and etoposide Subsequently Kim et al revealed that this protein functions as a transcriptional coactivator of nuclear receptors In human breast cancer cells as CCAR1 interacts and cooperates with the coactivators of estrogen receptor signaling it promotes the estrogendependent proliferation of cancer cells In CRC cells Ou reported that CCAR1 can be recruited by βcatenin to act as a coactivator for the transcriptional activation of lymphoid enhancer binding factor CCAR1 is essential for the expression of Wnt target genes as well as the neoplastic transformation of CRC cells In gastric cancer cells researchers have revealed the cooperation between CCAR1 and βcatenin which leads to the promotion of the proliferation and migration of cancer cells In lung cancer CCAR1 was reported to be an effector of Doxorubicininduced apoptosis Moreover Muthu demonstrated that certain chemical compounds that bind with CCAR1 can increase the expression of CCAR1 and induce apoptosis However a contradictory conclusion was reported in a recent study which observed that CCAR1 was promoted by serine and arginine rich splicing factor which is activated by glucose intake and further enhanced tumorigenesis by increasing the glucose consumption rate Corroborating this finding in the current study via the targeting of miR3p the expression of CCA | 2 |
"The Center for Functional Cancer Epigenetics Dana-Farber Cancer Institute Boston MA 02215 14USC Epigenome Center and USC/Norris Comprehensive Cancer Center Los Angeles CA 90089 USA Correspondence: landimmail.nih.gov 4 4 2014 27 2 2014 27 8 2014 5 3365 3365 The genetic regulation of the human epigenome is not fully appreciated. Here we describe the effects of genetic variants on the DNA methylome in human lung based on methylation-quantitative trait loci (meQTL) analyses. We report 34304 cis- and 585 trans-meQTLs a genetic-epigenetic interaction of surprising magnitude including a regulatory hotspot. These findings are replicated in both breast and kidney tissues and show distinct patterns: cis-meQTLs mostly localize to CpG sites outside of genes promoters and CpG islands (CGIs) while trans-meQTLs are over-represented in promoter CGIs. meQTL SNPs are enriched in CTCF binding sites DNaseI hypersensitivity regions and histone marks. Importantly 4 of the 5 established lung cancer risk loci in European ancestry are cis-meQTLs and in aggregate cis-meQTLs are enriched for lung cancer risk in a genome-wide analysis of 11587 subjects. Thus inherited genetic variation may affect lung carcinogenesis by regulating the human methylome. Introduction DNA methylation plays a central role in epigenetic regulation. Twin studies have suggested that DNA methylation at specific CpG sites can be heritable12; however the genetic effects on DNA methylation have been investigated only in brain tissues34 adipose tissues56 and lymphoblastoid cell lines (LCL)7. Most studies were based on the Illumina HumanMethylation27 array which has a low density and mainly focuses on CpG-sites mapping to gene promoter regions. While the functional role of DNA methylation in non-promoter or non-CpG Island (CGI) regions remains largely unknown evidence shows roles in regulating gene splicing8 and alternative promoters9 silencing of intragenic repetitive DNA sequences10 and predisposing to germline and somatic mutations that could contribute to cancer development1112. Notably a recent study13 suggests that most DNA methylation alterations in colon cancer occur outside of promoters or CGIs in so called CpG island shores and shelves and the Cancer Genome Project has reported high mutation rates in CpG regions outside CGI in multiple cancers14. Although expression QTLs (eQTLs) have been extensively studied in different cell lines and tissues15 the minimal overlap observed between cis-acting meQTLs and eQTLs (?510%)347 emphasizes the necessity of mapping meQTLs that may function independently of nearby gene expression. This might reveal novel mechanisms for genetic effects on cancer risk particularly since many of the established cancer susceptibility SNPs map to non-genic regions. Lung diseases constitute a significant public health burden. About 10 million Americans had chronic obstructive pulmonary disease in 201216 and lung cancer continues to be the leading cancer-related cause of mortality worldwide17. To provide functional annotation of SNPs particularly those relevant to lung diseases and traits we systematically mapped meQTLs in 210 histologically normal human lung tissues using Illumina Infinium HumanMethylation450 BeadChip arrays which provide a comprehensive platform to interrogate the DNA methylation status of 485512 cytosine targets with excellent coverage in both promoter and non-promoter regions (Fig. 1a) CGI and non-CGI regions (Fig. 1b) and gene and non-gene regions. Thus our study enables the characterization of genetic effects across the methylome in unprecedented detail. Moreover since DNA methylation exhibits tissue specific features18 we investigated whether similar meQTLs could be identified in other tissues. Results Identification of cis-acting meQTLs We profiled DNA methylation for 244 fresh-frozen histologically normal lung samples from non-small cell lung cancer (NSCLC) patients from the Environment and Genetics in Lung cancer Etiology (EAGLE) study19. A subset of 210 tissue samples that passed quality control and had germline genotype data from blood samples20 was used for meQTL analysis. The analysis was restricted to 338456 autosomal CpG probes after excluding those annotated in repetitive genomic regions or that harbored genetic variants. The distribution of methylation levels differed strongly across distinct types of genomic regions (Supplementary Fig. 1ab). Consistent with previous studies21 CpG sites in promoter or CGI regions were largely unmethylated while those in other regions were largely methylated (Supplementary Fig. 1ab). We performed cis-meQTL analysis for each methylation trait by searching for SNPs within 500kb of the target CpG-site in each direction (1Mb overall). The genetic association was tested under an additive model between each SNP and each normalized methylation probe adjusting for sex age plate population stratification and methylation-based principal component analysis (PCA) scores. Controlling FDR at 5% (P=4.010?5) we detected cis-meQTLs for 34304 (10.1% of 338456) CpG probes (Supplementary ) mapping to 9330 genes. A more stringent threshold (P=6.010?6) at FDR=1% detected cis-meQTLs for 27043 CpG probes mapping to 8479 genes. Moreover with a 200kb window (100kb from both sides) instead than 1Mb we detected 40650 cis-meQTLs (P=2.010?4) controlling for FDR=5%. The methylation distribution in CpG sites detected with meQTLs differed substantially from those without meQTLs (Supplementary Fig. 1ab). The peak SNPs were equally distributed on either side of the target CpG-sites with a median distance (?) of 11.8 kb. The proportion of explained phenotypic variance (h2) ranged from 7.7% to 79.8% (Supplementary Fig. 1c) and inversely depended on ? (Supplementary Fig. 1d). We detected strong cis-meQTLs for DNMT1 a gene known for establishment and regulation of tissue-specific patterns of methylated cytosine residues and for DNMT3A/B two genes involved in de novo methylation in mammals but not for MTHFR which affects global methylation (Supplementary Fig. 1e). The likelihood of detecting cis-meQTLs varied across CpG regions and strongly depended on the variability of the methylation levels (Fig. 1d e). CpG probes in non-CGI regions were twice as likely to harbor cis-meQTLs than CpG probes in CGI regions (11.5% v.s. 4.8% t-test P<10?100); similarly CpG probes located in CGI of non-gene regions were twice as likely to harbor cis meQTL than those in gene regions (14.6% v.s. 6.6% t-test P<10?100). To verify the cis-meQTLs we analyzed data from The Cancer Genome Atlas (TCGA)22 NSCLC patients (n=65) for whom both DNA methylation data from llumina HumanMethylation450 BeadChip of histologically normal lung tissue and germline genotypes from Affymetrix Genome-Wide Human SNP Array 6.0 were available. Genetic associations were tested using the imputed genotypic dosages. EAGLE findings were strongly replicated in TCGA lung data: for the 34304 associations detected in EAGLE32128 (93.8%) had the same direction and 22441 (65.4%) had FDR<0.05 based on single-sided P-values (). For 34304 CpG probes detected with cis-meQTLs we searched for secondary independently associated SNPs in cis regions by conditioning on the primary cis-meQTL SNPs. We detected secondary cis-meQTL SNPs for 3546 CpG probes (FDR=5% P=410?5) 61.5% of which were replicated in TCGA lung data. Identification of trans-acting meQTLs Identification of trans-meQTLs was performed by searching for SNPs that were on different chromosomes from the target CpG-sites or on the same chromosome but more than 500kb away. We detected 615 CpG-probes with trans-meQTLs (FDR=5% P=2.510?10) including 438 interchromosomal and 177 intrachromosomal trans-meQTLs. Among 177 intrachromosomal trans-associations 30 lost significance after conditioning on the corresponding cis-regulating SNPs suggesting that these trans-associations were caused by cis-acting regulations through long range linkage disequilibrium (LD). Thus we detected 585 traits with true trans-meQTLs (Fig. 2a) mapping to 373 genes. The number of trans-meQTLs was reduced to 500 if controlling for FDR=1% (P=4.010?11). We replicated 79.8% of the 585 trans-associations in TCGA lung data. Interestingly trans-meQTLs were strongly enriched in CGI sites in contrast to the observation that cis-meQTLs were strongly enriched in non-CGI sites (Fig. 2b). CpG dinucleotides in 3UTR regions where microRNA target sites are typically located showed an opposite trend in both cis- and trans-meQTLs (Fig. 2b). In 62.8% of the trans-associations the SNPs involved were also detected to have cis-acting effects. We investigated whether trans-associations were mediated by these cis-regulated proximal CpG sites (Fig. 2cd). We found that 30 and 166 trans-associations had full and partial mediation respectively while 389 had no significant mediation. The trans-associations involving SNPs in gene desert regions are less likely to be mediated by proximal CpG probes (15.7% v.s. 34.3%; P=0.0067 Fishers exact test). To obtain mechanistic insight into the trans-associations showing mediation effects (n=196) we used the DAVID tool23 to characterize the function of genes harboring the mediating cis-CpG probes. The analysis was performed for 115 genes after excluding the major histocompatibility complex (MHC) region because of long range complex LD patterns. The GO analysis revealed three top gene categories with nominal significance involved in DNA methylation regulation including GTPase-activity related genes (P=0.004 Fishers exact test) genes regulating transcription (P=0.02) and genetic imprinting (P=0.04 Fishers exact test Supplementary Table 2). Notably 106 trans SNPs with P<2.510?10 were associated with multiple distal CpG probes suggesting that they are multi-CpG regulators. In particular we detected one master regulatory SNP rs12933229 located at 16p11.2 in a very large intron of the NPIPL1 gene which was associated with the methylation of CpG sites annotated to five genes on different chromosomes (Fig. 2a Supplementary Fig. 2 and Supplementary Table 3). These associations were partially mediated by a proximal CpG probe cg06871736. All five trans-associations were replicated in TCGA. The trans-associations show a consistent direction with the C allele associated with higher methylation levels. All five regulated target sites are in CGIs and three are in gene promoter regions. We evaluated the association with gene expression for these three CpG probes using 28 TCGA histologically normal lung tissue samples with RNA sequencing data. Based on this limited sample size two of the target genes PABPC4 and STARD3 showed decreased expression with increased methylation (FDR=10%). Enrichment of meQTLs in DNA regulatory regions SNPs associated with complex diseases in GWAS or with eQTLs have been reported to be enriched in ENCODE-annotated regulatory regions2425. These include DNaseI hypersensitivity sites CCTC-binding factor (CTCF) binding sites and regions enriched in- active and repressive histone modification marks. The large number of meQTLs detected in our study both cis and trans enabled us to systematically investigate their enrichment in regulatory regions. We performed enrichment analysis using Chip-Seq data in small airway epithelial cells (SAEC) from the ENCODE project for histone marks26 CTCF occupancy27 and DNaseI hypersensitivity sites28; and histone marks in primary human alveolar epithelial cells (hAEC) from our own laboratory29. Compared to the control SNP set not associated with the methylation of CpG sites (with minor allele frequency and CpG probe density matched with meQTL SNPs) the meQTL SNPs were strongly enriched for sites of CTCF DNaseI hypersensitivity and histone marks (H3K4me3 H3K9-14Ac and H3K36me3) associated with active promoters enhancers and active transcription and to a lesser extent for the repressive mark H3K27me3 (Table 2). Enrichment of all regulatory regions became stronger with increasing significance of association with the exception of the H3K27me3 repressive mark (Fig. 3). Using SAEC CTCF ChIP data we found that meQTL SNPs or associated SNPs in high LD located within CTCF consensus sequences can affect allele-specific binding of CTCF (see two examples in Supplementary Fig. 3 and 4). Lung cancer risk SNPs affect methylation in human lung tissue To determine whether the identified meQTLs might provide functional annotation to the established genetic associations with lung cancer risks we examined SNPs in five genomic regions reported to be associated with lung cancer risk in genome-wide association studies (GWAS) of populations of European ancestry: 15q25.13032 (CHRNA5-CHRNA3-CHRNB4) 5p15.33203334 6p21.3333 (BAT3 most strongly associated with squamous cell carcinoma or SQ) 12p13.335 (RAD52 for SQ) and 9p21.336 (CDKN2A/CDKN2B particularly for SQ). The GWAS SNPs at 15q25.1 were reported to be associated with total expression levels and multiple isoforms of CHRNA5 in normal lung tissue samples3738. The GWAS SNPs at the other four loci have not been reported to be associated with the total expression of nearby genes. Consistently we did not observe an association in RNA-seq data from TCGA lung normal tissue samples (n=59) although a detailed investigation of alternative promoters splice sites and allele-specific gene expression in larger studies is warranted. Here we investigated whether these SNPs contributed to lung cancer risk with epigenetic regulation by examining their associations with DNA methylation levels. The top GWAS SNPs located at 15q25.1 5p15.33 6p21.33 and 12p13.3 were all strongly associated with the methylation of the nearby CpG probes and the associations were replicated in TCGA lung data (Fig. 4). Importantly five of the six GWAS SNPs at these loci excluding the RAD52 locus were also the SNPs with the strongest association with the corresponding CpG probes. For the cg22937753 probe located in the RAD52 locus another SNP rs724709 with weak correlation with the GWAS SNP (r2=0.1) had the strongest association with meQTL. All involved CpG sites are located within gene bodies (which may affect gene splicing39) or the 3UTR regions. No meQTL was detected for 9p21.3 (Supplementary Fig. 5) possibly because of fewer CpG dinucleotide probes available in this gene region on the Illumina platform. The location of these lung cancer GWAS-associated CpG sites might identify which genes within the relevant regions are more likely associated with the risk SNPs something that is particularly important for regions with complex LD structure as the MHC region on 6p21. In MHC two GWAS SNPs in complete LD (r2=1) rs3117582 (BAT3) and rs3131379 (MSH5) were most strongly associated with the methylation of CpG sites located nearby of MSH5 (involved in DNA mismatch repair and meiotic recombination process) suggesting that MSH5 (P=5.410?13 t-test) is more likely to be involved in lung carcinogenesis than BAT3 (P=8.810?5 t-test) or that the SNP closer to MSH5 (rs3131379) is more likely to be the SNP most responsible of the GWAS association with lung cancer risk (Fig. 4b). Our meQTL data also show that rs3131379 trans-regulated the methylation level of CpG probe cg12093005 located in the body of FBRSL1 at 12q24 (PEAGLE=4.010?9 PTCGA=7.210?4 and Pcombined=5. 410?11 t-test). Thus this known GWAS locus might affect lung cancer risk through a gene located on a different chromosome. Of note on the 15q25.1 locus two independent lung cancer risk SNPs rs2036534 and rs1051730 were associated with CpG probes not linked with CHRNA5 expression. In Supplementary Fig. 6 we show that the two SNPs jointly regulated another methylation probe cg22563815 within the CHRNA5 promoter which is associated with CHRNA5 expression. This extends and further confirms the complex regulatory pattern with multiple SNPs previously observed for this locus35. Most subjects in the analyses were smokers (n=206). Adjustment for smoking status (former and current) or intensity (pack/years) did not change the results. cis-meQTLs are enriched in lung squamous cell carcinoma risk We investigated whether the identified cis-meQTL SNPs were enriched in the National Cancer Institute (NCI) lung cancer GWAS including 5739 cases and 5848 controls of European ancestry19. To focus on potentially new genetic risk associations we excluded the top lung cancer GWAS SNPs mentioned above and their surrounding regions. We tested the enrichment by examining whether the GWAS P-values for the LD-pruned cis-meQTL SNPs deviated from the uniform distribution i.e. no enrichment. When all cis-meQTL SNPs were analyzed together we detected a strong enrichment for overall lung cancer risk (P<10?4 based on 10000 permutations) which was primarily driven by the enrichment in SQ (P<10?4 based on 10000 permutations) (Fig. 5a). The genomic control ?-values based on genome-wide SNPs showed that the type-I error rates of our enrichment test were not inflated (?=1.01 and 1.00 for overall lung cancer and SQ respectively). Stratified analyses further refined the enrichment to the cis-meQTL SNPs regulating CpG-sites mapping to north shore (Fig. 5b) and gene body (Fig. 5c) regions (see Supplementary Fig. 7 for the quantile-quantile plot). These gene bodies and north shores were enriched for genes involved in cancer pathways (P=2.510?4 Fishers exact test) and particularly those in NSCLC pathway (e.g. AKT1 MAPK1 RASSF5 etc. Supplementary Table 4). In contrast cis-meQTLs related with CGI regions or promoters were not enriched with the risk of overall lung cancer or any lung cancer subtype further emphasizing the need to comprehensively study the methylome to identify functional mechanisms for GWAS findings and identify new genetic loci. " | 1 |
s case reports duplicate studies and those with insufficient available data were excludeddata extraction and managementdata were independently extracted by two investigators yao xl and wu ww according to the same inclusionand exclusion criteria disagreements were adjudicated by a third reviewer qu kthe following data will be extracted from eligible literatures 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 bsr20202509101042bsr20202509¢¢¢study characteristics name of the first author year of publication and sample size of included studiesparticipant characteristics tumor stage and age of patientsinterventions intervening methods and dosage administration route cycles and duration of treatment of huaiergranule¢ outcome and other data overall response rate orr disease control rate dcr overall survival osdiseasefree survival dfs quality of life qol immune indexes [cd3 cd4 cd8 natural killer cellsnk percentage and cd4cd8 cell ratios] and adverse effects we will attempt to contact the authors to request the missing or incomplete data if those relevant data are notacquired they will be excluded from the analysisquality assessmentto ensure the quality of the metaanalysis the quality of the included randomized and nonrandomized controlledtrials was evaluated according to the cochrane handbook tool and methodological index for nonrandomizedstudies minrrs supplementary table s2 respectively types of outcome measuresmain outcomesthe primary outcomes in present analysis included shortterm and longterm clinical efficacy and adverse effectsaccording to response evaluation criteria in solid tumors recist criteria i shortterm clinical efficacy the shortterm tumor response included orr and dcr orr was defined as thesum of complete and partial response rates and dcr was defined as the sum of complete response partialresponse and stable disease ratesii longterm clinical efficacy year os the time from the date of randomization to death from any cause year dfs the time from date of random assignment to date of recurrence or deathiii adverse events gastrointestinal adverse effects myelosuppression and hepatotoxicity secondary outcomesi qol qol was evaluated using the qualityoflife improved rate qir and karnofsky score kpsii immune function indicators the immune function of breast cancer patients was assessed in terms of cd3cd4 cd8 nk cells percentage and cd4cd8 cell ratiosstatistical analysisstata stata corp college station tx usa and review manager nordic cochran centre copenhagendenmark statistical software were used for statistical analyses cochranes q test and i2 statistics were used to assessheterogeneity among the studies if p01 or i2 a fixed effects model was used for the metaanalysisotherwise a random effects model was used the mantelhaenszel method will be applied for pooling of dichotomous data and results will be presented as risk ratio rr with their confidence intervals cis inverse variancemethod will be used for pooling of continuous data and results will be presented as standardized mean differencesmd with their cis a twotailed p005 was considered statistically significantthe presence of publication bias was investigated using the funnel plots beggs and eggers test if or more studiesare included in the metaanalysis [] if publication bias existed a trimandfill method should be applied tocoordinate the estimates from unpublished studies and the adjusted results were compared with the original pooledrr sensitivity analysis was performed to explore an individual studys influence on the pooled results by deleting onesingle study each time from pooled analysisresultssearch resultsthe initial search retrieved a total of s of which were excluded due to duplication after title and review s were further excluded because they were noncomparative clinical trials n19 were 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 bsr20202509101042bsr20202509figure study selection process for the metaanalysisnot related to huaier granule n9 were nonpeer reviewed s n8 were literature review or metaanalysisn3 and were case report and series n5 leaving studies as potentially eligible after detailed assessment offull texts studies with breast cancer patients n5 trials with insufficient data n8 and inappropriate criteriafor the experimental or control groups n11 were excluded ultimately trials [] involving patients with breast cancer were included in the final analysis figure patient characteristicsall included studies were performed in different medical centers in china in total patients with breast cancerwere treated using conventional methods in combination with huaier granule while patients were treated usingconventional methods alone huaier granule was manufactured by qidong gaitianli pharmaceutical co ltd andgranted a manufacturing approval number issued by the chinese sfda z20000109 study and patient characteristics are summarized in table quality assessmentquality assessment of the risk of bias is shown in figure and table the results revealed that the literature retrievedfor the present study was of medium and high quality 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 bsr20202509101042bsr20202509table clinical information from the eligible trials in the metaanalysisincluded studieschen qj chen y dai yg guo fd han sj lei ss liang yq li zh lu mq2017lu y qun sx ren xb shan cy tan zd tang y wang mh wang w wu yb xiong y xu f yang z2017yin x iiiiiiiviiiiinotprovidediiiiiiiiiviiiiiiviivnotprovidediiiiiiiiiiiiiiiiiiiiiiiiiviiiiiiiiiiiinotprovidedtumorstagepatientsconexpage year control vsexperimentalintervening methods range median ct vs cthuaier granules vs ct vs cthuaier granulesoa meanoadosage ofhuaiergranulesduration oftreatments20gtime times monthcourse daycourses20gtime times weeks for a course daycourses months vs range vs vs meanmeanct vs cthuaier granules20gtime timesoadayct vs cthuaier granules20gtime times monthoadayct vs cthuaier granules20gtime times weeks for a course oadaycourses yearsnot providedct vs cthuaier granules20gtime timesoaday vs range vs vs meanmeanct vs cthuaier granules20gtime times monthsoadayct vs cthuaier granules20gtime times months for a course oadaycoursesct vs cthuaier granules20gtime times weeks for a course oadaycourses vs medianct vs cthuaier granules20gtime times weeks for a course not providedct vs cthuaier granules20gtime timesoadaycourses years mean vs vs vs meanmeanoadayct vs cthuaier granules20gtime times weeksoadayct vs cthuaier granules20gtime times weeks for a course oadayct vs cthuaier granules20gtime timesoadaycoursesnot providednot providedct vs cthuaier granules20gtime times monthoadaynot providedct vs cthuaier granules20gtime times months vs meanoadayct vs cthuaier granules20gtime times monthsoaday range mean ct vs cthuaier granules20gtime times monthsoaday vs rangect vs cthuaier granules20gtime times weeks for a course vs range vs vs meanmeanoadayct vs cthuaier granules20gtime timesoadaycoursesnot providedct vs cthuaier granules20gtime times weeks for a course oadayct vs cthuaier granules20gtime timesoadaycourses monthparametertypes1cid2 3cid21cid2 3cid25cid22cid22cid2 3cid25cid24cid2 5cid22cid2 3cid25cid22cid24cid2 5cid23cid2 4cid25cid24cid2 5cid21cid2 2cid24cid22cid2 3cid22cid2 3cid24cid25cid22cid23cid22cid22cid2 5cid25cid24cid2 5cid22cid22cid22cid21cid2 5cid22cid22cid2 3cid2zhang jg iiii vs rangect vs cthuaier granules20gtime times monthsoadayzhang y notprovidedzhao zw iiiiv vs range vs meanct vs cthuaier granules20gtime times monthsoadayct vs cthuaier granules20gtime times weeksoadayzhong sw zhou p iviiii74range median ct vs cthuaier granules20gtime times monthsoaday vs rangect vs cthuaier granules20gtime times monthsoadaynotes control group conventional treatments alone group experimental group conventional treatments and huaier granule combined group1cid2 overall response rate and disease control rate 2cid2 overall survival or diseasefree survival 3cid2 adverse events 4cid2 quality of life 5cid2 immunefunction indexabbreviations ct conventional treatments oa oral administration 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 bsr20202509101042bsr20202509figure risk of bias summaryreview of authors judgments about each risk of bias item for included studies note each color represents a different level ofbias red for highrisk green for lowrisk and yellow for unclearrisk of biastable quality assessment of nonrandomized comparative studiesnonrandomized studiesadditional criteria in comparativestudytotalabcdefghijklstudyguo fdhan sjlei ssren xbwu ybyin xzhang yzhongsw zhou pa a clearly stated aim b inclusion of consecutive patients c prospective collection of data d endpoints appropriate to the aim of the study eunbiased assessment of the study endpoint f followup period appropriate to the aim of the study g loss to follow up less than h prospectivecalculation of the study size i an adequate control group j contemporary groups k baseline equivalence of groups l adequate statistical analysesnotes the items are scored not reported reported but inadequate and reported and adequatetherapeutic efï¬cacy assessmentsorr and dcrfour clinical trials involving patients compared orr and dcr between the two groups as shown in figure the pooled results revealed that patients who underwent combination therapy experienced improved orr rr ci p002 and dcr rr ci p019 compared with those whoreceived conventional treatments alone although the dcr did not reach significant difference dcr p095 i2 was not heterogeneous among the studies therefore a fixedeffect model was used to analyze rr otherwise arandomeffect model was used 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 bsr20202509101042bsr20202509figure comparisons of orr and dcr between experimental and control groupforest plot of the comparison of orr a and dcr b between the experimental and control group control group conventionaltreatment alone group experimental group conventional treatment and huaier granule combined grouplongterm survival1year 2year 3year and 5year oseleven clinical trials with breast cancer patients reported os figure metaanalysis revealed that the 2yearrr ci p002 3year rr ci p00001 and 5year os rr ci p0004 of patients in the combined treatment group were significantly prolongedcompared with the control group there was statistical heterogeneity in 1year os p009 i2 and 2year osp00001 i2 according to the heterogeneity test therefore a randomeffect model was used to pool thismetaanalysis otherwise the fixedeffect model was used1year 2year 3year and 5year dfsten clinical trials with breast cancer patients reported dfs figure metaanalysis revealed that the 1yearrr ci p0003 2year rr ci p000001 3year rr ci p000001 and 5year dfs rr ci p003 of patients in thecombined treatment group were all significantly prolonged compared with the control group there was statisticalheterogeneity in 5year dfs p005 i2 according to the heterogeneity test therefore a random effectsmodel was used to pool this metaanalysis otherwise the fixedeffect model was usedqol assessmentfour trials with participants evaluated qir and three trials including patients reported kps data figure results demonstrated that the qol of breast cancer patients in the combined group was significantly better than thatof the control group indicated by significantly increased qir rr ci p000001 and kpsrr ci p000001 qir p084 i2 was not heterogeneous among the studiestherefore a fixedeffect model was used to analyze rr otherwise a randomeffect model was usedimmune function evaluationimmune status of the patients was examined between the two groups in eleven controlled studies including patients figure the percentages of cd3 cd3 rr ci p005 cd4 rr ci p000001 and nk cells rr ci p00001 and cd4cd8 ratio rr ci p000001 in the combined treatment group were significantly increased compared with 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 bsr20202509101042bsr20202509figure comparisons of os between experimental and control groupforest plot of the comparison of 1year a 2year b 3year c and 5year os d between the experimental and control groupcontrol group conventional treatment alone group experimental group conventional treatment and huaier granule combinedgroup 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 bsr20202509101042bsr20202509figure comparisons of dfs between experimental and control groupforest plot of the comparison of 1year a 2year b 3year c and 5year dfs d between the experimental and control groupcontrol group conventional treatment alone group experimental group conventional treatment and huaier granule combinedgroup 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 bsr20202509101042bsr20202509figure comparisons of qol between experimental and control groupforest plot of the comparison of qir a and kps b between the experimental and control group control group conventionaltreatment alone group experimental group conventional treatment and huaier granule combined groupthose in the conventional treatment alone group whereas the proportions of cd8 rr ci to p033 did not differ significantly between the two groups a randomeffect model was used to pool thismetaanalysis due to significant heterogeneityassessment of adverse eventsas shown in figure patients treated with huaier granule and conventional methods exhibited lower incidences ofmyelosuppression rr ci p0001 and hepatotoxicity rr ci p005 whereas analysis of gastrointestinal adverse effects rr ci p014 leukopeniarr ci p006 nausea and vomiting rr ci p052 andalopecia rr ci p020 did not differ significantly between the two groups there wasstatistical heterogeneity in gastrointestinal adverse effects p006 i2 according to the heterogeneity testand a random effects model was used to pool this metaanalysis otherwise the fixedeffect model was usedpublication biasas shown in figure the funnel plots beggs and eggers regression tests results showed that there was publicationbias in cd4cd8 ratio begg egger to determine whether bias affected the pooled risk ofcd4cd8 ratio a trimandfill analysis was performed the adjusted rr indicated a trend similar to the resultsof the primary analysis before p00001 after p00001 reflecting the reliability of the primary conclusionsparameters discussed less than papers were not conducted publication bias analysessensitivity analysisas figure signified the results revealed that no individual studies significantly affected the primary indicatorscd4 and cd4cd8 ratio which indicated statistically robust results parameters discussed less than paperswere not conducted sensitivity analysesdiscussionhuaier granule the active ingredient of huaier extract appears as a lightyellow powder through hotwater extractionethanol precipitation deproteinization and lyophilization procedures as a type of tcbp huaier granule hasbeen clinically applied as an effective adjuvant drug in cancer treatment for decades although several studies havereported that addition of huaier granule could be beneficial to patients with advanced breast cancer but the 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 bsr20202509101042bsr20202509figure comparisons of immune function between experimental and control groupforest plot of the comparison of immune function indicators including cd3 a cd4 b cd8 c and nk d cells percentage andcd4cd8 ratio e between the experimental and control group control group conventional treatment alone group experimentalgroup conventional treatment and huaier granule combined group the random effects metaanalysis model inverse variancemethod was used 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 bsr20202509101042bsr20202509figure comparisons of adverse effects between experimental and control groupforest plot of the comparison of adverse effects including gastrointestinal adverse effects a myelosuppression b hepatotoxicity c leukopenia d nausea and vomiting e and alopecia f between the experimental and control group control groupconventional treatment alone group experimental group conventional treatment and huaier granule combined group 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 bsr20202509101042bsr20202509figure funnel plot of cd4 a and cd4cd8 bexact therapeutic effects have yet to be systematically evaluated thus indepth knowledge of the efficacy and safetyof huaier granule is needed this systematic review will provide a helpful evidence for clinicians to formulate thebest postoperative adjuvant treatment strategy for patients with breast cancer and also provide scientific clues forresearchers in this fielddata from trials [] including patients with breast cancer were included in our metaanalysishuaier granule in all of the included studies was manufactured by qidong gaitianli pharmaceutical co ltd thedosages of huaier granule were g per day via oral administration the pooled results revealed that the combinationof huaier granule and conventional treatment for breast cancer achieved more beneficial effects compared withthose treated solely with conventional therapy compared with conventional treatment alone huaier granule couldsignificantly improve orr and qol in patients with breast cancer p005 the study also assessed whether huaiergranule could prolong the longterm survival rates of breast cancer patients and the results showed that the and 5year os and and 5year dfs of patients were all significantly prolonged compared with the controlgroup these results indicated that using huaier granule could improve the short and longterm curative effects ofconventional treatment for breast cancert lymphocyte subsets cd3 cd4 cd8 cell subsets and cd4cd8 ratio and nk cells play an importantrole in antitumor immunity studies have shown that patients with advanced cancer showed decreased immunefunction and nk activity and exhibiting imbalance of t lymphocytes percentage many studies have reportedthat huaier granule can enhance the ability of the bodys immunity and resistance to tumors our analysisdemonstrated that the percentages of cd3 cd4 and nk cells and cd4cd8 ratio were all significantly increasedin breast cancer patients treated with huaier granule indicating that immune function of breast cancer patients wasimproved after huaier granule adjuvant therapysafety is the top priority of clinical treatment seven clinical trials with breast cancer patients reported adverse events according to world health anization standards metaanalysis revealed that patients who underwent huaier granule plus conventional treatment demonstrated a lower risk for myelosuppression and hepatotoxicitycompared with conventional treatment alone whereas analysis of other toxic side effects did not differ significantlytherefore huaier granule appears to be a safe auxiliary antitumor medicine for individuals with breast cancerthere were some limitations to our analysis currently five clinical trials table in which breast cancer are being treated by huaier granule in conjunction with conventional regimens have been registered onclinicaltrialsgov nct02615457 and nct02627248 and chinese clinical trial register chictr1800015390chictroic16007737 and chictrtrc11001250 however except for two studies most of the includedtrials were not registered before the first participant enrolled second as an important chinese patent medicinehuaier granule was mainly applied in china which may bring an unavoidable regional bias and subsequently influence the clinical application of huaier granule worldwide third different trials evaluated the treatment efficacy withdifferent outcomes resulting in a reduction in the size of the statistical sample making it difficult to summarize theresults at the same scale fourth several results demonstrated significant heterogeneity among the included trialswhich may be due to the different tumor stage tumor subtypes ages of the breast cancer patients and duration oftreatment however based on the currently available literature there are insufficient data to perform more statistical analysis to evaluate correlations in addition the efficacy of monotherapy of huaier granule in the treatment of 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 bsr20202509101042bsr20202509figure sensitivity analysis for cd4 a and cd4cd8 b 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 bsr20202509101042bsr20202509table search results of clinical trial registrationregistrationnumbernct02615457nct02627248chictr1800015390chictroic16007737chictrtrc11001250titlephaseconditionsinterventionslocationshuaier granule intreating women withtriple negative breastcancerneoadjuvantchemotherapy with orwithout huaiergranule in treatingwomen with locallyadvanced breastcancer that can beremoved by surgeryhuaier granule forstage ii and iii triplenegative breastcancer with lymphnode metastasis amulticenterrandomizeddoubleblindplacebocontrolledclinical triala multicenter doubleblind randomizedplacebocontrolledstudy on stage iiiiitriplenegative breastcancer with lymphnode metastasistreated by huaiergranulesextract of fungi ofhuaier used for triplenegtive breastcancera prospectiverandomized controlledtrialivivtriple negative breasthuaier granuleqilu hospital ofcancershandong universityjinan shandong chinabreast cancerhuaier granule otherqilu hospital ofchemotherapyshandong universityjinan shandong chinaivtriple negative breasthuaier granulecancerthe first aï¬iliatedhospital of amusouthwest hospitalchongqing chinaibreast cancerhuaier granuleivtriple negative breasthuaier granulecancersouthwest hospital thethird military medicaluniversity chongqingchinasouthwest hospital thethird military medicaluniversity chongqingchinabreast cancer also needs highquality evidence to verify however up to now huaier granule is mainly combinedwith radiotherapy chemotherapy or surgery and other conventional treatment methods for breast cancer we willkeep paying close attention to upcoming highquality clinical trials in our later studies and carry out further analyseson studies conducted huaier granule monotherapy against breast cancer finally publication bias was exists in someindicators which might because some authors tended to deliver positive results of s to editors therefore anyconclusions need to be made with cautionconclusionin summary findings of this metaanalysis indicate that the combination of huaier granule and conventional treatment is effective in treating patients with breast cancer the clinical application of huaier granule not only clearlyenhanced the therapeutic effects of conventional treatment but also effectively improved qol and immune functionin patients with breast cancer thus we anticipate that our study will provide valuable evidence for further evaluationof huaier granule on the other hand the low quality of some of the included publications increased the risk of biaswhich to some extent affects the reliability of this research therefore additional studies with highquality evidenceto verify the effectiveness of huaier granulemediated therapy for breast cancer are warrantedcompeting intereststhe authors declare that there are no competing interests associated with the manuscriptfundingthis study was supported by grants from national science foundation of china [grant numbers and ] 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 bsr20202509101042bsr20202509author contributionxi w and yao xl conceived and designed the methods yao xl and wu ww extracted the original data and drafted themanuscript yao xl wu ww and qu k performed statistical analysis xi w and yao xl interpreted results xi w and quk revised the manuscript all authors had full access to all data in the study and take responsibility for the integrity of the data andthe accuracy of data analysisabbreviationscbm chinese biological medicine database ci confidence interval cnki china national knowledge infrastructure csjdchinese scientific journal database dcr disease control rate dfs diseasefree survival kps karnofsky performance scoreminrrs methodological index for nonrandomized studies nk natural killer cells orr overall response rate os overallsurvival prisma preferred reporting items for systematic reviews and metaanalyses qir qualityoflife improved rate qolquality of life rct randomized controlled trial recist response evaluation criteria in solid tumors rr risk ratio sfdastate food and drug administration smd standardized mean difference tcbp traditional chinese biomedical preparationreferences ferlay j colombet m soerjomataram i mathers c parkin dm pi Ëneros m estimating the global cancer incidence and mortality in globocan sources and methods int j cancer 101002ijc31937 bray f ferlay j soerjomataram i siegel rl torre la and jemal a global cancer statistics globocan estimates of incidenceand mortality worldwide for cancers in countries ca cancer j clin 103322caac21492 watkins ej overview of breast cancer jaapa 10109701jaa0000580524957333d akram m iqbal m daniyal m and khan au 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"Oral squamous cell carcinoma is one of the most common causes of malignancyassociated death Early diagnosis of oral squamous cell carcinoma OSCC is important in patient treatment and prognostic evaluation Due to the lack of significant therapeutic benefit the year survival rate has not improved Therefore effective novel markers are needed to improve diagnosis To determine novel promising diagnostic biomarkers for OSCC upregulated and downregulated differentially expressed genes were screened from OSCC tissues using an RNA microarray The results suggested that minichromosome maintenance protein MCM5 mRNA was significantly overexpressed in OSCC tissues compared with that in adjacent normal tissues Moreover silencing of MCM5 expression an OSCC cell line SCC significantly impaired proliferation and colony formation Furthermore negative regulation of the mRNA and protein expression of MCM5 and demonstrated that MCM5 served as a cancerpromoting gene modulating OSCC cell proliferation through induced G2M phase arrest In this process the mRNA expression of cyclin E and cyclindependent kinase was downregulated while p21 expression was upregulated These results suggested that MCM5 may be an important pathogenic factor of OSCC High expression levels of MCM5 may serve as a marker for the early diagnosis of OSCC IntroductionOral squamous cell carcinoma OSCC affects individuals worldwide annually Presently the clinical treatment of OSCC is primarily surgery radiotherapy or chemotherapy Over the past decades the overall survival Correspondence to Dr Xiaofeng Wang Department of Stomatology ChinaJapan Union Hospital of Jilin University Xiantai Changchun Jilin PR ChinaEmail wangxiaofengjlueducnContributed equallyKey words minichromosome maintenance protein cell cycle oral squamous cell carcinoma biomarker microarrayOS rate of patients with OSCC has not significantly improved with a year survival rate of Insufficient sensitive and specific biomarkers may lead to the diagnosis of OSCC at advanced stages Therefore it is necessary to identify novel biomarkers for the early diagnosis and treatment of OSCCRecently with the continuous development of sequencing technology researchers can efficiently distinguish differentially expressed genes DEGs by transcriptome sequencing which allows screening of potential tumor markers or therapeutic drug targets For example a number of new potential tumor markers have been found in human malignancies such as breast cancer epithelial ovarian cancer and glioma Minichromosome maintenance protein MCM5 a member of minichromosome maintenance family of proteins plays an important role in cell proliferation and DNA replication Some studies have confirmed that MCM5 is highly expressed in numerous human malignancies such as renal cell carcinoma pancreatic ductal adenocarcinoma cervical cancer and skin cancer Further studies have found that high expression of MCM5 is closely associated with the clinicopathological features of specific cancer types For example overexpression of MCM5 is significantly associated with overall survival rate OS in hepatocellular carcinoma Moreover increased expression of MCM5 is positively correlated with larger tumor size positive lymph node metastasis more advanced clinical stage higher histological grade deeper invasion depth and perineural invasion of OSCC However thus far the expression function and potential mechanisms of MCM5 in OSCC are still unclear Therefore the present study aimed to analyze the DEGs in OSCC using a microarray screen for MCM5 and further evaluate the possible functions of MCM5 in OSCC The present results may provide evidence to support the value of MCM5 as a biomarker or a therapeutic target of OSCC Materials and methodsTissue sampling Pairs of OSCC tissues and adjacent normal tissues were obtained from patients undergoing resection operations at the ChinaJapan Union Hospital of Jilin University Changchun China Clinicopathological data were also collected No patient received preoperative treatment including radiotherapy or chemotherapy No other inclusionexclusion criteria were used Matched normal OSCC 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAtissues were obtained from a segment of the resected specimens cm away from the tumor Pathological analysis was used to identify surgically resected specimens Pathological analysis was performed by our group with no specific diagnostic guidelines Three paired samples were obtained for transcriptome sequencing Then to confirm the reliability of sequencing data the samples size was increased using the remaining paired tissues and analyzed using quantitative qPCR All comparisons between OSCC tissues and adjacent normal tissues were performed simultaneously The KaplanMeier analysis of OS and survival curves were from the Cancer Genome Atlas database TCGA wwwcancergovThe study was approved by the Ethics Committee of ChinaJapan Union Hospital of Jilin University Written informed consent was obtained from all patients who participated in this studyTranscriptome sequencing and functional annotation analysis Total RNA extraction RNA library construction and transcriptome sequencing were performed at Sangon Biotech Co Ltd The biological relevance of unique genes in expression profiles of DEGs were screened according to the threshold values of log2foldchange¥ and P005 Then the differentially expressed mRNAs were analyzed by Gene Ontology GO whose annotations were downloaded from Gene Ontology httpgeneontology UniProt sparqluniprot and NCBI wwwncbinlmnihgov Significant GO categories were identified using Fisher's exact test with a P005 which indicated that significantly upregulated genes in the set of DEGs were assigned to a specific functional category more often than expected by chance Significant pathways of the DEGs were then analyzed and identified according to the Kyoto Encyclopedia of Genes and Genomes KEGG database wwwkeggjpCell lines The human tongue squamous cell carcinoma SCC and CAL were obtained from the American Type Culture Collection CAL cells were cultured in DMEM medium with fetal bovine serum FBS Gibco Thermo Fisher Scientific Inc Uml penicillin and streptomycin at ËC in a humidified atmosphere containing CO2 SCC cells were cultured in MEM medium with FBS NEAA Uml penicillin and streptomycin at ËC in a humidified atmosphere containing CO2MCM5specific siRNA and transfection Three MCM5 siRNA sequences were synthesized by Suzhou GenePharma Co Ltd The sequences were as follows '' siRNA Forward CCG ACU ACU UGU ACA AGC ATT and reverse UGC UUG UAC AAG UAG UCG GTT siRNA forward CCA AAU GUC UAU GAG GUC ATT and reverse UGA CCU CAU AGA CAU UUG GTT siRNA forward GUC GUC UGU AUU GAC GAG UTT and reverse ACU CGU CAA UAC AGA CGA CTT and scrambled forward UUC UCC GAA CGU GUC ACG UTT and reverse ACG UGA CAC GUU CGG AGA ATT The mock was an untransfected empty vector serving as the controlSCC cells 45x104 cellswell were cultured in well plates overnight at ËC Then cells were transfected with nM negative control siRNA or MCM5 siRNA using Lipofectamine® Transfection Reagent Invitrogen Thermo Fisher Scientific Inc according to the manufacturer's protocol After h transfection cells were collected and then RNA was extracted by TRIzol® regent Invitrogen Thermo Fisher Scientific Inc for further experiments as indicatedReverse transcription RTqPCR RNA extracted from tissues samples were reverse transcribed into cDNA using a GoScript Reverse Transcription System kit Monad httpwwwmonadbiotechcom according to the manufacturer's instructions Relative mRNA expressions were quantified by qPCR using the QuantiTect SYBR Green PCR kit Roche Diagnostics and normalized to GAPDH using primers listed in Table I The cycling parameters were cycles of ËC for sec ËC for sec and ËC for sec Relative mRNA levels were assessed by the comparative ÎÎCq method All analyses of the samples were conducted in triplicateFor association of MCM5 expression levels with clinicopathologic features of OSCC the relative expression levels of MCM5 were evaluated using qPCR as aforementioned Relative mRNA levels of paired samples adjacent vs cancer tissues were assessed by the comparative ÎÎCq method A ratio was considered to have high MCM5 expression whereas a ratio ¤ was considered to have low MCM5 expression Cell proliferation assay To analyze cell proliferation a Cell Counting Kit CCK Dojindo Molecular Technologies Inc was used according to the manufacturer's instructions In total cells were cultured to each well of well plates After h post siRNA transfection cells were incubated for and h CCK reagent was added h prior to detection The OD was measured at nm using a microplate reader BioTek The experiment was performed three timesColony formation assay This assay was performed according to a previous study Briefly cells were cultured in well plates at cellswell followed by culture in complete medium DMEM supplemented with FBS Uml penicillin and streptomycin for weeks The colonies were fixed with methanol for min at room temperature and washed with PBS and stained with crystal violet at room temperature Beyotime Institute of Biotechnology solution for min A cell colony was defied as a group formation of at least cells Finally formed colonies were observed and images were captured under a light microscope at magnification x200 Cell migration analysis using scratching assays SCC cells were cultured in a well plate at 5x105 cellswell overnight at ËC Then the cells were scratched and scraped with fresh DMEM Cells were observed and images were captured under a light microscope magnification x200 at h The width of the scratch was measured and referred to as Wbefore Then the cells were starved with no FBS and returned to the incubator for h at ËC The width of the same scratch was measured and referred to as Wafter Migrating distance was calculated by subtracting Wafter from Wbefore The migration of the control was set as Western blot analysis The protein extractions from the cells were isolated using RIPA Lysis Buffer P0013B Beyotime Institute of Biotechnology Then µg protein was loaded per lane on a gel resolved using SDSPAGE and electroblotted onto 0cONCOLOGY LETTERS Table I Primers used for reverse transcription quantitativePCRmRNA MCM5 P21 CyclinE CDK2 GAPDH MCM5 minichromosome maintenance protein cyclindependent kinase Forward primer '' GATCCTGGCATTTTCTACAG GGAGACTCTCAGGGTCGAAA TTCTTGAGCAACACCCTCTTCTGCAGCC GCTAGCAGACTTTGGACTAGCCAG AGAAGGCTGGGGCTCATTTG Reverse primer ''CCCTGTATTTGAAGGTGAAGGGATTAGGGCTTCCTCTTGGTCGCCATATACCGGTCAAAGAAATCTTGTGCCAGCTCGGTACCACAGGGTCAAGGGGCCATCCACAGTCTTCFigure Volcano plots and KEGG pathway analysis of differentially expressed genes between OSCC cancer tissue group and adjacent normal tissue group A Differences in gene expression profiles between OSCC cancer tissue group and adjacent normal tissue group The horizontal line corresponds to a fold log2 scaled change up or down and the vertical line represents P005 The red points on the plot represent the differentially expressed genes with a fold change upregulation while the green points represent downregulation with P005 B Top KEGG enrichment terms of DEG in OSCC The vertical axis represents the pathway category and the horizontal axis represents the enrichment score [lgPvalue] of the pathway LgP was the logarithm of Pvalue and P005 was considered significant KEGG Kyoto Encyclopedia of Genes and Genomes OSCC oral squamous cell carcinoma DEG different expressed genes OSCC oral squamous cell carcinomaPVDF membranes Roche Diagnostics After blocking at ËC for h nonfat milk in PBS plus Tween the blots were incubated with primary antibodies against antiMCM5 D220960 BBI Life Sciences antip21 Proteintech anticyclin E ProteinTech Group Inc and antiβactin D16H11 CST Biological Reagents Co Ltd at ËC for h Western blots were probed with secondary antibodies and detected using the Odyssey infrared imaging system LICOR BiosciencesCell cycle analysis SCC cells were harvested and fixed with ethanol on ice for min and then washed with PBS to decant the ethanol solution Then the cells were suspended and stained by PI and RNase A treatment Cell cycle analysis was performed using a flow cytometer FACSARIA
¡ BD Biosciences The data was performed using CXP Analysis software Beckman Coulter Inc Statistical analysis All the data analysis was performed using SPSS version SPSS Inc The results are presented as mean SD Associations between MCM5 mRNA expression and clinicopathological factors were analyzed using the Pearson's Ï2 test or Fisher's exact test The differences in MCM5 mRNA expression between carcinoma and adjacent normal tissues were evaluated by a paired ttest Oneway ANOVA followed by Tukey's post hoc test was used to determine the differences between groups and unpaired ttests for the rest of the data The survival rate was calculated by the KaplanMeier method and compared using the logrank test P005 was considered to indicate a statistically significant difference All experiments were performed in triplicateResultsRNA sequencing and functional annotation analysis To explore novel biomarkers for OSCC the RNAs derived from tissue samples by sequencing were detected Three matched primary OSCC tissues and adjacent normal tissues were randomly selected As shown in Fig 1A the aberrant expression of genes was detected in tissue samples To screen 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMATable II Twenty randomly selected differentially expressed genes between oral squamous cell carcinoma and adjacent tissue samples The genes selected randomly instead of listing based on rank or foldchange in expressionGene ID ENSG00000160182 ENSG00000205592 ENSG00000171195 ENSG00000126549 ENSG00000090382 ENSG00000161798 ENSG00000161055 ENSG00000107562 ENSG00000214711 ENSG00000106066 ENSG00000184330 ENSG00000137745 ENSG00000243207 ENSG00000107159 ENSG00000183072 ENSG00000196611 ENSG00000171217 ENSG00000178445 ENSG00000100297 ENSG00000127564 Log2 foldchange Gene nameTFF1MUC19MUC7STATHLYZAQP5SCGB3A1CXCL12CAPN14CPVLS100A7AMMP13PPANP2RY11CA9NKX2MMP1CLDN20GLDCMCM5PKMYT1Pvalue 881x10 468x10 144x10 140x10 209x10 528x10 959x10 604x10 601x10 846x10 234x10 753x10 222x10 161x10 380x10 154x10 705x10 751x10 565x10 240x10 Result Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Downregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation Upregulation the candidate biomarkers for diagnosing OSCC the DEGs were selected when changes in RNA expression were foldchanges As shown in Fig 1A aberrant RNAs were significantly downregulated while RNAs were upregulated In order to represent all differentially expressed genes twenty randomly selected dysregulated genes between OSCC and adjacent tissue samples are summarized in Table II The Pvalue and log2 foldchanges of all aberrant expression genes are shown in Table SI The DEGs were selected randomly instead of listing based on rank or foldchange in expression To explore the role of differentially expressed RNAs in OSCC KEGG pathway analysis was performed Depending on the Pvalue and enrichment signal pathways associated with OSCC were identified Table SII The top KEGG enrichment terms of DEGs are shown in Fig 1B including cell cycle pathways in cancer cell cycleyeast meiosisyeast and cytokinecytokine receptor interaction Among these it was reported that some genes such as MCM5 cell division cycle related protein kinase and Cyclindependent kinase homolog were primarily enriched in the cell cycle pathway Some genes such as lysozyme C statherin and aquaporin were enriched in the saliva secretion pathway MCM5 which participated in cell cycle regulation and had high expression in OSCC was selected for further study and it was hypothesized that MCM5 might be a candidate tumor marker for OSCCValidation of MCM5 using RTqPCR To further verify the aforementioned expression profile data MCM5 expression levels were investigated using RTqPCR in tumor and Figure Relative expression levels of MCM5 mRNA in paired adjacent normal tissues and oral squamous cell carcinoma tissues using quantitative PCR The relative expression data were analyzed by the ÎÎCq method GAPDH was used as an internal control P005 P001 vs adjacent normal tissue MCM5 minichromosome maintenance proteinadjacent normal tissues As shown in Fig MCM5 mRNA was significantly upregulated in of tumor tissues compared with that in matched normal tissues These results showed that MCM5 was highly expressed in OSCC tissues which was in line with the sequencing data Association of MCM5 expression levels with clinicopathologic features of OSCC and survival analysis The results of the potential association between MCM5 expression and clinicopathological features in patients with OSCC are presented in Table III No significant association with MCM5 expression was found for age sex histological differentiation metastasisrecurrence and survival status P05 0cONCOLOGY LETTERS Value n MCM5 expressionLow n3 High n12 Table III Association between expression of MCM5 and clinicopathologic features of patients with oral squamous cell carcinoma Characteristic Age years ¥ Sex Male Female Histological differentiation Well and Moderate Poor MetastasisRecurrence Yes No Survival status Death Survival PvalueA KaplanMeier analysis of OS is shown in Fig Analysis of clinical data from TCGA showed that high MCM5 expression was no associated with a shorter OS in patients with OSCC logrank P062 These results suggested that MCM5 might not be a prognostic biomarker for OSCC Inhibitory effect of MCM5 in OSCC cell lines To determine the functional role of MCM5 first MCM5 expression was analyzed using RTqPCR in two OSCC cell lines Notably SCC cells expressed significantly higher levels of MCM5 compared with Cal cells P001 Fig 4A Considering that knockdown of MCM5 in the cell line with high MCM5 expression may bring about more significant changes the SCC cell line was selected for further investigation of the functional role of MCM5 Three specific siRNA sequences were designed to inhibit MCM5 expression and transfected in SCC cells and the impact on MCM5 expression was determined using RTqPCR As shown in Fig 4B siRNA1 siRNA2 and siRNA3 transfection decreased MCM5 expression by 651P001 P001 and P001 respectively compared with the negative control Then the efficiencies were confirmed using western blotting Fig 4C The inhibitory effect of siRNA1 and siRNA2 was significant but not found in siRNA3 The results were consistent with those of RNA expression siRNA1 transfection reduced MCM5 expression significantly in SCC cells Therefore siRNA1 was used for subsequent experiments Effect of MCM5 inhibition on proliferation colony formation and migration To determine whether MCM5 regulates cell cycle and modulates cell proliferation in OSCC the effect of inhibiting MCM5 expression on SCC cell proliferation was investigated As shown in Fig 5A the results showed that downregulation of MCM5 had significant antiproliferative Figure Survival curves from The Cancer Genome Atlas datasets n518 containing high and low MCM5 expression levels MCM5 minichromosome maintenance protein HR hazard ratio TPM transcripts per millioneffect compared with the negative control P001 Colony formation assays were performed and the results revealed that compared with the number of colonies in the control group downregulation of MCM5 significantly reduced colony formation P001 Fig 5B and C To estimate the impact of MCM5 on OSCC migration scratching assays were conducted and inhibition of MCM5 showed no significant impact on the migration of SCC cells P005 Fig 5D These results suggested that inhibiting MCM5 expression inhibited cell proliferation and colony formation but had no effect on migration in SCC cells\x0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAFigure Inhibition of MCM5 expression in oral squamous cell lines A MCM5 expression in Cal and SCC cells B Efficiency of siRNAMCM5 was confirmed by reverse transcription quantitativePCR in SCC cells C The efficiency of siRNAMCM5 was confirmed by western blot in SCC cells MCM5 minichromosome maintenance protein si small interfering NC negative controlFigure Effects of MCM5 inhibition on antiproliferation colony formation and migration capacity in SCC cells A Downregulation of MCM5 expression suppressed SCC cell proliferation compared with the corresponding control at different time points B Downregulation of MCM5 expression inhibited SCC cells colony formation compared with the corresponding control C Quantification of MCM5 inhibition on colony formation compared with the corresponding control D Cells scratching wounds observed by microscopy E Downregulation of MCM5 had no effect on the migration capacity of SCC cells P001 vs NC MCM5 minichromosome maintenance protein si small interfering NC negative controlEffect of MCM5 inhibition on cell cycle To determine the potential mechanism for the observed proliferation inhibition of SCC cells by MCM5 inhibition cell cycle analysis was performed using flow cytometry As shown in Fig 6A and B after MCM5 inhibition the number of cells were decreased in the G0G1 phase and the S phase but significantly increased in the G2M phase compared with the negative control These results indicated that MCM5 inhibition significantly induced G2M phase arrest compared with that in the control group\x0cONCOLOGY LETTERS Figure Effects of MCM5 inhibition on cell cycle regulation in SCC cells A Flow cytometry assays were performed to analysis the cell cycle progression when SCC cells transfected with siRNAMCM5 B The bar chart represented the percentage of cells in G0G1 S or G2M phase as indicated C Expression levels of cell cycleregulated genes detected by quantitative PCR and normalized to GAPDH D Expression levels of cell cycleregulated proteins determined using western blotting βactin was used as the loading control Data are presented as mean ± SD P005 P001 vs control group MCM5 minichromosome maintenance protein si small interfering NC negative control CDK2 cyclindependent kinase To elucidate the mechanism underlying this effect the expressions levels of cyclin E cyclindependent kinase CDK2 and p21 related to cell cycle arrest were determined using RTqPCR As shown in Fig 6D MCM5 inhibition decreased both cyclin E and CDK2 mRNA levels but increased the mRNA expression of p21 significantly Then cyclin E and p21 were selected to detect the protein levels using western blotting As shown in Fig 6C cyclin E levels decreased while p21 levels increased in MCM5downregulated SCC cells which was consistent with the RTqPCR results DiscussionDespite notable progress in cancer research and treatment the survival rate of patients with OSCC has not significantly improved in the past few decades To date there are no effective tumorspecific biomarkers for the early detection and prognosis prediction of OSCC Several studies have shown that DEGs serve an important role in the development of tumors in different cancer types However few studies have reported differentially expressed genes in OSCC The present screened genes that regulate the progression of oral cancer by gene expression profiling and found that genes were dysregulated of which DEGs were upregulated and were downregulated DEGs significantly affected GO terms and KEGG pathways MCM5 did not have one of the highest log2 foldchange values and log10 qValues however MCM5 is one of the differentially expressed genes in cell cycle signaling pathway which was the most significant enrichment of differentially expressed genes Therefore MCM5 which regulates the cell cycle was selected for further investigation However previously published studies on biomarkers in OSCC mainly focused on pathological studies The present study not only verified the overexpression of MCM5 in OSCC but also confirmed using cell experiments that MCM5 affects cell proliferation by regulating the cell cycle MCM5 is a member of the MCM family of proteins and is a component of the starting complex for DNA synthesis MCM5 has been identified as a cell cycle biomarker of aberrant proliferation which is associated with the progression of various cancer types Previously MCM5 has been found to be overexpressed in numerous human malignancies such as esophageal thyroid and ovarian cancer For example increased MCM5 levels in urine sediment cells predicts the presence of bladder cancer Inhibition of transcription factor SOX can inhibit the proliferation of skin melanocytes and MCM5 expression is significantly decreased following downregulation of SOX Moreover high expression of MCM5 is associated with poor prognosis and poor malignant status in patients with cervical adenocarcinoma It is well known that immunohistochemistry and western blotting are necessary methods to evaluate protein expression 0cHAO MCM5 IS A POTENTIAL EARLY DIAGNOSTIC MARKER FOR ORAL SQUAMOUS CELL CARCINOMAHowever due to a limited number of tissue samples the present study did not have enough samples for simultaneous qPCR western blotting and immunohistochemistry detection Therefore this is a limitation of the present study However the study did perform qPCR to evaluate the expression of MCM5 at the mRNA level The results demonstrated that of patients with OSCC have high MCM5 expression which is consistent with the results of the aforementioned studies indicating that high MCM5 expression may play an important role in the pathogenesis of OSCC In addition other members of the MCM family of proteins have also served as biological markers of dysplasia and malignancy such as glioma cervical colorectal breast prostate and lung cancer Therefore some researchers even suggested that changes in MCM5 expression may be a sign of cell cycle disorders It is worth noting that some researchers reported that the high expression of MCM family members may be closely related to tumorigenesis and prognosis For example MCM2 MCM4 and MCM6 are overexpressed in breast cancer of high histological grades MCM7 expression is a potent prognostic marker in nonsmall cell lung cancer while MCM5 may be an independent adverse prognostic marker in lung squamous cell carcinoma It is well known that the cell cycle is related to cell proliferation signaling pathways In most tumors an increase in the expression level of genes encoding proteins that regulate cell proliferation is observed The abnormal expression of cellcyclerelated genes is associated with infinite proliferation of tumors and poor prognosis Thus far only Yu reported the relationship between MCM5 and OSCC The study reported that overexpression of MCM5 in patients with OSCC was significantly associated with tumor site tumor size positive lymph node metastasis later clinical stage higher histological grade deeper infiltration depth and peripheral nerve infiltration However in the present study association between high expression of MCM5 with survival metastasis and poor histologic differentiation was not observed A KaplanMeier analysis of the overall survival rate was not significantly changed in patients with high MCM5 expression compared with patients with low MCM5 expression It was speculated that due to the small number of samples that there were large differences between individuals Therefore in future research a larger sample size should be used to clarify the relationship between high expression of MCM5 and prognosis of OSCC Little is known about the role and potential function of MCM5 in OSCC In the present study a lossoffunction analysis was conducted and it was demonstrated that MCM5 participated in regulating cell cycle and cell proliferation in OSCC cells In fact inhibiting the expression of MCM5 in SCC cells resulted in the downregulation of cyclin E and CDK expression and upregulation of p21 expression which ultimately led to G2M phase arrest in oral cancer cells These results further verified that MCM5 is highly expressed in patients with OSCC which promotes the proliferation of OSCC cells and regulates cell cycle In addition it was observed that MCM5 was not only expressed in SCC cells but also expressed in CAL cells Fig 4A Notably according to the ATCC the MCM5 gene had no mutations in either of the two cell lines indicating that the two cell lines selected in this study have similar genetic backgrounds and could be used for the study of MCM5 cell functions Considering MCM5knockdown experiments in SCC cells with high MCM5 expression received more significant results SCC cells were selected for followup studies However analyzing the functional effects of MCM5knockdown in CAL27 cell lines may provide more information In addition both SCC and CAL cells are transformed cell lines In future investigations untransformed cell lines for multiple comparisons should be used to clarify the role of MCM5 in OSCC Surgical resection is currently the main method to treat OSCC However considering the particularity of the oral structure surgical resection will lead to a huge impact on patients' quality of life Therefore it is important to find effective diagnostic biomarkers for early detection or to develop targeted drugs for OSCC In the present study it was reported that MCM5 is overexpressed in OSCC and that MCM5 can affect cell proliferation by regulating cell cycle Therefore the results suggested that MCM5 might be one of the important pathogenic factors of OSCC and is expected to be used as a potential tumor marker for OSCC target drugs The specific mechanism of action of MCM5 is still worthy of further investigationOverall the present study evaluated differentially expressed genes using sequencing patterns in OSCC tumor tissues and further validated MCM5 upregulated expression in OSCC tissues By knocking down MCM5 expression in SCC cells it was revealed that cell proliferation and colony formation was significantly inhibited by inducing G2M phase arrest The results also suggested that during this process cyclin E and cell cyclerelated gene expression levels were decreased while p21 was significantly upregulated Therefore MCM5 may modulate OSCC cell proliferation by regulating the cell cycle MCM5 is an important pathogenic factor and might have important role as a potential diagnostic marker or drug target for OSCCAcknowledgementsNot applicableFunding This study was funded by the National Natural Science Foundation of China grant no the Bethune Project of Jilin University of China grant no 2018B02 the Education Department of Jilin Province grant no JJKH20190074KJ and Department of Science and Technology of Jilin Province grant no 20190103086JH and 20200201398JC Availability of data and materials The datasets used andor analyzed during the present study are available from the corresponding author on reasonable request The other datasets generated andor analyzed during the current study are available in The Cancer Genome Atlas wwwcancergov Gene Ontology httpgeneontology UniProt sparqluniprot NCBI wwwncbinlmnihgov and Kyoto Encyclopedia of Genes and Genomes wwwkeggjp databases\x0cONCOLOGY LETTERS Authors' contributions HW CZ and CL performed the experiments HW MH and XW analyzed the data MH and HW wrote the manuscript MH and XW designed and supervised | 2 |
" 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 | 0 |
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