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Genetic alterations in the 3q263132 locus conferan aggressive prostate cancer phenotypeBenjamin S SimpsonSusan Heavey Jason Pitt5 Caroline M Moore6 Hayley C Whitakerœ‰ 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 ofspecific changes in many identified 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 gainsamplifications are more prevalent in aggressivesubtypes of prostate cancer when compared to primary cohorts In primary disease gainsamplifications occurred in CI“ and CI“ for NAALADL2 and TBL1XR1 respectively increasing in frequency in higherGleason grade and stage tumours Gainsamplifications 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 UKœ‰email HayleyWhitakeruclacukCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xProstate cancer PCa is the most common noncutaneouscancer in developed countries12 and is defined 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 definedOverall copynumber alteration CNA burden has been linkedto poorer prognosis in prostate cancer associating with Gleasongrade biochemical recurrence and prostate cancer specific deathhowever the exact mechanism driving these prognostic changes isunknown and thought to be primarily driven by general chromosomal instability4“ Changes in specific 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 identified 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 severalcellularand‚ammation17 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 gainsamplifications 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 subtypes19“Alteration 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 Significantcopynumber 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 goodnessoffittestFig 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 goodnessoffit 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 — ˆ’ and p — ˆ’ 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 CNA™s 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 confineddisease 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 Identification of Significant Targets in Cancer GISTIC2 methodfavoured by the broad institute andTCGA27 as it distinguishes between lowlevel copy numberincreases gains and highlevel copynumber increases amplifications Within the three cohorts we found that copynumbergains across both genes were frequent with gains in NAALADL2ranging from Canada to UK and between“ for amplifications Table TBL1XR1 had an almostidentical CNA frequency of between UK to Canada Table We fitted a randomeffects model to more accurately estimatethe frequency of NAALADL2 and TBL1XR1 gainamplificationscombining 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 refit The final estimated frequency ofNAALADL2 and TBL1XR1 gainsamplifications was CI “ and CI “ respectivelyin primary prostate cancerDue to their close proximity in the genome we investigated ifgainsamplifications in NAALADL2 and TBL1XR1 cooccurred inthe same patients using a genomewide Fisher™s exact test with afalse discovery rate correction NAALADL2 and TBL1XR1significantly coamplified in all three cohorts ICGC UK p — ˆ’ ICGC Canada p 158e — ˆ’ and TCGA p — ˆ’testingconfirmed that widespanning gainsamplifications occurred inneighbouring regions in the majority of patients In the ICGC UKSupplementary Fig Additionallycohort n there was a significant cooccurrence of somaticcopynumber gainsamplifications in NAALADL2 with TBL1XR1FDRcorrected Fisher™s exact test — ˆ’ 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 gainsamplifications 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 coamplification 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 Fisher™s 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 defined region bycomparing the overlapping genes that coamplified 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 resultsconfirm 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 findings we also found NAALADL2 andTBL1XR1 amplifications 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 Chisquaredgoodnessoffit test showed that the distribution of gainsamplifications between Grade groups was significantly different to thedistribution of diploid patientsfor both NAALADL2 andTBL1XR1 p — ˆ’ and p — ˆ’ WhenTable Alteration frequency of NAALADL2 and TBL1XR1 called via the GISTIC2 method in three nonoverlapping primary anconfined radical prostatectomy cohorts from the International Cancer Genome Consortium ICGC and The Cancer GenomeAtlas TCGAICGC UKICGC CANADATCGANAALADL2TBL1XR1NAALADL2TBL1XR1NAALADL2TBL1XR1AlterationDeep DelShallow DelDiploidGainAmplificationTotalnnnnnnThe degree of copy number alteration is discretised into five categories amplification 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 positivitydefined 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 goodnessoffit test p and p Finally while only one man in the cohorthad evidence of positive findings 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 gainsChisquaredgoodnessoffit 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 gainsamplifications 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 gainsamplifications 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 confirmed that carrying a gainamplification 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 stratified 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 stratified byNAALADL2 gain TBL1XR1 gain or both NAALADL2 andTBL1XR1 gain showed significant differences in survival Logrank MantelCox p Supplementary Fig 5A“E 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 gainsamplifications The Y axis shows “log10 qvalues from a Fishers exact testbetween gainamplifications in NAALADL2 and cooccuring genes Thedotted line represents the threshold for statistical significance aftercorrection for multiple testing a Significantly cooccurring gains across thegenome in the ICGC UK cohort b Significantly cooccurring gains acrossthe genome in the ICGC Canada cohort c Significantly cooccurring gainsacross the genome in the TCGA cohort NAALADL2 and TBL1XR1 cytobandpositions are labelled All Fisher tests use NAALADL2 gain or amplificationas 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 goodnessoffit 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 goodnessoffit testp and p There was also an increased relativenumber of positive surgical margins Chisquared goodnessoffittest 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 confirm 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 stratified byCOMMUNICATIONS BIOLOGY 101038s4200302001175x wwwnaturecomcommsbio 0cCOMMUNICATIONS BIOLOGY 101038s4200302001175xTable The frequency of NAALADL2 and TBL1XR1 gainamplifications by Gleason Grade Group in the TCGA cohortGrade groupGG1ObservedExpected within GGGG2ObservedExpected within GGGG3ObservedExpected within GGGG4ObservedExpected within GGGG5ObservedExpected within GGTotal nNAALADL2DiploidGainTBL1XR1DiploidGainTotalGrade groups defined 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 gainamplification 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 gainamplifications 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 gainamplification 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 101038s4200302001175xgainamplification 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 profiles 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 NAALADL2identified 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 model2634“When comparing the DEGs between patients with and withoutin either NAALADL2 or TBL1XR1 wegainsamplificationsobserved that of the DEGs overlapped between NAALADL2 and TBL1XR1 Fig 3a of the geneswere located within the locus we identified as coamplified 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 significanceFDR 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 gainsamplifications 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 filamentbased 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 gainsamplification 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 findings 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 identified CNAs in this region inmCRPC however to our knowledge this is the first 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 sufficientsequencing 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 gainsamplifications in a larger regionsurvivalto diseasefreeIn primary prostate cancer Gainsamplifications in this regionassociated with Gleason grade tumour stage number of positivelymph nodes bone scan results and as these variables contributetostratified byNAALADL2TBL1XR1 status also have altered diseasefree survival times Our work is supported by previous studies that haveeluded to the clinical significance 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 identified 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 gainsamplifications 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 gainamplification and TBL1XR1 gainamplification overlap b Venn diagram showing the number ofNAALADL2 and TBL1XR1 DEGs and genes with identified 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 gainamplification 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 PAPSS210q232“10q2331 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 amplification wehave observed that Gainsamplifications in NAALADL2TBL1XR1equally or better segregate patients who will have reduced diseasefree survivalThe gainsamplifications 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 gainamplification in NAALADL2TBL1XR1 have been shown to beandrogen regulated however further work is required to determine if gainsamplifications 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 finding that gains in the 3q26 locus result in concurrentexpression of oncogenes located within this region and theirdownstream targets identifies 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 specificity 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 PIK3CBspecific inhibitors may have utility in patients with mutationsamplifications andor fusion of this gene59 These findings mayhave clinical relevence as it has been reported by de Bono et althat many individuals who had durable year responses toPIK3CBspecific inhibition harboured activating mutation oramplification 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 gainsamplifications occur in the 3q26 region in a hi
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pancreatic cancer is a devastating malignancy with a 5year relative survival rate of only “ dependenton the geographical location surveyed [“] with these statistics exhibiting only modest improvementover the last four decades [“] the median survival postdiagnosis ranges from just “ months forlocally advanced disease and “ months for metastatic disease it was estimated by the world healthanisation that pancreatic cancer is currently the 7th leading cause of cancerrelated death being responsible for over deaths worldwide in with incidence increasing pancreatic cancerhas been predicted to be the third leading cause of cancerrelated death in the european union by and the second leading cause of cancerrelated death in the united states of america and germanyby several factors contribute to the poor survival of pancreatic cancer patients a current lack of reliablediagnostic markers that would enable early screening coupled with largely nonspecific symptoms ofdisease results in over of patients presenting with metastatic disease at diagnosis this subgroupof patients have limited therapeutic options and are thus typically administered palliative chemotherapyaimed at prolonging survival and reducing symptoms during endoflife care [“] moreover whilstapproximately “ of patients present with localised disease that is eligible for potentially curativesurgery disease recurs in over of patients postresection ultimately these factorsculminate in more than of patients diagnosed with pancreatic cancer succumbing to disease these harrowing statistics highlight that despite research efforts there remains a lack of understandingof the pathogenesis of disease which in turn limits the development of new therapeuticsreceived march revised july accepted august version of record published august the authors this is an open access published by portland press limited on behalf of the biochemical society and distributed under the creative commonsattribution license cc byncnd 0cclinical science “101042cs20191211pancreatic ductal adenocarcinomapancreatic ductal adenocarcinoma is the predominant pancreaticmalignancypancreatic cancer can arise from either the endocrine or the exocrine region of the pancreas tumours arising fromthe exocrine compartment are termed pancreatic ductal adenocarcinoma pdac and account for over of allpancreatic cancers pdac develops via a stepwise progression from normal tissue through to invasive lesions which is associated withdistinct morphological characteristics [“] it has been proposed that this process begins with a phenomenontermed acinartoductal metaplasia adm which is a normal homeostatic mechanism whereby acinar cells transdifferentiate into a ductal phenotype in response to particular stimuli however if compounded by an oncogenic˜hit™ cells are pushed towards a pathogenic phenotype that develops into pancreatic intraepithelial neoplasia panin[“] disease progresses through preinvasive stages termed panin1a panin1b panin2 and panin3 priorto invasive pdac this progression is associated with increasing nuclear atypia whereby the nuclei are no longerpositioned basally and loss of normal architecture as cells become more papillary in nature with panin3 lesionsdemonstrating increased mitoses as disease progresses to pdac cells become invasive and breach the basement membrane growing through the extracellular matrix and metastasising to distant ans figure less common precursor lesions include intraductal papillary mucinous neoplasms ipmns and mucinous cysticneoplasms mcns that also develop through multistep processes whilst they share some common featureseach lesion is morphologically and genetically distinct in contrast with panins that form within small ducts ipmnsdevelop within the primary or secondary branches of the main pancreatic duct whilst mcns lack ductal involvement an ‚ammatory tumour microenvironment contributes to pdacpathogenesisan archetypal feature of pdac is the development of extensive stromal networks within the tumour microenvironment tme that can account for up to of the total tumour volume [“] this unique characteristic drives theinflammatory nature of pdac that contributes to its aggressive phenotype desmoplasia is driven by pancreaticstellate cells pscs and cancerassociated fibroblasts cafs that upon activation produce a range of extracellularmatrix ecm components such as collagen laminin and fibronectin which in turn form a physical barrier preventing the penetration of therapeutics [“] though pscs and cafs have been shown to support cancer metastasis and drug resistance they interact with cancer cells in a bidirectional manner with each promoting the survivalgrowth and proliferation of the other [“] quiescent fibroblasts are able to differentiate into two unique subtypes termed myofibroblastic cafs mycafs and inflammatory cafs icafs these two subtypes are distinct whereby mycafs express high levels of αsmooth muscle actin αsma and are located adjacent to cancercells while icafs express low levels of αsma and instead secrete high levels of inflammatory mediators including il6 and are distributed distant from cancer cells within desmoplastic tumour regions broadly mycafsappear to have roles in epithelialtomesenchymal transition emt and ecm remodelling whilst icafs appear tobe involved in inflammation and ecm deposition a third less abundant subtype termed antigenpresentingcafs apcafs has more recently been defined these cells express low levels of both αsma and il6 andinstead express high levels of major histocompatibility complex class ii mhcii and related genes as such allthree subtypes are transcriptionally and functionally distinctthe wider tme contains a plethora of other cell types including endothelial cells tumourassociated macrophagestams and neutrophils tans mast cells regulatory tcells myeloidderived suppressor cells mdscs dendriticcells natural killer nk cells and nerve cells interactions between various cells within the tme can driveeither proor antitumorigenic functions of others for example cancer cells can induce pscs to secrete inflammatorycytokines that drive immune cells towards an immunosuppressive phenotype and also form a positive feedback loopby increasing the tumorigenic potential of cancer cells the ecm itself has also been suggested to modifypsc behaviour in particular that ecm stiffness promotes the mycaf phenotype indicated by increased αsmaexpression this results in substantial complexity that ultimately determines tumour phenotype the components of the microenvironment modify tumour behaviour through the production of cytokines growthfactors and other signalling molecules that predominantly drive a proinflammatory and immunosuppressive program that enhances pdac tumour growth and progression [“] figure the ability of the tme toinhibit therapeutic efficacy through both molecular mechanisms and physical fibrotic barriers contributes to the 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 byncnd 0cclinical science “101042cs20191211figure our current understanding of the contribution of il6 family cytokines to panin and pdac developmentpreinvasive panin lesions develop from normal ductal epithelia through panin stages 1a 1b and to stage invasive andormetastatic pdac this process is associated with acinartoductal metaplasia adm early in disease combined with an accumulation of oncogenic mutations most common mutations are indicated a number of cells within the tumour microenvironment havebeen shown to secrete il6 family cytokines which in turn results in the activation of a protumorigenic signalling cascade a betterunderstanding of the relationship between each of these cells within the tumour microenvironment may reveal new therapeuticopportunities to manage cancer progressionintrinsic resistance of disease thus dual targeting of cancer cells and the tme may be required to induce afavourable therapeutic response although this poses a signficant scientific and clinical challenge [“]molecular basis of pathogenesispdac development is associated with accumulation of mutationsthe progression of tumorigenesis through panin and pdac stages is associated with the stepwise accumulation ofspecific genetic mutations that drive malignant transformation the most frequent genetic alteration is an activatingkras point mutation codon that occurs early on in tumour development and is detected in over ofpdac tumours [“] mutations in kras have been shown to drive development of precursor panin lesions andwhen combined with an appropriate tumour suppressor mutation these lesions progress to invasive or metastaticpdac figure patient tumours harbouring wildtype wt kras often carry activating mutations indownstream effector molecules such as braf or pik3ca 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 byncnd 0cclinical science “101042cs20191211inactivation of a range of tumour suppressor proteins is also common including mutations in tp53 cdkn2aand smad4 in approximately and of tumours respectively whilst each mutation has uniquemechanistic outcomes all three proteins are either directly or indirectly involved in the regulation of the g1s cellcycle checkpoint analysis of patient tumours indicates that two or more of these mutations often occur together withcdkn2a mutation being combined with either tp53 smad4 or both usually in the background of kras mutation this suggests that by disrupting this checkpoint cancer cells are able to overcome inhibitory mechanismsallowing continued progression to invasive diseaseunbiased sequencing efforts have also enabled identification of low prevalence pdac mutations observed in lessthan of cases these include mutations in genes involved in chromatin modification kdm6a dnadamage repair atm and other tumourrelated processes such as growth tgfbr1 or tgfbr2 furthermore it is important to note that technical advances are continuously uncovering epigenetic mechanisms thatfurther modulate the pdac transcriptional landscape and ultimately influence disease heterogeneity and tumourprogression molecular subtypesthe pdac epithelial compartment is typically divided into two subtypes including a classical subtype exhibiting anepitheliallike expression profile and a squamous or mesenchymallike subtype an additional third exocrinesubtype is outlined in some analyses and is characterised by a gene expression profile related to digestive enzymeproduction the classical or epithelial subtype has also been further divided into a pancreatic progenitor andan immunogenic subtype whereby the immunogenic subtype is distinguished by significant immune infiltration andassociated gene programmes though there is no consensus on which classification system will allow the mostvaluable stratification of patients the mesenchymal subtype is invariably associated with a poor prognosis the stromal compartment has also been classified into either normal or activated subtypes reflecting the proandantitumorigenic capabilities of the tme with the activated subtype associated with reduced survival this isparticularly valuable as the extensive heterogeneity of pdac complicates clinically relevant stratification of patientsthus the identification of molecularly unique subtypes may enable development of tailored therapeutic regimensthat will provide improved clinical outcomescurrent treatment optionsregardless of disease stage at time of diagnosis patients have relatively limited treatment options for the majorityof patients disease will be locally advanced or metastatic disqualifying them from undergoing potentially curativesurgery in these cases patients are typically offered chemotherapy with palliative intent [“]surgery provides the only potentially curative treatmentsurgical resection remains the only potentially curative treatment option due to minimal efficacy of standardofcarechemotherapy and radiotherapy due to its aggressive nature the majority of patients present to clinic with locallyadvanced or metastatic disease with only “ of patients presenting with localised tumours that are eligiblefor surgical resection even for those able to undergo surgical intervention over of patients relapsepostresection with median survival improving to months and 5year relative survival rate increasingmodestly to “ the use of neoadjuvant therapy is generally reserved for borderline resectable disease inan effort to enable patients to become eligible for surgery however a range of recent trials have shown improved clinical outcomes including overall survival for neoadjuvant treatment of patients with resectable tumours following surgical resection patients are typically treated with adjuvant gemcitabinebased chemotherapy although a recent study showed improved diseasefree survival and overall survival with a modified folfirinoxtherapy combination of oxaliplatin irinotecan leucovorin and fluorouracil radiotherapy provides variable clinical outcomewhilst the use of radiotherapy and chemoradiotherapy combination chemo and radiotherapy in the neoadjuvantand adjuvant settings have been investigated there remains a lack of consensus regarding therapeutic benefit this is due to issues such as insufficient radiation dose and low participant numbers as well as low uptake of moderntechniques in the neoadjuvant setting preliminary studies reported reduced lymph node positivity and rates oflocal recurrence for chemoradiotherapy compared to surgery with adjuvant chemotherapy however the useof radiotherapy in the palliative setting was reported to modestly reduce overall survival more recent studiesusing ablative doses of radiation have shown a survival benefit highlighting that technological advancements mayprovide an avenue for improved clinical outcomes following radiotherapy these contrasting results highlight the 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 byncnd 0cclinical science “101042cs20191211need to determine which subset of patients may benefit from the inclusion of radiotherapy approaches in standardtreatment regimenschemotherapy remains the cornerstone of treatmentdespite modest improvements in overall survival palliative chemotherapy remains the standard treatment optionfor patients with locally advanced or metastatic disease gemcitabine monotherapy has been the mainstay treatmentfor pancreatic cancer since when it was demonstrated to improve median survival by just over month compared with fluorouracil within the last decade there have been some further improvements in clinical outcomewith combination chemotherapies gemcitabinenabpaclitaxel and folfirinox providing median overall survivalbenefits of and months respectively compared with gemcitabine alone although folfirinox treatment resulted in a lower percentage of patients experiencing reduced quality of life it also had increased toxicity andadverse events thus preventing its administration to patients with multiple comorbidities therapeutic resistance remains a significant barrier to patient survivaldespite advances in chemotherapeutic options treatment efficacy and patient prognosis remain poor due to the inherent therapeutic resistance of pancreatic cancer it has been proposed that this drug resistance may be driven by thetme including changes to cytokine signalling and metabolic pathways [“] this intrinsic resistance is demonstrated by patients experiencing similar overall survival for chemotherapy treatment “ months compared withbest supportive care “ months which encompasses the use of palliative surgery psychological support painmanagement and other methods of symptom control whilst a range of targeted treatments such as egfr orcheckpoint inhibitors have been trialled with or without chemotherapy they have shown limited success [“]emerging roles for the il6 family of cytokines in pdaccytokines are soluble molecular messengers that enable efficient communication between a range of cell types andhave been recognised to be major contributors to the growth and metastasis of cancers [“] in pancreatic cancer cytokines mediate signalling between cancer cells and the cells of the tme including pscs cafs endothelialcells and a range of immune cells including macrophages mast cells neutrophils and regulatory tcells [“]it is the specific signalling pathways active within this community of cells that dictates the balance of pro andantitumorigenic functions the il6 family of cytokinesthe interleukin il6 family of cytokines includes il6 il11 leukaemia inhibitory factor lif oncostatin mosm ciliary neurotrophic factor cntf cardiotrophin1 ct1 cardiotrophinlike cytokine clc neuropoietin np il27 and il31 [“] these cytokines are grouped as they share structural similarity forming a fourαhelical bundle termed helices ad with an upupdowndown topology il6 and il11 utilise a hexameric signalling complex consisting of two molecules each of the cytokine αreceptoreither il6r or il11r respectively and βreceptor glycoprotein gp130 [“] il6 and il11 are ableto signal via two distinct mechanisms termed classic and transsignalling classic signalling involves the formation of a complex including membranebound il6r or il11r with gp130 and the respective cytokine converselytranssignalling utilises soluble il6r or il11r molecules which are able to form a signalling complex with gp130and the respective cytokine [“] in this way classic signalling relies on the responding cell™s intrinsic expressionof il6r or il11r whilst transsignalling is able to activate any cell expressing gp130 lif osm il27 and il31 signal through trimeric complexes with a single cytokine molecule engagingthe respective receptor lifr osmr il27r wsx1 or il31r and either gp130 or osmr for il31[“] cntf ct1 clc and np form tetrameric signalling complexes composed of one cytokinemolecule one lifr one cntfr and one gp130 receptor in each case the active signalling complex consists of two chains that are signalling competent with a combination of either gp130 lifr osmr il27r or il31r the requirement for multiple receptor subunits means that although gp130 is ubiquitously expressed the expression of other receptor subunits dictates the ability for any given cell to respond to cytokine as signalling initiationrequires the presence of cytokine and a compatible receptor complex figure 2asignalling complex assembly leads to transphosphorylation and activation of receptorassociated janus tyrosinekinases jaks largely jak1 and to a lesser extent jak2 and tyk2 in the case of gp130mediated signalling this results in phosphorylation of the cytoplasmic domain of gp130 at tyrosine y and phosphotyrosine py and of gp130 provide docking sites for signal transducer 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 byncnd 0cclinical science “101042cs20191211figure il6 family cytokine signalling pathwaya schematic representation of the stepwise binding process for the il6 family members with il6 as an example the site interaction involves cytokine binding to the respective receptor with the site interaction generally between the cytokine and thecommon gp130 receptor chain finally site interactions involve formation of the final active signalling complex in this case formation of the il6il6rgp130 hexameric complex b general outline of the il6 family cytokine signalling pathway formation ofan active hexameric complex leads to activation of jaks with subsequent activation of the stat3 mapk and pi3k pathways leftthis results in upregulation of the negative regulator socs3 as well as a range of inflammatory and protumorigenic moleculesthe pathway is inhibited by socs3 pias3 and ptps via dephosphorylation ubiquitinmediated proteasomal degradation andsumoylation right 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 byncnd 0cclinical science “101042cs20191211activator of transcription stat molecules leading to their subsequent phosphorylation by jak1 and formation ofactive stat dimers [“] phosphorylated stat pstat dimers then translocate to the nucleus and modulatetarget gene expression including upregulation of a range of genes involved in inflammatory and protumorigenicpathways [“] figure 2b broadly these stat3regulated genes can be categorised into pathways associatedwith inhibition of apoptosis increased cell proliferation modulation of immunity and inflammation increased angiogenesis and increased invasive and metastatic potential [“]although jakstat signalling is the predominant pathway activated downstream of il6 family cytokines themitogenactivated protein kinase mapk and phosphoinositide 3kinase pi3k pathways can also be activated the mapk pathway has been suggested to be activated by a src homology domain 2containing phosphatase shp2mediated mechanism whereby shp2 is recruited to py759 on gp130 allowing jakmediated phosphorylation of shp2 this promotes association with the adaptor protein growth factor receptor bound protein grb2 leading to activation of the gprotein ras via son of sevenless sos with a subsequent phosphorylationcascade including raf mek and erk12 activity following this a mapkdependent phosphorylationevent leads to the recruitment of grb2associated binding protein gab1 to the plasma membrane where gab1 issuggested to act as a scaffold or adaptor protein to allow binding of pi3k and shp2 leading to activation of the pi3kand mapk pathways respectively figure 2bthe suppressor of cytokine signalling socs3 is largely responsible for regulation of signalling and is directlyupregulated by stat3 socs3 contains an sh2 domain allowing it to bind to py residues within the gp130receptor with preferential binding to y759 once bound socs3 recruits an e3 ubiquitin ligasecomplex containing cullin5 rbx2 and adaptors elongin b and c via its socs box domain this complexubiquitinates the gp130 receptor inducing its internalisation and targeting it for proteasomal degradation [“]and is also able to ubiquitinate jak2 in vitro socs3 also mediates direct inhibition of the kinase activityof jak12 via its kinase inhibitory region [“] thus socs3 is able to downregulate il6 family cytokinesignalling pathways through two distinct mechanismsthe phosphotyrosine phosphatases ptps and protein inhibitors of activated stats piass also limit the strengthand duration of cytokine signalling a range of ptps including shp2 are responsible for dephosphorylatingtyrosine phosphorylated substrates including jaks stats and other shp2 molecules pias3 preferentially binds pstat3 and inhibits activity either by preventing stat3 interaction with dna by recruiting transcriptional repressors to stat3 target genes or by sumoylating stat3 to prevent its activity figure 2binterleukin in pdacelevation of serum il6 is a negative prognostic marker in human pdacserum il6 levels were increased in pdac patients compared with healthy patients [“] or those withchronic pancreatitis and were also increased in patients with metastatic pdac compared to thosewith locally advanced disease [“] moreover elevated serum il6 positively correlated with increased diseaseburden weight losscachexia and metastasis [““] however there are conflicting observations inthe literature regarding il6 and cachexia although increased serum il6 levels correlate with increased disease stage and in metastatic patients correlates with poor overall survival as such it has been suggestedthat il6 may be a superior marker for diagnostic and prognostic purposes compared with the standard creactiveprotein crp carcinoembryonic antigen cea and carbohydrate antigen ca199 markers il6 is expressed within the tmell6il6 was overexpressed in human pdac tumours in comparison with adjacent normal tissue whilstthis tumourspecific elevation has been correlated with reduced survival in some studies othersshowed no significant correlation with survival similar to the data available in the cancer genome atlastcga dataset for both il6 and il6r figure 3ab the tcga comprise aggregate sequencing data which doeshave limitations regarding interpretation of contributions of individual cell populations to disease outcome howeverit remains a widely used resource for exploratory investigations however overexpression of il6 has been observedat the mrna and protein level in the pancreata of pdac mice with il6 expression increasing with agewhich is indicative of disease stage in these models despite the presence of il6 in tumours primary human and commercial pancreatic cancer cell lines have been reported to exhibit variable expression levels of il6 and secreted cytokine albeit consistently higher than normal pancreatic ductal epithelial cells in an anoid model minimal il6 was expressed by pancreaticcancer cells pccs or pscs in monoculture however in coculture pccs expressed only il6ra whilst icafs expressedhigh levels of il6 with this activating stat3 within pccs icafs also demonstrate an upregulation of 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 byncnd 0cclinical science “101042cs20191211figure il6 family cytokine expression in pdac patientsoverall survival for patients with high top quartile and low bottom quartile level expression of a il6 b il6r c il11 d il11re lif f osm g cntf h ctf1 ct1 i clcf1 clc and j il27 n per group data and graphs obtained fromoncolnc using data from the cancer genome atlas tcga statistical significance determined by mantelcox logranktest 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 byncnd 0cclinical science “101042cs20191211the jakstat pathway with expression of il6 being dramatically increased in vitro when incubated with pcc conditioned media indicating that soluble factors trigger il6 production more recently pccderived il1αhas been shown to induce autocrine lif secretion and thereby promote the icaf phenotype including activation ofthe jakstat signalling pathway and il6 production in addition tams have been identified as producers of il6 in pancreatic cancer by correlative immunohistochemistry and expression analysis of isolated cell populations production of il6 by tams was shownto influence tumour development via bonemarrow chimeras as mice reconstituted with il6 knockout ko il6myeloid cells developed lowgrade panins whilst those reconstituted with il6 wt cells developed panin3 lesions il6 is a driver of pdac pathogenesisboth in vitro and in vivo studies suggest that the presence of il6 in the tme can drive activation of stat3 with il6 inhibition reducing stat3 phosphorylation this il6stat3 program has been proposed tobe a driver of pdac pathogenesis by enhancing tumour initiation and progression angiogenesis regulation of cytokine expression and immune cell behaviour resistance to apoptosis and promotion of metastasis [“] in aninducible krasdriven mouse model genetic deletion of il6 resulted in a reduction of adm and panin formationwhen kras mutation was initiated embryonically compared with controls suggesting a role for il6 in tumour initiation this was also observed in a constitutive kras mutant model where genetic deletion of il6 preventedtumour initiation in vivo with a reduction in the number of panin and lesions interestingly oncogenickras and hypoxic conditions both features of pdac tumours were shown to induce il6 production perhaps representing a feedforward pathway enhancing tumorigenesis however il6 is notabsolutely required for panin formation as induction of kras mutation at weeks of age in conjunction with anexperimental pancreatitis model drove formation of panin lesions that were not significantly different between il6wt and ko mice il6 mice exhibited reduced tumour progression with decreased proliferative capacity of both cancer and stromal cells enabling regression of precursor lesions furthermore this inhibition of tumour progression by il6deletion was due at least in part to the reversal of adm with ductal cells reverting to an acinarlike phenotype increased apoptosis of cancer and stromal cells was also shown to contribute to this reduced tumour progression as demonstrated by appropriate immunohistochemical analyses with upregulation of proapoptotic anddownregulation of antiapoptotic bcl2 family members this is mirrored by in vitro data whereby il6 stimulation increased the expression of antiapoptotic bcl2bcl2 and bcl2l1bclxl with blockade of il6signalling or stat3 activation inducing apoptosis collectively these data suggest that whilst il6 contributes it is not required for pdac initiation and progressionthe process of angiogenesis supports tumour growth and progression by enabling adequate blood supply whichis enhanced by il6 signalling upon il6 stimulation pdac cell lines upregulate key angiogenic factors such asvascular endothelial growth factor vegfvegf and neurophilin1 nrp1nrp1 with significant correlation observed between the expression of il6r and vegf on human pdac sections il6inducedupregulation of vegf correlated with a growth advantage in pccs with both features inhibited by treatment witha jak2 inhibitor another facet of the protumorigenic effects of il6 is the regulation of cytokine expression that enables modulationof the immune system in particular it has been shown that il6 is able to upregulate a type cytokine profile invitro that may inhibit antitumour immunity in disease il6 suppressed the differentiation of human cd14cells into dendritic cells dcs in vitro whilst combination treatment with il6 and granulocyte colonystimulatingfactor gcsf inhibited the ability of dcs to respond to alloantigen a process that is required for dc maturationand antigen presentation where these effects were reversed by blockade of il6 andor gcsf il6 has alsobeen implicated in driving increased apoptosis of type i conventional dcs cdc1s leading to cdc1 dysfunctionearly in tumorigenesis and thereby decreased cd8 tcell activation this notion is further supported by invivo studies whereby genetic deletion of il6 in a krasdriven pdac mouse model exhibited a significant decreasein the percentage of intratumoral cancerpromoting macrophages and mdscs utilising primary human pscsmdsc differentiation was shown to be driven by pscderived il6 in a stat3dependent manner the resultantmdscs were able to suppress tcell proliferation suggesting a role for il6 in promoting an immunosuppressivetme correlative analysis indicates that il6 through the generation of metabolic stress indirectly causes a reductionin the percentage of intratumoral natural killer nk cells cd4 and cd8 tcells in precachectic and cachecticmice this effect was coupled to a reduction in the expression of an array of genes involved in immune cell 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 byncnd 0cclinical science “101042cs20191211recruitment and tcell effector function indicating that il6 is able to directly and indirectly modulate immuneregulation to enhance tumorigenesisemt migration and invasion are prerequisite abilities that are required for tumour metastasis stimulation of pccswith il6 modulated the expression of a range of proteins that drive emt and migration including upregulationof snai1snai1 snail snai2 slug cdh2 ncadherin vim vimentin fn1 fibronectin col1a1 collagen and twist2 and downregulation of cdh1cdh1 ecadherin with these effects mitigated by il6 orstat3 inhibition il6treated pccs and preinvasive panins exhibited morphological cha
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Circulating tumor cells CTCs derived from primary tumors andor metastatic tumors aremarkers for tumor prognosis and can also be used to monitor therapeutic efficacy andtumor recurrence Circulating tumor cells enrichment and screening can be automatedbut the final counting of CTCs currently requires manualintervention This not onlyrequires the participation of experienced pathologists but also easily causes artificialmisjudgment Medicalimage recognition based on machine learning can effectivelyreduce the workload and improve the level of automation So we use machinelearning to identify CTCs First we collected the CTC test results of patientsAfter immunofluorescence staining each picture presented a positive CTC cell nucleusand several negative controls The images of CTCs were then segmented by imagedenoising image filtering edge detection image expansion and contraction techniquesusing python™s CV scheme Subsequently traditional image recognition methodsand machine learning were used to identify CTCs Machine learning algorithms areimplemented using convolutional neural network deep learning networks for trainingWe took cells from patients for training and testing About cells wereused for training and the others were used for testing The sensitivity and specificity ofrecognition reached and respectively We will further revise our modelshoping to achieve a higher sensitivity and specificityKeywords circulating tumor cells CTCs imFISH machine learning image segmentation CNN networkINTRODUCTIONThe metastasis of cancers is a complex and multistage process The circulating tumor cells CTCsare the œseeds shed from the primary tumor andor metastatic lesions and rooted in a new œsoiltransferred by the circulatory system Paget Circulating tumor cell is an intermediate stageof cancer metastasis correlated with cancer aggressiveness and the likelihood of metastasis andtherefore can be used to predict disease progression and survival on a realtime basis by liquidbiopsy Lindsay Praharaj Anand and Roszik Baek Maly Marcuello Pan Riebensahm The molecular subtypesof CTCs not only the CTCs count are interrelated with the prognosis BanysPaluchowski Cristofanilli Dong Stefanovic What™s more the PDL1Edited byCheng GuoColumbia University United StatesReviewed byJuanying XieShaanxi Normal University ChinaKhanh N Q LeTaipei Medical University TaiwanCorrespondenceBinsheng Hehbscsmu163comQiliang Zhou13974942986163comYuebin LiangliangybgeneiscnGeng Tiantianggeneiscn These authors have contributedequally to this workSpecialty sectionThis was submitted toComputational Genomicsa section of the journalFrontiers in Bioengineering andBiotechnologyReceived March Accepted July Published August CitationHe B Lu Q Lang J Yu HPeng C Bing P Li S Zhou Q Liang Yand Tian G A New Methodfor CTC Images Recognition Basedon Machine LearningFront Bioeng Biotechnol 103389fbioe202000897Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine Learningexpression in CTCsis correlated with the response toimmunity inhibitors Kloten PDL1EMTCTCs were associated with significantly poorer survival aftercurative surgery showing that PDL1 expression and EpithelialMesenchymal Transition EMT of CTCs are negative survivalpredictors for Nonsmall celllung cancer NSCLC patientsJanning Manjunath Pretreatment PDL1 CTCs are usually associated with a bad prognosis in patientstreated with PD1 inhibitors in NSCLC such as nivolumabGuibert The liquid biopsies worked as an ongoing monitoring systemto assess tumor heterogeneity and make it possible to detect asingle CTC or clusters of cells Wan Merker Praharaj Asante The breakthroughfor CTCdetection is the application of immunomagnetic CTCenrichment combined with flow cytometry which is still theœgold standard of CTCdetection Racila Howeverthis method that lack of the cancer specific markers still remainslots of limitation Grover Ferreira Gabriel Keller Thusthe multimarker immunofluorescence staining is required for recognizeCTCs Antibodies against chromosome centromere duplicationCEP8chromosome centromere duplication CEP17 areused to mark the rapidly dividing tumor cells antibodies againstCD45 as typical leukocytes filaments as well as 4cid486diamidino2phenylindole DAPI for labeling nuclears Koudelakova Lu Liu Lee Although there are great advantages in enrichment technologythe automatic recognition of CTCs still remains problemsManual identification is very timeconsuming and unreliableWith the continuous deepening of the application of CTCsrecognition in various cancer diseases the demand for rapidand automatic identification and counting methods of CTCs isincreasing Several studies have reported the automated screeningprocess Nagrath Yang Kraeft performed a fluorescencebased automated microscope systemREIS for cell detection This scanning can quantify the numberof cells reliably and reproducibly and categorize positive cellsbased on the marker expression profile Ligthart redefined the CTCs by computer algorithms after the manualcounting The stricter definition with the standard deviationof the signal in the CKPE channel the peak signal value inboth the DNADAPI and CD45APC channels and the size ofthe objects used as classifier was well validated CTC by clinicaloutcome using a perfectly reproducing automated algorithmMingxing reported an automated CTC enumerationZhou Allimages with diï¬erent colors weretransferred to a grayscale image and the grayscale images wereused to identify the position and outline of cells Howeverdespite the widely accepted these classification methods stillremain subjective as the rules are set artificially The fixedconditions may not identify the morphologically heterogeneousCTCs integrally What™s more diï¬erent technologies usually usediï¬erent antibodies making comparison and standardizationacross diï¬erent platforms challenging Marcuello With the maturity of artificial intelligence AI recent yearsmachine learning become an exciting field for research TheUS Food and Drug Administration FDA has approved severalcommercial products using machinelearning algorithms in themedical diagnosis and research The cardiovascular MRI analysissoftware of Arterys was the world™s first internet platform formedical imaging AI powered and FDA cleared This software isable to analyze multiple multiperiod MR images to determineblood flow in heart and main vessels The cloud platformwill enable software to collect and analyze the vast amount ofcardiovascular data from MR scanners in real time which willspeed up doctors™ diagnosis This artificial machine is consistentand tireless and is able to identify characters beyond humanperception which provided a substantial interest in the fieldof medical research specifically medical images Dominguez Erickson Lundervold and Lundervold Maier Many algorithms are developed forselecting the best weights for features involving neural networksHornik decision trees Quinlan supportvector machines Cristianini and ShaweTaylor the na¯veBayes Lowd and Domingos knearest neighbors Zhouand Chen and deep learning McBee Wainberg Zou Deep learning as wellas deep neural network learning refers to the use of neuralnetworks with more than layers able to integrate vast datasetslearn arbitrarily complex relationships and incorporate existingknowledge Convolutional neural networks CNNs is a powerfulalgorithm for advancing biomedical image analysis as it assumesthat the input layer has a geometric relationship such as the rowsand columns of images Anthimopoulos Poplin It has been successfully applied in the cancer diagnosisand nuclei or tissue identification Le Le Xing present a novel method for automatednucleus segmentation powered by CNNs The features involvedin the images are considered as a part of the search processand there is no need to limit the features compared to thetraditional machine learning methods which will eliminate thebias created subjective Here we apply deep learning to therecognition of CTCs in order to reduce the artificial errors andimprove accuracyMATERIALS AND METHODSPatients and Samples PreparationA cohort of patients with cancers were enrolled inthis study during “ which was approved by theethics committee of Chifeng Municipal Hospital The clinicalpathological characteristics of patients including age genderCTC number and cancer type are summarized in Table Fourmilliliter of peripheral venous blood was routinely collectedfor every patient The first ml blood samples obtainedafter puncture was discarded in order to avoid the skinepithelial cells contamination Then the blood was placed inanticoagulation tubes and store at room temperature The testwas completed within hAll the patients were divided into two parts according tothe collecting date The earlier patients we collected wereused as the training data the others were used as the independentFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Clinical pathological characteristicsClinicopathologic variableAgeGenderSamples typeCTC numberCancer typeCategoryMeanMaleFemaleUnknownPeripheral bloodMeanLung cancerLiver cancerGastrointestinal cancerBreast cancerCarcinoma of thyroidNPCOtherClinical level““testing data Thousand three hundred cells images in the earlierreceived patients were selected to build the CTC recognitionmodel which will be further tested by the cells images of thetest dataset There was no cross part between the two datasets inorder to avoiding the overfittingEnrichment and imFISH Identification ofCTCsThe Cyttel method was used to isolate and enumerate CTCsThe peripheral blood was first centrifuged at g for minto get the precipitation and then washed by CS1 buï¬er CyttelBiosciences Co Ltd Beijing China Then the red blood cellswere lysed by CS2 buï¬er Cyttel After centrifuged at gfor min the precipitate was washed by CS1 buï¬er Thenthe cells were incubated completely with antiCD45 monoclonalantibodyconjugated beads Cyttel for min Three milliliterseparation medium was used to separate the beads and the CTCsby gradient centrifugation at g for min Then the upper rarecell layer was centrifuged at g for min and resuspendedby CS1 The tube was put on a magnetic stand for min Aftersmeared fixed and dried cells were used to perform the imFISHThe slides were fixed dehydrated and then dried at roomtemperature µl CEP8CEP17 antibody was added to the cellsand the slides were placed in a hybridization and denatured for h at —¦C The probe was eluted and the slides were washedtwice in — SSC Then the CD45 fluorescent antibody was addedto the sample area and the slides were put in a wet box andincubate for h at —¦C After incubation CD45 fluorescentantibody was aspirated and µl mounting media containingDAPI was added to the sample area After mounted the cells canbe observed and counted under a fluorescence microscopeThe Manual Interpretation Standard ofCTCs CountingAfter imFISHlots ofimages were acquired with diï¬erentfluorescent colors Usually manual counting is the œgoldstandard but it™s a time consuming and exhausted processionThe Manual interpretation standard of CTCs counting is Eliminates the aggregation superposition and interference ofnucleus or impurity DAPI positive CD45 negative and Three or more than three CEP8CEP17 signal points Itwill be regarded as one signal point if the distance between twosignal points is smaller than the diameter of one pointThe Image Segmentation Method WasUsed to Segment Single Nucleus andGive Labels of Cells Instead of ManualSince the obtained microscopic image is very huge the algorithmwill be limited by the memory and cannot be executed normallyon a conventional computer We first selected part of the imagecontaining one CTC cell and several nonCTC cells around toperform the following test The chosen resolution is — The CV package of python was used to process theCTCs images including conversion of color and morphologicaltransformations The RGB image was converted to the gray image The derivatives were calculated using the CVfunction Sobel from an image Morphological transformations operations based on theimage shapeThe Morphological package of python was used to segmentthe images of CTCs by image denoising image filtering edgedetection image expansion and contractionNuclei were segmented in the blue channel DAPI and theproportion of red in the red channel was detected based onthe position of the nucleus The nucleus with proportion of redhigher than was defined as having a common leukocyteantigen The orange channel was used to detect the number ofCEP8 chromosomes and the green channel was used to detectthe number of centromere probes extracted by CEP17 Diï¬erentcell types were distinguished by diï¬erent colors Figure The CNN Deep Learning Method WasUsed for CTCs IdentificationWith the development of AI machine learning has been wildlyused in the procession of medical images Deep learning is a bigimprovement on artificial neural networks allowing higherlevelfeature extraction and better data prediction with more layersAfter segmentation CNN network were used to identify CTCcells in single nucleus Finally it enters the output layer andoutput the result ie CTCs or nonCTCsOur CNN model was built based on AlexNet which wasfirst introduced in Krizhevsky The networkconsists of eight weighted layers Figure the first five layersare convolution layers and the remaining three layers are fullconnection layers The output of the last full connection layeris the input of the dimensional softmax values which willgenerate the distribution network of two types of labelsThe fivefold cross validation was used to prevent overfittingand select hyperparameters of the model The best crossvalidation score was obtained by searching the hyperparameterspace round and round The final hyperparameters involved inFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The imFISH result and the segmentation of chromosome and nuclear A“C The imFISH result of CEP8 CD45 and DAPI D The merge of panelsA“C E The CTCs were identified by CV segmentation method and marked in red box a“c The CEP8 signal points were identified by CVsegmentation method and marked in red boxour model are activation function kernel regularizer type andregularization factor The workflow is shown belowSp The grid was defined on 3dimensions with eachofthese maps for hyperparameter sets eg hyperparameters activation function kernel regularizer typeregularization factor activation function œsoftmaxœReLU œtanh kernel regularizer type œl1 œl2regularization factor œ œ The range of possible values were defined of eachdimension Allestablishing the best onethe possibleconfigurations weresearched forEvaluation Criteria for ClassificationModelsAfter segmentation some performance evaluation criteria Xie were involved in to evaluate the performance of theclassification model such as sensitivity Se or recall specificitySp precision F1 score and area under the receiver operatingcharacteristic curve AUCSerecall TPTP FNTNTN FPTPprecision TP FPF1 — precision — recallprecision recallIn the equations TP stands for the number of positive CTCcells which are correctly recognized as positive CTC cells FPstands for the number of negative CTC cells that are incorrectlyrecognized as positive CTC cells FN stands for the numberof positive CTC cells incorrectly recognized as negative CTCcells TN stands for the number of negative CTC cells correctlyrecognized as negative CTC cells Table RESULTSPatient CharacteristicsA total of patients were enrolled in this study from January to June The average age is years old Patients withlung cancer count of all patients and the next is breastcancer and gastrointestinal cancer Table Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The layers of the CNN model The first five layers are convolution layers and the remaining layers are full connection layersThree SubImages Were Required forManual CountingWe performed imFISH for all the patients and required images of CTCs cells Every image was divided into or channels with diï¬erent color The orange channel representedthe chromosome with CEP8 Figure 1A the green channelthecentromereofthechromosomerepresentedCEP17 Supplementary Figure S1represented the whitethe blueFigure 1C The mergence wasWe then manually labeled all withred channelcell with CD45 Figure 1Brepresented the nuclei with DAPIshown in Figure 1Dthese subimages accordingchannelFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningTABLE Confusion matrix definitionsTABLE The confusion matrix of the models for test datasetConfusion MatrixPredictionMethodConfusion MatrixPredictionPositiveNegativePositiveNegativeTruePositiveNegativeTrue positive TPFalse positive FPFalse Negative FNTrue Negative TN CVALexNetTrueTruePositiveNegativePositiveNegativethetoare CTCs positivestandard AmongourresultspatientsTABLE Tuning of the hyperparameters of AlexNetActivation functionKernel regularizer typeRegularization factorThe Segmentation of Nuclear andIdentifying CTCs by CVSegmentation MethodIn order to avoid the artificial error and save costs we performedthe traditional image identification method for CTCs countingFigure The nucleus was separated in the blue channelDAPI Figure 1E and the red proportion of the red channelwas detected according to the location of the cell nucleus Theproportion higher than was defined as the number of theCEP8 chromosome detected by the common antigen orangechannel of white blood cells Figures 1A“C the number ofcentromeric probes detected by the green channel such as CEP17Supplementary Figure S1After segmentation of nuclear we used CV segmentationmethod to identify CTC cells from single nucleus regions in testing dataset by the manual interpretation standard ofCTCs counting After identification and judgment cells of negative nuclei were recognized as CTC negative About cells of positive nuclei were recognized as CTCnegative The sensitivity and specificity were and while the precision and F1 score reached and respectively Table We also applied the regionbased image segmentationalgorithm such as watershed algorithm in the segmentationprocess The watershed algorithm was implemented the bywatershed function in CV python and CV In this method optimal threshold value was used respectivelyin binaryzation process by setting THRESH_OTSU mode Thetraditional watershed algorithm was sensitive to noise and theaccuracy was lower than our segmentation method on CTCnegative data set in size of Supplementary Table S3The HyperParameters Selected forEvaluating the CNN MethodWe used GridSearchCV class in scikitlearn by providinga dictionary of hyperparameters to determine the hyperparameters of the model After the crossvalidation processactivation function was set to ReLU kernel regularizer type wasset to l2 and regularization factor was set to as shown inTable with the best performance Further the hyperparameterswe selected were used to construct the model on the wholetraining datasetsoftmaxReLUtanhl1l2l1l2l1l2The underline value shows the best result of AUC value in the tuning process of thehyperparameters of AlexNetThe Identification of CTCs by CNNMethodWe got nuclei of patients by segmentation processFigure showed the whole flowchart of the experiment About nuclei were used for training the left were usedfor testing We use the same images for testing cells of negative nuclei were recognized as CTC negative and cells of were recognized as CTC positive The sensitivityand specificity were and while the precisionand F1 score reached and respectively Table and Figure Before that we also compared the performance of AlexNetmodel with others such as ResNet and Xception All ofthem have close AUC values Figure butthe AlexNetwas less timeconsuming in the training and test processSupplementary Table S1DISCUSSIONThis study showed a method for CTC counting powered bymachine learning The use of machine learning for imageinterpretation can capture important image features reduceerrors caused by manually setting interpretation standardsand save time and labor costs Although this method showsa higher sensitivity and specificity in CTC countingisslightly worse than the first method for the data used inthis study Actually we have analyzed that the main reasonis that there are fewer positive samples for training and thealgorithm cannot extract features of more positive samplesin the group were excludedIn additiondue to quality problems Unfortunatelythe CTC imagesincluded in the group doesn™t cover the whole film but asome picturesitFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningFIGURE The flowchart of the whole experimentFIGURE The ROC curve of AlexNet ResNet and Xception modelpicture just focused on a certain CTCpositive cell under themicroscope which results in that the machine learning methodhas no advantage in recognition speed compared with thetraditional image recognition method Enlarging the scope ofimages and collected more samples is also that need to beimproved in the futureDeep learning has already been shown to be suitablefor detection of CTCs because of the high sensitivity andspecificity in CTC counting We had changed the filter sizeand number in all convolution layers in order to find thebest CNN parameters We found diï¬erent filter size andnumber will influence the results largely We changed filternumber from range to in our training process Wefound that the training result was not convergence when thenumber was less than It showed that the range of thefeature number of the image is about “ We tried toincrease the filter size from to but the result was notchanged a lot and the convergence speed even became slowerwhen the filter size higher than From this process wesummarized that the feature size in CTCs could not be greaterthan pixels Furthermore there are many appropriately AImodels such as VGG InceptionV14 We will apply themon the CTCs dataset to establish a more suitable model inthe later testingtumorsCirculating tumor cellis an important marker for earlyscreening and prognosis ofIn addition CTCsoriginating from the primary tumor may be more eï¬ectivefor tumor tissue tracing and molecular classification Imagerecognition can only obtain the characteristics ofthe cellsurface If strict tissue tracing is required other molecularbiological experimental data such as the isolation of CTCcells and single cell sequencing may be required Besidesin this study we also evaluated the performance of AlexNetmodel in variant types of cancers Supplementary Table S2and Figure S2 showed that our model presents a betterperformance in Lung cancer than Gastrointestinal cancer andBreast cancer One of the reasons may be that the trainingdata size of Lung cancer is much larger than those ofGastrointestinal cancer and Breast cancer Furtherpostoperative recurrence may occur in approximately of patients even after complete resection of NSCLC Yano These proteins especially epithelial proteinssuch as EpCAM PIK3CA AKT2 TWIST and ALDH1may have more activitiesHanssen whichwilllead more influence in the morphology of cells andaï¬ecting the recognition performance thereby Therefore themultiimage omicsincluding CT images HE staining andimmunohistochemical images as well as the sequencing datamay be urgently needed at this stageFrontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCONCLUSIONIn orderIn the present study we established a CTC cell recognitionsoftware based on deep learningto make itmore practical we collected samples from the real worldinstead of using the public databases We performed theCTC enrichment and imFISH experiments and screened thefluorescence images according to the figure™s quality In order toimprove the efficiency we used the machine instead of ngmanual screening First the python™s package was used to mage segmentation The obtained recognition sensitivity andspecificity are and respectively In addition therecognition sensitivity and specificity can also reach to and respectively using CNN instead of manual interventionIn the future studies we willfocus on the improvementof the accuracy and sensitivity with a more suitable deeplearning model promoting this technology to the clinic assoon as possibleDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorCTCs Recognition by Machine Learninglegal guardiannextstudy was provided by the participants™of kin Written informed consent was obtained from theindividuals and minors™ legal guardiannext of kin for thepublication of any potentially identifiable images or data includedin this AUTHOR CONTRIBUTIONSGT YL BH and QZ conceived the concept of the work BH QLJL PB HY and SL performed the experiments QL and BH wrotethe manuscript CP and HY reviewed the manuscript All authorsapproved the final version of this manuscriptFUNDINGThis research was funded by Hunan Provincial Innovation2018RS3105Platform and Talents Program NotheNaturalNo Science Foundation of Chinathe Natural Science Foundation of Hunan Province No2018JJ3570 and the Project of Scientific Research Fundof Hunan Provincial Education Department Nos 19A060and 19C0185ETHICS STATEMENTSUPPLEMENTARY MATERIALThe studies involving human participants were reviewed andapproved by The Ethics Committee of Chifeng MunicipalHospital Written informed consentto participate in thisThe Supplementary Materialonline202000897fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389fbioeatREFERENCESAnand K and Roszik J Pilot study of circulating tumor cells in earlystage and metastatic uveal melanoma Cancers Basel cancers11060856Anthimopoulos M Christodoulidis S Ebner L Christe A and MougiakakouS Lung pattern classification for interstitial lung diseases using a deepconvolutional neural network IEEE Trans Med Imaging “ 101109tmi20162535865Asante D B Calapre L Ziman M Meniawy T M and Gray E S Liquid biopsy in ovarian cancer using circulating tumor DNA and cellsready for prime time Cancer Lett “ 101016jcanlet2019Baek D H Kim G H Song G A Han I S Park E Y Kim H S Clinical potential of circulating tumor cells in colorectal cancer aprospective study Clin Transl Gastroenterol 10e00055 1014309ctgBanysPaluchowski M Schneck H Blassl C Schultz S MeierStiegen FNiederacher D Prognostic relevance of circulating tumor cellsin molecular subtypes of breast cancer Geburtshilfe Frauenheilkd “ 101055s00351545788Cristianini N and ShaweTaylor J An Introduction to Support VectorMachines and Other KernelBased Learning Methods Cambridge Cambridgeuniversity pressCristofanilli M Pierga J Y Reuben J Rademaker A Davis A A Peeters D J The clinical use of circulating tumor cells CTCs enumerationfor staging of metastatic breast cancer MBC International expert consensuspaper Crit Rev Oncol Hematol “ 101016jcritrevonc201812Dominguez C Heras J and Pascual V IJ CV combining ImageJand CV for processing images in biomedicine Comput Biol Med “ 101016jcompbiomed201703027Dong J Zhu D Tang X Qiu X Lu D Li B Detectionof circulating tumor cell molecular subtype in pulmonary vein predictingprognosis of stage IIII nonsmall cell lung cancer patients Front Oncol 103389fonc201901139Erickson B J Korfiatis P Akkus Z and Kline T L Machinelearning for medical imaging Radiographics “ 101148rg20171Ferreira M M Ramani V C and Jeï¬rey S S Circulating tumortechnologies Mol Oncol “ 101016jmolonc2016cellGabriel M T Calleja L R Chalopin A Ory B and Heymann D Circulating tumor cells a review of nonEpCAMbased approaches for cellenrichment and isolation Clin Chem “ 101373clinchem2015Grover P K Cummins A G Price T J RobertsThomson I C andHardingham J E Circulating tumour cells the evolving concept andthe inadequacy of their enrichment by EpCAMbased methodology for basicand clinical cancer research Ann Oncol “ 101093annoncmdu018Guibert N Delaunay M Lusque A Boubekeur N Rouquette I Clermont E PDL1 expression in circulating tumor cells of advanced nonsmallcell lung cancer patients treated with nivolumab Lung Cancer “ 101016jlungcan201804001Hanssen A Wagner J Ges T M Taenzer A Uzunoglu F G Driemel C Characterization of diï¬erent CTC subpopulations in nonsmallcell lung cancer Sci Rep 101038srep28010Frontiers in Bioengineering and Biotechnology wwwfrontiersinAugust Volume 0cHe et alCTCs Recognition by Machine LearningHornik K Stinchcombe M and White H Multilayer feedforwardnetworks are universal approximators Neural Netw “ Janning M Kobus F Babayan A and Wikman H Determination ofPDL1 expression in circulating tumor cells of NSCLC patients and correlationwith response to PD1PDL1 inhibitors Cancers Basel cancers11060835Keller L Wernerand clinicalrelevance of EpCAM Cell Stress “ 1015698cst2019and Pantel K BiologySKloten V Lampignano R Krahn T and Schlange T Circulatingtumor Cell PDL1 expression as biomarker for therapeutic efficacy ofimmune checkpoint inhibition in NSCLC Cells 103390cells808Koudelakova V Trojanec R Vrbkova J Donevska S Bouchalova K KolarZ Frequency of chromosome polysomy in relation to CEP17copy number in a large breast cancer cohort Genes Chromosomes Cancer “ 101002gcc22337Kraeft S K Ladanyi A Galiger K Herlitz A Sher A C Bergsrud D E Reliable and sensitive identification of occult tumor cells usingthe improved rare event imaging system Clin Cancer Res “ 10115810780432ccr030361Krizhevsky A Sutskever I and Hinton G œImageNet classification withdeep convolutional neural networks in Paper Presented at the NIPS LakeTahoe Harrahs and HarveysLe N Q Ho Q T and Ou Y Y Incorporating deep learning withconvolutional neural networks and position specific scoring matrices 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" many suggest that shared decisionmaking sdm is the most effective approach to clinical counseling itis unclear if this applies to surgical decisionmakingespecially regarding urgent highlymorbid operations in thisscoping review we identify s that address patient and surgeon preferences toward sdm in surgerymethods we used the preferred reporting items for systematic reviews and metaanalyses extension for scopingreviews prismascr to develop our protocol medline embase and cochrane databases were searched frominception through title review identified peerreviewed empirical s that addressed patientsurgeon preferences toward sdm in surgery identified s underwent full review by two independentinvestigators we addressed the following questions what is known from existing empirical evidence aboutpatients™ andor surgeons™ surgical decisionmaking preferences why might patients andor surgeons prefer sdm does acuity of intervention impact surgical decisionmaking preferences outcome measures included studymethods surgical specialty diagnosis study locationsetting typenumber of subjects acuity of intervention surgeonpatient decisionmaking preferences and factors associated with favoring sdm data was analyzed in microsoft excelresults s were identified with duplicates yielding s for title review swere included in final analysis of s discussed oncologic decisionmaking of these focused on breastcancer of s included patients included surgeons of s found surgeons favored sdm demonstrated surgeons favored surgeon guidance of s demonstrated patients favored sdm showedpatients favored surgeon guidance showed patients preferred independent decisionmaking the most commonfactors for patients favoring sdm included female gender higher education and younger age for surgeons the mostcommon factors for favoring sdm included limited evidence for a given treatment plan multiple treatment optionsand impact on patient lifestyle no s evaluated decisionmaking preferences in an emergent settings there has been limited evaluation of patient and surgeon preferences toward sdm in surgical decisionmaking generally patients and surgeons expressed preference toward sdm none of the s evaluated decisionmaking preferences in an emergent setting so assessment of the impact of acuity on decisionmaking preferences islimited extension of research to complex emergent clinical settings is neededkeywords surgery shared decision making ethics correspondence ericacarlisleuiowaedu1program in bioethics and humanities university of iowa carver college ofmedicine iowa city usa3department of surgery university of iowa hospitals and clinics iowa cityusafull 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 0cshinkunas bmc medical informatics and decision making page of over the past several decades physician paternalism hasbeen systematically rejected and respect for patient autonomy has emerged as a leading ethical priority in clinical counseling shared decisionmaking sdm aprocess by which physicians and patients actively worktogether to integrate care plans that are responsive topatient goals and values has been advocated as a clinicalcounseling approach that promotes patient autonomy byencouraging patients to participate in clinical decisionmaking [“] along with its presumed promotion ofpatient autonomy data suggesting that sdm reduceshealth care costs and improves quality of care have ledto relatively widespread incorporation of sdm intohealth policy despite this implicit acceptance ofsdm relatively limited data exist regarding patient orphysician preferences toward sdm such data seem tobe especially lacking in surgical decisionmakingby supporting patient autonomy sdm places somelimits on the extent to which a physician™s influenceguides a patient™s decisions some ethicists have arguedthat such prioritization of patient autonomy is criticallyimportant and that even subtle attempts by a physicianto sway a patient toward a particular decision violates respect for patient autonomy however others have argued that if attempts to promote patient autonomy aretoo strong or rigid the emphasis on selfdeterminationmay be inconsistent with patients™ wishes for more professional guidance [ ] in fact there is an emergingbody of literature that suggests that patients may prefermore physician guidance during medical decision making [ “] these findings prompt one to questionwhether autonomyheavy approaches to sdm in clinicalcounseling are always consistent with patient preferencesor whether patients would at least sometimes prefer aless autonomous and more guided approach to clinicalcounselingwith respect to the physician™s perspective it is important to note that studies have shown physicians to besomewhat reluctant to incorporate sdm into clinicalpractice one reason for this may be a sense thatwhen a physician overly prioritizes patient autonomythere is lessening of the physician™s role such that the fiduciary nature of the patientphysician relationship isundermined prioritization of patient autonomy and integration of sdm into clinical counseling has left somephysicians feeling that their role has become one ofmerely offering patients the information necessary tomake their own œinformed decisions rather than trulyengaging in a fiduciary relationship with the patient this is illustrated in a recent narrative that describes anencounter in which a physician reviewed all options fortreatment of nonischemic cardiomyopathy with her patient but was stopped by the patient before she couldmake a recommendation with the request that the patient be allowed time to independently reflect and makea decision that was best for him in the physician™s reflection on the encounter she notes œsince the decisionwas his it was no longer mine i had informed him buthad i been his doctor perhaps such efforts to assure patient autonomy and sdm limit the role of thephysician in patient counseling these types of reportscall for further investigation so we can better understandphysician preferences toward shared decision makingconcerns about the appropriateness of sdm may beparticularly pronounced in surgical decision making giventhe often dramatic and irreversible outcomes associatedwith surgery these concerns may further escalate whenconsidering emergent highly complex operations that areassociated with a high risk of mortality or morbidity in aninitial effortto better understand preferences towardsdm in surgical decision making we reviewed the literature regarding parent and surgeon preferences towardsdm in pediatric surgery we found that there wasmarkedly limited data available of the existing sthe predominant focus was on parent preferences towarddecision making in elective nonurgent procedures therewas limited data regarding surgeon preferences and virtually no discussion of preferences for decision making inmore urgent settings the purpose of this review is to gain a more thorough understanding of patient and surgeon preferences toward sdmin adult surgery we chose to conduct a scoping review because there is limited published data on patient and surgeondecision making preferences particularly when surgery isconsidered urgent or emergent scoping reviews are a valuable methodology because they allow for the mapping of important concepts and research gaps in a defined area ofstudy by comprehensively identifying reviewing and summarizing the existing information from the literature specific research questions addressed in our scoping reviewincluded what is known from existing empirical evidence about patients™ andor surgeons™ surgical decisionmaking preferences why might patients andor surgeonsprefer sdm does acuity of intervention impact surgicaldecisionmaking preferencesmethodsprotocol designour scoping review protocol follows arksey and o™malley™s methodological framework as well as the preferred reporting items for systematic reviews andmetaanalyses extension for scoping reviews prismascr this protocol has not been registeredidentifying relevant studiesafter ascertaining our research questions we worked inconjunction with an experienced medicallibrarian to 0cshinkunas bmc medical informatics and decision making page of identify relevant studies we followed the preferredreporting items for systematic reviews and metaanalyses prisma guidelines for reporting the identified screened eligible and included studies fig after drafting refining and finalizing our search strategies we searched three bibliographic databases from inception through november medline embaseand cochrane databases the final search strategies forall three databases are outlined in additional file thefinal search results were imported into endnote versionx91 and yielded sstudy designeligibility criteriainclusion and exclusion criteria were defined a priorireview was limited to english language no translatorsavailable peerreviewed published literature only empirical studies were included review was limited todecision making preferences of surgeons andor adultpatients decision making preferences were loosely defined and included œpreferred role œperceived role œexpectationsœdesires and œsatisfaction with actualdecisionmaking role s in the following categories were excluded reviews letters to the editor editorialssuggested models of care patient educationhandouts decision making tools animal studies and s related to pediatric surgery in addition we excluded s without accessible full textliterature reviewafter duplicates were removed by the primary authorlas we were left with s to screen twoof the authors las and emc independently reviewedall titles and s and jointly decided to exclude s based on the eligibility criteria theremaining s were selected for full text reviewfig flow diagram of study selection 0cshinkunas bmc medical informatics and decision making page of following full text review additional s were excluded because they either did not pertain to an adultsurgical population or to decisionmaking preferences inthe surgery setting disagreements were resolved by discussion between the two authorssubjects gender acuity ofsurgeryothercharting the datafor each of the included s two of the authorslas and cjk independently ed the followingoutcome measures study methods quantitativequalitativemixed methods surgical specialty cancer diagnosisyesnounclear study location usnonus study setting inpatientoutpatient type of subject patientsurgeon number oftheintervention electiveurgentemergentunclear surgicaldecision to be made surgery v nonoperative managementchoice among different surgical proceduresdecision on timing ofsurgeonpatientdecision making preferences shared decision makingsurgeon guided decision making independent decisionmaking and surgeonpatient factors associated with favoring sdmacuity ofthe intervention was defined as followsemergent immediate need for surgery to preserve lifeurgentsurgery is required within the next days orweeks and elective surgery is not required notablycancer resections were considered urgent however subsequentreconstruction was considered elective iebreast cancer resection with subsequent reconstructionthe control preferences scale which is a fivepointmeasure used to gauge preferred involvement in medicaldecision making was adapted to define surgeonpatient preferences as follows shared decision makingsdm the patient and surgeon prefer to make the decision regarding surgery together surgeon guided decisionmaking sg the preference is for the surgeon to guidedecision making either entirely or in part while the patient takes a more œpassive role independent decisionmaking idm the preference is for the patient to take amore œactive role in decision making either partly orentirely independent from the surgeonthe data ion form was a modified version ofthe one we used for a literature review we conducted ondecision making preferences in the pediatric surgical setting discordant opinions were discussed at weeklymeetings the third author emc was available to mediate if consensus could not be reached data was analyzed in microsoft excel resultssummarizing collating and reporting the resultssummarizing the results s were identified medline n embase n cochrane n duplicateswere removed and s underwent title review seventyfour s were included in thefinal analysis because they specifically addressed existingempirical evidence about patient andor surgeon decision making preferences toward sdm in adult surgerysetting table provides a summary of allincludedscollating and reporting the resultstable provides frequencies for the characteristics of allincluded s over half of the s were quantitative n and performed outside of the us n sixtyseven included outpatient surgeries fourteen surgical subspecialties were representedwith the most s originating from surgical oncology n general surgery n orthopedic surgery n and urology n fifty s discussed decision making for patients with cancer and of these focused onbreast cancer most s assessed a choice betweenoperative and nonoperative management n or an option among different surgical procedures n sixtyeight of the s included patients ofthese demonstrated that patients preferredsdm showed that patients favored a surgeonguided approach and revealed a patient preference for independent decision making the most common factors for patients favoring sdm included femalegender higher education and younger ageonly of the s assessed surgeons™ preferences of these found that surgeons preferredsdm while demonstrated that surgeons favoreda more surgeonguided decisionmaking approach thefactors most commonly listed for surgeons favoringsdm included limited evidence for a given treatmentplan multiple treatment options and impact on patientlifestylenone of the s evaluated patient decisionmakingpreferences in an emergent setting out of the sthat assessed patient decisionmaking preferences in theelective surgery setting preferred sdm threeout of four of the s assessing surgeon decision making preferences in the elective surgery settingreported that surgeons preferred sdm in six out of nine of the s surgeons also preferred sdm in theurgent surgery setting in s patients were fairlysplit on their decisionmaking preference when it cameto urgent surgeries with desiring sdm and favoring a more surgeonguided approachonly s looked at both patient and surgeon decision making preferences in a little over half ofthese s n there was discordance between patient and surgeon decision making preferences 0cshinkunas bmc medical informatics and decision making page of table characteristics of included sacuity of andthedate ofpublicationinterventionstudy populationmajor findings related to decisionmaking dm preferencesalmyroudi ananian andersen asghari ashraf avis ballinger breast cancerpatients breast cancerpatientsurgentelective breast cancersurvivorsurgent hospitalized patients on surgical wardsunclear patients undergoingeither immediate ordelayed breastreconstructionelective hernia repair patientselective breast cancerpatientsurgentbeaver colorectal cancerpatientsurgentbeaver belue health professionalscaring for colorectalcancer patients weresurgeons cardiologists making adecision about surgery patients with coronaryartery diseaseblumenthalbarby left ventricular assistdevice patients andcandidatesburton older breast cancerpatientsbutow patient advocates breast cancersurgeonsurgenturgenturgenturgenturgent preferred a passive role acollaborative role46 an active role of women choosing breastreconstruction œdecided with surgeon of these patients were satisfied withthe information receivedon average reported being œveryinvolved i made all the decisions myself were content with dm roleœstrongly desire to receive informationand participate in decisionmaking were in the œinformedconsumeristgroup when it came to actual dm of these patients were satisfied with theinformation receivedœexpectations of participation can besummarized as ˜being told™ and ˜going into get it fixed™ œfelt their healthcare professionalshad surgical preferences for thembelieved that clinical issues determinedthese preferences but still knew thechoice was theirsœwanted to be well informed andinvolved in the consultation process butdid not necessarily want to use theinformation they received to makedecisionsœshared decision making was favored byhealth professionalsphysicians œprefer patients whoactively participate in the decisionpatients œprefer the physician tomake the decision sdm œprefer to make the decision on theirownœdeferred heavily to clinicians preferred œpatientcentred œdoctorcentred sdm of surgeons and of patientadvocates preferred sdm breast cancer survivors urgentcampesino cohen patients with localizedprostate cancerurgentcorriere patients undergoingelectivespanishspeaking latinas preferredœphysician treatment recommendationsenglishspeaking latinas and africanamericans preferred sdmmost viewed the surgeonguided approach as œappropriate and welcome preferred œchoosing together withdm themerelated tomajorfindingsaptsgsurg“factors associated with favoringsdmyounger age higher educationsdm “type of procedureidm“younger age level of educationincomesdm “female level of educationidmsg““sdm “sg““sdmyounger patient agesgsdm““sgsgidmsdm sdmsdmsgsg““englishspeakingsdm “multiple treatment options type 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferencesfactors associated with favoringsdmdm themerelated tomajorfindingsaptsurg elective vascularprocedures prostate cancersurvivors hand surgeons patients with triggerfinger breast cancerpatientsurgentelectiveurgentcuypers doring durifbruckert gainer the provider preferred œhaving theprovider choose for them preferred a collaborative role an active role a passive roleof proceduresdm “higher education younger agehigher sespatients œpreferred to decide forthemselves surgeons preferred sdmidmsdmwanted to participate in decisions butœperceived sdm as an obligation becauseit did not seem to fit with their idea of aproper doctorpatient relationshipsg“trust in surgeon support fromfamily written information fromsurgeon frail and olderpatients care teammembers includessurgeonsunclearboth patients and care team membersœsupported a formal approach to sdmsdm sdmghane general surgerypatientsgolden clinicians weresurgeonsgong patients with carpaltunnel syndromeelectiveurgentœpreferred relatively high levels ofdecisional control on averagem out of sd most felt that they practiced sdm eventhough they did not tend to distinctlyprompt patient dm preferencesidm“male good health high healthliteracy“sdmelective preferred sdmsdm “hack breast cancerpatientshageman hand surgeons patients with carpaltunnel syndromehawley breast cancerpatientsheggland hausken health professionalsfrom surgical wards patients who underwentsurgical treatmenturgentelectiveurgentelectiveheggland hausken surgical patients surgeonselectiveurgentheggland [ ]henderson shum physicians workingin surgical wardsunclear surgical and medicalpatientselectiveurgent preferred a collaborative role an active role a passive rolesurgeons preferred œpatient andprovider make a shared decisionpatients preferred that œthe patientdecidesactual dm role sdm œpatientbased œsurgeonbased preferreddm role content with level of dminvolvementhealth professionals majority preferred aœshared or œinformed model patientsabout half preferred a œshared orœinformed model and the other halfpreferred a œpaternalistic modelsurgeons the majority preferred anœinformed model ¦ patient is giveninformation and left to make thedecision patients preferred aœpaternalistic model and preferredsharedphysicians on average rated decisionmaking control a which means thatœphysicians were not reluctant to involvepatients in decisionmaking processeswhere active role shared and passive “ the mean dm value for thesevere scenario was moderatescenario was mild scenario was history of surgical procedureimportance of family memberopinions having privateinsuranceage nonwidowed longerduration postopsdm “idmsdm“sdmidmsdmfemalesdmsgsgidm“sdmsdm “younger age noncriticalcondition 0cdm themerelated tomajorfindingsaptsdm “surgfactors associated with favoringsdmfemale higher educationsgsg“““female no stomasdmmore years in practice morecomfort in pulmonary nodulemanagementcollege degree higher selfefficacysdm “sdm “sg“sdm “œfemales indicated that they would like tohave more input in the decisionmakingprocess than the males v onthe controlled preferences scaleœguidance by the clinician was identifiedas most important œactive role of patientin treatment decision making regardedas less important preferred a passive role sdm an active roleperceived role œshare decisionsequally with the patient œallowthe patient to decide œdecide forthemselves after considering the patient™sopinionœmost patients wanted to decide on theirtreatment options together with theirphysician preferred sdm preferred tomake the decision œwith physician inputœ wanted their surgeons to make arecommendation and when given followed the recommended treatmentplan preferred a collaborative role inpatients in twosurgical unitsunclear lung cancer patientsurgenthenderson hopmans hou colorectal cancerpatientsiaccarino clinician members ofthe american thoracicsocietyurgenturgentihrig prostate cancerpatientsjanz breast cancerpatients newly diagnosedbreast cancer patientsurgenturgenturgent breast cancerpatientsurgentjohnson keating keating lally shinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferences lung or colorectalcancer patientsurgent œpatient controlled sdm œphysician controlledsdm “married better prediagnosishealth status caucasian strongevidence for procedure breast cancer patients urgentœwomen™s lack of sharing theirpreferences with their surgeons and thesurgeons™ lack of making treatmentrecommendations resulted in what wasmore likely informed than shared decisionmaking preferred sdm preferred œthechoice to be their own preferred œtodelegate the decisionactual role sdm madedecision with œsurgeon input weresatisfied with dm levelactual dm œjoint patientdoctor decision œdoctor advocated œpatient asked preferred dm contentwith level of dm involvementthe surgical group showed a morepassive role in both their preferred andactual dm roleœsurgeon™s recommendationand fear of dying from cancer played themost important role in dmin surgery preferred a œdirectivecommunication style a œnondirective communication styleidm“sdm “younger agesdm “sdm “femalesgsg““sdmsglam breast cancerpatientslantz breast cancerpatientsurgenturgentlarsson patients scheduledfor invasive surgeryelectiveurgenturgentlee patients with earlygastric cancermarkovic martinez newly diagnosedgynecologic cancerpatients newly diagnosedbreast cancer patientsurgent 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferences surgeonsunclearmcguire mendick breast cancer patients surgeonsurgentmeredith surgical patients surgeonsunclear older breast cancerpatientsurgent patients withirritable bowel diseaseelective breast cancer patients urgentman morishige moumjid nam patients with carpaltunnel syndromeelectiveomar op dendries consecutive patientsbeing seen in a multidisciplinary stone clinicelective liver transplantcandidates and recipientsurgentorsino end stage renaldisease patientselectiveœmany physicians saw their role as anexpert who educates the patient butretains control over the decisionmakingprocessothers took a more collaborativeapproach encouraging patients toassume decisional prioritysurgeons œmade most decisions forpatients patients œgenerally lacked trustin their own decisions and usually soughtsurgeons™ guidancepatients œmajority agreed that thesurgeon should supply them with the˜pros™ and ˜cons™ of all measures toaddress the problem and it was for themultimately to decide what was right forthem surgeons œnot enthusiastic at theprospect of devoting more time todiscussing surgical alternatives risks andcomplications and outlook indicators fortheir patients benefitin surgery preferred sdm aœdoctorcentered approach œ aœpatientcentered approach œthought having a physician involvethem in the decisions concerning theirtreatment was very importantœmost were satisfied with the informationgiven and the possibility of participatingto the treatment decisionmakingprocessœi prefer that my doctor and i shareresponsibility œœi prefer that mydoctor makes the final decision aboutwhich treatment will be used butseriously considers my opinion œwould rely on the physician™srecommendationœ wished to be involved in makingthe decision to accept or not accept aliver for transplantation preferred œequal responsibility an œautonomous role adecision driven by the health care teamdm themerelated tomajorfindingsapt“surgsdmsgfactors associated with favoringsdmmultiple treatment optionsincreased risk impact ofprocedure on patient lifestylemoral contentsgsgsdm sgpatients strong evidence forprocedure surgeons multipletreatment options impact ofprocedure on patient lifestylesdm “older agecomorbidities surgical historyuse of biologics treated at anacademic hospital being marriedsdm “sdm “sdm “sg“sdm “sdm “younger agepieterse ramfelt rectal cancer patients surgical oncologistsurgentthe majority of patients and clinicianspreferred sdmsdm sdmpatients female highereducation rectal or colon cancerpatientsurgent of rectal cancer patients ofcolon cancer patients preferred acollaborative rolesdm “younger ageratsep patients with lumbardisc herniationelective preferred sdmsalkeld rectal or coloncancer patientsurgent preferred a surgeonguided approach sdm a more independent dm rolesdm “sg“desire for more disease specificinformationfemale younger age history ofradiation 0cshinkunas bmc medical informatics and decision making page of table characteristics of included s continued anddate ofpublicationacuity oftheinterventionstudy populationmajor findings related to decisionmaking dm preferencessantema patients with eitherabdominal aorticaneurysm or peripheralarterial occlusive diseaseelective preferred sdmseror young breast cancerpatientsurgentsidana young prostatecancer patientsurgentsnijders stiggelbout kiebert gi surgeonsurgent cancer patients surgical patientsunclearpreferred a more passive approach preferred œfully passive and preferred fairly passive preferred sdm an œinformeddecision made by myself based oninformation a passive roleœmost patients were offered only onetreatment option and little sdm wasseenœthe physician should make the decisionsbut strongly consider my opinion wasselected most frequentlyfactors associated with favoringsdmtrust in doctor doctor has aclear communication styledoctor listens enough time forconsultationhigher education type ofproceduredm themerelated tomajorfindingsaptsdm “surgsg“sdm ““sgsg“younger age femalesung patients with pelvicfloor disordertyler ellis newly diagnosedrectal cancer patientsuldry patients undergoingelective gi surgeryvogel breast cancerpatientswang breast cancerpatientsurgenturgent spine clinic patientselectiveweiner essis wilson patients undergoingmajor thoracicabdominal operationsurgentelectivewoltz patients withdisplaced midshaftclavicular fractureelectiveurgent preferred a collaborative role an active role a passive rolesdm “ of total mesorectal excision patientsand of local excision patientspreferred sdmsdm “higher education younger ageelective preferred an active roleidmyounger age male level ofeducationhigher anxiety scores multipletreatment options preferred a passive role anactive role sdm preferred a collaborative role a passive role an active roleœthe majority of patients felt that thephysician rather than the patient shouldmake the basic treatment decision preferred a œpatientdriven role sdm a œsurgeondriven rolesg“sdm “sgidm““ preferred sdm œautonomousrole a passive rolesdm “ ovarian cancerpatientsziebland adecision making preference dm decision making sg surgeonguided sdm shared decision making idm independent decision makingdx diagnosis pt patient surg surgeonœpreferred their medical team to decideon their behalf or œ˜going along with™their doctor™s recommendationurgentsg“out of these s three focused on elective surgeriesin orthopedics one on urgent surgeries in cardiac surgery one on both elective and urgent surgeries in general surgery and one was unclear on the acuity of theinvention but occurred in general surgerydiscussionshared decision making has been highlighted as a desirable approach to clinical counseling however it isunclear if this applies to surgical decision making particularly when considering surgical counseling in settingsof emergent complex highlymorbid operations inour scoping review of the adult surgical literature wefound relatively few studies that address patient and surgeon preferences toward sdm in surgery we found thata large proportion of existing s on preferences toward sdm address elective outpatient procedureswhile patients did seem to prefer sdm in these controlled settings it is possible that patients and surgeonsmay prefer more surgeon guidance when discussingemergent complex operations that have a high risk ofmorbidity or mortality further studies that specifically 0cshinkunas bmc medical informatics and decision making page of table frequencies for characteristics of all included sn variablesurgical specialtyastudies noncologygeneral surgeryorthopedicsurologygynecologycolorectalthoraciccardiacplastic surgerytransplantationvascularneurosurgeryentotolaryngologyophthalmologycancer diagnosisyesnounclearstudy methodsqualitativequantitativemixed methodsstudy locationusnonusstudy settinginpatientoutpatientbothtype of subjectspatients onlysurgeons onlybo
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coronavirus disease COVID19 pandemic access to surgical care for patients with head and neck cancer HNC is limited and unpredictable Determining which patients should be prioritized is inherently subjective and difficult to assess The authors have proposed an algorithm to fairly and consistently triage patients and mitigate the risk of adverse outcomes METHODS Two separate expert panels a consensus panel participants and a validation panel participants were constructed among international HNC surgeons Using a modified Delphi process and RAND CorporationUniversity of California at Los Angeles methodology with consensus rounds and meetings groupings of highpriority intermediatepriority and lowpriority indications for surgery were established and subdivided A pointbased scoring algorithm was developed the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN Agreement was measured during consensus and for algorithm scoring using the Krippendorff alpha Rankings from the algorithm were compared with expert rankings of case vignettes using the Spearman rank correlation coefficient RESULTS A total of indications for surgical priority were rated Weights for each indication ranged from ˆ’ to scale range ˆ’ to The response rate for the validation exercise was The SPARTANHN demonstrated excellent agreement and correlation with expert rankings Krippendorff alpha [ CI ] and rho [ CI ] S The SPARTANHN surgical prioritization algorithm consistently stratifies patients requiring HNC surgical care in the COVID19 era Formal evaluation and implementation are required Cancer American Cancer Society LAY SUMMARY ¢ Many countries have enacted strict rules regarding the use of hospital resources during the coronavirus disease COVID19 pandemic Facing delays in surgery patients may experience worse functional outcomes stage migration and eventual inoperability¢ Treatment prioritization tools have shown benefit in helping to triage patients equitably with minimal provider cognitive burden¢ The current study sought to develop what to the authors™ knowledge is the first cancer“specific surgical prioritization tool for use in the COVID19 era the Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN This algorithm consistently stratifies patients requiring head and neck cancer surgery in the COVID19 era and provides evidence for the initial uptake of the SPARTANHN KEYWORDS coronavirus disease COVID19 delivery of health care head and neck cancer health priorities patient selection surgical procedures waiting listsCorresponding Author John R de Almeida MD MSc Division of Surgical Oncology Department of Otolaryngology“Head and Neck Surgery 8NU883 Toronto General Hospital University Health Network Elizabeth St Toronto ON M5G 2C4 Canada Johndealmeidauhnca Division of Surgical Oncology Department of Otolaryngology“Head and Neck Surgery Princess Margaret Cancer Center University Health Network University of Toronto Toronto Ontario Canada Institute of Health Policy Management and Evaluation Dalla Lana School of Public Health University of Toronto Toronto Ontario Canada Division of Otolaryngology“Head and Neck Surgery Dalhousie University Halifax Nova Scotia Canada Division of Otolaryngology“Head and Neck Surgery McMaster University Hamilton Ontario Canada Department of Otolaryngology“Head and Neck Surgery Western University London Ontario Canada Department of Otolaryngology“Head and Neck Surgery Memorial Sloan Kettering Cancer Center New York New York Head and Neck“Endocrine Oncology Moffitt Cancer Center Tampa Florida Department of Otolaryngology“Head and Neck Surgery Medical University of South Carolina Charleston South Carolina Department of Otolaryngology“Head and Neck Surgery Stanford University Palo Alto California Department of Otolaryngology“Head and Neck Surgery University of Michigan Ann Arbor Michigan Department of Otolaryngology“Head and Neck Surgery The University of Texas MD Anderson Cancer Center Houston Texas Head and Neck Unit The Royal Marsden Hospital London United Kingdom Department of Otolaryngology“Head and Neck Surgery Icahn School of Medicine at Mount Sinai New York New York Department of Otolaryngology“Head and Neck Surgery Sunnybrook Health Sciences Centre University of Toronto Toronto Ontario Canada Department of Otolaryngology“Head and Neck Surgery Sinai Health System University of Toronto Toronto Ontario CanadaThe first authors contributed equally to this Additional supporting information may be found in the online version of this 101002cncr33114 Received June Revised June Accepted June Published online Month in Wiley Online Library wileyonlinelibrarycomCancer Month 0cOriginal INTRODUCTIONOn March the World Health anization declared a global pandemic due to the novel coronavirus severe acute respiratory syndrome coronavirus SARSCoV2 and the resulting coronavirus disease COVID191 As a result in many jurisdictions operating room capacity has been limited to only emergent or urgent surgical procedures2 Several advisory bodies have issued recommendations to safeguard access to oncologic surgery while still acknowledging that treatment delays may be necessary The American College of Surgeons has recommended postponing elective surgery including for patients with lowrisk cancers while recommending that other urgent cancer surgeries proceed34 Cancer Care Ontario has issued similar guidance recommending that hospitals include cancer surgery in their care delivery plan5The time from the diagnosis of head and neck cancer HNC to surgery is a metric with prognostic importance with treatment delays portending poorer oncologic outcomes68 In a recent systematic review evaluating delays in time from diagnosis to treatment initiation of studies demonstrated a decrease in survival to be associated with treatment delays68 These data support the urgency of initiating treatment for patients with HNC but to our knowledge do not inform a stratification schema when operating room access is not available for all patientsAs a result of these new imposed constraints difficult decisions regarding prioritization for cancer surgery are obligatory and require the consideration of broader principles regarding scarce resource allocation9 Key among these is the need for consistency and transparency to achieve fairness and to avoid engendering disparities in both access and outcomes1011 Prioritization on a casebycase basis using expert clinical judgment can be logistically challenging carries a cognitive burden and is susceptible to the biases of practitionersSurgical prioritization tools or algorithms offer decisionmaking transparency and provide equitable and timesensitive access to care to the patients who need it most1213 Although tools for surgical prioritization in the era of COVID19 continue to emerge to our knowledge oncology patients have not been explicitly considered14 Herein we have presented the development and validation of a novel algorithm Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN for the prioritization of surgery for patients with HNCMATERIALS AND METHODSThe current study was granted a waiver from the research ethics board at the University Health NetworkParticipants and SettingFor instrument development a group of expert HNC surgeons JRD DPG RG JCI DBC DB AE DJE KMH EM and IJW from institutions University Health Network Sinai Health Systems and Sunnybrook Health Sciences Centre at the University of Toronto participated in the consensus process consensus panel At the time of the consensus process all institutions were operating under significant resource constraints with limited availability of operating room time For instrument validation a group of participants JRD CWN DF DPG and EM completed the scoring algorithm designed after the consensus process Fifteen external head and neck surgeons HZ ACN RJW MAC CM EMG VD AGS AJR CML EYH JM VP BM and EG from institutions across Canada institutions the United States institutions and the United Kingdom institution participated in a ranking exercise of clinical vignettes validation panelScopeThe scope of variables considered in the prioritization algorithm was established and vetted by the consensus panel see Supporting Information All indications for prioritization were presented to the consensus panel using an online survey platform Google Forms httpsdocsgooglecomforms With exceptions survey respondents were asked to consider each of the indications in isolation For wait times panel members were asked to also consider histologic grade Similarly for surgical site the panel was asked to simultaneously consider extent of surgery Related indications were presented sequentially to facilitate pairwise comparison eg stage I and II vs stage III and IV were presented in sequence AJCC 8th edition The list of indications was pilot tested by surgeons JRD DPG EM and RG for sensibility readability content validity language and comprehensibilityConsensus ProcessThe consensus panel participated in a Delphi consensus process with rounds of rating see Supporting Information The first rounds aimed to achieve consensus regarding the priority grouping high intermediate or low High priority was defined as an indication to Cancer Month 0cproceed to surgery within weeks The second rounds of rating involved ranking each indication less important neutral or more important within their respective priority grouping Two teleconference meetings were conducted between the first and second rounds and between the third and fourth rounds with anonymized results from the prior round presented for discussion and to address inconsistencies and misinterpretationsA modification of the RANDUniversity of California at Los Angeles UCLA method was used to achieve consensus15 This methodology typically is used to determine the appropriateness of an intervention but in this setting was used to determine surgical priority We used a scale ranging from to in rounds and to indicate the decision to not operate or low priority scores intermediate priority scores or high priority scores For rounds and we used a scale from to to rate each indication compared with other indications within each of the priority groupings as either less important neutral or more important Consensus was determined based on RANDUCLA criteria15 For the first rounds to determine surgical priority a hierarchical logic was adopted to determine consensus regarding whether surgery should be performed and to then determine the priority of surgery based on the given indication Agreement on the decision to not operate was defined as a minimum of of the panelists rating a given indication with a zero score If there was no agreement to avoid surgery agreement for surgical priority then was defined as ‰¤ panelists rating the indication outside the 3point range containing the median as per RANDUCLA guidelines15 For rounds and any indication that failed to achieve consensus was classified as being of intermediate priority and for rounds and any indication failing to achieve consensus was classified as neutral within the priority groupingDevelopment of the SPARTANHNThe algorithm uses a pointbased system to assign a total score based on the sum of the individual indication scores see Supporting Information with higher scores corresponding to higher priority Scoring weights were based on consensus from both sets of rounds such that highpriority indications were assigned scores ranging from to intermediatepriority indications were assigned scores ranging from ˆ’ to and lowpriority indications were assigned scores ranging from ˆ’ to ˆ’ Within each priority grouping 3point range the scores were assigned based on the consensus ratings from the third and fourth rounds For any patients with the same total score the SPARTANHNde Almeida et alpatient with the longer surgical wait time was assigned the higher priority rankClinical VignettesTwelve clinical vignettes were constructed see Supporting Information after the consensus rounds to validate the SPARTANHN The vignettes described a variety of clinical scenarios incorporating multiple prioritization indications and additional clinical information Experts were asked to consider only the patientlevel information provided to them and not their own unique clinical and community practice environments Twelve scenarios were selected for diversity of cases The number was considered appropriate while avoiding the excessive cognitive burden associated with ranking too many scenariosStatistical AnalysisAgreementAgreement between raters during the Delphi process was calculated at each round and within each priority grouping using the Krippendorff alpha Kalpha Because typical coefficients of reliability are not suitable for coded data agreement for the rank orders generated by coders JRD CWN DF EM and DPG applying the SPARTANHN algorithm to the clinical vignettes was assessed using Kalpha calculated with bootstrap samples16 The Kalpha allows for estimation of reliability for any number of raters and categories and may be used when there are missing data17Validity of the SPARTANHN AlgorithmConvergent validity of the median rankings from the coders of each of the vignettes using the SPARTANHN and the expert panel rankings were assessed using the Spearman rank correlation coefficient The strength of the correlation was considered weak if the rho was moderate if the rho was between and and strong if the rho was In addition to SPARTANHN a second algorithm using a decisionmaking flowchart was developed SPARTANHN2 The tool and associated performance characteristics are included in Supporting Information Sample Size ConsiderationsFor determination of an adequate sample size for the expert panel we assumed that for model validity there was a strong correlation between the model rank order and expert rank order ie rho ‰¥ an alpha of power of and a nonresponse rate of Therefore the calculated sample size requirement was participantsCancer Month 0cOriginal All analyses were 2sided and statistical significance was set at P\xa0‰¤\xa0 Analyses were conducted using SAS University Edition statistical software SAS Institute Inc Cary North CarolinaRESULTSEstablishing Consensus Priority Groupings First Consensus RoundsAfter the first rounds the panel failed to achieve consensus for any indications that would result in a decision to not operate More than respondents indicated that they would not operate for the following indications the availability of alternative nonsurgical treatment with a similar prognosis respondents poor performance status ie Eastern Cooperative Oncology Group [ECOG] performance status of respondents and very severe comorbidity as indicated by a non“cancerspecific survival rate of at year respondents In the first round consensus was achieved for indications for surgical prioritization of which were considered high priority of which were considered intermediate priority and of which were considered low priority After review of firstround results consensus was achieved for an additional indications indications were rated as being of intermediate priority and indications were rated as low priority Table Establishing Ranking Within Each Priority Grouping Second Consensus RoundsOf the lowpriority indications consensus for the importance of factors was achieved for scenarios both of which were deemed less important Table Of the intermediatepriority indications consensus for the importance of factors was achieved for of scenarios Of highpriority factors consensus for the importance of factors was achieved for scenarios all of which were deemed to be more importantAgreement during consensus rounds was found to be weak to moderate for all rounds ranging from to The agreement was similar when measured as per priority grouping in which the Kalpha ranged from to Table SPARTANHN Surgical Prioritization Scoring SystemPriority weights for each indication ranged from ˆ’ to spanning a 9point range and translated from the rounds of priority groupings into categories Four indications were assigned a weight of based on consensus that these factors were both high priority and more important Supporting Information Table All other highpriority indications were assigned a weighted score because there was no consensus that they were either less or more important For intermediatepriority indications a weighted score of was assigned for of the indications deemed to be more important by consensus The other indication deemed to be more important thyroid cancer with tracheal invasion was assigned a score of because of the fact that this indication can be associated with lowgrade histology which is assigned a negative weighted score Three intermediatepriority indications that were rated as more important were resource use indications which generally are colinear As such the decision was made to assign a maximum score of for the presence of any or all of these indications One intermediatepriority indication was deemed to be less important by consensus and was assigned a score of ˆ’ All other intermediatepriority indications were assigned scores of For the lowpriority indications those deemed to be less important were assigned a weight of ˆ’ and all other indications were assigned a weight of ˆ’ The total scale score ranged from ˆ’ to Fig Reliability and Validity AssessmentAgreement between the coders for the SPARTANHN was excellent Kalpha Agreement between the expert raters was moderate Kalpha Convergent validity was demonstrated by a strong correlation between the rank orders generated by the SPARTANHN and external experts rho CI [P\xa0 a0 ] Agreement between expert rankings and SPARTANHN rankings for the vignettes is shown in Figure DISCUSSIONIn the setting of the COVID19 pandemic in which the availability of operating room time as well as hospital and intensive care unit beds is limited the prioritization of surgical oncology cases is imperative to mitigate downstream adverse outcomes1920 The current methodology was adopted based on expert consensus In the current study we have proposed the SPARTANHN with the objective of providing transparency and facilitating surgical prioritization for treatment providersCreating COVID19“era allocation schemas that are ethically sound is both critical and challenging Emanuel et al have advocated ethical principles with which to Cancer Month 0cSPARTANHNde Almeida et ali ytidbrom lanoitcnufi tnacifings laitnetoP fi ytilibareponi rohtw romut fi tnemriapmi citem etaredom laitnetoPsoc ro lanoitcnuf htiw recnac doryhTiinosavni laehcarthtw romut inossergorp esaeisd citamotpmySENEtsil tiawno e lihwTR suoverPi dedeecxe emit tiaW dedeecxe emit tiaWihgh rof kw‰¥ yb liygootsh edargihgh rof kw yb liygootsh edarg igngami ro lacniilC hpmyl decnavdAegats gncnavdai ei inossergorpi cpocsorcam roN ge esaesd edoni esaesdV inoitceserI ot III egatSnoitceser enob htiw recnac ytivac larOnoitide ht CCJA yregrus fo htgneL latot ro latotraen gniriuqer recnac ytivac larO yats fo htgnel latipsoHh tinu erac evsnetni ioNtinu nwodpets rod yregrus larosnart htiw recnac laegnyrahporOymotcessogllymotoubdnam htiiw recnac laegnyrahporO ymotcegnyral latot htiw recnac laegnyrahpopyHymotcegnyral latot gniriuqer recnac laegnyraLi cpocsodne gniriuqer recnac laegnyrahposaNymotcegnyrahp laitrapdna odne gniriuqer recnac suns lasanarap roi lasaNymotolli xamgniriuqer recnac laegnyrahposaNnoitceser cpocsinoitceseri noitceser nks gniriuqer recnac nks decnavdAinoitcurtsnocer palf lanoger dnai edon hpmyl detimil htiw renac kcen dna daeH edon hpmyl on htiw recnac kcen dna daeHycnangilam enob laropmeTesaesdiII ot I egatSy egAy egAy egAesaesdi SPGOCE y ‰¥ egAycnangil amditorap edarghgHinoitcurtsnocer palfeerf gniriuqer recnac nks decnavdAi laitrapgniriuqeryregrus laegnyral recnac laegnyraL gniriuqer recnac laegnyrahpopyHnyrahpognyral latotymotcegriuqer recnac sunsi roiretna nepogn i lasanarap ro lasaN ‰¥i lacgrus lacafonarciili ygootsh edargwol rof dedeecxe emit tiaWkwnoitceser eussittfos htiw recnac ytivac larOi rof gnhcaorppa tubdedeecxe ton em it tiaW dedeecxe emit tiaWliygootsh edarghghiwoliygootsh edargl rof kw deecxe ton emit tiaWromoc ereves yreV SP GOCE ei sutats ecnamrofrep rooP ypareht evitanretlAlebaliava ni dehcaorppa tub edargwol rof kw ditorap edargwoLycnangilam edon hpmyl htiw recnac doryhTiesaesdilygootshi recnacnonge ytidb i ta i lavvrusy srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLˆ’ˆ’ˆ’ˆ’i gnknaR fo sdnuoR retfA serocSdna snoitacdn iI noitazitiroirP ELBATCancer Month 0cOriginal TRj tnavuda dna esaesddecnavda hti iw tneitaPderiuqer tinu nwodpets ro tinu erac evsnetniIderiuqer ebut ymotsoehcart oNderiuqer palf eerf oNd yats fo htgnel latipsoHh yregrus fo htgneLd yats fo htgnel latipsoHderiuqer palf eerFh yregrus fo htgneLnoitpo na si SPGOCEˆ’ˆ’ˆ’ˆ’srotcaF ytiroirPhgHisrotcaF ytiroirPetademretniIsrotcaF ytiroirPwoLdeunitnoC ELBATyparehtodar iTRi nosnetxe ladonartxe ENE sutats ecnamrofreppu ygoocnO evitarepooCnretsaE l SPGOCE snoitaverbbAiTABLE Agreement Between Experts During the Delphi ProcessRoundOrdinal ScaleaLCL UCLPer Priority GroupLCL UCLAbbreviations LCL lower confidence limit UCL upper confidence limitThere were raters and agreement was measured using the Krippendorff alphaaœOrdinal scale refers to the scale of to used to rate priority of surgery and œPer Priority Group refers to the lowpriority mediumpriority and highpriority groups related to the scoring scaleguide the allocation of scarce resources maximizing the benefits produced by scarce resources treating people equally promoting and rewarding instrumental value and giving priority to those patients who are worst off9 These have been contextualized for cancer care more broadly and are manifest in the SPARTANHN algorithm21 The highpriority indications implicitly embrace an underlying premise of saving the most lives andor preserving the most lifeyears Many procedures for patients with HNC are aerosolgenerating and increase the risk to health care workers and other hospitalized patients22 Our process accounted for these by giving consideration to these factors during the consensus process although indications associated with potential exposure to health care workers did not emerge as lowpriority ones Indications associated with lower resource use did achieve consensus for higher importance This may help to avoid the opportunity cost of treating fewer patients with longer surgeriesAnecdotal and institutionspecific prioritization schemas for patients with HNC and general otolaryngology have been suggested213 These parallel similar efforts for general surgery cardiac surgery and orthopedic surgery12132328 In many of these patients are prioritized by the scoring of several criteria and summing of the scores to achieve a total patient score Many of these systems have been validated against expert rankings of surgical priority2728We used a methodology for developing a pointbased prioritization system similar to those previously described29 To the best of our knowledge pointbased surgical prioritization systems have been very well studied to date Hansen et al previously proposed a methodology for developing a pointbased prioritization system using the following steps ranking patient case vignettes Cancer Month 0cSPARTANHNde Almeida et alFIGURE The Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancer SPARTANHN scoring system ECOG indicates Eastern Cooperative Oncology Group ENE extranodal extensionusing clinical judgment drafting the criteria and categories within each criteria pretesting the criteria and categories consulting with patient groups and other clinicians determining point values for criteria and categories checking the testretest reliability and face validity and revising the points system as new evidence emerges29 Our approach to the development of the SPARTANHN was similar However given the relatively expedited nature of the process we did not directly involve patientsOne method proposed for establishing the priority of all indications in a pointbased scoring system is known as Potentially All Pairwise Rankings of all Alternatives PAPRIKA30 In the current study we chose to use the RANDUCLA process instead of pairwise comparison to minimize computational burden We established Cancer Month 0cOriginal FIGURE External validation rank results A total of experts were asked to rate the scenarios provided shown on the xaxis and the results were compared with the rank generated by models and shown on the yaxis Green shading reflects high priority ranked yellow shading indicates medium priority ranked and red shading indicates low priority ranked Asterisk denotes ties from the algorithm SPARTANHN indicates Surgical Prioritization and Ranking Tool and Navigation Aid for Head and Neck Cancerindications for surgical prioritization that would create an enormous computational burden using pairwise comparison methodology One problem inherent in the PAPRIKA methodology is the assumption that all indications are not equal and can be ranked However clinically certain indications may be equivalent in priority Furthermore pairwise comparisons assume mutual exclusivity of each of the indications which is not always the case Use of the RANDUCLA consensus process avoids the need for multiple pairwise comparison and allows for consideration of each factor in isolation The goal of the consensus rounds was not to establish a rank order for all indications but mainly to understand which indications result in high intermediate or low priorityThe SPARTANHN algorithm has demonstrated preliminary reliability and validity We demonstrated good agreement between raters and the SPARTANHN algorithm suggesting minimal interpretive error Many of the highpriority indications accounted for some component of interpretation because raters were forced to consider imminent disease progression that may result in an adverse outcome Despite the subjective decisions that must be made as part of SPARTANHN agreement remained high In fact true interrater reliability was found to be higher because the Kalpha is a conservative measure of reliability Other measures of reliability such as the Kendall coefficient of concordance tend to overestimate reliability and cannot be applied to missing data31 Perhaps most important the SPARTANHN correlated highly with expert rankings With established validity this algorithm may be ready for preliminary clinical use although further testing against realworld data to validate it with other cancer outcomes such as survival is neededThe results of the current study must be interpreted within the context of the study design Although externally validated by other surgeons across North America and the United Kingdom the criteria for which consensus was achieved for prioritization were not vetted by patients advocacy groups or other stakeholders such as medical or radiation oncologists The latter groups represent essential providers in the multidisciplinary care of patients with HNC and may have important insight into the availability and effectiveness of nonsurgical treatments1920 Nonetheless the actual prioritization of surgical waitlists remains the sole responsibility of surgeons and their practice partners In addition the SPARTANHN algorithm is intended to assist in making difficult prioritization decisions and is not intended to make recommendations for the time frame in which patients should receive treatment Instead established guidelines should be adhered to for treatment targets Patient wait times as they relate to those targets should be considered when using the SPARTANHN algorithm The validation process in the current study used expert opinion as the gold standard of prioritization which is potentially biased and reflected the opinions of surgeons practicing in academic medical centers from resourcerich nations Subsequently use of the SPARTANHN algorithm in other geographic regions Cancer Month 0cand health care systems requires additional investigation because local treatment paradigms and risk factors may vary substantiallyThe current study has presented the development and validation of a novel algorithm for the prioritization of surgery for patients with HNC Further evaluation of its implementation in various practice settings will be obligatory However the results of the current study have provided data with which to inform realworld use as the current pandemic has obviated our ability to more rigorously study the instrument prior to making necessary and difficult realtime allocation decisionsFUNDING SUPPORTNo specific funding was disclosedCONFLICT OF INTEREST DISCLOSURESEvan M Graboyes has received grants from the National Cancer Institute and the Doris Duke Charitable Foundation for work performed outside of the current study Vinidh Paleri offers his services as a proctor for a transoral robotic surgery proctoring program run by Intuitive Surgical and has been remunerated for his time Antoine Eskander has received a research grant from Merck and acted as a paid consultant for BristolMyers Squibb for work performed outside of the current study Ian J Witterick has stock in Proteocyte Diagnostics Inc and has received honoraria from Sanofi Genzyme and Medtronic Canada for work performed outside of the current study The other authors made no disclosuresAUTHOR CONTRIBUTIONSJohn R de Almeida Study idea and design writing and editing Christopher W Noel Study design writing data analysis and editing David Forner Study design writing data analysis and editing Han Zhang Data acquisition and editing Anthony C Nichols Data acquisition and editing Marc A Cohen Data acquisition and editing Richard J Wong Data acquisition and editing Caitlin McMullen Data acquisition and editing Evan M Graboyes Data acquisition and editing Vasu Divi Data acquisition and editing Andrew G Shuman Writing data acquisition and editing Andrew J Rosko Data acquisition and editing Carol M Lewis Data acquisition and editing Ehab Y Hanna Data acquisition and editing Jeffrey Myers Data acquisition and editing Vinidh Paleri Data acquisition and editing Brett Miles Data acquisition and editing Eric Genden Data acquisition and editing Antoine Eskander Data acquisition and editing Danny J Enepekides Data acquisition and editing Kevin M Higgins Data acquisition and editing Dale Brown Data acquisition and editing Douglas B Chepeha Data acquisition and editing Ian J Witterick Data acquisition
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the incidence and death rate of nonsmall cell lung cancer nsclc in china ranks the first among the malignant tumors circular rna circrna was reported to be involved in the progression of nsclc our study aimed to investigate the underlying mechanism of circ_0020123 in nsclc progressionmethods quantitative realtime polymerase chain reaction qrtpcr was used to detect the expression of circ_0020123 mir5905p and thrombospondin thbs2 in nsclc tissues and cells cell proliferation and migration were examined by cell counting kit8 cck8 assay and transwell assay respectively flow cytometry assay was used to detect the apoptosis of nsclc cells the protein levels of ki67 matrix metalloprotein9 mmp9 cleavedcaspase9 cleavedcasp9 and thbs2 were detected by western blot the targets of circ_0020123 and mir5905p were predicted by starbase and targetscan and then confirmed by dualluciferase reporter assay and rna immunoprecipitation rip assay the animal experiment showed the effect of circ_0020123 on tumor growth in vivoresults the expression of circ_0020123 was upregulated in nsclc tissues and cells functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of nsclc cells interestingly circ_0020123 directly targeted mir5905p and inhibition of mir5905p reversed the knockdown effects of circ_0020123 on nsclc cells more importantly thbs2 was a target of mir5905p and thbs2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in nsclc cells finally suppression of circ_0020123 inhibited tumor growth in vivo through mir5905pthbs2 axis circular rna circ_0020123 regulated thbs2 by sponging mir5905p to promote cell proliferation and migration and inhibit cell apoptosis in nsclc cellskeywords nsclc circ_0020123 mir5905p thbs2highlights circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in nsclc cellscorrespondence bskrju163comdepartment of thoracic surgery lianyungang second people™s hospital no hailian east road haizhou district lianyungang jiangsu china circ_0020123 directly targeted mir5905p and mir5905p downregulation reversed the knockdown effects of circ_0020123 on nsclc progression thbs2 acted as a target of mir5905p and overthe effects of expression of thbs2 reversed circ_0020123 knockdown on nsclc progression downregulation of circ_0020123 suppressed tumor growth in vivo through mir5905pthbs2 axis the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwang a0et a0al cancer cell int page of lung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis small cell lung cancer sclc accounts for about of all lung cancer cases however nonsmall cell lung cancer nsclc accounts for of lung cancer and the a0years overall survival rate os is only about therefore it is important to find the effective treatment and potential molecular targets of nsclc progressioncircular rna circrna is a single stranded rna molecule with a closed circular structure recently amounts of circular dna have been discovered and most of which were thought to be the byproducts of typical splicing [ ] previous reports indicated that the expression of circrna was tissuespecific and the change of its expression intensity was associated with some diseases [“] furthermore circrna was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] for example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging mir34a5p to regulate cc chemokine ligand ccl22 meanwhile hsa_circ_0043256 participated in the progression of nsclc cells by mediating the cinnamaldehyde treatment a previous report suggested that circ_0020123 acted as an oncogene in nsclc and circ_0020123 regulated zincfingerenhancer binding protein zeb1 and enhancer of zeste homolog ezh2 by competitively binding with mir144 to induce cell progression and migration these reports suggested that circ_0020123 was a vital factor in the pathogenesis of nsclc and its function and molecular mechanism need to be further studiedas a small endogenous rna microrna mirna is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers recently some aggregated mirnas have been found in prostate cancer such as mir221222 mir143145 mir23b27b241 and mir1133a which were downregulated and had tumor inhibiting functions a previous study found that circulating mir5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy besides overexpression of mir5905p reduced the development of nsclc cells and regulated the expression of epithelialmesenchymal transformation emtrelated proteins by targeting the signal transducers and activators of transcription stat3 however the precise mechanism by which mir5905p affects nsclc needs further investigationthrombospondin thbs2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer colorectal cancer and nsclc a previous report suggested that thbs2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by mir20a tian et a0al found the expression and clinicopathological features of thbs2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis however the molecular function of thbs2 in nsclc remains poorly definedin this study the targeting relationship between circ_0020123 and mir5905p was firstly detected the effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesmaterials and a0methodspatients and a0specimensnsclc tissues and the adjacent healthy lung tissues were taken from nsclc patients in the lianyungang second people™s hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °c for further experiments this research was approved by the ethics committee of lianyungang second people™s hospitalcell culture and a0cell transfectiontwo nsclc cell lines a549 and h1299 and one normal lung cell line imr90 were obtained from the beijing concorde cell library beijing china a549 h1299 and imr90 cells were cultivated in dulbecco™s modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0„ƒ with co2small interfering rna sirna targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin rna shrna targeting circ_0020123 shcirc_0020123 mir5905pinhibitors sirna negative control sinc shnc and ncinhibitors were all obtained from biomics biotech jiangsu china full length of thbs2 cdna sangon biotech shanghai china was subcloned into pcdna31 plasmid ekbioscience shanghai china then cell transfection was performed by lipofectamine thermo fisher scientific waltham ma usa 0cwang a0et a0al cancer cell int page of rna isolation and a0quantitative real‘time polymerase chain reaction qrt‘pcrthe trizol reagent invitrogen carlsbad ca usa was used for extracting the total rnas next the reversed transcription was carried out by rtpcr kit invitrogen the qrtpcr was performed using the abi sybr green master mix invitrogen the primers in our study were as follows f5²ttc gga cga ccg tca aac at3² and r5²agg atc cct gca cca caa tg3² for circ_0020123 f5²tga aag acg tga tgg cac ac3² and r5²ctt cca ttt tgg for mir5905p f5²aga agg ggt ttt tgg3 ² ctg ggg ctc att tg3² r5²agg ggc cat cca cag tct tc3² for glyceraldehyde3phosphate dehydrogenase gapdh f5²gcg gct ggg tct att tgt c3² and r5²gca gga ggt gaa gaa cca tc3² for thbs2 f5²att gga acg ata cag aga agatt3² and r5²gga acg ctt cac gaa ttt g3² for u6 gapdh and u6 were the internal parameterscell counting kit‘ cck‘ assaythe proliferation viability of a549 and h1299 cells were detected by the cellcounting kit8 msk wuhan china a549 and h1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°c for or a0h then a0μl fresh medium and cck8 solution was added after incubation at a0°c for a0h the od values were detected by the multiskan ascent microplate reader abcam cambridge ma usatranswell assaytranswell chamber corning life sciences corning ny usa was used to detect cell migration firstly the serumfree dmem thermo fisher scientific was fixed with cell suspension cells and seeded into the upper chamber and the dmem containing serum was put into the lower chamber after incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed finally the migrated cells were stained with crystal violet corning life sciences and observed by using a microscopeflow cytometryfirstly a549 and h1299 cells were cultured and pbs was used for washing cells then the binding buffer was used to resuspend cells and the annexin vfluorescein isothiocyanate vfitcpropidium iodide pi apoptosis detection kit thermo fisher scientific was used to stain cells finally cell apoptosis was detected by flow cytometry thermo fisher scientificwestern blot analysisthe total proteins of nsclc tumors or cells were collected by ripa lysis buffer sangon biotech then the proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis sdspage and transferred to polyvinylidene fluoride pvdf membranes thermo fisher scientific the skimmed milk was added and incubated with primary antigapdh antibody invitrogen carlsbad ca usa antiβactin antibody invitrogen antiki67 antibody invitrogen antimatrix metalloprotein9 mmp9 antibody invitrogen anticleavedcaspase9 cleavedcasp9 antibody invitrogen or antithbs2 antibody invitrogen at a0°c overnight finally the membranes were incubated with the secondary antibody for a0 h at room temperature the results were viewed using kodak film developer fujifilm japandual‘luciferase reporter assaysthe wild type circ_0020123 sequences circ_0020123wt mutant circ_0020123 sequences circ_0020123mut wild type thbs2 ²utr sequences thbs2wt mutant thbs2 ²utr sequences thbs2mut were cloned into pgl3 luciferase reporter plasmid promega madison wi usa then the plasmid and mir5905p or mirnc were cotransfected into a549 and h1299 cells by lipofectamine thermo fisher scientific after transfection for a0h the dualluciferase reporter assay system promega was performed to detect the luciferase activityrna immunoprecipitation ripfirstly the magna rip rnabinding protein immunoprecipitation kit gzscbio guangzhou china was performed to verify the relationship between circ_0020123 and mir5905p in brief the magnetic beads and antiago2 antibody abcam were added into cells and incubated for a0h then the proteinase k and the phenol“chloroformisoamyl alcohol reagent were added for purifying rnas finally qrtpcr was used to measure circ_0020123 enrichmentanimal experimentsthe 4weekold balbc male nude mice vitalriver beijing china were raised in a sterile environment for 0cwang a0et a0al cancer cell int page of experiments then pbs was used to suspend a549 cells × transfected with shcirc_0020123 or shnc next the nude mice were divided into two groups n a549 cells transfected with shcirc_0020123 or shnc were shcirc_0020123 or shnc inoculated into the nude mice the tumor volume was detected every a0 days after a0days the nude mice were euthanatized and the tumor weight was detected besides the tumor tissues from each group were collected to detect the expression of circ_0020123 mir5905p and thbs2 the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalstatistical analysisthe software graphpad prism was performed for statistical analysis the data was displayed as mean ± standard deviation sd the significant difference was calculated by student™s t test and oneway analysis of variance anova p was considered as statistically significantresultscirc_0020123 was a0upregulated in a0nsclc tissues and a0cellsto begin with qrtpcr was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in nsclc tissues compared with the adjacent healthy tissues fig a0 1a similarly the expression of circ_0020123 in nsclc cells a549 and h1299 was markedly higher than that in normal cells imr90 fig a01b from these data it is speculated that circ_0020123 might be acted as an oncogene in nsclcfig circ_0020123 was upregulated in nsclc tissues and cells a qrtpcr was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b the expression of circ_0020123 in normal cell line imr90 and nsclc cell lines a549 and h1299 was detected by qrtpcr p downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0nsclc cellsto investigate the functional effects of circ_0020123 on nsclc cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into a549 and h1299 cells firstly the transfection efficiency was detected by qrtpcr fig a02a next cck8 was used to detect the proliferation and the results showed that the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced fig a0 2b moreover the migration of a549 and h1299 cells was significantly downregulated by circ_0020123 knockdown fig a02c in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with sinc fig a02d finally the protein levels of cell proliferationrelated protein ki67 and cell migrationrelated protein mmp9 were inhibited while cell apoptosisrelated protein cleavedcasp9 was upregulated in nsclc cells transfected with sicirc_00201231 or sicirc_00201232 fig a02e these data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in nsclc cellscirc_0020123 directly targeted mir‘‘5pby searching in the online software starbase the potential binding sites between circ_0020123 and mir5905p were detected fig a0 3a to confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123wt reporter plasmid was reduced by mir5905p mimic while the circ_0020123mut reporter plasmid activity was not changed in a549 and h1299 cells fig a03b furthermore the expression of mir5905p was lower in a549 and h1299 cells compared with that in imr90 cells fig a0 3c in contrast mir5905p expression was elevated in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a0 3d finally the rip assay was also used to confirm the targeting relationship between circ_0020123 and mir5905p and the results showed that circ_0020123 and mir5905p were enriched in antiago2 group fig a03emir‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0nsclc cellsto further explore the functional effects between circ_0020123 and mir5905p mir5905pinhibitor was established qrtpcr was used to detect the transfection efficiency fig a0 4a interestingly mir5905p was upregulated in a549 and h1299 cells transfected with sicirc_00201231 while the expression of mir5905p was recovered in cells transfected with 0cwang a0et a0al cancer cell int page of fig downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of nsclc cells a the transfection efficiency of sicirc_00201231 and sicirc_00201232 in a549 and h1299 cells was detected by qrtpcr b cck8 assay was used to detect the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 c the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by transwell assay d flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 e the protein levels of cell proliferation related protein ki67 cell migration related protein mmp9 and cell apoptosis related protein cleavedcasp9 were detected by western blot p fig a0sicirc_00201231 mir5905pinhibitors 4b moreover circ_00201231 knockdown inhibited cell proliferation and migration while the mir5905p inhibitor reversed these effects fig a0 4c d in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 was increased which was abolished by mir5905pinhibitor fig a0 4e similarly mir5905p inhibitors reversed the effects on the protein levels of ki67 mmp9 and cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 fig a0 4f these results that mir5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of nsclc cellsindicated mir‘‘5p targeted thbs2 in a0nsclc cellsthe thbs2 ²utr was predicted to contain the binding sites of mir5905p through the online software targetscan fig a05a then the dualluciferase reporter assay was used to confirm the targeting relationship the results showed that cotransfection of mir5905p and thbs2wt significantly limited the luciferase activity in both a549 and h1299 cells the luciferase activity was not altered in cells cotransfected with mir5905p and thbs2mut fig a05b importantly the mrna and protein level of thbs2 was enahnced in nsclc cells fig a05c d we further explored whether circ_0020123 affected the functions of thbs2 in nsclc cells the mrna and protein expression of thbs2 were repressed in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a05e f 0cwang a0et a0al cancer cell int page of fig circ_0020123 directly targeted mir5905p a the binding site between circ_0020123 and mir5905p was detected by the online software starbase b the luciferase activity of circ_0020123wt or circ_0020123mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p was detected by dualluciferase reporter assay c qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells d the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr e rip assay was used to confirm the relationship between circ_0020123 and mir5905p p thbs2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0nsclc cellsbased on the work ahead of us the pcdna31thbs2 was constructed then the qrtpcr and western blot were used to detect the transfection efficiency and the thbs2 expression was increased in a549 and h1299 cells transfected with pcdna31thbs2 fig a0 6a b in addition the proliferation and migration rates of a549 and h1299 cells transfected with sicirc_00201231 pcdna31thbs2 were higher than that transfected with sicirc_00201231 fig a0 6c d meanwhile a similarly phenomenon was also observed in cell apoptosis the pcdna31thbs2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis fig a0 6e furthermore the effects of circ_0020123 deletion on ki67 mmp9 and cleavedcasp9 protein levels were also reversed by thbs2 overexpression fig a0 6f these data suggested that overexpression of thbs2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisreduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123mir‘‘5pthbs2 axisto further explore the function of circ_0020123 in nsclc cells the shcirc_0020123 was constructed and the xenograft tumor was established then a549 cells transfected with shcirc_0020123 or shnc were injected into the nude mice the xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shnc group fig a07a moreover tumor weight was inhibited by circ_0020123 knockdown fig a0 7b furthermore the expression circ_0020123 and thbs2 was decreased while the mir5905p was increased in xenograft tumor transfected with shcirc_0020123 fig a0 7c western blot assay also revealed that the protein level of thbs2 was repressed by circ_0020123 knockdown fig a07d finally the digital tomosynthesis dts was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group fig a07e the results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivodiscussionclinically only a few nsclc patients were diagnosed at an early stage and treated by surgical resection more than of nsclc patients were diagnosed with the advanced stage or metastatic tumors thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of nsclcrecently circrna was no longer considered as a random product in the rna shearing process and its biological significance and function in malignant tumors 0cwang a0et a0al cancer cell int page of fig mir5905p downregulation reversed circ_0020123 knockdown effects in nsclc cells a qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells transfected with mir5905pinhibitors b the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was detected by qrtpcr c the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was tested by cck8 assay d transwell assay was used to measure the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors e flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors were detected by western blot p had received more and more attention previous reports revealed that circ_0020123 was involved in the development of nsclc moreover the level of circ_0020123 was elevated in nsclc cells consistently we found that the expression of circ_0020123 was markedly higher in nsclc tissues and cells moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of nsclc cells in a0 vitro besides circ_0020123 promoted tumor growth in a0vivoendogenous circrnas could act as microrna sponges to inhibit their function and some studies linked mirna sponges to human diseases including cancer a previous study indicated that circrna ctransferrin receptor ctfrc regulated tfrc by sponging mir107 to facilitate bladder carcinoma development mir5905p was studied in different cells such as airway smooth muscle cells colon epithelial cells and nsclc cells however the potential relationship between mir5905p and circrna has not been researched in this study circ_0020123 directly targeted mir5905p and mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc progression these data provided a clue to the therapeutic strategy for nsclc 0cwang a0et a0al cancer cell int page of fig mir5905p targeted thbs2 in nsclc cells a the potential binding site between thbs2 ²utr and mir5905p was predicted by the online software targetscan b dualluciferase reporter assay was used to measure the luciferase activity of thbs2wt or thbs2mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p c qrtpcr was used to detect the mrna expression of thbs2 in nsclc cells d the protein level of thbs2 in nsclc cells was tested by western blot e the mrna expression of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr f western blot was used to measure the protein level of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 p our study also confirmed that mir5905p could target thbs2 directly in nsclc cells thbs2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase mmp genes involved in tissue formation and repair [ ] a previous document suggested that thbs2 acted as a target of mir2213p and participated in lymph node metastasis in cervical cancer the data in this research showed that the expression of thbs2 in nsclc cells was markedly higher than normal healthy cells furthermore overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells suggesting that circ_0020123 promoted the progression of nsclc cells through mir5905pthbs2 axisin our research showed that the expression of circ_0020123 was higher in nsclc tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of nsclc cells and also suppressed tumor growth in a0 vivo moreover circ_0020123 directly targeted mir5905p while mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc cells more importantly circ_0020123 regulated the expression of thbs2 by sponging mir5905p and upregulation of thbs2 reversed the effects of circ_0020123 knockdown on nsclc cells therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cwang a0et a0al cancer cell int page of fig overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells a b the mrna and protein expression of thbs2 in a549 and h1299 cells transfected with pcdna31thbs2 was detected by qrtpcr and western blot c cck8 assay indicated the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 d the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was measured by transwell assay e the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was detected by flow cytolysis assay f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 were detected by western blot p apoptosis of nsclc cells by sponging mir5905p to regulate thbs2results and develop the manuscript all authors read and approved the final manuscriptabbreviationsnsclc nonsmall cell lung cancer circrna circular rna qrtpcr quantitative realtime polymerase chain reaction cck8 cell counting kit8 mmp9 matrix metalloprotein9 cleavedcasp9 cleavedcaspase9 cleavedcasp9 cleavedcaspase9 rip rna immunoprecipitation zeb1 zincfingerenhancer binding protein ezh2 zeste homolog stat3 signal transducers and activators of transcription thbs2 thrombospondin acknowledgementsnot applicableauthors™ contributionslw collaborated to design the study lz were responsible for experiments analyzed the data yw wrote the paper all authors collaborated to interpret fundingnoneavailability of data and materialsplease contact corresponding author for data requestsethics approval and consent to participatethis research was approved by the ethics committee of lianyungang second people™s hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalconsent for publicationall listed authors have actively participated in the study and have read and approved the submitted manuscript 0cwang a0et a0al cancer cell int page of fig reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123mir5905pthbs2 axis a a total of × a549 cells transfected with shcirc_0020123 or shnc were injected into nude mice to establish the xenograft tumor tumor volume was measured every d after injection b tumor weight was measured on d c the expression of circ_0020123 mir5905p and thbs2 in xenograft tumor was measured by qrtpcr d the protein level of thbs2 in xenograft tumor was evaluated by western blot e the number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis dts p competing intereststhe authors declare that they have no competing interestsreceived april accepted july references bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ abe h takase y sadashima e fukumitsu c murata k ito t kawahara a naito y akiba j insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value cancer cytopathol “li c zhang l meng g wang q lv x zhang j li j circular rnas pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer j cancer res clin oncol “ belousova ea filipenko ml kushlinskii ne circular rna new regulatory molecules bull exp biol med “ zhang z xie q he d ling y li y li j zhang h circular rna new star new hope in cancer bmc cancer li l chen y nie l ding x zhang x zhao w xu x kyei b dai d zhan s guo j zhong t wang l zhang h myodinduced circular rna cdr1as promotes myogenic differentiation of skeletal muscle satellite cells biochim biophys acta gene regul mech “ greco s cardinali b falcone g martelli f circular rnas in muscle function and disease int j mol sci weng xd yan t liu cl circular rna_larp4 inhibits cell migration and invasion of prostate cancer by targeting foxo3a eur rev med pharmacol sci “ deng n lei d huang j yang z fan c wang s circular rna expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsi
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" rectus sheath block rsb is known to attenuate postoperative pain and reduce perioperative opioidconsumption thus a retrospective study was performed to examine the effects of bilateral rectus sheath blockbrsb in cytoreductive surgery crs combined with hyperthermic intraperitoneal chemotherapy hipecmethods a total of patients undergoing crshipec at our hospital were included patient information andanaesthesiarelated indicators were collected from the electronic medical record emr system all subjects weredivided into the following two groups the g group general anaesthesia and the gr group rsb combined withgeneral anaesthesia patients in the gr group received ropivacaine for brsb before surgery the primaryoutcomes included the total amount of remifentanil and rocuronium the total consumption of dezocine aftersurgery the visual analogue scale vas score and the patientcontrolled intravenous analgesia pcia input dose at h t6 h t7 h t8 h t9 and h t10 after surgery other outcomes were also recorded such aspatient demographic data the intraoperative heart rate hr and mean arterial pressure map and postoperativecomplicationsresults compared with the g group the gr group showed a shorter time to tracheal extubation p adecreased total amount of remifentanil and rocuronium p and a reduced vas score pcia input dose andnumber of pcia boluses at h h and h after surgery p however at h and h after surgery therewere no differences in the vas score of pain at rest or during motion between the two groups p moreoverthe incidence of hypertension emergence agitation delayed recovery hypercapnia and nausea and vomiting waslower in the gr group than in the g group p there were no differences in the changes in map and hrduring the surgery between the two groups p no complications associated with nerve block occurred brsb could provide shortterm postoperative analgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complications it is an effective and safe procedure in crshipeckeywords cytoreductive surgery hyperthermic intraperitoneal chemotherapy rectus sheath block generalanaesthesia analgesia correspondence trmzltz126comdepartment of anesthesiology beijing shijitan hospital capital medicaluniversity beijing china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc anesthesiology page of radical cytoreductive surgery crs combined withhyperthermic intraperitoneal chemotherapy hipec isconsidered a standard for the treatment of peritonealcancer such as rectal cancer ovarian cancer peritonealpseudomyxoma and peritoneal mesothelioma thistechnique could prolong the longterm survival of patients with a decreased recurrence rate although thepositive results of this treatment have been proven inprevious studies [“] because of the large peritonealsurface area involved in this kind of surgery crshipecis time consuming and complex which presents agreat challenge for the anaesthesiologist in terms of perioperative managementdue to the stable respiratory and circulatory supportgeneral anaesthesia is the preferred choice in this surgery however long periods of general anaesthesia leadto drug accumulation in the body followed by increasedanaesthesiarelated complications including delayed recovery respiratory inhibition and cognitive dysfunction consequently exploring better anaesthesia methodsfor this surgery is still a major concerna new approach called ultrasoundguided bilateral rectus sheath block brsb has been proven to amelioratepostoperative pain and reduce the consumption of morphine [“] nonetheless there have been no reportson the application of general anaesthesia combined withbrsb in patients undergoing crshipec based on theinformation presented above this retrospective observational study was conducted to examine the efficacy andsafety of brsb in patients treated with crs and hipecmethodssubjectsall patients who underwent crs and hipec at beijingshijitan hospital between august and december were retrieved from the institutional database inthis study the exclusion criteria were as follows laparoscopic surgery with crshipec intraoperativeblood loss volume greater than ml mechanicventilation required after surgery and use of analgesictechniques apart from brsb and general anaesthesiaaccording to this standard a total of patients wereincluded and divided into the following groups generalanaesthesia g group n and general anaesthesiacombined with posterior rsb gr group n anaesthesia methodgeneral anaesthesia was consistently induced in all patients with intravenous propofol mgkg sufentanil μgkg and rocuronium mgkg invasive arterialpressure and central venous pressure were monitored byradial artery puncture flotracvigileo® edwards lifesciences irvine ca usa and internaljugular veinsitetargeteffectpuncture respectively after anaesthesia induction anaesthesia was maintained with sevoflurane and remifenconcentration “ ngmltanilkeeping the bispectral index bis between and rocuronium mgkg wasintermittently used tomaintain muscle relaxation in the gr group before anaesthesia induction patients received brsb under ultrasound guidance the puncture site was placed at theouter edge of the bilateral rectus abdominis at the levelof the umbilicus fig a a total of ropivacaine ml was injected into each side the spindleshapedspread of ropivacaine was observed between the posterior sheath of the rectus abdominis and the rectus abdominis itself implying success of the procedure fig b c patientcontrolled intravenous analgesia pciawas applied in both groups after the surgery sufentanil μgkg palonosetron hydrochloride mg was diluted to ml the dose was mlh and asingle dose was mlh with a 15min lockout intervalafter the surgery all patients were sent to the surgicalintensive care unit sicu if the visual analogue scalevas score at rest after surgery was ‰¥ dezocine mgwas used as a rescue analgesicdata collectionall the indicators we needed were obtained from theemr system the records included patient demographicdata patient medical history american society of anesthesiologists asa grade and new york heart association nyha grade the hr and map were recordedat the time before brsb t1 the time of anaesthesiat2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgeryt5 in addition the duration of the surgery time totracheal extubation the time after skin closure totalamount of remifentanil and muscle relaxants total fluidvolume urine volume and the total volume of allogeneicerythrocytes and plasma infused during the surgery wereall recorded moreover after surgery the occurrence ofhypertension the systolic blood pressure dropped bymore than of baseline blood pressure beforeanesthesia or the sbp mmhg during surgery nausea and vomiting hypoxemia spo2 or pao2 mmhg hypercapnia paco2 mmhg and emergence agitation during the recovery period were recorded the recovery period was considered as the timefrom switching off inhalation anaesthetics remifentaniland muscle relaxant to recovery of the patients™ abilitiesto command movement orientation as well as conscious state when the recovery period of patients is beyond min it was considered as delayed recovery thevas score for pain at rest and during motion the pciainput dose and the number of boluses at h t6 ht7 h t8 h t9 and h t10 after surgery 0cwang bmc anesthesiology page of the incomplete datachemotherapy crshipec during the year from to in our hospital one hundred and six patientsreceived brsb seventeen patients were excluded because ofincluding patientsundergoing intraoperative haemorrhage blood ml and other patients received mechanic ventilationbecause of acute respiratory distress syndrome ardsallergic shock and cardiac insufficiency thus patients with brsb were eventually obtained finally patients without brsb were randomly selected to analysis in this study fig statistical analysisspss software was used for statistical analysisnormal distribution data were recorded as the mean ±standard deviation sd and analysed by independentsamples t test for comparison between the two groupsnonnormally distributed data are presented as themedian range and were analysed by kruskalwallistest chisquared test or fisher™s exact test was used forcategorical data a p value of was considered statistically significantresultscharacteristics of study populationin total patients were included in the study thebaseline demographic and surgical variables of patientsare presented in table there were no significant differences in age sex body mass index bmi basic diseases asa grade nyha grade total surgery time totalfluid volume urine volume total volume of allogeneicerythrocyte infusion or total volume of plasma p however the time to tracheal extubation was shorter inthe gr group than in the g group p the totalamount of both remifentanil and rocuronium used wasless in the gr group than in the g group p thus posterior rsb could reduce the use of remifentaniland rocuronium during surgerychanges in haemodynamic parametersthe changes in hr and map are presented in table there were no significant differences in hr or map atany point in time t1 to t5 between the two groupsp the results suggest that brsb did not affectthe haemodynamics ofthe patient undergoing crshipecpainrelated indicatorstable shows the postoperative vas score the pca input dose and the number of pca boluses at h t6 ht7 h t8 h t9 and h t10 after surgeryas well as the dose of dezocine used as a rescue analgesic from t6 to t8 compared with the g group thegr group showed significantly decreased vas scores offig ultrasoundguided brsbas well as the dose of dezocine used as a rescue analgesic were also recordedin addition brsbrelatedcomplications such as peritoneal punctureinternalan injury and systemic toxicity were all recordeda total of patients underwent cytoreductive surintraperitonealcombined withgeryhyperthermic 0cwang bmc anesthesiology page of fig flow chart showing patient consecutive enrolment and analysis abbreviations crshipec cytoreductive surgery and hyperthermicintraperitoneal chemotherapy ga general anesthesia brsb bilateral rectus sheath block ards acute respiratory distress syndrome vas visualanalogue scalepain at rest and during motion p however at h and h after surgery there were no significant differences in the vas scores of pain at rest and duringmotion between the two groups p from t6 tot10 the pcia input dose and the number of pca boluses were also obviously reduced in the gr group compared with the g group p in addition as arescue analgesic the dose of dezocine after surgery inthe gr group was significantly lower than that in the ggroup p postoperative adverse eventsadverse events that occurred in the sicu are presentedin table after surgery there were cases withhypertension cases of emergence agitation cases ofdelayed recovery cases of hypercapnia and cases ofnausea and vomiting in the g group fewer cases of allof these events occurred in the gr group p there were no differences in the incidence of hypoxemiabetween the two groups p there were no complications associated with nerve block in either group 0cwang bmc anesthesiology page of table demographic and surgical variables mean ± sdage ysex malefemalebmi kgm2medical historydiabetes mellitus n yesnohypertension n yesnocoronary heart disease n yesnoasa grade iiiiiinyha grade iiitotal surgery time mintime to tracheal extubation minremifentanil mgrocuronium mgtotal fluid volume mlurine volume mltotal volume of allogeneic erythrocyte infusion mltotal volume of plasma mlg group n ± ± ± ± ± ± ± ± ± ± gr group n ± ± ± ± ± ± ± ± ± ± p value asa american society of anesthesiologists bmi body mass index calculated as weight in kilograms divided by height in metres squared nyha new york heartassociation g general anaesthesia gr bilateral rectus sheath block combined with general anaesthesia before bilateral rectus sheath block t1 the time ofanaesthesia t2 the time of skin incision t3 the time of peritoneal thermochemotherapy t4 and the end of surgery t5discussionin this retrospective study we examined the efficacy andsafety of brsb combined with general anaesthesia in patients undergoing crshipec regarding efficacy theresults show that ultrasoundguided brsb significantlyreduced the total dose of remifentanil used during thesurgery and shortened the time to tracheal catheter extraction which is consistent with the findings of previous studies of other surgeries [ ] in addition rsbreduced the total dose of rocuronium in this studytable haemodynamic parameters in both groups mean ± sdindextimepointgn ± grn ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± pvaluemmhghrbpmt1t2t3t4t5t1t2t3t4t5map mean arterial pressure hr heart rate g general anaesthesia gr bilateralrectus sheath block combined with general anaesthesiawhich may be associated with the high concentration ofropivacaine used in the studyrsb also effectively relieved postoperative pain in thisstudy we found that the vas scores of pain at rest andduring motion were all lower in the gr group than inthe g group at h after surgery however at h and h after surgery there were no differences in the vasscores of pain at rest and during motion between thetwo groups suggesting that the analgesic effects of asingle brsb remained within h after surgery thisresult may be different from the findings of others cho reported that at h after surgerythere were no differences in the vas scores of painat rest and during motion between the rsb and nonrsb groups the discrepant results may be related todifferences in the concentration of ropivacaine andthe physical constitution of patients a high concentration can prolong the duration of action of a localanaesthetic in this study we selected not ropivacaine additionallythese patientsundergoing crshipec may have been adaptive topain furthermore compared with the control groupthe rsb group showed a reduced totalinfused doseof sufentanil as pcia number of pca boluses within h after surgery and total dose of dezocine used asa rescue analgesic after surgery these results furtherprove the role of rsb in providing shortterm postoperative analgesia 0cwang bmc anesthesiology page of table painrelated indicators in both groups median [range]vas score of pain at rest [median range]t6t7t8t9t10vas score of pain during motion [median range]t6t7t8t9t10total infused dose of pcia [ml median range]t6t7t8t9t10cumulative number of pcia boluses [median range]t6t7t8t9t10total dose of dezocine as a rescue analgesic mggn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± grn [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ± p value vas visual analogue scale pcia patientcontrolled intravenous analgesia g general anaesthesia gr bilateral rectus sheath block combined with generalanaesthesia the time at h after surgery t6 h after surgery t7 h after surgery t8 h after surgery t9 and h after surgery t10table postoperative adverse events in both groupsadverse eventsn gn grn hypertensionemergence agitation delayed recoveryhypoxemiahypercapnia nausea and vomiting peritoneal punctureinternal an injurysystemic toxicity g general anaesthesia gr bilateral rectus sheath block combined withgeneral anaesthesiawe also examined the safety of rsb during the surgery ultrasoundguided brsb had no significant effectson the haemodynamics of patients during surgery compared with general anaesthesia alone in terms of postoperative adverse events the results show that comparedwith the control group the rsb group showed a reducedincidence of hypertension emergence agitation delayedrecovery hypercapnia and nausea and vomiting whichmight be correlated with the decreased analgesic andmuscle relaxant doses no rsbrelated complications occurred in any patient these data indicate that rsbcould reduce the risk of complications associated withgeneral anaesthesia and is safe for patientsrsb an established technique has regained popularityin clinical applications [“] previous studies havedemonstrated that this technique could achieve relaxation of the anterior abdominal wall [ ] bashandyreported that anterior branches of the t7t12 thoracicnerve and the l1 lumbar nerve travelled through thepvalue“““ 0cwang bmc anesthesiology page of plane of the transverse abdominis muscle entered the rectus abdominis sheath and distributed on the surface of theskin the main process of rsb is to inject local anaesthetics between the rectus abdominis and the posteriorsheath of the rectus abdominis therefore rsb exerteda good effect in terms of perioperative analgesia for medianabdominal incisions a midline incision is required inthis kind of surgery thus based on these results rsbcould meet the need for analgesia in these patientsin addition for a long time epidural analgesia eawas thought to be an effective method for abdominalsurgery [“] studies have proved that epidural analgesia could maintain a good analgesic effect and reduceperioperative opioid consumption including in this typeof surgery [ ] however the safety of ea in crshipec remains controversial especially regarding effectson coagulation and circulatory function coagulationdysfunction and profound fluid loss are the main characteristics of patients with peritoneal cancer [ ]which might limit the administration of eaalthough epidural catheter is standard of care insolanki™s guideline in our hospital epidural catheter in not the standard of care we performed generalanesthesia combined with epidural anesthesia in somepatients to reduce the consumption of intravenous drugsand provide perfect analgesia but coagulation dysfunction and profound fluid loss are the main characteristicsof patients with peritoneal cancer in our previous studywe found that the mean arterial pressure of patientsundergoing epidural anesthesia was difficult to be maintained in the surgery besides there were many patientswith coagulation dysfunction before surgery who werenot suitable for the epidural anesthesia these results wefound in clinical were similar with others™ researcheskajdi and colleagues reported a case of epidural haematoma in their study godden found that the incidence of hypotension in the ea group was obviouslyhigher than that in the rsb group consequentlyrsb could be a better choice than ea in crshipecadditionally there are some limitations to this studyfirst all the data of this study were collected from theemr system as this was a retrospective study the findings are not as persuasive as those of a randomized controlled study we plan to conduct prospective studies toexplore the comprehensive influence of rsb in this surgery second we only examined the application of brsbestablished with a single injection which provides only ashortterm analgesic effect the efficacy of continuousanalgesia with brs catheters in crshipec remains unclear and needs further exploration third in our the results are initially presented according to the different aspects the primary outcome of this study is thetotal consumption of remifentanil during the surgeryother indicators were belonged to second outcomessin brsb could provide good postoperativeanalgesia reduce perioperative opioid consumption andreduce the incidence of postoperative complicationsthis is an easily applicable and safe procedure in crshipecabbreviationsrsb rectus sheath block brsb bilateral rectus sheath blockcrs cytoreductive surgery hipec hyperthermic intraperitonealchemotherapy emr electronic medical record vas visual analogue scalepcia patientcontrolled intravenous analgesia hr heart rate map meanarterial pressure bis bispectral index sicu surgical intensive care unitasa american society of anesthesiologists nyha new york heartassociation spo2 pulse oximetry paco2 partial pressure of carbon dioxidepao2 oxygen partial pressure bmi body mass indexacknowledgementsi would like to express my heartfelt thanks to the staff of the informationdata center of beijing shijitan hospital affiliated to capital medical universityauthors™ contributionswsh study design data collection writing paper lpf gt data collectionand data analysis gl coordinated the study and manuscript revision ltzstudy design and manuscript revision all authors read and approved thefinal manuscript all authors ensure the accuracy of the manuscript andagree to take personal responsibility for their contributionsfundingno fundingavailability of data and materialsthe datasets used andor analyzed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethis study was approved by the ethics committee of beijing shijitan hospitalaffiliated to capital medical university approval code research ethicsno69 this study is retrospective only anonymous data sources were usedand informed consent was not requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived january accepted july referencesklaver ce musters gd bemelman wa punt cj verwaal vj dijkgraaf mgaalbers ag van der bilt jd boerma d bremers aj adjuvanthyperthermic intraperitoneal chemotherapy hipec in patients with coloncancer at high risk of peritoneal carcinomatosis the colopec randomizedmulticentre trial bmc cancer 201515undefined428van oudheusden tr braam hj nienhuijs sw wiezer mj van ramshorst bluyer md lemmens ve de hingh ih cytoreduction and hyperthermicintraperitoneal chemotherapy a feasible and effective option for colorectalcancer patients after emergency surgery in the presence of peritonealcarcinomatosis ann surg oncol “passot g vaudoyer d villeneuve l kepenekian v beaujard ac bakrin ncotte e gilly fn glehen o what made hyperthermic intraperitonealchemotherapy an effective curative treatment for peritoneal surfacemalignancy a 25year experience with procedures j surg oncol“arjonasánchez a barrios p boldoroda e camps b carrascocampos jmartín vc garcíafadrique a gutiérrezcalvo a morales r ortegapérez g hipect4 multicentre randomized clinical trial to evaluate safety andefficacy of hyperthermic intraperitoneal chemotherapy hipec with 0cwang bmc anesthesiology page of huepenbecker sp cusworth se kuroki lm lu p samen cd woolfolk cdeterding r wan l helsten dl bottros m continuous epiduralinfusion in gynecologic oncology patients undergoing exploratorylaparotomy the new standard for decreased postoperative pain and opioiduse gynecol oncol “teoh da hutton mj else s walker a lee a mack la epidural analgesia aprospective analysis of perioperative coagulation in cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy am j surg “schmidt c creutzenberg m piso p hobbhahn j bucher m perioperativeanaesthetic management of cytoreductive surgery with hyperthermicintraperitoneal chemotherapy anaesthesia “kajdi me beckschimmer b held u kofmehl r lehmann k ganter mtanaesthesia in patients undergoing cytoreductive surgery withhyperthermic intraperitoneal chemotherapy retrospective analysis of asingle centre threeyear experience world j surg oncol solanki sl mukherjee s agarwal v thota rs balakrishnan k shah sb desain garg r ambulkar rp bhorkar nm society of oncoanaesthesia andperioperative care consensus guidelines for perioperative management ofpatients for cytoreductive surgery and hyperthermic intraperitonealchemotherapy crshipec indian j anaesth “ godden ar marshall mj grice as daniels ir ultrasonography guidedrectus sheath catheters versus epidural analgesia for open colorectal cancersurgery in a single centre ann r coll surg engl “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsmitomycin c used during surgery for treatment of locally advancedcolorectal carcinoma bmc cancer baratti d kusamura s iusco d gimondi s pietrantonio f milione mguaglio m bonomi s grassi a virzì s hyperthermic intraperitonealchemotherapy hipec at the time of primary curative surgery in patientswith colorectal cancer at high risk for metachronous peritoneal metastasesann surg oncol “chua tc robertson g liauw w farrell r yan td morris dl intraoperativehyperthermic intraperitoneal chemotherapy after cytoreductive surgery inovarian cancer peritoneal carcinomatosis systematic review of currentresults j cancer res clin oncol “li y zhou yf liang h wang hq hao jh zhu zg wan ds qin lx cui szji jf chinese expert consensus on cytoreductive surgery andhyperthermic intraperitoneal chemotherapy for peritoneal malignanciesworld j gastroenterol “ willschke h bosenberg a marhofer p johnston s kettner sc wanzel o kaprals ultrasonographyguided rectus sheath block in paediatric anaesthesiaanew approach to an old technique br j anaesth “azemati s khosravi mb an assessment of the value of rectus sheath blockfor postlaparoscopic pain in gynecologic surgery j minim invasive gynecol“ dingeman rs barus lm chung hk clendenin dj lee cs tracy s johnsonvm dennett kv zurakowski d chen c ultrasonographyguided bilateralrectus sheath block vs local anesthetic infiltration after pediatric umbilicalhernia repair a prospective randomized clinical trial jama surg “ relland lm tobias jd martin d veneziano g beltran rj mckee c bhalla tultrasoundguided rectus sheath block caudal analgesia or surgical siteinfiltration for pediatric umbilical herniorrhaphy a prospective doubleblinded randomized comparison of three regional anesthetic techniques jpain res 201710undefined2629“ xu l hu z shen j pm mq efficacy of usguided transversus abdominisplane block and rectus sheath block with ropivacaine anddexmedetomidine in elderly highrisk patients minerva anestesiol “ cho s kim yj jeong k moon hs ultrasoundguided bilateral rectus sheathblock reduces early postoperative pain after laparoscopic gynecologicsurgery a randomized study j anesth “li t ye q wu d li j yu j doseresponse studies of ropivacaine in bloodflow of upper extremity after supraclavicular block a doubleblindrandomized controlled study bmc anesthesiol bell jc rylah bg chambers rw peet h mohamed f moran bjperioperative management of patients undergoing cytoreductive surgerycombined with heated intraperitoneal chemotherapy for peritoneal surfacemalignancy a multiinstitutional experience ann surg oncol “landmann a visoiu m malek mm development of a novel technique forbilateral rectus sheath nerve blocks under laparoscopicguidance j pediatrsurg “kumar a wilson ga engelhardt te ultrasound guided rectus sheathblockade compared to perioperative local anesthetic infiltration in infantsundergoing supraumbilical pyloromyotomy saudi journal of anaesthesia“ bashandy gm elkholy ah reducing postoperative opioid consumption byadding an ultrasoundguided rectus sheath block to multimodal analgesiafor abdominal cancer surgery with midline incision anesthesiol pain med201443e18263 dowidar aerm ezz haa shama aae eloraby ma postoperative analgesiaof ultrasound guided rectus sheath catheters versus continuous woundcatheters for colorectal surgery a randomized clinical trial ˜† egypt journalof anaesth “karaarslan e topal a avci o tuncer uzun s research on the efficacy of therectus sheath block method agri “ piccioni f casiraghi c fumagalli l kusamura s baratti d deraco m arientif langer m epidural analgesia for cytoreductive surgery withperitonectomy and heated intraperitoneal chemotherapy int j surg 16pt a99“ vesterandersen m lundstrøm lh møller mh the association betweenepidural analgesia and mortality in emergency abdominal surgery apopulationbased cohort study acta anaesthesiol scand httpsdoi101111aas13461 0c"
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EndocrinesyPostoperative vocal fold dysfunction in covid19 era are we still intime for a recoveryElena Bonati Elena Giovanna Bignami2 Paolo Del Rio1Received May Accepted July Springer ScienceBusiness Media LLC part of Springer Nature To the EditorThe novel coronavirus COVID19 is a highly contagious zoonosis produced by SARSCoV2 which arose inChina and spread all over the world transmitting from manto man through respiratory secretions In March itwas defined by the World Health anization WHO as apandemic to underline its spread and severityHealthcare professionals are one of the categories most atrisk of contracting the infection in particular when theiractivity involves the direct management of the patient™sairways Among these categories we can count anesthetistshead and neck surgeons otolaryngologists maxillofacialsurgeons ophthalmologists and dentists For these reasonsthe latest evidencebased recommendations for otolaryngology and head and neck surgery practice suggest thathealthcare facilities should prioritize urgent and emergencyvisits and procedures until this condition stabilizes ceasingelective care []Nevertheless oncological surgical activity althoughslowed down did not stop in most hub hospitals Regardingthyroid cancer thyroid surgery is complex and the rate ofnerve damage is still considerable Immediate postoperativevocal fold rate is “ in our case study and decrease to“ after months Postoperative dysphonia can becaused by several factors other than nerve damage such astracheal intubation or scarring in the thyroid lodge It istherefore important to identify the cause of vocal corddysfunction and treat it correctly at the right time If anunilateral vocal fold paresisparalysis is diagnosedthetreatment consist in improving the speech while in case of Elena Bonatiebonati86gmailcom General Surgery Unit Department of Medicine and SurgeryParma University Hospital Parma Italy Unit of Anesthesiology Department of Medicine and SurgeryParma University Hospital Parma Italybilateral vocal fold paresisparalysis respiratory obstructionalso needs to be urgently treated Fortunately we havebroughtthe incidence of this last and most dangerouscomplication to at our Clinic since the introduction in of the routine use of intraoperative neuromonitoringduring thyroidectomyThe latest guidelines published by the Americanin March Association of Endocrine Surgeonsrecommend laryngeal examination in patients with knownor suspected new recurrent laryngeal nerve dysfunctionafter thyroidectomy for additional evaluation and possibletreatment with a speech pathologist According to theAmerican Academy of Otolaryngology”Head and NeckSurgery they assert that early referral “ weeks postsurgery to a laryngologistin combination with earlyintervention results in superior voice outcomes since theideal time for vocal fold augmentation is months afterthyroidectomy []A metaanalysis about therapy for vocal fold paresisparalysis after thyroidectomy concluded that the timingof therapy for unilateral vocal fold paralysis after thyroidectomy has a significant impact on the effect sizebeing significantly greater if therapy is performed within months This may be explained by progressive atrofolds and disappearance of nervephy offunction so that vocalfold movements cannot berecovered []the vocalPatients who underwent thyroid surgery from February and who had experienced a vocal fold disfunctionVFD were unable to undergo a laryngoscopy nor muchless a speech therapy according to health measuresnecessary to contain the spread of the virus This unfortunately causes a progressively reduced possibility ofrecovery increasing the specific morbidity related to surgery for thyroid cancer in this period The only indicationthat we can give to patients is the rest of the voice to avoidthe establishment of compensation mechanisms worseningthe clinical picture waiting to be able to resume the correcttreatment 0cTherapeutic diagnostic pathways in the COVID19 erahave become difficult and dangerous logarithms that mustconsider the need for patient care and the possibility oftreatments delay in safety but also the risk of contagion ofthe patientsleast protection ofhealthcare personnel The hospital setup has been significantly changed and much of the economic structuraland human health resources have been dedicated to themanagement of the COVID pandemicthemselves and notIn parallel with the COVID19 emergency we areexperiencing another health emergencythe one thatinvolves the management of nonCOVID19 patients Evenin the second phase of the pandemic only urgent healthservices are provided A reanizing effort within theindividual healthcare companies is required to guaranteetreatment even for nonCOVID19 patientsMoreoverThe COVID19 pandemic highlighted the limits andweaknesses of our health system and now that the correctprotocols for the protection of healthcare personnel havebeen described allthe all healthcare companies shouldequip their staff with the appropriate materials such as N95masks hair cover protective coverall gown gloves faceshields goggles and shoe covers In the face of higherexpenses this would allow the resumption of activitiesminimizing the risk of an increase in the rate of infectionroutine health practices must be reconsidered preferring less invasive techniques in order toscreen patients who need secondlevel examination Evenif not used yet in our hospital transcutaneous laryngealultrasonography is a valid noninvasive and painlessalternative method in the assessment of vocal cords It hasbeen demonstrated in a recent prospective multicentricstudy that it has concordance with laryngoscopy in themajority of cases and so it can be a valid alternative asfirstline exam for vocalfold examination pre andpostoperativelyEndocrineFinally the growing use of virtual platforms for the needof social distancing could encourage their application evenin healthcare services that can be performed by teleconference such as speech therapyWe can assess that COVID19 pandemic is causingdirect morbidity and mortality and even a related one dueto missed or delayed treatment of multiple nonCOVID19diseases The delivery of the health service should beimproved and the health system itself must be modernizedto adapt to new needsCompliance with ethical standardsConflict of interest The authors declare that they have no conflict ofinterestPublisher™s note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliationsReferences LP Kowalski A Sanabria JA Ridge WT Ng R de BreeA Rinaldo RP Takes A A Mkitie AL Carvalho CR Bradford V Paleri DM Hartl V Vander Poorten IJ Nixon C PiazzaPD Lacy JP Rodrigo O GuntinasLichius WM MendenhallA D™Cruz AWM Lee A Ferlito COVID19 pandemic effectsand evidencebased recommendations for otolaryngology and headand neck surgery practice Head Neck 101002hed26164 KN Patel L Yip CC Lubitz EG Grubbs BS Miller W ShenP Angelos H Chen GM Doherty TJ Fahey 3rd E KebebewVA Livolsi ND Perrier JA Sipos JA Sosa D StewardRP Tufano CR McHenry SE Carty The American Associationof Endocrine Surgeons Guidelines for the definitive surgical management of thyroid disease in adults Ann Surg e21“e93 X Chen P Wan Y Yu M Li Y Xu P Huang Z Huang Typesand timing of therapy for vocal fold paresisparalysis after thyroidectomy a systematic review and metaanalysis J Voice “ 0c'
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"SARSCoV2 infection associated respiratory disease COVID19 has evolved into a pandemic but being anew form of virus pathogenesis of disease causation is not fully understood and drugs and vaccinesagainst this virus are still being tested so that no effective drugs or vaccines have been advised byregulatory authority In this context the Ministry of AYUSH Government of India has recommended˜Ayush Kwath™ to improve the immunity and combat the infection Our objective of this literature reviewis to review the role of immunity in pathogenesis of COVID19 and role of Ayush Kwath against the virusand regulation of immunity Current review was conducted using a search of available literature onCOVID19 and immunity Vyadhikshamatwa Ayurveda and COVID19 Rasayana Coronavirus SARSCoV immunomodulatory effects of medicinal plants TulsiHoly BasilOcimum sanctum DalchiniCinnamonCinnamomum zeylanicum SunthiGingerZingiber officinale and MarichBlack PepperPiper nigrum Ayurveda being an ancient science have both medicinal and cultural values and had stimulated our kitchenand ‚uenced what we ate in different seasons and the remedies we used for common ailments Herbssuch as Tulsi Marich Sunthi Dalchini are the most commonly used and easily available drugs in homeThus Ayush Kwath due to its immunemodulatory antiviral antioxidant anti‚ammatory antiplatelet antiatherosclerotic hepatoprotective renoprotective properties seems to be effective inimmunoregulation for controlling viral infections like COVID19 Further preclinical and clinical trialsneed to be done for the evaluation of safety and efficacy of this polyherbal formulation The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciencesand Technology and World Ayurveda Foundation This is an access under the CC BYNCNDlicense httpcreativecommonslicensesbyncnd40 IntroductionCOVID19 also known as severe acute respiratory syndromecorona virus SARSCoV2 is an infectious disease believed to beoriginated from bats and transmitted to human beings [] Being anew form of virus pathogenesis of disease causation is not fullyunderstood and drugs and vaccines against this virus are still beingtested so that no effective drugs or vaccines have been advised byregulatory authority Not only for Coronavirus have many otherviruses also lack preventive vaccines and effective antiviral medications Studies have explored that these viruses can form drugresistant mutants which decrease the existing drug™s efficacy Sothese viruses can be a threat to the mankind for long time [] Corresponding authorEmail shankargautammohpgovnpPeer review under responsibility of Transdisciplinary University BangaloreHigh mortality among immunecompromised and those withsome underlying pathology implies that the factors that improveimmunity can prevent serious manifestations due to COVID19infection [] Many herbal products are found to have immunemodulatory and antiviral property so their discovery can be amilestone in the prevention and control of COVID19 [] In thiscontext the Government of India has recommended to take ˜AyushKwath™ in order to boost the immunity As this is a new formulationthis needs to be validated scientifically We have made an attemptto review the immunepathogenesis of COVID19 and the role ofeach herb over it Immunopathogenesis of COVID19The ˜S™ protein of coronavirus can bind to host cells through theACE2 receptor found in the oral and nasal mucosa [] Other siteswhere ACE2 receptors are found are lungs stomach intestinebladder heart and kidney [] Variable presentation of disease in101016jjaim202008003 The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciences and Technology and World Ayurveda Foundation This isan access under the CC BYNCND license httpcreativecommonslicensesbyncnd40Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxdifferent age groups serious manifestations that are seen morecommonly in immunecompromised old aged and in those withunderlying pathology many asymptomatic cases in pediatric agegroup and presence of lymph ia in the majority of the casesthese factors implies that immunity has a vital role in the pathogenesis of COVID19 [16e8] It is assumed that our immune systemhas lack of memory against such a virus that gave it an edge overhumans []Viruses cause cell destruction mainly in two ways direct cytopathic effects ofthe virus and immune response mediateddestruction [] COVID19 cannot lyse the cells directly as the majorpathway of cell destruction is due to immunemediated destruction[] It has been mentioned that unlike adults less vigorous cellmediated immune response in alveoli of children results in beingasymptomatic in the majority of cases []The pathogenesis can be split into two stages Nonsevere andSevere [] Nonsevere stageThe virus fuses with the host cell membrane and enters insidethe host cell through airway epithelium [] The virus propagates and multiplies inside the host cell and can reach lower airwayand alveoli In adults with good innate cellular and humoral immunity propagation of virus can be limited and viral load reachingalveoli can be reduced thus recovery can take place within 2e3weeks with mild symptoms []Humoral immunity prevents the viruses to enter new cells whilecellmediated immunity targets on eradicating virusinfected cells[] In this stage a strong immune system can be helpful inpreventing the propagation of the virus thus reducing the severityof the disease [] Severe stageOnce the immune system is breached the virus propagates andreaches the lower respiratory tract and alveoli Then the virus canpenetrate alveoli and reaches systemic circulation causing viremia[] The virus binds to multiple ans having ACE2 receptor protein During this stage cellmediated immunity becomes robustand starts releasing various pro‚ammatory cytokines IFNaIFNg IL1B IL6 IL12 IL18 IL33 TNFa etc and chemokinesCCL2 CCL3 CCL5 CXCL8 CXCL9 CXCL10 etc causing damage tomultiple ans known as Cytokine storm [] We may need tosuppress the ‚ammation for improvement during this severestage []Tocilizumab and anti‚ammatory interleukin IL10 are proposed to have a therapeutic role in the reduction of severity and mortality of COVID19[] As increased risk of thromboembolic phenomena is alsofound to be associated with COVID19 prophylactic antithromboticmedications are advised during this stage []IL6 receptor antagonist Ayurveda purview Disease conceptIt seems that most early cases had a history of contact with theoriginal market for seafood but the disease has now advanced to betransmitted through human to human contact [] Thus this disease can be considered as Communicableboth contagious and infectious diseases In Ayurveda epidemics are discussed under theterm of Janapadodhwamsa [ CSVi ] by Charaka and Marakaby Sushruta [ SSSoo ] The symptoms like fever coughbreathing difficulty headache and vomiting resemble with clinicalfeatures of SARS [ SSSoo ] Dalhana in his commentary hasmentioned that symptoms like anosmia cough catarrh will occurafter the entry of contaminated air through the nasal ingwhich is similar to typical clinical features of COVID19 [ SSSoo] Furthermore this disease can be classified as Adidaivika BalaPravritta Vyadhi ABPV Sansargaja Upsargaja and Aupasargic RogaABPV are those diseases arising due to causes that cannot becontrolled by human intelligence Upasargaja Vyadhi are thosefeverlike diseases that manifest due to close contact with diseasedpersons [ SSSoo ] whereas Sansargaja Vyadhi resides withpeople who are cursed by almighty god ie due to ‚uence ofinvisible forcesforces behind human control [ SSSoo ]Aupasargic Vyadhi is defined in two different ways by Sushruta oneas a disease which spreads from one person to another person [SSNi ] and another as ˜¦Upadravasangyah™ ie complications or associated diseases that manifest after primary disease [SSSoo ] Susruta mentions the diseases like Jwara Kusthaskin diseases Shosha tuberculosis Netrabhisyandi conjunctivitis and other Aupasargika roga alike communicable diseasescan be spread through Prasanga intimate relationship GatraSansparsha direct contact Nishwasa breathing or airborneSahabhojana eating together Sahashayana sleeping togethersharing and using of others™ clothes ornaments ointments etc [SSNi ]Agantuja Vyadhi Ì´ diseases of exogenous origin occurs due tophysicalexternal factors like Bhuta Visha Vayu Agni and Praharatrauma etc without any involvement of Vataadi Dosha initiallyhowever in later stage dosha are involved in the disease process[ CSSoo1145] Cakrapaṇidatta clarifies that Bhuta meansVisakṛimi or a virulent anism [ CSSa1121] Krimi may beSahaja natural or Vaikarika pathogenic anisms that may bevisible macroscopic or invisible to the naked eye microscopic[ CSVi ]Thus it is difficult to correlate this disease with specific Ayurveda terminology but while interpreting the disease on the basis ofSamprapti by considering the causative agent and the clinical features like fever Jwara cough Kasa anorexia Aruchi fatigueTandra generalized body ache or myalgia Angamarda andTiredness it can be contemplated as an Agantuka Vyadhi whichlater on due to the involvement of dosha develops to Nija Vyadhi asKaphaVatolvana Hina Pitta Sannipataja Jwara Severe Vata andKapha with mild Pitta [ CSSoo CSChi ] Whiletalking about the pathogenesis of fever in Ayurveda Charakamentioned that when Vataadi dosha either singly or in Sansristatwo dosha or in Sannipataja all three dosha got aggravated thenit enters Amashaya and mixed with Rasa Dhatu causing obstructionof Rasavaha and Swedavaha Srotas resulting in the destruction ofAgni Agni then spreads out from its Sthana to whole over the bodycausing the febrile condition [ CSNi120 CSChi3129]Immunity concept in AyurvedaStrength health lifespan and vital breath are dependent on thecondition of Agni [ CSSoo ] Charaka has mentioned theterm Vyadhikshamatwa and states that during certain conditions ordue to certain factors even unwholesome unhealthy food doesnot produce disease immediately all unwholesome diet are notequally harmful all dosha are not equally powerful all persons arenot capable of resisting diseases [ CSSoo ] This suggeststhat the body™s immune system plays a crucial role in diseasedevelopment The equilibrium state of Dhatu is called Swasthya [CSSoo ] The person who is desirous to be healthy should adopthealthy practices related to diet conduct and activities [ CSSooImmunity can be considered in Ayurveda as] ThusPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxVyadhikshamatwa and Oja which depends on the condition of AgniDosha and DhatuThere are three factors Aahara Swapna and Brahmacharya dietsound sleep and celibacy that support the life with which the bodywill be endowed with strength complexion and development tilllife span [ CSSoo ] Bala Ì´ StrengthImmunity is of threetypescongenital time affected and acquired Congenital is thatwhich is developed naturally in the body and mind time affected isdue to seasonal variation and age factor and acquired one is produced by the proper application of diet and exercise [ CSSoo ] Thus not only diet but also performing yoga or exercises withproper methods by giving rest in between exercises as Rasayanatherapy will increase acquired strength [ CSSoo1136] Oja isalso called Bala is the essence of all Sapta Dhatu being located inHridaya combines with Rasa and circulates through the Dhamaniand performs Tarpana or Prinanam of the whole body [ SSSoo CS1774] The equilibrium state of Kapha promotesstrength that™s why normal Kapha is called Oja [ CSSoo ]Normal pure blood promotes strength complexion health andlifespan [ CSSoo ] While dealing with Sannipataja JwaraSusruta in Uttarsthana mentioned Abhinyasa Jwara also called asHataujasa Jwara indicating the loss or deranged condition of Oja[ SSUtt ]The word ˜Rasayana Rasa þ ayana™ refers to nutrition and itstransportation in the body for attaining excellent Rasadi Dhatuswhich leads to gain longevity freedom from disorders optimumstrength of physique and sense ans [ CSChi ]Rasayana promotes nutrition by explicitly enriching the nutritionalvalue of Rasa by enhancing Agni ie digestion metabolism andabsorption by Srotashodhana Consequently any medication thatimproves Rasa™s consistency would enhance the health of all bodytissues Role of Ayurveda and traditional medicineEvery society has its own medical system which is deeplyrooted in its culture and guided by its philosophy of life Beingculturally and linguistically diverse countries there developedseveral types of traditional medicines TM based on practices skilltraditional knowledge based on beliefs theories and experiencesindigenous to different cultures Ayurveda Traditional Chinesemedicine TCM Ancient Egyptian medicine Sowa Rigpa etc system of medicine remain the most ancient yet living traditions inSouth East Asia Western Pacific Eastern Mediterranean Africaregion Up to percent of the population in some Asian and African countries depend on TM for primary health care PHC needs[] Still there is a high trend of using many herbs in religious andcultural works therapeutically for common ailments and as spicesfor foods according to occasion specific and seasonal regimes Ayurveda and TM have made a significant contribution to the prevention and alleviation of various communicable and noncommunicable diseases for thousands of years A long history ofusing many herbal remedies and experiences passed from generation to generation has resulted in people relying on herbal remedies and some simple home remedies for common diseases can beused even by illiterate citizens The selfcare an integral part ofPHC with home remedies using various herbs is the most commontreatment for India Nepal Bhutan and China for different flucommon cold fever GI disorders etc Prevention of smallpox inChina has been an epochmaking effort in the period of mankind™spreventive care One observational study found that the prevalencebetween the total number of COVID19 cases per million populationand the grams of spice supply per capita per day is clearly interrelated Most nations with lower spice intake per capita reportedmore COVID19 cases per million population and vice versa []Nevertheless with the invention of drugs many herbal remediesused historically have become modern medicines Few notableexamples include morphine digoxin artemisinin and colchicine Asmany herbs are found to have immunomodulatory role and possessantiviral activity many people are being optimistic over the traditional system of Medicine Ayurveda and TCM have descriptions ofimmunomodulation along with antiviral treatments even targeted to the coronavirus family []The key factor for COVID19 to occur and evolve is the interaction between the virus and an individual™s immune system [] Asmedicinal plants enhance NK cell activity inhibit activated transcription factor ATF2 downregulate Th17related cytokinesincluding transcription factor RORc IL17A and Th2related cytokines including IL5 IL13 and IL6 inhibit GATA3 IL4 IL6 IL1bRt IL17A TNFa expression and increase the secretions of ILit shows that natural products have potentimmunemodulatory and immuneboosting effects that may behelpful during the infection course by increasing innate immuneresponse to infections []INFg etc Ayush KwathConsidering the importance of immunity boosting measures inthe wake of COVID19 outbreak the Ministry of AYUSH Government of India with the interest of health promotion of the massesrecommends ˜Ayush Kwath™ or ˜Ayush Kudineer™ or ˜Ayush Joshanda™which comprises of four medicinal herbs Table [] Theherbs like holy basil cinnamon ginger black pepper are highlyavailable accessible and widely used in the kitchen and areconvenient to educate and train about its use to community healthworkers community and even to all public that they can have costeffective treatment with herbal home remedies This will help topromote immunity and to lower the gatherings at hospitals andpharmacies in this pandemic This type of public health measurewould eventually promote ˜health for all™ with the theme ˜ourhealth in our own hands™ making responsible to each and everypeople by active involvement in their own health instead of relyingon mass distribution of some medicine As people leave theirhomes to earn a living this herbal decoction will ensure broadaccess to health care The WHO SEARO adopted a resolution torevitalize PHC through health systems strengthening to achievehealth for all with the emphasis on health promotion and diseaseprevention [] This Kwath is not just a mechanical mixtureinvented for the COVID19 pandemic but it is a revival of healthtraditionMethod of preparation and useTake all the ingredients in dry form as per standards laid downin Ayurvedic Pharmacopoeia and make coarse powder Make sachets or tea bags each of g of powder or mg tablet of aqueousextract to be consumed like tea or hot drink by dissolving in mlof boiled water once or twice daily Gud JaggeryDraksha Resinsandor Lemon Juice can be added while consuming the formulation TulsiMany invitro animal and human experimental scientificstudies showed that due to presence of eugenol phenolic compoundslinoleic acid etc compounds Tulsi has antimicrobialincluding antibacterial antiviral antimalarial antidiarrhealantioxidantcardioprotectiveimmunemodulatory properties and is thus recommended as a treatmentfor a range of diseases including features like cough fever asthmaanxiety diarrhea gastric cardiac and genitourinary disorders[32e36] Due to its anti‚ammatory and antioxidant properties itanti‚ammatoryhepatoprotectiverenoprotectiveanalgesicantipyreticPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxTable Contents and properties of Ayush KwathSN NameScientific name Parts used Main chemicalRasaVirya AyurvedicSansthanika KarmaProportion RemarksTulsiOcimumsanctum LinnLeavesconstituentsVolatile oil PhenolAldehyde EugenolAscorbic acid Linoleicacid CaroteneDosha karmaKatu Tikta Ushna KaphavatashamakaPittabardhakaDalchini CinnamomumStem Bark CinnamaldehydezeylanicumBreyncuminaldehydeEugenolKatu TiktaMadhuraUshna KaphavatashamakaPitta vardhakaSunthiZingiberofficinale RoscRhizomeZingiberene Zingiberol KatuUshna KaphavatashamakaVedanahara DeepanaPachana AnulomanaKrimighna HridhyaRaktashodhakaKasahara SwasaharaKshayanashakaMutrala VishaghnaJwaraghna esp useful inVatashlaishmikaVishama and Jirna JwaraDeepana PachanaVajikaranaVataanulomanaYakridutejaka GrahiHriyottejakaOjovardhakaRaktashodhakaShelshmaharaYakshmanashakaMutrajananaDeepana PachanaVrishyaShoolaprashamanaRaktashodhakaHridhyottejakaShothahara KaphaghnaSwasahara JwaraghnaAampachana partsPrabhava Specialaction Krimighna parts partsPrabhava KrimighnaContraindicationsPandu KushthaMutrakriccharaktapitta Grishma andSharada Ritu MarichPiper nigrumLinnFruitPiperine PiperidinePiperettine andChavicineKatuUshna Kaphashamaka Deepana Pachana partYakriduttejakaVatanulomanaKrimighnaHriddhyottejakaKaphaghnaKaphamissarakaJwaraghna espVishamjwarapratibandhakaprotects against toxic chemicalinduced injury enhance the antioxidant enzymes and protect cellular anelles and membranes byclearing damaged free radicals []The compounds such as ursolic acid carnosol rosmarinic acidcirsilineol apigenin eugenol and cirsimaritin present in O sanctumincrease haemoglobin concentration enhance SRBC agglutinin titers decrease cyclooxygenase CoX2 and lipoxygenase LOX5enzymes activity suppress NFkB classical pathway up regulationof IL2 IFNg and TNFa down regulation of IL1b and produce ofSRBC antigenspecific antibodies which represent a major defensemechanism to assess Tcelldependent antibody responses ie Tulsiby enhancing immune response boost the defense mechanismagainst the infection [38e40] Several studies have shown that Tulsiaqueous and methanol extract of leaf and seed oil besidesimproving vital capacity also is an immunemodulator and regulator as it enhances immune response by increasing Thelper andNK cells phagocytic activity and index with the rise in lymphocytecount neutrophil count and antibody titer []In an acute toxicity study it did not produce any hazardoussymptoms or CNS and ANS toxicities or death and did not show anychange in water and food consumption body weight and hematological and biochemical profiles [] DalchiniIt is a potent immune system booster and is used in variousailments like flu indigestion edema cough etc [] Cinnamonbark contains cinnamaldehyde benzaldehyde cuminaldehyde andterpenes [] In one study cinnamon at high dose mgkgshowed immunestimulant activity as it significantly increased thephagocytic index serum immunoglobulin levels and antibody titerand decreased the percentage reductions in neutrophil countCinnamon low dose mgkg increased serum immunoglobulinlevels only This showed that high dose increases both cell mediated and humoral immunity whereas low dose showed effect onlyon humoral immunity [] The studies also suggest that cinnamaldehyde can act as a strong regulator of monocytemacrophagemediated immune responses by inhibition of PI3K PDK1 and NFkBactivation of signaling components In addition to this by theactivation of CD29 and CD43 it blocked cell migration cellecelladhesion induced but not cellfibronectin adhesion and it wasable to suppress both the production of nitric oxide NO and upregulation of surface levels of costimulatory molecules CD69 andCD80 and pattern recognition receptors TLR2 and CR3 []Cinnamon bark decrease systemic levels ofIFNg withoutaltering the levels of IL4 or IL2 inhibit antiCD3 AbstimulatedIFNg and IL4 at the mRNA and secreted protein levels enhance IL protein secretion at the cellular level which helps to decrease celldeath inhibit IL2mRNA expression inhibit antiCD3induced p38JNK ERK12 and STAT4 activation but not IkBa degradation orSTAT6 and ultimately alter the ‚ammatory responses in T cellsThis shows the immunemodulatory effect of cinnamon on cytokine secretion and the involvement of intracellular signaling molecules in activated T cells It also causes a reduction in the subG1Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxphase accompanied by an increased ratio of apoptotic cells tonecrotic cells [] The constituents like cinnamaldehyde cinnamophilin etc are found to be a thromboxane A2 receptor antagonistanticoagulative antiatherosclerotic and thus prevents unnecessary clumping of platelets and atherosclerotic CVD []In a systematic review of its adverse events relatively few selflimiting adverse effects were reported like allergic reactions andgastrointestinal disorders on clinical trials case reports and caseseries The evidence available show that cinnamon is safe for use asspice in daily diets or as a medication [] However its use fortherapeutic reasons in high doses or for prolonged periods cancause some adverse effects and should be observed clinically SunthiAn alcohol extract increases the immunological status of micewith increased phagocytosis by macrophages whereas crudeextract was also shown to increase humoral and cellmediatedimmune responses [] The bioactive compounds of ginger suchas nevirapine bsitosterol 6gingediol germacrene methyl6shogaol 6gingerol alinalool 6shogaol gingerdion zingibereneetc are known to inhibit viral replication among these the mostpotent inhibitors of reverse transcriptase RT enzyme is bsitosterol which is predicted to be used as nonnucleoside reversetranscriptase NNRTIs HIV1 inhibitors [] It is reported thatGinger contains TNFa which is also known as an anti‚uenzacytokine [] The rhizome of Ginger and its main componentslike gingerols shogaols etc inhibit prostaglandin and leukotrienebiosynthesis inhibit cyclooxygenase and lipoxygenase activitiesinhibits the synthesis of pro‚ammatory cytokines such as IL1TNFa and IL8 without any significant effect in IL6 levelsinhibit the excessive production of NO PGE TNFa and IL1beta reduce the elevated expression of NFkB and TNFa downregulate ‚ammatory iNOS and COX2 gene expression inhibitthromboxane synthetase raise levels of prostacyclin without aconcomitant rise in PGE or PGE alpha inhibit platelet aggregation decrease agerelated oxidative stress markers and enhancefibrinolysis [53e58]The concentration of IgM and eosinophil count in nonsmokerswas significantly increased in a comparative study of the effect ofginger extract among male smokers and nonsmokers whereas theconcentration of hemoglobin and lymphocyte count in smokerswas strongly increased This indicates that in nonsmokers gingerresults in a stronger antibody response or humoral immunity thanin smokers []According to Ayurveda it is contraindicated to be used in a fewdiseases Kushtha Pandu Mutrakriccha Raktapitta and in Grishmasummer and Sharada autumn Ritu There are few minor adverseeffects recorded that did not need care such as mild gastrointestinal symptoms sleepiness mild diarrhea during prior few days oftreatment It is also explained that ginger has the ability to induceheartburn and as a gastric irritant with doses above g [] Duringpregnancy ginger did not pose a major risk for side effects oradverse events [] MarichIt has been also found to increase bioavailability thus enhancethe therapeutic efficacy of many drugs vaccines and nutrients andhave immunemodulatory antioxidant antiplatelets antihypertensive antiasthmatic antipyretic analgesic anticarcinogenicanti‚ammatory antidiarrheal antispasmodic anxiolytic antidepressants hepatoprotective antiulcer antithyroids antiapoptotic antimetastatic antimutagenic antibacterial antifungal[62e65] The extract and itsand antiamoebic propertiesconstituents like piperine regulate the balance of the cytokinesproduction of Th1 Th2 Th17 and Treg cells reduce the accumulation of ‚ammatory cells inhibit the expressions of GATA3 IL4IL6 IL1b Rt IL17A and TNFa increase INFg and IL10 secretions in BALF Bronchoalveolar lavage fluid and increasemacrophage activation and T and B cell proliferation []Beside this Marich possess cytotoxic activity suppresses thelevels of total IgE antiOVA IgE antiOVA IgG1 and histaminerelease in serum ameliorates fibrosis and ltration of ‚ammatory cells inhibits the allergic responses inhibitsTh2Th17 responses and mast cells activation inhibits NFkB cFos cAMPresponse elementbinding CREB and activated transcription factor ATF2 suppresses PMAinduced MMP9 expression inhibitsPKCaextracellular signalregulated kinase ERK and reducesNFkBAP1 activation In addition piperine also inhibits the Pglycoprotein Pgp and CYP3A4 functions [67e69] Piper nigrum isfound to have dose dependent antifertility effects on mice [] DiscussionAccording to Ayurveda therapeutics is of two types Swasthasyorjaskarawhich promotes strength immunity in the healthyand Roganutwhich alleviates disorders Both of these groupsperform both of these functions but Rasayana and Vajikarana aremostly used for promotive treatment CSChi [] AyushKwath has both immune promoting and disease alleviating properties which can be achieved by various treatment modalities likeRasayana Satwawajaya Yuktivyapashraya Vyadhi Viparitarthakarichikitsa etcThe Katu and Tikta Rasa Usna Virya and Deepana PachanaYakriduttejaka properties of Ayush Kwath help to improve Agni andSrotosodhana improves microcirculation and tissue perforationthus promotes proper digestion metabolism and absorption andacts as Rasayana for the development of preceding Dhatu andfinally form Oja Oja itself acts as immunity to prevent diseaseImmunity is dependent on the condition of Agni Ayush Kwath withits Agni promoting and Kaphashamaka properties balance Kaphaand with Raktashodhaka Hridhya Krimighna properties purify theblood It is already mentioned that natural Kapha and pure bloodpromote Oja and Bala respectively Krimighna is the Prabhavaspecial action of Tulsi and Sunthi which directly acts againstpathogens The properties like Jwaraghna esp VatashlaishmikaVishama Kasahara Swasahara Kshayanashaka ShoolaprashamanaSwothahara Kaphaghna Hridayaottejaka Yakridutejaka have directrole to alleviate various clinical signs symptoms and complicationsAs this disease is considered as KaphaVatolvana Hina PittaSannipataja Jwara the Kapha Vata Shamaka properties of AyushKwath can play a significant role in balancing the vitiated doshasAfter six days of Jwara Charaka suggests the decoction of Pachanadrugs in the case of Amdosha and Shamaniya drugs in Niramadosha[ CSChi ] This shows that Yuktivyapashraya and Vyadhiviparita chikitsa can be done even after the involvement of Dosha inlater stages Ayush Kwath has potential psychoneuroimmunemechanisms via evidence of a reduction in depression anxietyand stress in controlled trials and shows meaning response as it is aspecific remedy for cough and respiratory problems this shows therole of Satvawajaya Chikitsa in its management []Immunity plays a key role in the pathogenesis of COVID19 bothduring the early nonsevere stage and during the severe stage ofthe disease The earlystage strong immune response may preventthe propagation and spread of viruses inside the body thusreducing the severity of cases and early termination of infectionWhile during later stage strong cellmediated immunity of thebody against the virus itself can be a factor responsible for graveconsequences due to cytokine storm The target during the earlyPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxlikesteroidsand IL6 receptorstage should be to reduce viral propagation while at a later stageshould be to reduce the ‚ammatory response of the immunesystem Medicinal herbs with immune booster property can be anoption during the early nonsevere stage while herbs with anti‚ammatory and antithrombotic properties can be an optionduring a later or severe stage Cytokine storm that is believed as amajor factor responsible for complications and death of COVID19patients has been found to be reduced with anti‚ammatorydrugsantagonists Anti‚ammatory interleukins IL10 in modern medicine [] Therole of medicinal herbs with anti‚ammatory property on thecytokine storm is still lacking in research Like anti‚ammatoryinterleukins and IL6 receptor antagonist Tocilizumab IL thatare proposed in modern medicine to have a therapeutic role in thereduction of severity and mortality of COVID19 Cinnamon barkthat is found to decrease INFg and IL4 Its antiatheroscleroticanticoagulative and antiplate
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catalytic properties of key activesite mutants of flavocytochrome p450 lee ds crystallization and preliminary xray diffraction analysis of fattyacid hydroxylase cytochrome p450bsβ from bacillus subtilis acta crystallogr sect d biol crystallogr “ lee ds substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome p450 from bacillus subtilis crystallographic spectroscopic and mutational studies j biol chem “ girvan h m structural and catalytic properties of the peroxygenase p450 enzyme cyp152k6 from bacillus methanolicus bower s cloning sequencing and characterization of the bacillus subtilis biotin biosynthetic operon j bacteriol j in biochem “ “ cryle m j schlichting i structural insights from a p450 carrier protein complex reveal how specificity is achieved in the p450bioi acp complex proc natl acad sci “ cryle m j bell s g schlichting i structural and biochemical characterization of the cytochrome p450 cypx cyp134a1 from bacillus subtilis a cyclolleucyllleucyl dipeptide oxidase biochemistry “ cimermancic p insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters cell “ tran p n yen m r chiang c y lin h c chen p y detecting and prioritizing biosynthetic gene clusters for bioactive compounds in bacteria and fungi appl microbiol biotechnol “ s0025 z marchlerbauer a cddsparcle functional classification of proteins via subfamily domain architectures nucleic acids res d200d203 101093nargkw11 syed k mashele s s comparative analysis of p450 signature motifs exxr and cxg in the large and diverse kingdom of fungi identification of evolutionarily conserved amino acid patterns characteristic of p450 family one e95616 101371journ alpone00956 graham s e peterson j a how similar are p450s and what can their differences teach us arch biochem biophys “ katoh k kuma k toh h miyata t mafft version improvement in accuracy of multiple sequence alignment nucleic acids res “ 101093nargki19 boc a diallo a b makarenkov v trex a web server for inferring validating and visualizing phylogenetic trees and networks nucleic acids res w573“w579 101093nargks48 letunic i bork p interactive tree of life itol v4 recent updates and new developments nucleic acids res w256“w259 saeed a i tm4 a free opensource system for microarray data management and analysis biotechniques “ 10214403342 mt01 weber t antismash 30a comprehensive resource for the genome mining of biosynthetic gene clusters nucleic acids res “ 101093nargkv43 battistuzzi f u feijao a hedges s b a genomic timescale of prokaryote evolution insights into the origin of methanogenesis phototrophy and the colonization of land bmc evol biol acknowledgementstiara padayachee and nomfundo nzuza thank the department of science and technology”national research foundation dstnrf south africa for master™s scholarships grant numbers mnd190619448759 and mnd190626451135 respectively khajamohiddin syed expresses sincere gratitude to the nrf south africa for a research grant grant number and university of zululand grant number c686 the authors want to thank barbara bradley pretoria south africa for english language editingauthor contributionsks designed conceptualized and provided funding for the study all authors were involved in generation analysis and interpretation of data all authors reviewed and approved the manuscriptcompeting interests the authors declare no competing interestsadditional informationsupplementary information is available for this paper at 101038s4159 correspondence and requests for materials should be addressed to drn a0or a0ksreprints and permissions information is available at wwwnaturecomreprintspublisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsopen access this article is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or 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colorectal cancer crc is a malignant tumor in the gastrointestinal tract and it arises from theinner wall of the large intestine the colon crc is the third most common cancer worldwideaccounting for roughly million new cases per year and ˆ¼ deaths per year whichmakes it the fourth most common cause of cancerrelated death globally and remains a hugechallenge “ in order to identify eï¬ective molecular targets for crc diagnosis and potentialedited byzhonghua taofudan university shanghai cancercenter chinareviewed byshengli liuniversity of texas health sciencecenter at houston united statesrossano lattanziouniversity of studies g d™annunziochieti and pescara italycorrespondencejianhua wangwangjianhuaman163comyansong pudoctor_puyahoocom these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived september accepted june published august citationliu y cao j zhu yn ma ymurtaza g li y wang jh and pu ys c1222c deletion in exon ofabl1 is involved in carcinogenesisand cell cycle control of colorectalcancer through irs1pi3kaktpathway front oncol 103389fonc202001385frontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerinterventions for crc therapy indepth studies on the regulatorymechanism of crc progression should be conductedthe pathogenesis of crc accompanies with genetic orepigenetic changes numerous genes and pathways such aswnt tgf egfr“ras erk“mapk pi3k and p53 havebeen demonstrated to be associated with crc “ abl1a protooncogene of cabl encodes a nonreceptor tyrosinekinase plays an important role in carcinogenesis regulatingcell adhesion proliferation diï¬erentiation and apoptosis studies have characterized abl1 as an oncogene thatpromotes breast cancer cell proliferation and induces anchorageindependent growth under p53 deficiency in breast cancercells “ craig reported that inhibition of abl1by imatinib reduced the proliferation of lymphoma cells andprevented tumor formation in mice however the roleand mechanism of abl1 in crc development and progressionremain largely unclearthe aim of this study was to elucidate the role of abl1using highthroughput dna sequencing technology to obtaininformation on colon cancer gene mutation we analyzedthe variation in the expression of abl1 among patients withcrc and in crc celllines we additionally determinedthe eï¬ect of downregulating abl1 on the proliferation cellcycle progression and apoptosis of crc cells further theeï¬ects of knockout of abl1 in tumor and the molecularmechanisms of activated and suppressed downstream signalingpathways were assayed to elicit the mechanisms involved incrc carcinogenesismaterials and methodsadmitted atclinicalpathological data ofpatients and samplesthefortyeight patients with crc wereshaanxi people™s hospitalshaanxi china colorectalcancer was confirmed by histopathology or biopsy basedon which thesubjectswere evaluated formalxed paraffinembedded ffpetissues were used as the study material tumor contentsin the ffpe tissues werethedepartment of histopathology shaanxi people™s hospitaland only ffpe tissue blocks with tumor contentwere qualified the study was approved by the ethicscommittee of shaanxi people™s hospital written informedconsent was obtained from allsubjects participating inthis studythethoroughlychecked atcell culture and rna interferencecrc cell lines ncm460 lovo sw620 sw480 and hct116were purchased from the cell bank of the chinese academyof sciences shanghai china the cells were cultured inrp1640 medium supplemented with heatactivated fetalbovine serum gibco gaithersburg md and penicillinstreptomycin gibco at —¦c with co2abl1 protein phosphatase catalyticsubunit alphappp3ca and tgf1 knockdown kd lentiviruses weregenerated using pfugwgfprnai vector by insertingshabl1 shppp3ca and shtgf1 sequence empty pfugwgfp vector was used as vector control shctrl in crc cells or ncmice the rnai sequence of abl1 ppp3ca and tgf1 were²cgttctatatcatcactga3² ²atatacgcgttctgaatactt3² ²gattatcga catggagctg3² respectivelysw480 and hct116 cells were plated in a 24wellplate andincubated at —¦c with co2 for h a multiplicity ofinfection of was added to infect sw480 and hct116 cellsovernight the infection medium was then replaced with normalcomplete growth medium cells without infection were used ascorresponding controlsproliferation and colony formation assayproliferation rate was determined using bromodeoxyuridinebrdu cell proliferation elisa kit abcam boston ma theoptical density of each sample was measured at nm using asynergy h1 microplate reader biotek winooski vtfor the clonogenic assay sw480 and hct116 cells wereplated onto 6wellplate and incubated in culture medium for days the cells were then fixed with pfa and stained with crystal violet sigmaaldrich st louis mo for h at roomtemperature the total number of colonies was counted wheneach clone contained more than cells size “ mmflow cytometric analysisfor cell cycle analysis cells were fixed in pfa for minat —¦c and treated with propidium iodide pi µgmlsigmaaldrich at room temperature for min in dark atotal of cells were analyzed by flow cytometry using abd facscalibur system becton“dickinson el paso tx thedistribution of cell cycle phases was estimated using modfitlt in mac v30 software apoptosis was further determinedby annexin v fitcconjugated thermo fischer scientificmiami ok and pi staining cells were immediately counted byflow cytometrydna extraction and sequencingfortyeight specimens of colorectal cancer tissue with clinicalliver metastases were collected dna was isolated using allprep dna ffpe kit qiagen germantown md genomic dnawas extracted by fully automated purification using promegamaxwell promega madison wi the dna concentrationwas measured fluorimetrically using the qubit dna highsensitivity kit thermo fisher scientific waltham ma anion torrent semiconductor chip sequencer was used to sequencecommon gene mutations in the tumorsin vivo studybalbc nude mice female aged weeks were purchased fromshanghai ling chang biological technology co ltd shanghaichina the mice were housed in spflevel laboratories with freeaccess to food and water and accommodated for week prior toany experiments the animal study was performed in accordancewith iacuc guidelines shabl1hct116 kd or shctrlhct nc cells — µl were subcutaneously injected tothe left flank of the mice at day posttransplantation micewere sacrificed and tumors were excised and weighed the tumorfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancervolume was calculated using digital calipers with the followingformula tumor volume volume length width22ingenuity pathway analysisto elucidate the role and action mechanism of abl1 incrc after abl1 kd high throughput realtime pcr arraywas performed by shanghai genechem co ltd shanghaichina and the data were analyzed using ingenuity pathwayanalysis ipa software to elucidate the aï¬ected molecules andsignal pathwaysimmunohistochemistryfor crc 180point tissue microarray hcolade180sur07which contained crc tissuesfrom patients and thecorresponding adjacent tissues table s4 was purchased fromshanghai outdo biotech co ltd shanghai china briefly thetissue microarray block was constructed by embedding a singletissue core mm in diameter was taken from each region informalxed paraffinembedded crc or adjacent tissue blockusing a tissue microarrayer beecher instruments silver springmd usa and was set to a blank recipient block predrilled with mm holesthe tissue microarray blocks and paraffinembedded tumorsections were cut into 7µm sections for immunohistochemicalihc analysis slides were deparaffinized and rehydrated aspreviously described followed by antigen retrievalincitrate buï¬er mm citric acid tween ph for min in —¦c water bath after washing with pbsslides were incubated with pbst with bovine serumalbumin sigmaaldrich for h slides were then incubatedovernight at —¦c with antiabl1 antibody ab15130abcam cambridge ma and developed using mouse andrabbit specific hrpdab detection ihc kit ab64264 abcamfollowing the manufacturer™s instructionsthe selection of cutoï¬ value to dichotomize the expressionlevels of abl1 was based on previously reported method []briefly the high expression level of abl1 was defined from twocriteria dab staining showed equal or darker color comparedto positive control the population of abl1positive cells washigher than all cases were independently evaluated anddiagnosed by two senior pathologists y m and l y who wereblinded to the pathologic diagnosis cases with any disagreementwere reviewed simultaneously by the original two pathologistsand a senior pathologist j w until they reach a consensuswestern blotthe western blotting assay was performed by wellestablishedprotocols as previously described primary antibodiesused in this study were antiabl1 antibody ab85947abcam cambridge ma antibcl2 antibody bcl10c4biolegend san diego ca antibclxl antibody sc santa cruz biotechnology dallas tx antibaxantibody 2d2 biolegend antiactin antibody 2f11 biolegend antigapdh antibody ff26af9biolegend antip27 antibody sc56338 santa cruzbiotechnology anticyclind1 antibody sc8396 santacruz biotechnology antiirs1 antibody ab52167abcam antiakt2 antibody ab175354 abcam antippp3ca antibody ab52761 abcam antitgf1antibody ab92486 abcam antimap2k2 antibody sc81473 santa cruz biotechnology antipi3kp11aantibody ab151549 abcam secondary antibodiesused were antimouse igg hrpconjugated secondary antibody sc516102 santa cruz biotechnology and antirabbitigg hrpconjugated secondary antibody sc2357 santacruz biotechnologyreverse transcriptionpolymerase chainreactionthe mrna level was measured using realtime polymerasechain reaction briefly total rna was extracted from culturedcells using trizol reagent thermo fisher scientific andcdna synthesis was performed using the quantitect reversetranscription kit qiagen the primers used were as followsabl1andantisense ²acaccctcccttcgtatctcag3² gadphsense ²tgacttcaacagcgacaccca3² antisense ²caccctgttgctgtagccaaa3² the realtime pcr wascarried out by using rt2 sybr rcid13 green qpcr mastermixesqiagen according to the manufacturer™s instructions all pcrswere performed in triplicate 01 01ct method was used to calculatethe relative expression levels²catcacgccagtcaacagtct3²sensestatistical analysisstatistical analyses were performed using spss spss incchicago il χ 2test was used to investigate the possiblerelationships between abl1 expression and clinic pathologicalcharacteristics mann“whitney utest was used to compare thediï¬erence in abl1 protein expression between paired coloncancer and adjacent normal colon tissues survival analysiswas performed using the kaplan“meier curve and the logranktest the values are expressed as mean ± sd comparisonsbetween two groups were conducted using student™s ttest allexperiments were carried out in triplicate results with p were considered statistically significantresultshighly expressed abl1 in crc tissue isassociated with poor clinical outcometo verify the existence of abl1 in crc tissues we comparedcrc tissues and their adjacent noncancerous tissues by ihcstaining our results showed that the immunostaining of abl1was significantly higher in crc tissues compared with adjacentnormal colon tissues p figures 1a“d table ourwestern blot and realtime pcr results confirmed the muchhigher expression level of abl1 in crc tissues compared tonormal tissues figures 1ef similarly the expression of abl1was significantly increased in diï¬erent crc cell lines comparedwith that in the normal colon cell line ncm460 figures 1ghremarkably the expression of abl1 was significantly p increased in the advanced stages stage iiiiiiv of crccompared with the early stages stage i and noncancerousfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure abl1 is highly expressed colorectal cancer tissue and cell lines a“d representative ihc staining of abl1 in normal ab and crc cd tissuesac x bd x e western blot analysis of abl1 expression in crc tumor t and adjacent normal n tissues f realtime pcr analysis of abl1expression in crc tumor crc and adjacent normal n tissues p g western blot analysis of abl1 expression in crc cell lines h realtime pcranalysis of abl1 expression in crc cell lines relative expression was normalized to ncm460 cells p i realtime pcr analysis of abl1 expression incrc tumors at different clinical stages relative expression was normalized to adjacent normal n tissues p table expression of abl1 in colon cancer and adjacent tissuestable c1222c deletion in exon of abl1variablesnoexpression levelspvariablesexpression levelsor95 cipneglowhighno no mutation mutationnormalcolon cancergenderfemaleadjacent normal colon tissues neg negativetissues figure 1i with a median followup of monthsranging from to months our survival analysis showed thatthe patients with high abl1 expression death had a lowersurvival rate compared to patients with low abl1 expression death p figure s5 these results suggested thatabl1 is a potential oncogene abl1 and that its expression waspositively associated with the clinical stage in patients with crcc1222c deletion in exon of abl1 inrelation to the tnm stageprevious studies have shown the mutations of the abl1 geneare of major clinical relevance to study the possiblemutations in patients with crc we performed dna sequencingour results indicated that a mutation of abl1 was presentin males and females of patients with crc females and males this mutation occurred in exon ofthe abl1 gene and all mutations were found to be deletion““ maletnm stage““ofthe c1222c nucleotide sequence within this exon theincidence rate of mutation was in females and malestable figure additionally the analysis of patients withmutations and the corresponding stages revealed that patientswere in stages “ and in stages “ the tnm stage was asignificant risk factor for c1222c deletion the results suggestedthat c1222c deletion is involved in crc carcinogenesisinterference of abl1 decreased theproliferation and enhanced the apoptosisof sw480 and hct116 cellsto further investigate the role of abl1 in crc carcinogenesiswe used lentivirus vector to downregulate abl1 expression insw480 and hct116 cells after infection both cell lines showedfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure structural domain and c1222c deletion in exon of the abl1 genefigure depletion of abl1 decreases the proliferation of crc cells a representative fluorescent images showing gfppositive cells after infection images weretaken under x b western blot detects the abl1 expression in cells transfected infected with shctrl or shabl1 c representative images of colony formationassay using crc cells infected with shctrl or shabl1 cells without infection was used as control con d quantification of colony numbers data are shown asmean ± sd p compared with control e brdu assay detects the proliferation of sw480 and hct116 cell lines infected with shctrl or shabl1 cellswithout infection was used as control con p compared with control“ of average gfppositive rate figure 3a our westernblot results showed a significant decrease of abl1 protein levelfigure 3b indicating a successful downregulation after rnainterference to evaluate the proliferation of crc cells afterabl1 depletion we performed a clonogenic assay figure 3ccompared with the control group the number of clones inthe shabl1 group was obviously decreased figure 3d ourbrdu proliferation assay confirmed that the proliferation offrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure depletion of abl1 causes cell cycle arrest and apoptosis in sw480 and hct116 cells ab representative cell cycle analysis of sw480 a andhct116 b infected with shctrl or shabl1 cells without infection was used as control con c quantification of cell cycle distribution in g1 s g2m phases p p compared to control group d western blot analysis of p27 and cyclin d1 expressions in crc cells infected with shctrl or shabl1 cellswithout infection was used as control configure depletion of abl1 increases the apoptosis of crc cells flow cytometry detected apoptosis of sw480 a and hct116 b cells infected with shctrl orshabl1 cells without infection was used as control con c quantification of apoptotic cells data are shown as mean ± sd p compared with controld western blot analysis of bax bclxl and bcl2 expression in crc cells infected with shctrl or shabl1 cells without infection was used as control conshabl1 cells was obviously reduced compared with that ofthe control cells figure 3e additionally our flow cytometryresults showed more cells were arrested in s phase afterabl1 depletion while fewer cells in g2m phases were foundfigures 4a“c indicating downregulation of abl1 inhibitedcell cycle progression of crc cells to validate these data wedetected p27 a negative regulator of cell cycle progression andfound its expression was significantly increased in cells infectedwith shabl1 vector figure 4d on the contrary cyclin d1 wasdecreased in abl1depleted crc cells figure 4dnext we performed flow cytometric analysis to examinethe apoptosis of abl1depleted crc cells figures 5ab wefrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure depletion of abl1 inhibits crc tumor growth in vivo a representative image of mice injected with hct116 cells infected with shctrl nc or shabl1kd b representative tumor images showing depletion of abl1 decreased the size of crc tumors body weight c tumor volume d and tumor weight e weremeasured at day after inoculation p compared to nc groupfound downregulation of abl1 significantly increased apoptosisin crc cells as compared with the control group p figure 5c the expression of the apoptosisrelated proteinbcl2associated x bax was obviously increased while bcelllymphomaextralarge bclxl and bcell lymphoma bcl2were remarkably decreased in the shabl1 group figure 5dtaken together these results suggest depletion of abl1 increasesthe apoptosis of crc cellsdepletion of abl1 inhibited crc tumrowth in vivoin order to examine the involvement of abl1 in regulatingcrc tumor growth we inoculated hct116 cells infected withshctrl nc group or shabl1 kd group into balbc nudemice figure 6a as shown in figure 6b the tumor growth wasremarkably inhibited in the kd group compared with the ncgroup interestingly we also observed a significant bodyweightincrease in the kd group figure 6c as compared to controlabl1 depletion caused a significant reduction in tumor volumefigure 6d the average tumor weight in kd xenografts wasobviously lower than that in the nc group ± mgvs ± mg p figure 6e taken together ouranimal experiments demonstrated that abl1 knockdown couldinhibit crc tumor growth in vivoabl1 interference inhibited tgf1 via thepi3kaktirs1 pathway and ppp3cato elucidate the molecular pathways regulated by abl1 in crcwe performed high throughput pcr array from xenografts inabl1 kd or nc mice our ipa results identified upregulatedgenes and downregulated genes in xenografts from kdmice compared with those from nc mice further analysisrevealed that these diï¬erentially expressed genes were involvedin multiple biological functions and pathogenesis of multiplediseases figure s1as shown in figures s2 s3 and tables s1“s3 the tgfand pi3kakt pathways were inhibited by depletion of abl1the associated molecules of the two pathways including tlr4akt2 il4r camk2d ppp3cb map2k2 pdia3 irs1itpr3 abl1 atf4 ppp3ca ca2 cpt1a csrp1 ctsv fn1lamp2 ptgs2 runx2 s100a4 and spp1 were mapped withabl1 gene to show a predicted interaction network figure 7ato verify this interaction we examined the levels of proteins intgf and pi3kakt pathways in xenografts of nc and kd micefigure 7b our western blot results showed that knockdown offrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure abl1 interacts with pi3kakt and tgf1 pathways a molecule network generated by ipa showing interactions among abl1 pi3kakt and tgf1pathways up and downregulated genes are shown in red and green respectively b western blot analysis of predicted interactive proteins in xenografts fromabl1 knockdown kd or control nc mice the gene of œ is detected by multiple probes and it is statistically significantabl1 significantly decreased irs1 akt2 ppp3ca and tgf1 expression while did not change the expression of map2k2compared with those in the nc groupto further verify the involvement of tgf1 in the regulationof pi3kakt pathway we generated a tgf1depletion hct cell line by lentivirus infection figure 8a our western blotresult showed that the expression of tgf1 was significantlydownregulated after infection figure 8b as expected thekey proteins in pi3kakt pathways were deactivated upontgf1depletion figure 8c including irs1 phosphopi3kand akt according to the findings in the previous study thedownregulated gene ppp3ca found in abl1 kd mouse isinvolved in the regulation of the pi3kakt pathway wenext investigated the interaction between ppp3ca and abl1by establishing a ppp3ca knockdown cell line figures 8dewe found the depletion of ppp3ca significantly decreasedthe expression of abl1 figure 8f to validate the regulatoryrole of ppp3ca we also examined the abl1 expression inppp3ca overexpressed cells and found the expression of abl1was elevated by upregulated ppp3ca figure s4 indicatingppp3ca is a positive regulator of abl1discussionas a ubiquitously expressed nonreceptor tyrosine kinase abl1has been reported to be associated with glioblastoma and breastcancer in this study we examined the role of abl1 incrc progression the results indicated that abl1 might play animportant role in crc which is associated with the mutation andexpression of the abl1 genewe found the expression of abl1 was remarkably elevatedin crc tissues and cell lines figure which is correspondedto the survival rate among patients with crc figure s5indicating abl1 is a potential oncogene in crc “moreover the mutation of abl1 was also elevated in crcpatients based on the results from previous studies the mutationrate of the abl1 gene is relatively higher in men than in womenpatients with crc worldwide in a previous study theabl1 gene was found to be mutated in of patients withcrc at sir ganga ram hospital delhi india in the presentstudy the mutation rate was much higher in crcpatients accepted in our hospital the diï¬erent races of patientsfrom diï¬erent regions of the world reported in the two studiesfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure tgf1 knockdown deactivates pi3kakt pathway in hct116 cells a representative images of hct116 cells infected with gfpcontaining lentivirusexpressing shrna against tgf1 gfp positive lentivirus with scramble shrna was used as control images were captured under 200x magnification bknockdown of tgf1 was confirm by western blot proteins were extracted from hct116 cells at h postinfection actin was used as loading control cwestern blot examined irs1pi3kakt pathway in hct116 cells after knockdown of tgf1 d western blot examined irs1pi3kakt pathway in hct116 cellsinfected with gfpcontaining lentivirus expressing shrna against ppp3ca gfp positive lentivirus with scramble shrna was used as control e representativefluorescent images of hct116 cells at h postinfection images were captured under 200x magnification f expression of abl1 after ppp3ca knockdown wasdetermined by western blot proteins were extracted from hct116 cells at h postinfection actin was used as loading controlcould be a possible reason causes the diï¬erence of abl1 mutationrates “ gene mutations are often involved in tumorigenesisthe clustered deletions were found in abl1 notch1 retstk11 gna11 and jak3 genes in crc melanoma and nonsmall cell lung cancers additionally the abl1 mutationdata in tcga showed that an average mutation rate of abl1is in coad patients the high mutation rate isconsistent with the findings in this study that abl1 mutationcorrelates with the oncogenesis of crc to the best of ourknowledge the present study presents a novel mutation inexon in which c1222c deletion occurred this deletion wasrelatively higher in female patients than in male patients thehigher distribution of this deletion at the higher tnm stage inpatients with crc suggests that this deletion might be relatedto tumorigenesis of crc however further investigation withlarger sample size is needed to elucidate the relationship andmechanism between c1222c deletion and crc progressionto determine the role of abl1 in crc progression wedownregualted abl1 expression in crc cell lines and foundthat the cell cycle was arrested at s phase figure theseobservations are consistent with previous reports thatthenumber of cells in s phase was increased when abl1 wasinhibited by imatinib or sti571 in u2os hela and a549 cells it is wellreported that p27 inhibits g1s transition ofthe cell cycle while cyclin d1 is a key regulator of cell entryinto the s phase allowing cells to enter the s phase smoothlyfrom the g1 phase our study provides direct evidencethat abl1 interference increased p27 expression and decreased incyclin d1 expression figure which is similar to the increasedp27 expression and decreased cyclin d1 expression found incells treated with nilotinib an abl1specific inhibitor however previous studies showed that when abl1 expressionwas inhibited by nilotinib the number of cells in the g0g1 phasewas increased while the number of cells in s and g2m phases wasdecreased which is contradictory to the finding in this studythat downregulation of abl1 arrested crc cells at s phase thismight be due to the varied function of abl1 in diï¬erent tissuesand cell types abl1 controls cell apoptosis via downstream moleculessuch as puma bax and p73 as well as by changingfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancermembrane potential depletion of abl1 inducedapoptosis of crc cells observed in this study is consistentwith the findings ofthese studies studies have reportedthat abl inhibitor danusertib treatment significantly decreasedthe expression of bclxl and bcl2 while increasing theexpression of bax similarly we found increasedbax expression and decreased levels of bcl2 and bclxl after downregulation of abl1 in crc cells figure indicating abl1 is involved in the regulation of apoptosis incrc cellsinvolved in cell proliferation migrationfurthermore information obtained using the ipa indicatedthe expressions of numerousthat after abl1 knockdowngenesinvasiondiï¬erentiation death and survival were aï¬ected figures s1“s3 tables s1“s3 the results demonstrated that abl1 mightplay a pivotal role in crc progression especiallythe tgf and pi3kakt pathways were inhibited afterabl1 interferenceit has been reported that abl1 regulates tgf signaling which is associated with tumor progression by modulatingangiogenesis in crc resulting in poor prognostic outcome“ studies have demonstrated thattreatment withan abl1 inhibitor significantly reduced the tgf level similarly we found the expression of tgf1 wassignificantly inhibited after abl1depletion figure thisindicated that abl1 is a positive regulator of tgf signalpathways as one of the tgfaï¬ected downstream signals thepi3kakt pathway plays a crucial role in tumorigenesisitassociated withproliferation apoptosisinvasion and metastasis of cancercells insulin receptor substrates irs including irs1and irs1 are a downstream messenger of the pi3k pathway our study provides novel evidence that abl1 mightinteract with tgf1 via pi3kaktirs1 that is involved incrc progressionexpression of proteinsregulatestheca2aandcalcineurincalmodulindependentserinethreonine protein phosphatase has been reported topromote intestinal tumor development and crc tumorigenesis the expression of calcineurin a specifically increases inhuman crc cell lines in the present study we foundthat ppp3ca which is also known as an alpha isoform ofthe calcineurin catalytic subunit was inhibited afterknockdown of abl1 figure 7b this finding provides novelevidence that abl1 might interact with the ppp3ca oncogenein crc carcinogenesisconclusionin conclusion we found a high level of abl1 expression incrc tissue and cells which was associated with the tnmstages a novel mutation of c1222c deletion in exon of theabl1 gene was found and was associated with the crc stagedepletion of abl1 decreased the growth of crc cell lines bothin vitro and in vivo by inhibiting tgf pathway these resultsdemonstrated novel understandings of the function of abl1during the progression of crc thus provides a clinically viablestrategy for crc therapydata availability statementthe raw data supporting the conclusions of this will bemade available by the authors without undue reservationethics statementthe studies involving human participants were reviewed andapproved by ethics committee of shaanxi people™s hospital thepatientsparticipants provided their written informed consent toparticipate in this study the animal study was reviewed andapproved by ethics committee of shaanxi people™s hospitalauthor contributionsall authors have a significant scientific contribution to all aspectsof this studyfundingstudy wasthissupported by national natural sciencefoundation of china”youth projects grant no shaanxi natural science foundation grant nos 2015jq8321and 2019jm547 shaanxi innovative talents cultivate programgrant no 2017kct28 and operating expenses of basicscientific research project of xi™an jiaotong university grantno xzy012019112supplementary materialthe supplementary materialonline202001385fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatreferences ouerhani s bougatef k soltani i elgaaied ab abbes s menif s theprevalence and prognostic significance of kras mutation in bladder cancerchronic myeloid leukemia and colorectal cancer mol biol rep “ 101007s1103301325128 brenner h kloor m pox cp colorectal cancer lancet “ 101016s0140673613616499 navarro m nicolas a ferrandez a lanas a colorectal cancer populationscreening programs worldwide in an update world j gastroenterol “ 103748wjgv23i203632 favoriti p carbone g greco m pirozzi f pirozzi re corcione fworldwide burden of colorectal cancer a review updates surg “ 101007s133040160359yjauhri m bhatnagar a gupta s shokeen y minhas s aggarwal stargeted molecular profiling of rare genetic alterations in colorectalcancer 101007s1203201608202sequencing med oncolnextgenerationusing peng x luo z kang q deng d wang q peng h foxq1mediates the crosstalk between tgfbeta and wnt signaling pathways inthe progression of colorectal cancer cancer biol ther “ frontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancer rossner f gieseler c morkel m royer hd rivera m blaker h uncoupling of egfrras signaling and nuclear localization of ybx1in colorectal cancer oncogenesis 5e187 101038oncsis alpay k farshchian m tuomelainhibition ofsiljamaki ecancer9e105526 101371 pone0105526cells highly sensitivealetj sandholm j aittokallio krenderscabl kinaseactivityto mitoxantrone plos one tian xq guo ff sun df wang yc yang l chen sl downregulationof znf278 arrests the cell cycle and decreases the proliferation of colorectalcancer cells via inhibition of the erkmapk pathway oncol rep “ 103892or20176031 udden sm moritafujimura y satake m ikawa s cabl tyrosinefate “kinase modulates p53dependent p21 induction and ensuing celldecision in response to dna damage cell signal 101016jcellsig201310005 cai s cheng x liu y lin z zeng w yang c eya1 promotes tumorangiogenesis by activating the pi3k pathway in colorectal cancer exp cell res “ d
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introduction postoperative ileus poi a common complication after surgery severely affects postoperative recovery it is unclear whether pretreatment with transcutaneous electrical acupoint stimulation teas can improve recovery from poi this trial will evaluate the effects of pretreatment with teas on poimethods and analysis this will be a prospective randomised controlled trial american society of anesthesiologists asa physical status classification i“iii level patients aged “ years and scheduled for laparoscopic colon surgery will be included in the study it is planned that subjects will be randomised to the teas and sham teas steas groups the groups will undergo two sessions of teassteas daily for days before surgery with a final teassteas treatment min before anaesthesia the primary endpoint of the study will be time to first defaecation secondary endpoints will include time to first flatus time to tolerance of oral diet gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to independent walking length of hospital stay postoperative pain visual analogue scale score on the first days after surgery analgesic requirements complications and plasma concentrations of interferon ifn ifnÎ interleukin6 il6 and il1 multiple linear regression will be used to identify independent predictors of outcome measuresethics and dissemination this study has been approved by the chinese registered clinical trial ethics review committee no chiecrct20170084 the results of the trial will be published in an international peer reviewed trial registration number this study has been registered with the chinese clinical trial registry no chictr inr17013184trial status the study was in the recruitment phase at the time of manuscript submissionintroductionpostoperative dysfunction ileus poi of gastrointestinal is a transient gi strengths and limitations of this study –º this study aims to evaluate whether pretreatment with transcutaneous electrical acupoint stimulation teas can prevent postoperative ileus poi –º teas is a safe non invasive and easily accepted adjunctive intervention –º this study will provide deeper insights into the mechanism by which teas pretreatment reduces the inflammatory response –º this is a single centre study which is a potential limitationpropulsion that often occurs after abdominal surgery and may also occur after surgery at other sites1 the main symptoms of poi include abdominal pain and distention nausea vomiting difficult defaecation and intolerance to solid food poi is usually temporary but if prolonged may lead to surgical incision dehiscence intestinal anastomotic fistula abdominal cavity infection intestinal ischaemia aspiration pneumonia and other serious complications2“ a retrospective cohort study involving nearly hospitals in the usa showed that poi is a key reason for prolonged hospitalisation and increased medical costs for patients undergoing abdominal surgery1 the usa spends more than billion treating poi every year5 at present the most common methods used to treat poi include rational perioperative use of narcotic drugs and opioids eating as soon as possible after surgery avoidance of nasogastric tubes after the operation early ambulation postoperative epidural analgesia restriction of fluid intake the use wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access of minimally invasive surgery such as laparoscopic drug therapy and the use of chewing gum despite the numerous treatment strategies poi remains a difficult clinical challenge that compromises the rapid recovery of postoperative patients it is therefore necessary to find more effective convenient and economical treatment methods6“the main mechanism underlying poi may be activation of macrophages in the external muscular layer during the surgical procedure11 intestinal manipulation during surgery can activate macrophages in the outer muscle layer of the small intestine leading to release of inflammatory factors interleukin6 il6 il1 and the chemokine mip1α together with increased expression of the adhesion molecule icam1 on endothelial cells and induction of neutrophils and monocytes in the circulation into the small intestine muscle layer these cells and activated macrophages can release a large amount of inducible nitric oxide synthase and prostaglandin which inhibit the movement and contraction of the gi tract12 transport of these inflammatory mediators in the bloodstream causes activation of macrophages in the distal gi tract leading to poi over the entire intestinal tract14 it has been confirmed by a large number of animal experiments that reducing the inflammatory response is an effective way to treat poi15“there is a long history in traditional chinese medicine tcm of using acupuncture to treat functional gi diseases and in recent years there has been significant global interest in the beneficial effects of acupuncture on poi the positive effect of electroacupuncture ea on poi has been clearly demonstrated ng used ea to treat poi in patients undergoing laparoscopic colon surgery18 defaecation time and length of hospital stay were significantly shortened in patients who received ea compared with those who did not receive the treatment in patients undergoing hepatic resection you found a significant reduction in the incidence of poi in patients treated with a combination of acupuncture and chinese herbal medicine the length of hospitalisation was also significantly shortened in the treated group ± days vs ± days p001419in the previous studies we proved that pretreatment with acupuncture could reduce excessive activation of the innate immune system and inhibit the inflammatory response this effect may be achieved by activation of the vagal nervous system20 other studies have shown that transcutaneous electrical acupoint stimulation teas and ea have similar effects in the treatment of pain and alleviating the inflammatory response22 tcm holds that the best treatment for disease is prevention based on all of the above studies we hypothesise that the use of teas as a preoperative treatment may reduce the incidence of poi there have so far not been any studies that address this questionwe have therefore designed a randomised controlled trial to investigate whether pretreatment with teas can reduce the incidence of poi in patients undergoing laparoscopic colon resection the study is also designed to verify that the anti inflammatory effect is associated with the immunomodulatory function of teasmethods and analysisstudy objectivethe primary objective is to assess the effect of teas on clinical recovery of bowel function after laparoscopic colon surgery the secondary objective is to verify that suppression of overactivation of the innate immune system and reduction of the inflammatory response are the mechanisms underlying the ability of pretreatment of percutaneous acupuncture to prevent poistudy locationa prospective single centre double blinded randomised controlled trial will be conducted at shuguang hospital which is affiliated to the shanghai university of traditional chinese medicine chinastudy populationparticipants will be recruited according to the inclusion and exclusion criteriainclusion criteria male and female patients aged “ years patients undergoing elective laparoscopic colonic surgery and upper rectal resection such as left collect right colectomy and anterior resection of the upper part of the rectum and lower part of the sigmoid body mass index “ kgm2 asa classification i“iii patients provide signed informed consent the consent form can be viewed in online supplementary appendix exclusion criteria middle and lower rectal resection totalproctocolectomy or the need for complex endoscopic surgery need for abdominal wall fistula gi fistula fistula surgery or stoma creation history of abdominalpelvic operations or complications patients receiving epidural anaesthesia or epidural analgesia patients with skin infections surgical incision or scar at the point of application of acupuncture patients have a history of limb surgery spinal surgery or nerve injury patients who participated in other clinical trials or received other acupuncture therapy in the previous weeks patients with cardiac pacemakers patients have one of the following conditions before surgery chronic pain drug addiction or alcohol dependence patients with preoperative combination of severe central nervous system disease and severe mental illnesswang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cendpointsprimary endpointfirst defaecation time h that is time to first anal defaecation after laparoscopic surgerysecondary endpointstime to first flatus h time to tolerance of solid oral diet h gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to walk independently h length of hospital stay defined as the number of days from operation to discharge d criteria for hospital discharge include stability of vital signs with no fever achievement of flatus or defaecation ability to tolerate solid food without vomiting control of postoperative pain absence of other postoperative complications and ability to function at home independently or with home care provided pain will be assessed using the visual analogue scale vas on postoperative days and scale of to where represents complete absence of pain and represents the worst pain intensity postoperative requirements for analgesia will also be assessed inflammatory mediators interferon ifn ifnÎ interleukin6 il6 and il1 in blood will be measured before teassteas intervention and on days and after the operation postoperative complications will be recorded using the clavien dindo classification for complication assessment24 the follow up period will be at least monthswe add gi2 as a secondary outcome to the original protocol after recruitment of the study had already begun gi2 is a time indicator which will be calculated from two existing outcomes time to first defaecation and time to tolerance of oral diet there will be no harm to subjects no additional cost and no more workopen accessrandomisation and blindingpatients will be randomised to receive either teas or steas by stratified randomisation according to sex in a ratio figure using a computer generated random sequence a sealed envelope will be opened to determine to which group the patient has been assigned the acupuncturist will be aware of the treatment group patients as well as the outcome investigator nurse anaesthetist will be blinded to the treatment allocationcurrent sample size justificationaccording to wang jian and song jiangang™s preliminary study of teas pretreatment for prevention of poi in patients undergoing laparoscopic colon surgery in shuguang hospital the mean time to first defaecation following laparoscopic colon surgery was ± hours m±sd working on the assumption that a clinically meaningful difference in mean time to first defaecation between the teas and steas groups is day or hours patients would be needed in each group to reach a power of and a type i error rate if the dropout rate is a total sample size of patients for the two groups is needed for this studystatistical analysisdata for continuous variables ie first defaecation time first passage of flatus time to tolerance of oral diet time to walking independently length of hospital stay will be reported using the mean and sd m±sd for normally distributed data or median range for skewed data data for categorical variables will be expressed as a number percentage intergroup differences will be assessed using the student™s t test or mann whitney u test intergroup differences in inflammatory mediators at time points of pre teassteas treatment and on figure flowchart of the study protocolwang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access figure acupoints selected in this trial a hegu il4 and neiguan p6 b zusanli st36 and shangjuxu st37 c han™s acupoint nerve stimulatorpostoperative days and were assessed by two way repeated measures analysis of variance with bonferroni post hoc test the significance level will be set at all data will be analysed using spss v170 software or other appropriate statistical software packagespretreatmentpatients randomised to the teas and steas groups will undergo two treatment sessions daily for three consecutive days before surgery the patients will then be treated for a final time min before anaesthesiafor patients in the teas group the zusanli st36 shangjuxu st37 hegu li4 and neiguan p6 acupoints will be identified before electrical stimulation with surface electrodes figure selection of these acupoints is based on a consensus between the acupuncturists carrying out the study the acupuncturist will stimulate these acupoints using a han™s acupoint nerve stimulator hans200a nanjing jisheng medical technology nanjing china at a frequency of hz the intensity will be adjusted for each individual to maintain a slight twitching of the regional muscle and achieve de qi sensations such as soreness numbness distention and heaviness the steas group will receive a strong but comfortable current for s and the current will then gradually vanish over the next s25 the participants of both groups will be told that they are receiving current stimulation each session of acupoints treatment will last for min during the application of teas patients will be required not to change the current settings themselves a prompt beep at the end of teas will indicate the end of treatmentall surgery will be carried out under general anaesthesia using standardised anaesthetic procedures patients will be fasted for hours before surgery right upper extremity venous access will be established before the patients entering the operating theatre ringer™s lactate solution mlkg will be administered by intravenous infusion for compensatory expansion before induction of anaesthesia patients will then receive midazolam mgkg fentanyl µgkg vecuronium bromide mgkg and propofol “ mgkg intravenously for induction of anaesthesia anaesthesia will be maintained using a cp600 anaesthesia delivery system slgo medical technology beijing china the dose wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cof propofol will be adjusted to maintain the bispectral index in the range of “ after surgery all patients will remain in the post anaesthesia care unit and then return to the ward for recovery until dischargethe perioperative management of all patients will be standardised early ambulation will be encouraged and oral feeding will be resumed as early as possible all patients will be followed up for at least months after discharge from the hospitaladverse eventsall adverse reactions will be closely monitored through spontaneous reports by patients or direct observation by clinicians or by asking the patients about adverse events using open questions all adverse reactions will be recorded and appropriate treatment will be provided if necessary serious adverse events will be reported to the ethics committeedata collection and managementdemographic variables and clinical data will be collected from all patients during the study blood pressure heart rate and oxygen saturation will also be monitored any adverse events will be recorded data will be collected throughout the study and will be securely managed under conditions of confidentiality data collection will be performed by a nurse anaesthetist the participants will be referred to by their participant number rather than by their name throughout the study unless otherwise specified all relevant documents and files will be archived for years the data will be accessible only by investigators who sign the confidential disclosure agreement and by institutional or governmental auditors during the study data without patient identifiers will be publicly accessible after the study data collection and management will be monitored by the institutional ethics committee for clinical research of shuguang hospitalpatient and public involvementthis study is currently in the recruitment phase patients andor the public were not involved in study design or conduct of the study the participants will be able to access the study results through social mediadiscussionpoi continues to represent an important cause of morbidity after colon surgery the prevention of poi is thus of great importance in reducing perioperative complications and reducing hospitalisation costs although it has been shown that ea can shorten the duration of poi18 the effectiveness of teas which is a similar technique in preventing poi has not been investigated it is therefore important to assess the effectiveness of teas in preventing poi through a clinical studythis study has several strengths first the intervention strategy of the protocol will be pretreatment with teas previous studies have shown that pretreatment open accesshas a prophylactic effect for example pretreatment with teas has been shown to improve pain treatment26 and to improve resuscitation after anaesthesia with reduction of postoperative nausea and vomiting28 it is however unclear whether preoperative teas can prevent poi studies suggest that early preoperative intervention may be more beneficial in regulating physiological functions and preventing poi29 in an extension to these findings the present study will help to determine whether teas pretreatment could improvement poisecond the effectiveness of teas will be evaluated by assessing clinical function and by serological examination in this randomised controlled trial of patients undergoing laparoscopic colorectal surgery our aim is to assess the effects of preoperative teas on poi using relevant clinical parameters associated with bowel function these include time to first defaecation time to first flatus time to tolerance of oral diet and gi2 importantly we will also measure serum concentrations of inflammatory mediators associated with poi such as ifn ifnÎ il6 and il1 our findings may thus provide deeper insights into the mechanisms by which teas improves poithere are also limitations to this protocol various clinical indicators have been used in studies for the diagnosis of poi but there is no consensus on which clinical parameter is the best for assessment of gi transit9 two indicators that are widely used to assess bowel movement will be used in this study time to first defaecation will be the primary outcome and time to first flatus will be one of the secondary outcomes there is a possibility that we may observe conflicting results ie significant improvement in time to flatus but not defaecation because flatus can vary considerably between patients clinical trials support the time to tolerance of oral diet and gi2 defined as the later of the following two events time to first tolerance of solid food and time to first bowel movement as supplementary secondary outcomes to measure the recovery time of gi function and these will be used in this study32 other limitations of these indicators are that they require objective measurement of motility and are time consuming to measure34 recently this situation has been improved by the use of in vivo monitoring techniques to assess the function of gi movements innovative devices such as sitz markers have been used to evaluate postoperative recovery of small bowel movement by counting the number of sitz markers that did not pass through the ileocecal valve but remained in the small intestine using radiography36 the smartpill is a swallowable device that record parameters within the gi tract indicators such as ph temperature and intracavitary pressure can be collected to analyse gi transit times in vivo37 these devices acquire objective parameters to evaluate bowel movement and could save time research into the satisfaction of both doctors and patients with these device needs to be carried out furthermore this study is a single centre trial and because the therapeutic effect of teas may be affected by ethnicity and region it will wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access be necessary to conduct multicentre and large sample studies in the futurenotwithstanding its limitations this study can clearly indicate the overall effects of teas on postoperative recovery we hypothesise that pretreatment with teas could improve recovery of gi function in patients undergoing laparoscopic surgery if this study provides positive results it will be possible to recommend this pretreatment strategy for patients undergoing abdominal surgery relevant cost effectiveness studies are also worthy of considerationauthor affiliations1anesthesiology shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai china2anesthesiology wenzhou medical university the sixth affiliated hospital lishui china3research institute of acupuncture anesthesia shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai chinaacknowledgements we thank dr stanley tao from shanghai ruihui biotech for his valuable assistance in the statistical design of this studycontributors jw conceived the study dl wt jg and gf participated in its design and coordination wc yy ws and jg collected references and developed the protocol gy and ly will perform statistical analyses rf will follow up with patients and record data jw lf and js drafted the manuscript all authors have read and approved the final manuscriptfunding the present study is supported by the project of the national natural science foundation of china nos and and the commercial sponsorship of sinch pharmaceuticals techcompeting interests none declaredpatient consent for publication obtainedprovenance and peer review not commissioned externally peer reviewedopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idlihua a0fan http orcid org references iyer s saunders wb stemkowski s economic burden of postoperative ileus associated with colectomy in the united states j manag care pharm “ boelens pg heesakkers ffbm luyer mdp et a0al reduction of postoperative ileus by early enteral nutrition in patients undergoing major rectal surgery prospective randomized controlled trial ann surg “ melis m fichera a ferguson mk bowel necrosis associated with early jejunal tube feeding a complication of postoperative enteral nutrition arch surg “ moghadamyeghaneh z hwang gs hanna mh et a0al risk factors for prolonged ileus following 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" 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 didn™t 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 kaplan“meier 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 didn™t 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_amp“““““““““““““xue bmc cancer page of table the clinicopathological characteristics of dlbcl with jak2pdl2 amplification and pmbclmyd88_no diagnosis sitel265p“myc_ breakapartnormalhansalgorithmnon_gcbage ipi _riskspleenfemale lowsexpmbclna not applicablemediastinummalelowwhich was supported by the cancer genome atlas datatcga pancancer atlas from cbioportal [ ] fig 1ccategory and myc breakapart didn™t 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 he× and pdl1× 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_valueχ2test 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 didn™thave 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 wasn™t 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 wasn™t 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 a“swerdlow 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 oncol“lesokhin 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 “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"Accumulating evidence has revealed the critical role of long noncoding RNAs lncRNAs in cellularprocesses during tumor progression As documented in cancerrelated literatures LINC00992 expression isassociated with cancer progression whereas its function in tumors including prostate cancer has not beencharacterized yetMethods Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues The expressionlevels of RNAs were monitored via qRTPCR Western blot evaluated the levels of proteins The proliferationapoptosis and migration of prostate cancer cells were assessed by CCK8 EdU TUNEL Transwell and woundhealing assays Luciferase reporter RNA pull down and RIP assays were applied to detect the interplays amongLINC00992 miR3935 and GOLM1Results Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells LINC00992exerted facilitating functions in prostate cancer cell proliferation and migration Mechanically LINC00992 interactedwith and negatively regulated miR3935 to elevate GOLM1 expression in prostate cancer cells In addition thein vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed byGOLM1 upregulation Likewise LINC00992 depletion restrained tumor growth in vivo was offset by enhancedGOLM1 expressionConclusions LINC00992 competitively bound with miR3935 to elevate GOLM1 expression and therefore facilitatethe oncogenic phenotypes of prostate cancer cells implying a potential LINC00992targeted therapy for prostatecancerKeywords INC00992 miR3935 GOLM1 Prostate cancer Correspondence engineyangsinacom5Department of Urology the Second Affiliated Hospital of Bengbu MedicalCollege Hongye Road Bengbu Anhui 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 0cChen BMC Cancer Page of BackgroundClinically prostate cancer manifests as a dominatingcause of malerelated death worldwide and is characterized as the most continually occurred tumor amongmen in the United States [] The biggest challenge isthe detectable bone metastases in roughly advancedprostate cancer [] Virtually all prostate cancer patientsduring years™ androgen deprivation treatment inevitably undergo castrationresistance which contributes tothe poor clinical consequences in prostate cancer []However the mechanism underlaid prostate cancer remains mostly unknownThe widely studied long noncoding RNAs lncRNAsare transcribed from nonproteincoding human genomeand have more than nt in length [] LncRNAs are increasingly functionally identified and experimentally consolidated to be related to tumor neoplasia and progressionin diverse cancers [] Additionally lncRNAs with dysregulation can functionally modulate tumor developmentfrom multiple pathological aspects such as cell proliferation drugresistance and metastasis [“] For examplelncRNA A1BGAS1 inhibits cell proliferation and invasionin hepatocellular carcinoma via targeting miR216a5p []LncRNA LOC730100 sponges miR760 from FOXA1 toaccelerate cell proliferation and invasion in glioma []LncRNA SNHG16 functions as an oncogene in hepatocellular carcinoma [] Long intergenic nonprotein codingRNA LINC00992 is a novel lncRNA that has beenpreviously revealed to be elevated in tumors and substantiated as a master regulator for chemoresistance [] Besides LINC00992 has been uncovered as an elevatedlncRNA in prostate cancer [] which is consistent withthe detection from GEPIA database Despite that no previous study has given a comprehensive explanation aboutthe precise function or detailed mechanism of LINC00992in prostate cancerIn past decades the fact that lncRNAs function in tumors depending on their secondary or tertiary structureshas been reported in many cancerlinked studies For instance in the nucleus lncRNAs are entitled to work asmolecular scaffolds or alternative splicing assistants [] On the contrary lncRNAs dispersing in cytoplasm influence downstream mRNA translation or degradationthrough serving as miRNA sponges [ ] For exampleTNFαinduced lncRNA LOC105374902 promotes themalignant behaviors of cervical cancer cells by acting as asponge of miR12853p [] LncRNA TTNAS1 promotes papillary thyroid cancer tumorigenesis by regulatingmiR1533pZNRF2 axis[] Nevertheless whetherLINC00992 could exert its functions in prostate cancer viaits sponging role of certain miRNA remains unknownWe conducted this research aiming to explore thefunction or probable mechanism of LINC00992 in prostate tumor which might enrich the understanding interms of prostate tumor pathology and contribute to awider applied scopeMethodsTissue samplesThe prostate cancer tissue samples and matched peritumor tissue samples were collected from patientsdiagnosed with prostate cancer under the approval ofthe Ethics Committee of the First Affiliated Hospital ofKunming Medical University Each participant did notreceive radiotherapy and chemotherapy prior to tissuecollection and signed the written informed consentsbefore this study All samples were snapfrozen in liquidnitrogen and then stored at °C until required forfurther analysisCell cultureThe prostate epithelial cell line RWPE1 CRL11609and prostate cancer cellsincluding PC3 CRL1435LNCaP CRL1740 C4“ CRL3314 and DU145HTB81 were all purchased from American TypeCulture Collection ATCC Manassas VA USAinOctober All cells were cultured as recommendedin Dulbecco™s modified Eagle™s medium containing FBS GIBCO MA USA under the condition of a cellincubator with CO2 at °C Before using in thisstudy all cell lines were authenticated by STR profilingand tested for mycoplasma contamination in June Cell transfectionLINC00992 shRNA or negative control shRNA andpcDNA31LINC00992 pcDNA31GOLM1 or its emptycontrol pcDNA31 plasmid were chemically synthesizedand provided by Gene Pharma Shanghai China MiR mimics miR3935 inhibitor and theirrelatednegative controls NCmimics NCinhibitor were allpurchased for upregulating or downregulating miR3935from Ribobio Guangzhou China In line with the directions of LipofectamineTM RNAiMAX TransfectionReagent Thermo Fisher Scientific transfection of theseplasmids into DU145 PC3 and RWPE1 cells wasconducted and qRTPCR checked the transfection efficiency The sequences were as follows shNC ²CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG3²shLINC009921²CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG3² shLINC0 ²CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG3² s²CCGGTTACCTAATCAGTAGAThLINC009923GCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG3² NCmimics ²UCAGGUAGGGCUCAAACCAACC3² miR3935 mimics ²UGUAGAUACGAGCACCAGCCAC3² NCinhibitor²CUGGCUUUAG 0cChen BMC Cancer Page of GGUGCCACUUAG3² miR3935 inhibitor ²GUGGCUGGUGCUCGUAUCUACA3²Quantitative realtime PCR qRTPCROn the basis of the instructions of Trizol reagent Invitrogen USA RNA extraction was executed in prostatecancer cells After the examination of RNA purity withspectrophotometry cDNA was obtained from aboveRNA with reverse transcription kit ThermoFisher Scientific shanghai China qRTPCR analysiswas devised with the aid of a BioRad CFX96 system andSYBR green was applied for investigating the RNA levelsThe internal reference for LINC00992 and mRNAs wasGAPDH whereas that for miRNAs expression was U6Relative expression was assessed based on the method ofˆ’ΔΔCtab97779Western blotProtein content in cells was determined by western blotanalysis RIPA lysis buffer Beyotime Shanghai Chinawas adopted for cell lysing followed by the evaluation ofthe protein concentration with BCA Protein Assay KitP0011 Beyotime Tech SDSPAGE gel was applied for separating proteins μg protein per sampleand then proteins were transferred onto μm PVDFmembranes BioRad Hercules CA USA Antibodiesincluding antiGOLM1 ab109628 AbcamCambridge UK antiPCNA ab92552 AbcamantiCDK2 ab32147 Abcam antiCyclin D1ab40754 Abcam antiBax ab32503 Abcam antiBcl2 ab32124 Abcam antiMMP2antiMMP9ab38898 Abcam antipSrc ab40660 Abcam antiSrc ab47405 Abcam antipFAKab81298 Abcam antiFAK ab131435 Abcam antiGAPDH ab8245 Abcam andantiTubulin ab7291 Abcam were applied toprobe the membranes overnight at °C After that themembranes were further incubated for h with HRPconjugated secondary antibody Santa Cruz Co LtdSant Cruz CA USA atroom temperature ECLSubstrates Millipore Billerica MA USA was utilizedfor the visualization of signals followed by exposure toXfilm Kodak Rochester NY USA The quantificationof immunoblots was conducted with the aid of imageJsoftware National Institute of Health Bethesda MDUSA with GAPDH or Tubulin as the normalizer asneeded AbcamLuciferase reporter assayFragments of fulllength LINC00992 with wildtype ormutant binding sites for miR3935 and sequences ofGOLM1 ™UTR containing wildtype or mutated miR binding sites were inserted into the pmirGLOvectors Promega Madison WI USA for the construction of reporters LINC00992WT LINC00992MUTGOLM1WT GOLM1MUT Then the four reportersand miR3935 mimics or miR3935 inhibitor GenePharma were cotransfected into DU145 and PC3 cellsapplying lipofectamine2000 Invitrogen as neededFortyeight hours later DualLuciferase Reporter AssaySystem Promega was employed for the examination ofthe luciferase activity GloMax® Discover Multimode Microplate Reader Promega assessed the ratio of FireflyRenilla luciferase activity and the activity of Renilla wasthe normalized controlRNA immunoprecipitation RIP assayAccording to the direction for usage of Magna RIP„¢RNA Binding Protein Immunoprecipitation Kit “Millipore RIP assay was strictly performed RIP lysisbuffer was firstly applied to treat the transfected DU145and PC3 cells Afterwards the obtained cell lysates wereprocessed with magnetic beads integrated with humanantiAgo2 antibodies ab32381 Abcam MA USA orantiIgG AP162KC Millipore Following the recoveryof antibody by the protein AG beads qRTPCR detected the levels of LINC00992 miR3935 and GOLM1mRNA in the precipitates IgG worked as the negativecontrol for the normalization of RNAIPsRNA isolation of nuclear and cytoplasmic fractionsThe dispersion of LINC00992 in the prostate cancercells was assayed as described previously [] The isolation of cytosolic and nuclear sections was executed followingAM1921Invitrogen RNA levels of U1 nuclear control GAPDHcytoplasmic control and LINC00992 were all estimatedby qRTPCR analysisPARIS„¢ KittheprotocolofFluorescence in situ hybridization FISH assayIn line with the recommendation of Ribo„¢ FISH KitC10910 Ribobio Guangzhou China FISH analysiswas implemented for testing the presence of LINC00992in prostate tumor cells Ribobio Company synthesizedthe LINC00992 probes labeled by Cy3 fluorescent dyeFollowing the fixation by paraformaldehyde and Triton X100 permeabilization DU145 and PC3 cellswere subsequently blocked in prehybridization bufferblocking solution Then incubation of cells with probehybridization buffer was later performed Next day afterrinsing and Hoechst staining the fluorescence was measured under a confocal laser scanning microscope ZeissGermanyCell counting kit8 CCK8 assayFor the viability assessment in DU145 PC3 and RWPE cells CCK8 assay was implemented as described 0cChen BMC Cancer Page of previously [] Cell viability was monitored at and h In short after being seeded onto 96well platesand cultured for indicated times cells were processedwith μl of CCK8 solution Then a microplate readerexamined the absorbance values at the wavelength of nm²ethynyl2²deoxyuridine EdU incorporation assayCell proliferation was examined through EdU assay asdescribed previously [] by using ClickiT EdU AlexaFluor Imaging Kit C10086 Invitrogen After hoftransfection EdU staining was carried out asinstructed The observation and calculation of EdUpositive cells was proceeded under the fluorescencemicroscopyTransferasemediated dUTP nick end labeling TUNELstainingTUNEL assay was carried out as described previously[] for probing DU145 and PC3 cell apoptosis with theassistance of an In Situ Cell Death Detection Kit Roche Mannheim Germany TUNELpositivecells were recorded under a light microscope × from visual fields which were chosen at randomTranswell migration assayThe application of transwell chambers with pore size μm Corning Costar Cambridge MA USA was aimedfor detecting cell migration in strict line with the instructions Cells that were previously suspended inserumfree RPMI1640 media were seeded into theupper chamber RPMI1640 medium containing FBS was supplemented in lower chamber as a chemoattractant Cells in the filters following h incubationwere immobilized in methanol and went through crystal violet staining The images of cells migratedthrough the filters were obtained and counted under themicroscopeWound healing assayThe DU145 PC3 and RWPE1 cells × cellswellwere prepared on glass culture dishes and cultivated at °C for a whole night to allow cells adhered to theplates followed by the straight scratch made with a plastic pipette tip after cell samples reached confluenceLater cells were rinsed in PBS to clear the detachedcells Finally the wounds at and h were imaged viaa light microscopy Olympus Tokyo JapanIn vivo experimentSixteen sixweekold male BALBC athymic nude micewere commercially available from the National Laboratory Animal Center Beijing China and maintained inSPFgrade animal lab All animalrelated protocols wereapproved by the Animal Research Ethics Committee ofthe First Affiliated Hospital of Kunming Medical University The in vivo experiment was undertaken via subcutaneous injection of × DU145 cells into the nudemice while the DU145 cells injected into indicated fourshgroups of mice were transfected with shNCLINC009921shLINC009921 pcDNA31 orshLINC009921 pcDNA31GOLM1 Tumor volume wasmonitored every days 28day after injection nudemice were sacrificed via cervical dislocation and thentumor samples were carefully dissected for weight assessment and hematoxylin and eosin HE stainingImmunohistochemistry IHCThe tumor samples collected from in vivo experimentswere treated with PFA dehydrated and embedded inparaffin Afterwardsthe paraffinembedded sections μm were prepared for IHC assay as described previously [] by use of the antiKi67 and antiPCNA antibodies AbcamStatistical analysisSPSS statistical software SPSS Armonk NY USAwas employed in the processing of data from threebiological replicates and data were expressed as mean ±SD Significance of difference within two groups wasdetermined using Student™s ttest while that among noless than two groups was tested via oneway or twowayANOVA P was considered as the threshold ofsignificanceResultsLINC00992 is overexpressed in prostate cancer andregulates cell proliferation apoptosis and migrationLINC00992 expression pattern in prostate cancer wasacquired from online GEPIA database As a resultLINC00992 was considerably upregulated in PRADprostate adenocarcinomatissues relative to normalones Fig 1a After detecting LINC00992 expression intissue samples obtained from patients with prostate cancer we observed that LINC00992 expression was higherin prostate cancer tissues than that in peritumor tissuesFigure S1A Moreover clinical data showed that higherexpression of LINC00992 in prostate cancer patientswas associated with lower survival rate Figure S1BFurthermore LINC00992 expression in the prostate cancer cells and RWPE1 cells was evaluated by qRTPCRConsequently higher level of LINC00992 was exhibitedin prostate cancer cells than that in RWPE1 cells Fig1b which was completely consistent with the result presented in previous discovery [] Particularly DU145and PC3 cells expressed the highest level of LINC00992and was thereby chosen for the later assays For silencingLINC00992 special shRNAs targeting LINC00992 was 0cChen BMC Cancer Page of Fig LINC00992 was overexpressed in prostate cancer and regulates cell proliferation apoptosis and migration a GEPIA database demonstratedthe overexpression of LINC00992 in tumor tissues in contrast to adjacent normal ones b LINC00992 expression was detected by qRTPCR in fourprostate cancer cell lines and control RWPE1 cells c LINC00992 expression was monitored by qRTPCR in DU145 and PC3 cells after transfectionwith shRNAs targeting LINC00992 shNC was used as the negative control d The viability of DU145 and PC3 cells was estimated through CCK8assay following LINC00992 depletion e The proliferation of DU145 and PC3 cells was investigated after LINC00992 depletion via EdU assay Scalebar μm f The apoptosis of DU145 and PC3 cells transfected with shLINC0099212 or shNC was estimated via TUNEL assay Scale bar μm g Western blot analysis was applied to examine the expression of apoptosisrelated proteins hi The migration of DU145 and PC3 cellswas analyzed via Transwell migration assay scale bar μm and wound healing assay scale bar μm after inhibiting LINC00992expression The fulllength images for blots in Fig 1g were presented in Supplementary figure P p transfected into DU145 and PC3 cells and the efficiencywas corroborated in qRTPCR Fig 1c And then thedata from CCK8 assay revealed that LINC00992 depletion suppressed the proliferation of DU145 and PC3cells Fig 1d As expected a declined proportion ofEdU positive cells was observed after knocking downLINC00992 Fig 1e suggesting the suppressive effect ofLINC00992 deficiency on prostate cancer cell proliferation Additionally the expression levels of proliferationrelated proteins PCNA CDK2 and Cyclin D1 were allreduced by silenced LINC00992 Figure S1C On thecontrary TUNEL assay uncovered that LINC00992knockdown facilitated cell apoptosis Fig 1f Meanwhile western blot analysis revealed that LINC00992knockdown promoted the apoptosis of DU145 and PC3cells as Bax protein level was increased whereas Bcl2protein level was decreased after LINC00992 was silenced in these two cells Figs 1g Figure S1D FurtherTranswell and wound healing assays indicated that themigration of DU145 and PC3 cells was retarded byLINC00992 depletion Fig 1hi Likewise the expression of migrationrelated molecular markers MMP2MMP9 pSrc and pFAK was decreased by LINC00992inhibition Figure S1E To further verify the biological 0cChen BMC Cancer Page of role of LINC00992 in prostate cancer we carried outgainoffunction assays in RWPE1 cells After overexpressing LINC00992 in RWPE1 cells Figure S2A cellproliferation was promoted Figure S2BC As expectedthe expression of PCNA CDK2 and Cyclin D1 wasdecreased by upregulation of LINC00992 Figure S2DSimilarly LINC00992cellIn addition upregulatingmigration Figure S2EFLINC00992 resulted in the elevated protein levels ofMMP2 MMP9 pSrc and pFAK Figure S2G All thesedata elucidated that LINC00992 could facilitate cell proliferation and migration whereas suppress cell apoptosisin prostate cancerupregulationfacilitatedMiR3935 is targeted by LINC00992Given the high correlation of the sublocalization ofLINC00992 with its functional mechanism the predication of LINC00992 presence in cells was performed viaLncLocatorhttpwwwcsbiosjtueducnbioinflncLocator Result predicted that LINC00992 located mainlyin cytoplasm Fig 2a Likewise FISH assay and RNAisolation of nuclear and cytoplasmic fractions furtherverified the abundance of LINC00992 in the cytoplasmof prostate cancer cells Fig 2bc highlighting a posttranscriptional control of LINC00992 in such cellsHence we speculated that LINC00992 might act as aceRNA in prostate cancer regulation According toDIANAlncBase the top three potential miRNAs possessing the binding capacity with LINC00992 were listedFig 2d To targetthe highlymatched miRNA toLINC00992 qRTPCR analysis was conducted to testthe expression changes of these miRNAs following eitherLINC00992 depletion or augmentation The resultsdemonstrated that only miR3935 expression was increased by LINC00992 depletion Fig 2e but reducedby LINC00992 overexpression in the meantime Fig 2fThus miR3935 was chosen for further analysis Afterwards RNA pull down assay was implemented and theresult depicted that LINC00992 was pulled down byBiomiR3935WT Fig 2g which indicated the bindingof LINC00992 and miR3935 Later we observed thesatisfactory efficiency of miR3935 overexpression andmiR3935 inhibition through qRTPCR analysis Fig2h Thereafter RIP assay applying antiAgo2 was executed Results illustrated that LINC00992 and miR3935were highly enriched in antiAgo2 group in comparisonwith control antibody Fig 2i certifying the associationof LINC00992 with miR3935 in the RNAinduced silencing complexes RISCs To further explore the interaction between LINC00992 and miR3935 the bindingsites between LINC00992 and miR3935 were predictedat first and then data from luciferase reporter assayrevealed that miR3935 upregulation decreased theluciferase activity of LINC00992WT reporter whereasmiR3935 inhibition increased the luciferase activity ofLINC00992WT reporter Fig 2j Altogether LINC0 combined with miR3935 to act as a miRNA decoyin prostate cancerLINC00992 regulates the expression of GOLM1 a targetof miR3935Present evidence has suggested that miRNAs can bindwith downstream target genes to inhibit their expressionHerein we searched the miR3935 target genes andeight mRNAs were found out Subsequently we detectedtheir expression in prostate cancer cells and normalcells Interestingly we found that only Golgi membraneprotein GOLM1 was highly expressed in four prostate cancer cell lines relative to normal controls Fig 3aFurther we discovered that GOLM1 expression wasmarkedly upregulated in prostate cancer tissues according to data from GEPIA database Fig 3b SimilarlyGOLM1 expression was much higher in prostate cancertissue samples than in peritumor samples Figure S3AIn addition the mRNA and protein levels of GOLM1were overexpressed in prostate cancer cells in contrastto RWPE1 cells Fig 3c Figure S3B Besides GOLM1has been previously revealed as a prostate cancer facilitator and was metastasisrelated in prostate tumor [“] Thus we hypothesized that GOLM1 might act asthe downstream of LINC00992miR3935 signaling inprostate cancer Through TargetScan httpwwwtargetscanvert_72 the binding site between GOLM1and miR3935 was predicted Fig 3d After conductingluciferase reporter assay in DU145 and PC3 cells weobserved that upregulation of miR3935 specifically decreased the luciferase activity of GOLM1WT reporterFig 3e confirming the interaction between miR3935and GOLM1 relied on the putative binding sites Thenwe unveiled that GOLM1 mRNA and protein levels wereboth reduced by LINC00992 inhibition or miR3935upregulation according to qRTPCR and western blotanalyses Fig 3fg Figure S3C Moreover data fromRIP assay unveiled the binding of miR3935 to GOLM1in the RISCs Fig 3h Further we demonstrated thatthe decreased mRNA and protein levels of GOLM1 induced by LINC00992 depletion could be restored afterinhibiting miR3935 expression Fig 3ij Figure S3DAll the results showed that LINC00992 upregulatedGOLM1 expression via directly binding to miR3935LINC00992 promotes prostate cancer cell proliferationand migration via elevating GOLM1 expressionTo test whether LINC00992 affected prostate cancer cellproliferation apoptosis and migration via regulatingmiR3935targeted GOLM1 we executed the rescue experiments with the upregulation of GOLM1 To beginwiththe efficiency of overexpressing GOLM1 was 0cChen BMC Cancer Page of Fig MiR3935 was targeted by LINC00992 a LncLocator predicted LINC00992 subcellular location b FISH analysis of LINC00992 distribution inprostate cancer cells Scale bar μm c RNA isolation of nuclear and cytoplasmic fractions assayed the subcellular distribution of LIN00992 inprostate cancer cells d Top three miRNAs which might interact with LINC00992 were predicted by DIANAlncBase e After transfection ofLINC00992silencing plasmids the expression of miR31575p miR11783p and miR3935 was examined via qRTPCR f Following LINC00992upregulation qRTPCR tested the levels of miR31575p miR11783p and miR3935 in DU145 and PC3 cells g RNA pull down assay wasimplemented to testify the binding capacity between LINC00992 and miR3935 h miR3935 overexpression efficiency and inhibition efficiencywere examined by qRTPCR i RIP assay disclosed the binding of miR3935 to LINC00992 in the antiAgo2 group j The potential binding sitebetween LINC00992 and miR3935 was shown And the luciferase activity of LINC00992WT or LINC00992MUT reporter was assessed vialuciferase reporter assay in DU145 and PC3 cells after transfection with miR3935mimics miR3935inhibitor NCinhibitor or NCmimics P p p analyzed through qRTPCR and western blot analysesand the outcome turned out to be satisfactory Fig 4abFigure S3E Then we observed that overexpression ofGOLM1 could significantly elevate the mRNA and protein expression of GOLM1 in shLINC009921transfected cells Figure S3F Afterwards data from CCK8revealed that the viability of DU145 cells was firstly hindered due to LINC00992 depletion while subsequentGOLM1 elevation reversed the inhibitory trend onDU145 cell viability Fig 4c Results from EdU assayalso exposed similar trends that GOLM1 upregulationimpactposedthesuppressivecountervailedbyLINC00992 downregulation on DU145 cell proliferationFig 4d Similarly the restraining effect of silencedLINC00992 on the expression of proliferationrelatedproteins could be reversed by GOLM1 upregulationFigure S3G Later TUNEL assay revealed that cellapoptosis rate was elevated by LINC00992 depletion andthen overexpressing GOLM1 reduced the increasedapoptosis rate of LINC00992depleted cells Fig 4eLikewise western blot analysis uncovered that overexpressing GOLM1 could offset the effect of LINC00992 0cChen BMC Cancer Page of Fig LINC00992 regulated the expression of GOLM1 a target of miR3935 a The expression of eight mRNAs in four prostate cancer cell linesand RWPE1 cells was detected by qRTPCR b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database c The mRNAand protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE1 cell line by qRTPCR and western blot respectively d Thebinding sites between GOLM1 and miR3935 were predicted via TargetScan e Luciferase reporter assay presented the inhibited luciferase activityof GOLM1WT reporter in the presence of miR3935 mimics not NCmimics fg GOLM1 expression in transfected cells was tested by qRTPCR andwestern blot analyses h The combination of GOLM1 with miR3935 in the antiAgo2 group was validated by RIP assay ij The mRNA and proteinlevels of GOLM1 in different groups were examined via qRTPCR and western blot The fulllength gels for western blot data in Fig 3c g and jwere presented in Supplementary Figure P p p downregulation on the expression of apoptosisrelatedproteins Fig 4f Figure S3H Moreover Transwellmigration and wound healing assays illuminated thatthe retarding influence of silenced LINC00992 on cellmigration could be rescued by GOLM1 overexpression Fig 4gh As expected the inhibitory effect ofLINC00992 depletion on the expression of migrationrelated molecular markers MMP2 MMP9 pSrc andpFAK could be countervailed by GOLM1 overexpression Figure S3I Collectively GOLM1 was requiredcancercellular processesLINC00992regulatedinprostateLINC00992 contributes to tumor growth via upregulatingGOLM1 expressionAfter the in vitro exploration of LINC00992 performance in prostate cancer we applied the in vivo assays tofurther validate above findings As shown in Fig 5a tumors derived from LINC00992silenced DU145 cellswere smaller with the growth rate quite slower thanthose from control cells more importantly such blockage on tumor growth was obviously countervailed afterGOLM1 overexpression Besides elevating GOLM1 expression could recover the lessened tumor volume anddeclined tumor weight induced by LINC00992 deficiency 0cChen BMC Cancer Page of Fig LINC00992 promoted prostate cancer cell proliferation and migration via elevating GOLM1 expression ab GOLM1 mRNA and proteinlevels in DU145 cells transfected with pcDNA31 or pcDNA31GOLM1 were detected via qRTPCR and western blot pcDNA31 served as thenegative control c The viability of DU145 cells was determined via CCK8 following transfection of different plasmids d The proliferation oftransfected cells was evaluated via EdU assay e The apoptosis of transfected cells was monitored via TUNEL assay Scale bar μm f Theprotein levels of Bax and Bcl2 in different groups were detected via western blot gh The migration of transfected cells was measured viaTranswell migration assay scale bar μm and wound healing assay scale bar μm The fulllength gels for western blot data in Fig 4band f were presented in Supplementary Figure P p Fig 5bc Of note we discovered decreased level ofLINC00992 and enhanced level of miR3935 in tumorsfrom latter three groups compared to control group whilethe lowered expression of GOLM1 in tumors withLINC00992 inhibition was normalized under GOLM1overexpression Fig 5d In addition the inhibitory impactof silenced LINC00992 on the positivity of proliferationassociated proteins PCNA and Ki67 could be reversedby upregulation of GOLM1 Fig 5e Taken togetherLINC00992 promoted the tumorigenesis of prostate cancer through upregulating GOLM1 expressionDiscussionAs documented the aberrant regulation of lncRNAs is afrequent event in diversified tumor types Besides thecorrelation between abnormallncRNA expression andprostate cancer oncogenesis has also been extensivelyexplored For example lncRNA SNHG7 facilitates prostate cancer carcinogenesis via cyclin D1 by spongingmiR503 [] LncRNA SChLAP1 aggravates prostatecancer cell proliferation and metastasis by targetingmiR198 [] LncRNA PCAT1 contributes to prostatecancer tumorigenesis through modulating FSCN1 andsponging miR1455p [] In our work LINC00992 wasrevealed to be highly expressed in prostate cancer tissuesand cells but unlike former investigations our studygave a precise explanation about its role in prostate cancer Our study unveiled that LINC00992 promoted cellproliferation and migration whereas suppressed cellapoptosis in prostate cancer Abovementioned data 0cChen BMC Cancer Page of Fig LINC00992 contributes to tumor growth via upregulating GOLM1 expression a Representative images and the growth curves of tumorsfrom indicated groups bc The volume and weight of tumors from above groups d The expression of LINC00992 miR3935 and GOLM1 intumors from different groups was detected via qRTPCR analysis e The staining of PCNA and Ki67 in different groups was measured via IHCScale bar μm p 0cChen BMC Cancer Page of validated that LINC00992 elicited a tumorpromotingfunction in prostate cancerPresently accumulating evidence has indicated thatcytoplasmic lncRNAs assisted the expression of downstream miRNAtargeted mRNAs via sponging the specific miRNAs Before exploring LINC00992mediatedmechanism in prostate cancer herein we firstly discovered its subcellular distribution in prostate cancer cellswith both aids from online prediction tool LncLocatorand experimental data FISH and RNA isolation of nuclear and cytoplasmic fractions Our study for the firsttime uncovered that LINC00992 located mainly in thecytoplasm of prostate cancer cells Besides our studyalso completed LINC00992modulated mechanism bydisclosing the downstream target miR3935 The directinteraction between LINC00992 and miR3935
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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 confirmed 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 defined 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 [“] Reflux 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 preoperativereflux symptom index RSI and reflux finding score RFSassessments Patients with an RSI score points andoran RFS ‰¥ points were considered to have laryngopharyngeal reflux 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 providedspecific written informed consent Surgical Procedure According to the European Laryngological Society classification 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 first 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 defined 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 laser™s 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 thatreflux 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 findings suggest that although PPI treatment cannot reduce the incidence of TG it can significantlyshorten the duration of granulomas a finding that is similarto the study by Wang [] Additionally we did notobserve any severe cases of TG that were complicated with 0cBioMed Research InternationalabcdefghiFigure a“f 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 fibrolaryngoscope 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 g“i 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 fibrolaryngoscope 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 month”LPRD 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 reflux disease J R Lechien™s report in andChinse experts consensus on diagnosis and treatment of laryngopharyngeal and reflux 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 reflux hasa little eï¬ect Acid reflux 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 reflux 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 findings should be confirmedby 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 conflicts of interestfinancial or nonfinancial 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 œIncidence 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 œGlobal 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 œGlobal 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 œSystematic 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 œResults of curative laser microsurgery of laryngealcarcinomas American Journal of Otolaryngology vol no pp “ [] M S Strong œLaser excision of carcinoma of the larynxLaryngoscope vol no pp “ [] A S Carney M S Timms C N Marnane S KrishnanG Rees and S Mirza œRadiofrequency 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 œComplications after CO2 laser surgery for early glottic cancer an 0cBioMed Research Internationalinstitutional experience Head Neck vol no S1 pp E987“E990 [] B Liu L Cheng H Ming and C Zhong œTreatment 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 LiangœCarbon 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 œEndoscopic cordectomy A proposal for a classification by the Working Committee European Laryngological Society European Archivesof OtoRhinoLaryngology vol no pp “ [] L Wang S Sun S Wang D Liang and W Ji œClinical observation of traumatic granuloma after CO‚‚ laser cordectomy andlaryngopharyngeal reflux Zhonghua Er Bi Yan Hou Tou JingWai Ke Za Zhi vol no pp “ [] M Canis F Ihler A Martin C Matthias and W SteinerœTransoral laser microsurgery for T1a glottic cancer reviewof cases Head Neck vol no pp “ [] A Galli L Giordano D Sarandria D di Santo and M BussiœOncological 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 œFactors 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 œThe validityand reliability of the reflux finding score RFS Laryngoscopevol no pp “ [] P C Belafsky G N Postma and J A Koufman œValidity andreliability of the reflux symptom index RSI Journal of Voicevol no pp “ [] M Remacle C van Haverbeke H Eckel œProposal forrevision of the European Laryngological Society classificationof 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 œRole of endolaryngealsurgery with or without laser versus radiotherapy in themanagement of early T1 glottic cancer a systematic reviewHead Neck vol no pp “ [] C M Chiesa Estomba F A Reinoso A O Velasquez J LFernandez J L Conde and C S Hidalgo œComplications inCO2 laser transoral microsurgery for larynx carcinomas IntArch Otorhinolaryngol vol no pp “ [] J R Lechien F Mouawad M R Barillari œTreatment oflaryngopharyngeal reflux disease a systematic review WorldJournal of Clinical Cases vol no pp “ [] M K Wani and G E Woodson œLaryngeal contact granuloma Laryngoscope vol no pp “ [] C Ling M Yamashita J Zhang D M Bless and N V Welham œReactive response of fibrocytes to vocal fold mucosalinjury in rat Wound Repair and Regeneration vol no pp “ 0c'
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breast cancer bc is the most common malignant tumour in women worldwide and one of the most common fataltumours in women deltanotchlike epidermal growth factor egfrelated receptor dner is a transmembraneprotein involved in the development of tumours the role and potential mechanism of dner inepithelial“mesenchymal transition emt and apoptosis in bc are not fully understood we find that dner isoverexpressed in bc tissue especially triplenegative breast cancer tnbc tissue and related to the survival of bc andtnbc patients in addition dner regulates cell emt to enhance the proliferation and metastasis of bc cells via thewntcatenin pathway in vitro and in vivo moreover the expression levels of catenin and dner in bd tissue arepositively correlated the simultaneously high expression of dner and catenin contributes to poor prognosis in bcpatients finally dner protects bc cells from epirubicininduced growth inhibition and apoptosis via the wntcatenin pathway in these results suggest that dner induces emt and prevents apoptosis by the wntcatenin pathway ultimately promoting the malignant progression of bc in our study demonstrates thatdner functions as an oncogene and potentially valuable therapeutic target for bcintroductionbreast cancer bc is the most common malignanttumour in women worldwide and one of the most common fatal tumours in women12 bc treatments can beused to improve patient outcome3 however tumourrecurrence and metastasis and chemotherapeutic resistance are the most common causes of cancer treatmentfailure therefore the need to screen and identify keyregulatory factors in the process of tumour recurrenceand metastasis for the treatment of bc is urgentcorrespondence si sun karensisi126com or shengrong sun sun137sinacom1department of breast and thyroid surgery renmin hospital of wuhanuniversity wuhan hubei china2department of pathophysiology wuhan university school of basic medicalsciences wuhan hubei chinafull list of author information is available at the end of the these authors contributed equally zhong wang zhiyu liedited by s taittumour emt is a multifactorial and complex event inwhich epithelial properties and the ability to adhere toadjacent cells are lost and mesenchymal and stem cellphenotypes are eventually obtained4“ emt a crucialregulatory mechanism by which tumours acquire invasiveand metastatic abilities and the ability to resist apoptosisplays an irreplaceable role in the development of malignant tumours8“ recent studies upon activation of theclassical wntcatenin pathway catenin enters andaccumulates in the nucleus which induces the transcription and translation of downstream target genes thusaccelerating emt10 therefore maintaining cateninactivity is important for the wntcatenin pathway andtumour progressiondner a neuronspecific transmembrane protein foundin a variety of peripheral cells11“ is a member of theatypical notch ligand family and binds to notch1 receptor1115 dner is expressed at abnormally high levels in the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cwang cell death and disease page of various cancer tissues16 and promotes the proliferationmigration and invasion of cancer cells1617 but has aninhibitory effect on cell proliferation in glioma14 nevertheless the precise function and underlying molecularmechanisms of emt and chemosensitivity in bc areunclearin this study we have revealed the previously unrecognized role of dner in cancer progression emt andthe apoptosis of bc cells furthermore we investigatedthe expression of dner and its relationship with survivalin bc and tnbc patients in addition we have providedevidence for the correlation between dner and cateninand the prognostic value of the highlevel expression ofdner and catenin in bc patients finally the crucial roleof catenin in dnerinduced emt and the inhibitoryeffect of dner on apoptosis have been revealed takentogether our results elucidate the potential functions andmechanism of dner in emt and apoptosis in bc cellsand provide a new therapeutic pathway for the recurrence metastasis and chemotherapy resistance of bcmaterials and methodsethics statementtwo groups of the same human tissue specimens wereacquired from patients of renmin hospital of wuhanuniversity who were diagnosed with bc from to one group of specimens was promptly stored atˆ’ °c for western blotting and pcr analysis the othergroup of specimens was fixed in formalin and paraffinizedfor immunohistochemistry ihc all patients did notreceive chemotherapy radiotherapy or immunotherapythis research was approved by the ethics committee ofrenmin hospital of wuhan university and informedconsent was obtained from all patientscell culture and reagentshuman bc cell lines mcf7 and mdamb468 cellswere obtained from american type culture collectionand incubated by their corresponding recommendedmethod all celllines were mycoplasmafree by morphological examination and verified for their authenticities by str profiling epirubicin was purchased frompfizer pharmaceutical co ltd wuxi china and dissolved in physiological saline chir catenininhibitor and xav939 catenin agonist were purchased from selleck shanghai china and dissolvedin dmso “ “ and the stainingintensity was evaluated as follows no staining weak staining moderate staining and strongstaining the final protein staining score was the percentage score multiplied by the intensity score finalprotein staining scores were divided into three categoriesas follows negative “ low expression and “ high expressionsirna and plasmid transfectionscrambledner sirna ²gcuuugccaguccaagauuttsirna ²uucuccgaacguguandcacgutt were synthesized from genepharma coshanghai china flagdner and flagnc werepurchased from genechem co shanghai china whencells in a sixwell plate had grown to the appropriatedensity sirna and plasmids were transiently transfectedwith lipofectamine3000 invitrogen usa and rnaimax invitrogen usa respectively according to themanufacturer™s instructions after h of transfection thecells were used for subsequent experimentsqrtpcrtotal rna from tissue specimens and cell samples wasextracted by using trizol invitrogen usa according tothe protocol and then reverse transcribed to cdna usinga transscript firststand cdna synthesis kit takarajapan qrtpcr was implemented by using sybr greenmastermix takara japan with an abi 7900ht realtime pcr system usa the primer sequences areshown in supplemental table cell counting kit cck8 assayafter a series of interventions equal numbers of bccells were plated into 96well plates and cultured for days ten microlitres of cck8 ck04 dojindo japansolution was added to each well and the cells wereincubated at °c for h the absorbance was determined at nmwound healing assayafter intervention the cells were seeded into sixwellplates when the cell density exceeded the cells werewashed twice with pbs and scratches were made with ayellow plastic pipette tip cells were cultured in serumfree medium for h and photographed under amicroscopeimmunohistochemical staininginvasion assayihc staining was performed as previously described18the results of ihc staining were evaluated by two independent pathologists and scored according to the percentage of positive tumour cells and staining intensitythe percentage of positive cells was scored as follows after a series of treatments × cells in serumfreemedium were plated in the upper chambers of a transwell apparatus with matrigel corning ny usa medium in the bottom chambers containing fbs servedas an attractant after h of incubation cells that passedofficial of the cell death differentiation association 0cwang cell death and disease page of through the chamber membrane were fixed with precooled formaldehyde and stained with crystal violetc0121 beyotime the cells were counted and photographed under a microscopewestern blottingthe prepared tissue and cell samples were separated byprotein sdspage and transferred to a nitrocellulosenc membrane the membrane was blocked in skimmilk powder for h at room temperature and immunoblotted with primary antibody at °c overnight afterincubation with secondary antibody at room temperaturefor h protein expression was detected with corresponding protein development instrument and quantifiedby imagej software w s rasband image j nih theantibodies used are listed in supplementary table nuclear and cytoplasmic protein extractionnuclear and cytoplasmic extraction reagent p0027was purchased beyotime biotechnology the nuclear andcytoplasmic proteins were extracted according to theinstructions and then used for subsequent experimentsflow cytometry to detect apoptosisa fitc annexin v apoptosis detection kit i bdpharmingen usa was used to detect cell apoptosis the cellswere seeded in sixwell plates after a series of interventionscells were processed following the manufacturer™s protocolfig dner is upregulated in bc tissues and correlated with poor prognosis in bc and tnbc patients a the expression levels of dner inluminal a and tnbc tumour tissues compared with adjacent tissue by ihc magnification × b the mrna levels of dner in luminal a and tnbctumour tissues compared with adjacent tissue c the dner protein expression in bc tissues and adjacent tissues by western blotting d thekaplan“meier analysis showed the rfs of bc and tnbc patients with dner high expression or dner low expression e the staining of dner ecadherin and ncadherin in bc tissue by ihc magnification × f correlation analyses of protein expression levels between ecadherin ncadherinand dner p p vs the control groupofficial of the cell death differentiation association 0cwang cell death and disease page of and the cell fluorescence was measured with a facscan flowcytometer facscan becton dickinsontable clinicopathological associations of dnerexpression in breast canceranimal experimentsto acquire mdamb468 cells with dner stablyknocked down and mcf7 cells stably overexpressingdner cells were transfected with dner knockdown andoverexpression lentivirus genechem shanghai chinaand then selected with puromycin when the transfectionefficiency approached the dner protein level wasdetected with western blotting all experimental procedures were conducted according to the regulations ofexperimental animal administration issued by the animal committee of wuhan university the mice wererandomly divided into two groups a total of × stable cells in μl pbs were subcutaneously inoculatedinto the right iliac fossa of to 5weekold femaleathymic nude mice balbc after a certain period ofintervention the mice were sacrificed by anaesthesia andxenografts were removed for weighing and photographing the expression of relative proteins was detected bywestern blotting and ihcfor mammaryfatpad tumour assays we establishedmdamb231 cells with dner stably knocked downthe mice were randomly divided into two groups × stable cells were resuspended in a mixture of pbs andmatrigel and then injected into the fourth mammaryfat pad on the same side of nude mice to observe lungmetastasis tumours were excised by surgical operationwhen they reached about mm3 ten days after theoperation the mice were sacrificed by anaesthesia and thenumber of metastatic tumours per lung were determinedthe entire lung tissues were fixed with formalin andsectioned for haematoxylin and eosin he staining todetermine the presence of lung metastasis the entirelung tissues were fixed with formalin and sectionedfor haematoxylin and eosin he staining to determinethe presence of lung metastasisimmunofluorescenceimmunofluorescence staining was performed as previously described19 in brief after corresponding treatments the cells fixed with paraformaldehyde wereperforated by tritonx for min and blockedwith bsa for h next the cells were incubated withcatenin dilution overnight at °c and thenincubated for min with 488conjugated antibodyinvitrogen a11034 finally the slides were stained withdapi for min the images of sample were analyzed bylaser confocal microscopy zeiss lsm statistical analysisstatisticalspss software spss inc chicago il and graphpadanalyses were performed usingofficial of the cell death differentiation associationvariableslown highn p valueage at diagnosis years‰¤gradewellmoderatelypoorlytumour size cm‰¤lymph node metastasisnegativepositivevascular invasionnegativepositiveernegativepositiveprnegativepositiveher2negativepositiveki67 ‰¥ recurrencenoyes p values calculated by logrank testing bold if statistically significant p er oestrogen receptor pr progesterone receptor her2 human epithelial growthfactor receptor2prism graphpad software la jolla ca usa all datawere analyzed with at least three independent experiments and are presented as the mean ± sd a survivalcurve was prepared by kaplan“meier analysis and thelogrank test was used to compare survival differencesbetween groups pearson™s correlation method was used 0cwang cell death and disease page of table clinicopathological associations of dnerexpression in triple negative breast cancervariableslown highn p valueage at diagnosis years‰¤gradewellmoderatelypoorlytumour size cm‰¤lymph node metastasisnegativepositivevascular invasionnegativepositiveki67 ‰¥ recurrencenoyes p values calculated by logrank testing bold if statistically significant p to analyze the correlation between dner and catenina chisquare test was used to analyze associationsbetween dner expression levels and clinical characteristics oneway anova was used to compare differencesin three or more groups differences in which p were considered statistically significantresultsdner is upregulated in bc tissues and correlated withpoor prognosis in bc and tnbc patientsto determine the role of dner in development of bcwe first measured the expression levels of dner in bctissue and matched adjacent normal breast tissue by ihcthe expression level of dner in bc tissue was markedlyhighertheexpression in tnbc was higher than that in luminal a bcfig 1a we also detected the expression of dner in bctissue by pcr the results of which were consistent withthose of ihc experiments fig 1b to further verifytissue moreoverthan thatin adjacentofficial of the cell death differentiation associationdner expression in bc we utilized western blotting todetect dner protein expression in bc and adjacent tissues as expected compared with dner expression inadjacent tissues dner expression in bc tissues wassignificantly elevated fig 1c furthermore the highestdner expression level was found in tnbc tissue theclinicopathological characteristics with different expression of dner in all bc and tnbc patients were shown intables and kaplan“meier analysis of rfs showed thatthe group expressing high levels of dner had a worseprognosis than the group expressing low levels of dnerthe results of survival analysis of tnbc patients were thesame as that of bc patients and tnbc patients had ashorter rfs than bc patients fig 1d next to verifywhether the poor prognosis of bc patients caused bydner is related to emt we detected the correlationbetween dner and emtrelated markers the resultsshowed that dner expression was negatively correlatedwith the expression of ecadherin while positively correlated with ncadherin expression fig 1e f in addition we found that high expression of mesenchymalmarkers was significantly associated with high expressionof dner in bc through the tcga database httpgepiacancerpkucn although the negativecorrelationbetween ecadherin and dner in tcga database wasnot significant it also presented a negative trend supplementary fig 2a the results therefore suggested thatdner is highly expressed in bc and that elevated dnerprotein expression contributes to the progression of bcespecially tnbcdner increases the biological functions of bc cells in vitroto evaluate the effect of dner on bc cell proliferationmigration and invasion we used sirna to suppressdner expression in both mcf7 and mdamb468cells compared with dner expression in the control andscramble sirna groups dner was silenced by almost and in mcf7 and mdamb468 cells transfected with sirna respectively fig 2a b as shown infig 2c dner knockdown visibly downregulated thegrowth rate of bc cells by cck8 assay next a woundhealing assay was used to evaluate cell migration capacitycompared with wound closure in the scramble sirnagroup dner knockdown significantly inhibited woundclosure after h in bc cells fig 2d in addition thetranswell assay revealed that dner knockdown clearlyreduced bc cell invasion fig 2e these results suggestthat dner acts as a cancerpromoting gene in bc cellsto further confirm the role of dner in bc progressiondner was overexpressed by transfection with the flagdner plasmid for h as shown in supplementary fig1a dner was successfully overexpressed in the two bccell lines in striking contrast with the effects of dnerknockdown the ability of cell proliferation migration and 0cwang cell death and disease page of fig dner knockdown inhibits cell proliferation and metastasis of bc cells a b the knockdown efficiency of dner in mcf7 and mdamb cells c cell growth was measured by cck8 assay after dner knockdown in two bc cell lines d wound healing assay was used to determine themigratory ability of bc cells with dner knockdown e the invasion capacity of bc cells with knockdown of dner was confirmed by transwell assaydown quantitative analysis of invasion ratio was shown the values are the mean ± sd from three independent experiments nsp p p p p vs the control groupinvasion was markedly enhanced after dner overexpression supplementary fig 1b“e taken togetherthese results indicated that dner plays a crucial role inbc growth and metastatic potentialdner induces emt in bc cellstumour cell emt promotes the malignant progressionand metastasis of tumour cells10 we next examinedwhether dner has a regulatory effect on bc cell emtto assess this function we detected emtrelated proteinexpression by western blotting dner knockdown significantly upregulated epitheliallike marker ecadherinexpression and downregulated mesenchymal marker ncadherin vimentin snail expression fig 3a b conversely overexpression of dner dramatically shown theopposite effect fig 3c d these results indicate thatdner drives emt in bc cells to provide further evidence of this effect of dner on emt we suppresseddner expression and then transfected cells with theflagdner plasmid to restore the dner protein levelwe then determined whether dner overexpression couldreverse changes in the expression of emtrelated proteins as shown in fig 3e f dner knockdown alone hadan inhibitory effect on emt whereas dner knockdownand flagdner transfection suppressed the effect ofdner knockdown on ecadherin and partially restoredthe expression of ncadherin vimentin and snail theseresults suggest that dner plays a pivotal role in inducingemt in bc cellsofficial of the cell death differentiation association 0cwang cell death and disease page of fig dner induces emt in bc cells a b emtrelated proteins ecadherin ncadherin vimentin and snail were detected by western blotting indner knockdown cells right quantitative analysis of the optical density ratio of ecadherin ncadherin vimentin and snail compared with actinare shown c d emtrelated protein levels were measured by western blotting after dner overexpression in bc cells right quantitative analysis ofthe optical density ratio of ecadherin ncadherin vimentin and snail compared with actin are shown e f dner was overexpressed in dnerknockdown cells and then western blotting detected the expression of emtrelated proteins the values are the mean ± sd from three independentexperiments p p p vs the corresponding groupdner activates the wntcatenin signalling pathway andis positively correlated with cateninprevious reports have shown that the wntcateninsignalling pathway plays a crucial role in cancer cellmetastasis and emt2021 therefore we examined whether dner mediates the canonical wntcatenin signalling pathway as shown in fig 4a b compared withcontrol cells in dner knockdown cells the protein levelsof notch1 pgsk3 and catenin were increased andthose of gsk3 were unchanged conversely dneroverexpression dramatically shown the opposite effectnext we investigate whether there is a relationshipbetween notch signal and catenin in the case of dneroverexpressioncells wein dneroverexpressingknocked down notch1 and found that catenin expression was decreased compared with dner overexpressionalone supplementary fig 2b notch1 functioned as animportant role in the wntcatenin pathway and theactivation of notch1 was positively related to the nucleartranslocation of catenin22 theaccumulation ofcatenin in the nucleus plays an important role in themalignant progression of tumours we assessed the effectof dner knockdown on nuclear catenin accumulationby western blotting and observed that upon the knockdown of dner the levels of nuclear catenin and snailwere reduced in bc cell lines fig 4c and supplementaryfig 2c the nuclear location of catenin detected byimmunofluorescence showed the same results as thoseofficial of the cell death differentiation association 0cwang cell death and disease page of fig dner activates the wntcatenin signalling pathway and is positively correlated with catenin a b western blotting detected theexpression of notch1 pgsk3 gsk3 and catenin after dner knockdown or dneroverexpressing in bc cells c total proteins catenin andsnail nuclear proteins catenin and snail in dner knockdown cells were assayed with western blotting d the mrna levels of survivin cmyc andlef1 were detected by qrtpcr e the staining of dner and catenin in bc tissue by ihc magnification × f correlation analyses of proteinexpression levels between dner and catenin g kaplan“meier survival analysis of bc patients was performed with dnerhighcateninhigh anddnerlowcateninlow expression the values are the mean ± sd from three independent experiments p p vs thecorresponding groupdetermined by western blotting supplementary fig 2dto further confirm the decrease in nuclear cateninaccumulation following dner knockdown we examinedthe expression levels of catenin downstream targetgenes in bc cells by pcr consistent with the westernblotting results the mrna expression levels of survivincmyc and lef1 were significantly downregulated upondner knockdown fig 4d these data indicated thatdner knockdown can inhibit nuclear translocation andtranscriptional activity of catenin thereby controllingthe wntcatenin signalling pathwayto verify the relationship between dner and cateninwe measured the protein expression levels of dner andcatenin in bc tissues ihc showed that catenin washighly expressed when dner was overexpressed whilecatenin levels were low when dner was knocked downfig 4e interestingly correlation analyses showed thatcatenin expression was positively correlated with theexpression of dner fig 4f we also found a strongpositive correlation between dner expression andnuclear catenin expression supplementary fig 2efurthermore immunofluorescence analysis showed thatdner overexpression promoted more nuclear accumulation of catenin in bc cells supplementary fig 2ffinally kaplan“meier analysis showed that the prognosisof bc patients with high levels of dner and cateninwas worse than the prognosis of bc patients with lowlevels of both dner and catenin fig 4g in additionofficial of the cell death differentiation association 0cwang cell death and disease page of table clinicopathological associations of both dnerand catenin expression in breast cancervariableslown highn p valueage at diagnosis years‰¤gradewellmoderatelypoorlytumour size cm‰¤lymph node metastasisnegativepositivevascular invasionnegativepositiveernegativepositiveprnegativepositiveher2negativepositiveki67 ‰¥ recurrencenoyes p values calculated by logrank testing bold if statistically significant p er oestrogen receptor pr progesterone receptor her2 human epithelial growthfactor receptor2we continued to show the correlation between the highlevel expression of both dner and catenin and bcpatient clinicopathologic features as shown in table these data suggest a strong correlation between theexpression of dner with that of catenin and high levelsof dnercatenin with poor prognosis in bcofficial of the cell death differentiation associationthe wntcatenin signalling pathway is involved in dnerinduced emt and prometastatic phenotypesto determine whether the wntcatenin pathwayfunctions in dnerinduced emt we assessed whetherchir a specific wntcatenin pathway activator23 and xav939 a wntcatenin pathway inhibitor24 could reverse the effect of dner overexpressionand dner knockdown in bc cells catenin levels in thetwo bc cell lines were significantly elevated after chir treatment and markedly suppressed after xav939treatment fig 5a b compared with dner knockdownalone levels of the emtrelated proteins were dramatically exhibited the opposite effect after of the treatment ofdner knockdown cells with chir fig 5a thetreatment of dneroverexpressing cells with xav939clearly show similar results fig 5b these findingsindicated that chir partly rescued the inhibitoryeffect of dner knockdown on emt progression and thatxav939 suppressed the activation of emt induced bydner overexpression to investigate the role of the wntcatenin pathway in dnermediated cell proliferationmigration and invasion we performed rescue experimentsby activating or inhibiting catenin in dner knockdownor dneroverexpressing cells respectively consistentwith the effects of wntcatenin pathway activation andinhibition on emt in the presence of chir theproliferation migration and invasion of dner knockdown cells were clearly elevated fig 5c e f similarlyinhibition ofin dneroverexpressing cells distinctly decreased metastatic ability as shown by changes in cell growth migration andinvasion fig 5d g h altogether these data suggestedthat catenin is indispensable for dnerinduced bc cellemt and prometastatic phenotypescatenin by xav939dner enhances the tumorigenic and metastatic ability ofbc cells in vivoto verify our results in vitro we next examined the roleof dner in vivo to that end mdamb468 cells inwhich dner was stably knocked down and mcf7 cellsstably overexpressing dner were successfully establishedto use to establish xenograft models in mice fig 6a b fg after a period of time the xenografts were removedphotographed and weighed dner knockdown significantly inhibited tumour size and weight comparedwith those in nc group fig 6c d consistent with theeffect of dner knockdown xenografts from dneroverexpressing group were larger and heavier than thosefrom nc group more importantly xav939 reversedchanges in the size and weight of xenografts fig 6h ithe dner catenin cmyc and snail protein levels inxenograft tissue were measured to confirm the upregulation and downregulation by western blotting fig 6e jsupplementary fig 3a moreover ihc results found 0cwang cell death and disease page of fig the wntcatenin signalling pathway is involved in dnerinduced emt and metastasis a b the expression of emtrelated proteinsand catenin were detected by western blotting in dner knockdown or dneroverexpressing cells with chir μm h or xav939 μm h treatment respectively c d cell growth was measured by cck8 in bc cells treated as described above e g wound healing assay was used toexamined migration ability in bc cells treated as described above f h transwell assay showed the cell invasion abilities in bc cells treated asdescribed above right quantitative analysis of invasion ratio was shown the values are the mean ± sd from three independent experiments p p vs the corresponding groupthat dner knockdown reduced nuclear location ofcatenin while dner overexpression promoted thisnuclear translocation effect supplementary fig 3c inaddition as shown in supplementary fig 3a c thewestern blotting and ihc results showed that dnerimpacted the tumour growth in vivo was related to thelevel of ki67 which is consistent with the positive correlation between dner expression and ki67 expression inbc patients of tcga database supplementary fig 3bto explore the role of dner in bc metastasis to lungmdamb231 cells with stably dner knockdown wassuccessfully established fig 6k as shown in fig 6l theofficial of the cell death differentiation association 0cwang cell death and disease page of fig dner enhances the tumorigenic ability of bc cells in vivo a f k the transfection efficiency of dner knockdown or expression in mdamb468 mcf7 or mdamb231 cells respectively b g the knockdown or overexpression efficiency of dner in mdamb468 cells or mcf7 cellsrespectively c h the xenograft pictures of shdner and ncdner in mdamb468 cells n d i comparison of tumour weights from variousgroups e j the expression of dner and catenin in xenograft tissue by western blotting h the xenograft pictures of ncdner group oednergroup and oedner treated with xav939 group in mcf7 cells n l schematic diagram of in vivo experimental procedure for lung metastasispotential in situ of bc m bright imaging of the lungs metastasis left and quantification of the metastases tumour right generated by mdamb231cells n p vs the corresponding groupofficial of the cell death differentiation association 0cwang cell death and disease page of fig see legend on next pageofficial of the cell death differentiation association 0cwang cell death and disease page of see figure on previous pagefig dner reduces the chemosensitivity of bc cells to epirubicin in vitro a cell proliferation was detected by cck8 after treated withdifferent concentrations of epirubicin in two bc cell lines b c dner was analyzed by western blotting in bc cells treated as described above rightquantitative analysis of the optical density ratio of dner compared with actin are shown d expression of epirubicininduced dner was detectedby pcr e cell viability was assessed by cck8 after dner knockdown treated with epirubicin or not f analysis of apoptosis with facs in mdamb cells treated as described in e right quantitative analysis of apoptosis ratio g the expression of parp was detected by western blotting in bccells treated as described above right quantitative analysis of the optical density ratio of cparp compared with actin are shown h cell growthwas measured by cck8 after dner overexpression treated with epirubicin or not i analysis of apoptosis with facs in mdamb468 cells treated asdescribed in h right quantitative analysis of apoptosis ratio j the expression of parp was detected by western blotting in bc cells treated asdescribed above right quantitative analysis of the optical density ratio of cparp compared with actin are shown the values are the mean ± sdfrom three independent experiments p p p vs the corresponding groupcorresponding treated mdamb231 cells were injectedinto the fourth mammary fat pad and tumours wereexcised when they reached about mm3 lung metastasis was observed in each group after days brightfieldpicture demonstrated that more lung metastasis wasfound in the ncdner group compared with the shdner group fig 6m similar trends were observable inhe staining analysis supplementary fig 3e moreoverihc results of xenografts showed that dner knockdownobservably upregulated ecadherin expression anddownregulated ncadherin expres
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"Insulin shares a limited physiological concentration range with other endocrine hormones Not onlytoo low but also too high systemic insulin levels are detrimental for body functionsMain body The physiological function and clinical relevance of insulin are usually seen in association with its rolein maintaining glucose homeostasis However insulin is an anabolic hormone which stimulates a large number ofcellular responses Not only too low but also excess insulin concentrations are detrimental to the physiologicalbalance Although the glucoregulatory activity of insulin is mitigated during hyperinsulinemia by dampening theefficiency of insulin signaling œinsulin resistance this is not the case for most other hormonal actions of insulinincluding the promotion of protein synthesis de novo lipogenesis and cell proliferation the inhibition of lipolysisof autophagydependent cellular turnover and of nuclear factor E2related factor2 Nrf2dependent antioxidativeand other defense mechanisms Hence there is no general insulin resistance but selective impairment of insulinsignaling which causes less glucose uptake from the blood and reduced activation of endothelial NO synthaseeNOS Because of the largely unrestricted insulin signaling hyperinsulinemia increases the risk of obesity type diabetes and cardiovascular disease and decreases health span and life expectancy In epidemiological studieshighdose insulin therapy is associated with an increased risk of cardiovascular disease Randomized controlled trialsof insulin treatment did not observe any effect on disease risk but these trials only studied low insulin doses up to IUday Proof for a causal link between elevated insulin levels and cardiovascular disease risk comes fromMendelian randomization studies comparing individuals with genetically controlled low or high insulin productionConclusions The detrimental actions of prolonged high insulin concentrations seen also in cell culture argue infavor of a lifestyle that limits circadian insulin levels The health risks associated with hyperinsulinemia may haveimplications for treatment regimens used in type diabetesKeywords Hyperinsulinemia Insulin resistance Nrf2 Autophagy eNOS Obesity Type diabetes mellitusInflammation Oxidative stress Cardiovascular morbidity and mortalityBackgroundMost endocrine hormones exhibit a window of optimalphysiological concentrations with compromised function of the anism at levels below or above that rangeFor instance subnormal levels of thyroid hormone define the clinical condition of hypothyroidism above normalrepresent hyperthyroidism which usuallyrequires therapy Addison™s disease is characterized bylevels Correspondence kerstinkempfwdgzde2WestGerman Centre of Diabetes and Health Duesseldorf Catholic HospitalGroup Hohensandweg Duesseldorf GermanyFull list of author information is available at the end of the insufficient cortisol production while excess synthesis isseen in Cushing syndromeFor insulin we argue here that not only hypoinsulinemiabut also hyperinsulinemia is detrimental to body functionsHypoinsulinemia causes insulindeficient diabetes and thehormonal actions of insulin are necessary for the life of complex anisms [] On the other hand permanently elevatedlevels of insulin may cause disturbance of normal cellularphysiology and an function We describe the molecularbasis of these undesired insulin actions and consequences ofhyperinsulinemia for healthrelevant endpoints such as obesity or cardiovascular diseases 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 0cKolb BMC Medicine Page of transformingproteins345trisphosphateMain textInsulin signaling pathwaysBinding of insulin to its cognate cell surfacebound receptor causes a conformational change which initiatesa cascade of signaling events Autophosphorylation bythe insulin receptor tyrosine kinase is accompanied bytyrosine phosphorylation of receptor substrates suchas insulin receptor substrate IRS and Src homology domaincontainingSHCproteins Phosphorylation of IRS allows binding ofphosphatidylinositol3kinase PI3K and synthesis ofphosphatidylinositolPIP3which eventually leads to the phosphorylation and activation of the serinethreoninespecific protein kinaseB AKT Upon activation AKT interacts with severalsubstrates which mediate anabolic effects of insulinthese include glucose uptake glycogen synthesis denovo lipogenesis and protein synthesis [] Additionalpathways triggered by the activated insulin receptorcomprise phosphorylation of SHC followed by activathe Rat sarcoma Ras“rapidly acceleratedtion offibrosarcoma Raf“mitogenactivated protein kinasesignalregulated kinasekinaseERK pathway Theamitogenactivated kinase promoting cell proliferationand further cellular activities including protein synthesis [] Another pathway triggered by the engaged insulin receptor involves activation of NADPH oxidase and subsequent hydrogen peroxidemediated inhibition of phosphatase and tensin homolog PTENwhich is an important negative regulator of PI3Ksignaling [] Fig terminal kinase ERK isMEK“extracellularInsulin secretionInsulin secretion by pancreatic islet cells responds tothe level of circulating nutrients such as glucose aminoacids and free fatty acids Sweeteners may further increase carbohydrateinduced insulin secretion A largenumber of endogenous factors contribute to the regulation of cell activity either stimulatory inhibitory orboth contextdependent These include hormones neurotransmitters and immune mediators [“] Insulin isessential for maintaining glucose homeostasis primarilyby facilitating the postmeal uptake of glucose intomuscle and fat cells via translocation of the glucosetransporter [] In the absence of dietary glucose supply and after depletion of glycogen stores glucose in circulation primarily comes from gluconeogenesis in theliver If circulating insulin levels are below the concentrations required for stimulating glucose uptake fromthe blood endogenous stores of fat and protein must beused for energy production For the maintenance of lifein the fasting state circulating insulin levels range between approx and pmoll “ percentile asdetermined for healthy adult persons in the NationalHealth and Nutrition Examination Survey NHANES[] In response to meals with varying carbohydratecontent insulin levels may rise to the range of approx“ pmoll []Insulin promotes obesityAlmost years ago insulin injections were one of theoptions of therapy in nondiabetic persons suffering fromundernutrition in the context of various diseases Insulindoses were in the range of those applied in type Fig Metabolic signaling of insulin is anabolic Insulin signaling through the insulin receptor engages several pathways and results in ananabolic state of metabolism The canonical pathway via phosphokinases PI3K and AKTPKB promotes glucose uptake and glycogen and lipidsyntheses whereas lipolysis is inhibited in adipocytes as well as hepatic gluconeogenesis In addition AKT kinases activate mTORC1 whichsupports de novo lipogenesis and protein synthesis The insulin signaling pathway via SHC and the MAP kinases MEK and ERK promotes cellproliferation and protein synthesis Another insulin signaling pathway involves NOX4 and the inhibition of PTEN an inhibitor of the PI3KAKT pathway 0cKolb BMC Medicine Page of diabetes and led to increased appetite and weight gain[] Indeed one major function of insulin as an anabolic hormone is to favor energy storage over usageThis is reflected by the finding that insulin infusion mUkgmin significantly inhibits lipolysis in the skeletalmuscle about and even more effective in adiposetissue about [] Doubling fasting insulin levelssuffices to inhibit lipolysis by approx and to promote lipogenesis for both mean insulin concentrationfor effect EC50 of approx pmoll [] At thisinsulin level gluconeogenesis is still ongoing For halfmaximal inhibition of gluconeogenesis insulin concentrations must rise to approx pmoll in arterial circulation In order to stimulate glucose uptake to halfmaximum insulin levels must rise to even higher levelsapprox ten times the fasting insulin concentrations “ percentiles for stimulating glucose uptake approx“ pmoll [] Thus a modest rise doubling offasting insulin levels will already substantially inhibit lipolysis and promote lipogenesis while gluconeogenesis isnot yet inhibited Since such small increases of systemicinsulin concentrations are enough for favoring adipogenesis fasting and diurnal insulin levels are a determinantof obesity risk Indeed several data support the obesitypromoting role of insulin for a detailed review see []Fig These include epidemiological studies which foundhigh fasting insulin levels and concomitant insulin resistance in children and adolescents to be associatedwith higher weight gain in later years [] Studies inadults are less consistent [] Pharmaceutical interventions that lower insulin secretion such as treatment withdiazoxide or octreotide led to significant body weightloss [“] This fits with the observation that insulintherapy promotes weight gain [] One probable reasonis that insulin levels in the high normal range are closeto EC50 concentrations for inhibition of lipolysis []In mice modest lowering of circulating insulin concentrations by genetic manipulation ofinsulin genescaused resistance to weight gain despite a highfat diet[] Decreasing insulin gene expression in adult micevia partial gene ablation reversed dietinduced obesity[] In men the Hph1 œT polymorphism in the insulingene region was found to be associated with higher fasting insulin levels and a more rapid weight gain in obesepersons[] A Mendelian randomization analysisshowed that persons with genetically determined higherinsulin secretion to oral glucose exhibited a higher bodymass index BMI [] supporting a causal relationshipbetween insulin and obesity riskTaken together moderate to high normal levels of insulin in metabolic healthy persons appear to be a riskfactor for the development of obesitytransientElevated insulin concentrations impair cellularfunctions”insulin œtoxicityThere is ample evidence thatincreases ofmetabolic or immune mediator levels are benign physiological responses to biochemical challenges such as therise of systemic glucose or cytokines following mealsHowever chronic elevations of such mediators evenwhen modest in amplitude are usually detrimental tocellular functions [] In the case of glucose the termglucose toxicity was coined to describe this phenomenon[] Prolonged conditions of elevated glucose concentrations cause dysfunction of numerous cell types in thebody including beta cells neurons and the endothelium via several pathways including increased oxidative stress and activation of the sorbitol pathway [“] As described below there seems to be a similardetrimental outcome oflongterm elevated insulinconcentrations on cellular functions a correspondingterm would be insulin toxicityFig Insulin promotes obesity Several independent types of observations support the conclusion that insulin promotes adipogenesis andobesity For details see description in the general text 0cKolb BMC Medicine Page of When cells are exposed to continuously elevated insulin levels there is a partial downregulation of insulin signaling The resulting œinsulin resistance is not primarilydue to less insulin receptor expression on the cell surface but due to impaired insulin signal transduction as aresult of receptor dysfunction In response to prolongedhyperinsulinemia there is diminished autophosphorylation of the insulin receptor compared to that observedafter shortterm exposure to insulin and subsequentsteps of the PI3K“AKT signaling pathway are affected[ ] Consequently in muscle and fat cells there isless AKTstimulated translocation of GLUT to the cellsurface Fig Thus insulin resistance can be seen as aprotective mechanism for preventing excess activation ofglucose transport from the blood despite chronically elevated insulin levels for maintaining glucose homeostasisin vivo and for mitigating metabolic and oxidative stressdue to excess glucose influx [“] Limiting glucoseexportfrom the blood does not necessarily requiredampening of insulin signaling During the early weeksof feeding with a high caloric diet mice show decreasedinsulindependent glucose uptake despite unperturbedinsulinstimulated AKT phosphorylation [ ] Fig An interesting aspect is that the partitioning of insulinreceptor isoforms A and B and of hybrid insulininsulinlike growth factor1 receptors among cell types maycontribute to insulin resistance in some tissues but thepathophysiological relevance is unknown []The phenomenon of insulin toxicity partly arises fromthe fact that there are additional cellular responses to elevated insulin levels which are not toned down duringrole ofinsulin resistance Fig These comprise the upregulation of protein synthesis and the accumulation of ubiquitinated or otherwise modified proteins probably dueto insufficient degradation of these polypeptides [] Amajorinsulin signaling via the canonicalmitogenactivated protein MAP kinase pathway Ras“MEK“ERK as well as via activation of NADPH oxidase has been observed [] Even some AKTdependentpathways do not appear to be suppressed by insulin resistance such as de novo lipogenesis in hepatocytes orthe upregulation of mechanistic target of rapamycincomplex mTORC1 [“] Enhanced activity ofmTORC1 leads to increased protein synthesis and to deteriorated cell functions largely because of suppressedautophagy []Hence chronic exposure of cells to high ambient insulin concentrations causes an imbalance of cellular responses because of the downregulation of some insulinsignaling pathways œinsulin resistance but not ofothers The resulting functional state of cells is characterized by an unbalanced anabolic activity of insulin favoring protein synthesis while suppressing autophagyThe latter inhibits autophagic removal and turnover ofproteins and lipids which favors cell senescence [] Inshortterm experiments of exposure to high insulinlevels a protective cellular stress response is observedthe unfolded protein response probably due to the accumulation of derivatized proteins in the absence ofenough disposal In experimentally induced or diabetesassociated chronic insulin resistance and hyperinsulinemiathesuch a protectivestressresponse ofFig Signaling of insulin during insulin resistance During insulin resistance signaling through AKT kinases is partially impaired Not all AKTdependent pathways are affected as well as other signaling pathways indicating that insulin resistance is selective Therefore hyperinsulinemiain the presence of insulin resistance promotes anabolic cell activities via the MEK“ERK pathway and via mTORC1 Although the PI3KAKT pathwayis impaired during insulin resistance and provides only insufficient translocation of GLUT4 for glucose uptake and deficient activation of eNOSthere appears to be a normal activation of mTORC1 In addition to the anabolic consequences of signaling via the MEKERK pathway depicted inthe figure there is enhanced expression of ET1 and PAI1 not shown as well as inhibition of autophagy and of the nuclear factor Nrf2 whichcompromises cell constituent turnover and cell defense mechanisms to radical stress respectively Hyperinsulinemia downregulates glucoseuptake not only via dampening the PI3KAKT pathway œinsulin resistance but also via as yet unknown other pathways 0cKolb BMC Medicine Page of endoplasmic reticulum to high insulin levels is diminished or absent []Another activity of insulin is the suppression of transcription of the nuclear factor Nrf2 via induction of heterogeneous ribonucleoproteins F and K [] Nrf2 is thecentral regulator ofthe protective response of cellsagainst oxidative and other types of electrophile stress[] Suppression of Nrf2 expression is expected to impair the antioxidant and cytoprotective defense capacityof cells Insulin signaling required for Nrf2 inhibition occurs via the MAP kinase pathway and thus is not mitigated by insulin resistance [] Fig It therefore canbe assumed that hyperinsulinemia increases the susceptibility of cells against oxidative or other electrophilestress caused by environmental insults Prolonged exposure of cells to high insulin concentrations can thereforebe regarded as toxic Indeed exposure to nmoll insulin has been found to cause DNA damage in a numberof cell types including human lymphocytes [ ] Atthe only concentration tested nmoll insulin impairs oxygen radical defense and sensitizes apoptosispathways in human islets [] In the brain of micehyperinsulinemia impairs electrophysiological functionsof neurons and protein turnover causing a transition toa senescent cell state and an accompanying cognitive decline [] The direct toxic property of insulin deservesfurther studyChronically elevated insulin concentrations impair bodyfunctionsLongevityThe above list of detrimental cellular responses to highambientinsulin concentrations suggests concomitantfunctional impairments at the level of the anism Thisfits with the observed impact of insulin on longevityStudies in nonvertebrate model systems such as thenematode Caenorhabditis elegans or the fruit fly Drosophila melanogaster find that moderate to high insulinactivity shortens lifespan [ ] A consistent findingfrom mouse model studies is that decreased signaling ofanabolic hormones like insulin insulinlike growth factor or growth hormone results in a prolonged lifespan[] Disruption of the insulinreceptor substrate genecaused insulinresistance with defects in insulin signaling[] and led to an extension of lifespan by “ []A knockout of the insulin receptor in adipose tissue ofmice resulted in an increase of lifespan [] Disruption of the Ins1 gene and one of the two mouse Ins2alleles lowered insulin levels by “ Ins2ˆ’ miceversus Ins2 controls in aged female mice without altering circulating insulinlike growth factorIGF1levels These aged experimental mice exhibited lowerfasting glucose improved insulin sensitivity and “lifespan extension across[]two different dietsConcomitantly the proteome and transcriptome indicated a profile associated with healthy aging An important aspect is that this study selectively addressed insulinOther interventions for promoting longevity or extending healthspan such as caloric restriction not only lowercircadian insulin levels but several additional hormonesincluding IGF1 are also affected []InsulinIGF1 and hybrid insulinIGF1 receptorsshare signaling via PI3K and AKT The subsequent activation of the protein kinase mTORC1 is a major pathway for supporting somatic growth protein synthesisand fertility while impeding autophagy and lifespanSuppression of mTOR signaling by treatment with rapamycin prolongs life in model anisms and mice []In humans hyperinsulinemia in pre type diabetes isassociated with increased mTORC1 activity which mayhave a negative impact on beta cell survival healthspanand longevity []In the Leiden Longevity Studyfollowup of nonagenarians for years showed a strongassociation of low insulin and glucose levels with healthyaging []Since both IGF1 and insulin employ PI3K and AKTfor signal transduction it is difficult to disentangle thecontribution of insulin versus IGF1 to the modulationof longevity In animal models selective downregulationof circulating insulin levels improved the lifespan ofmice and in elderly persons of the Leiden LongevityStudy only insulin and glucose but not IGF1 consistently met all four predefined criteria of healthy aging[ ] Therefore it may be concluded that low circulating insulin concentrations are not only a marker oflongevity but are causally involved in promoting healthspan or lifespan extensionDetrimental combination of hyperinsulinemia with insulinresistanceInsulin resistance is defined as an attenuated effect of insulin on blood glucose homeostasis primarily by less efficient export of glucose from the blood into skeletalmuscle adipose and liver tissue Permanently elevatedinsulin concentrations in the blood are often consideredas an attempt to overcome insulin resistance Indeed induction of insulin resistance by genetic disruption of insulin signaling as well as by increased growth hormonelevels or an inflammatory milieu causes hyperinsulinemia [“] The opposite causality is of more relevanceHyperinsulinemia during insulin infusion in humansleads to systemic insulin resistance [] while in vitrohigh ambient insulin concentrations cause an increase ininsulin resistance in isolated adipocytes [] A summaryanalysis of nine studies in rodents and seven trials inhumans confirmed that the first detectable change in thefasting state after feeding a high caloric diet for severaldays is an increase of basal insulin concentrations but 0cKolb BMC Medicine Page of not of blood glucose concentrations or insulin resistance[] Both increased secretion of insulin by ß cells anddecreased insulin clearance in the liver contribute to elevated insulin levels postmeal the latter being of primaryimportance in the case of carbohydraterich food []functionincluding relaxation ofThe combination of hyperinsulinemia and insulin resistance appears to promote hypertension and atherogenesis Fig One important molecule for maintainingvesselthe arterialsmooth muscle layeris nitric oxide NO which isgenerated by endothelial NO synthase eNOS Insulinincreases NO production via posttranslational modification of eNOS via PI3KAKT activity howeverthismechanism is suppressed during insulin resistance [] Decreased local NO production impairs arterialsmooth muscle relaxation and concomitant vasodilatation An important factor in this context is the calciumion homeostasis of vascular smooth muscle cells Underphysiological conditions insulin promotes both calciuminflux into the cytoplasm of smooth muscle cells via several ion channels including Ltype and storeoperatedCa2 channels and counterregulatory NOmediated efflux of Ca2 and K ions which prevents calcium ioninduced myosin lightchain phosphorylation andFig Hyperinsulinemia insulin resistance and cardiovasculardisease High insulin concentrations in the blood may occur due togenetic predisposition overnutrition or highdose insulin treatmentof type diabetes Hyperinsulinemia induces œinsulin resistance as adefense response to maintain glucose homeostasis Converselyinsulin resistance may be directly induced such as by growthhormone or proinflammatory cytokines Hyperinsulinemia andinsulin resistance enhance the risk of cardiovascular disease byinducing endothelial dysfunction suppression of endothelial nitricoxide synthase eNOS and activation and promotion of calcium ioninflux into smooth muscle cells resulting in increased vascular toneenhanced reabsorption of sodium ions in renal tubules adhesion ofmacrophages to the vessel wall and development of arterial lesionswith increased lipoprotein lipase activity and cardiovascular diseaseconcomitant vascular contractility During insulin resistance NO production is impaired while the supportiveeffect of insulin on calcium ion influx via PI3K deltaand possibly the MEK“ERK pathway and vasoconstriction is still present Fig [ ]At the same time insulin signals through the mitogenactivated protein MAP kinase pathway to upregulatethe expression of endothelin1 ET1 plasminogen activator inhibitor1 PAI1 adhesion molecules and proinflammatory cytokines [ ] The reninangiotensinsystem is activated in the context of endothelial dysfunction and contributes together with decreased NO production and increased ET1 secretion to vascularstiffening and upregulation of vascular tone [“] Inthe absence of hyperinsulinemiainsulin resistance thelower insulin levels exert less potential proatherogenicactivities which are counteracted by insulinstimulatedlocal NO production [ ]Elevated insulin levels also increase the risk of hypertension by enhancing renal reabsorption of sodium ionsby several transport systems in different segments of thenephron Fig Signaling of insulin occurs via insulinreceptor substrate IRS2 and is not suppressed duringinsulin resistance while signaling via IRS1 for counterregulatory mechanisms including local NO production isimpaired [ ] These detrimental actions may be mitigated during chronic hyperinsulinemiainsulin resistance [] However a metaanalysis of prospectiveepidemiological studies showed that the pooled relativerisk of hypertension was when comparing the highest to the lowest category of fasting insulin levels and for comparing highest to lowest selective insulinresistance categories calculated as homeostasis modelassessment of insulin resistance HOMAIR []As a consequence of endothelial dysfunction duringprolonged treatment with insulin arterial lesions rich inlipids are formed [] The progression of early fattystreak lesions to plaques is accompanied by the adhesionand proinflammatory activity of macrophages whicheventually develop into foam cells This process is drivenby endothelial and macrophage lipoprotein lipase activity as demonstrated by the observation of less atherosclerosis in mice with inactivated lipoprotein lipase gene[“] Lipoprotein lipase activity in macrophages isenhanced with higher insulin levels in vivo but there isno direct stimulatory effect of insulin on isolated macrophages []The concern that hyperinsulinemia might promote arterial disease in diabetic persons developed in the late1960s due to the steady increase of incidences of atherosclerosis in diabetic persons despite improved glycemiaand decreased risk of ketosis due to insulin therapy []Since then a wealth of data supports the observationthatis ainsulin resistance and hyperinsulinemia 0cKolb BMC Medicine Page of marker of increased risk of cardiovascular disease in thegeneral population and in patients with diabetes [] Although observational studies suggested an approximatelylinear relation between the severity of hyperglycemiaand vascular damage severallarge randomized controlled trials have shown that intense glycemic controlper se does not decrease the risk of macrovascularcardiovascular events [] indeed insulin therapy may evenincrease the risk [ ] However these trials werenot randomized for insulin treatment and treatment ofCVD risk factors was not kept similar between patientsubgroups In the United Kingdom Prospective DiabetesStudy UKPDS hyperinsulinemia and insulin resistancewere not mitigated by insulin treatment and fastingplasma insulin levels even rose [] By contrastinUKPDS and other trials [ “] oral treatmentwith the biguanide metformin reduced the risk of cardiovascular events and in parallel decreased insulin resistance and hyperinsulinemiaIn epidemiological studies of type diabetesit hasbeen consistently observed that the addition of insulin tothe treatment regimen or the intensification of insulintreatment result in a higher rate of cardiovascular events[“] Fig Indeed it has been shown that therisk increases with increasing insulin dosage [ ]These epidemiological studies may suffer from residualFig Hazard ratio of insulin medication versus different reference medications Shown are adjusted hazard ratios HR for each study with confidence interval Moderate insulin exposure high insulin exposure moderate insulin dose to units per day §high insulin dose units per day 0cKolb BMC Medicine Page of confounding since it is difficult to account for the possibly more advanced disease stage of patients receivinginsulin A higher rate of hypoglycemic events may be anadditional confounder However covariates consideredin the statistical analyses cover a broad range of potential risk factors from different categories SupplementTable Large randomized controlled trials such asUKPDS [] or the Outcome Reduction With InitialGlargine Intervention ORIGIN Trial [] did not observe an increased incidence of cardiovascular diseasewith insulin therapy but these trials focused on lowdose insulin therapy of up to a median of IUday or IUkgday respectively Similar randomized trials ofhigherdose insulin therapy as typicalfor realworldconditions have not been conducted Recent studies ofrealworld clinical settings report mean daily basal insulin doses of close to IUkg in the Canadian REALITY Study for insulinexperienced patients with type diabetes [] and of IUkg in a physician survey inNew York [] In the European multicentre EUTREAT Study mean baseline insulin doses were between and U per day depending on the type of insulin therapy regimen applied [] It can be concludedthat under realworld conditions the majority of insulinexperienced patients with type diabetes receive higherinsulin doses per day than those tried in UKPDS orORIGINIn the absence of randomized controlled trials aMendelian randomization is an appropriate approach oftesting for a causal relationship in humans Mendelianrandomization studies made use of the finding that somegenotypes are associated with high or low fasting insulinlevels When comparing individuals carrying ‰¥ allelesthat raise fasting insulin levels with those exhibiting genetically determined low fasting insulin levels an increasedrisk of elevated blood pressure cardiovascular disease andtype diabetes was observed [] In two large recentMendelian randomization studies a genetic profile predicting high insulin levels in the blood after adjustmentfor BMI was also associated with increased systolic bloodpressure and risk of myocardial infarction []ConclusionsAs discussed aboveinsulin signaling engages at leastthree different pathways and modifies a large number ofcellular responses Table Transient elevations of systemic insulin concentrations are physiological responsesto dietary stimuli or other challenges such as environmental toxins [] In case of prolonged upregulationof insulin levels such as in response to overnutritionglucose homeostasis is maintained by mitigating insulinsignaling via PI3KAKT for glucose export from theblood into tissues Consequently insulin resistance hasbeen considered as a defense response in order to avoidTable Key messagescid129 Insulin employs at least three different pathways of signal transductionOne pathway involves phosphorylation steps via IRS“PI3K“AKT anotheris the MAP kinases Ras“MEP“ERK and third leads to the activation ofNOX4cid129 Insulin resistance is selective because it partially mitigates the PI3KAKTpathway for limiting glucose uptake and eNOS activation despitehyperinsulinemia but many other hormonal actions of insulin are notsuppressedcid129 Signaling via mTOR and the MEPERK pathway causes suppression ofautophagy and NRF2 leading to deficient turnover and impaired celldefensecid129 Moderate to high normal insulin levels inhibit lipolysis and promotelipogenesisobesitycid129 Insulin resistance and hyperinsulinemia are interdependent Dietinduced hyperinsulinemia precedes insulin resistancecid129 In epidemiological studies insulin therapy of type diabetes isassociated with a higher risk of cardiovascular events or deathcid129 Randomized trials of insulin therapy and associated risks only studieddosages up to IUdaycid129 Mendelian randomization studies found that genetically determinedhigh insulin levels lead to cardiovascular diseasecid129 Suppression of hyperinsulinemia and concomitant œinsulin resistanceprovides substantial health benefitshypoglycemia [] However other hormonal actions ofinsulin via the MAP kinase MEKERK pathway and inpart via PI3KAKT are no
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"However these results raised several specific questions. First tumor histology and progression risk may affect the response to TKIs. TKIs are associated with a good response in patients with CCRCC but are less effective against non-CCRCC [10]. Similarly patients with favorable risk factors have a greater chance of a good tumor response than those with poorer risk factors. Second there is the possibility of bias in terms of the types of TKI selected; given that sunitinib showed a higher response rate than sorafenib [78] patients with larger or more rapidly-growing tumors may be allocated sunitinib rather than sorafenib in clinical practice. Third efficacy based on initial tumor size may differ between different ans; although the previous study compared mean lesion-size reductions between different ans they did not compare the effect of initial tumor size in individual ans. It is therefore unclear if the association between initial lesion size and tumor response was observed in each an or if the association could be attributed to the fact that most of the small lesions were lung metastases which showed a good response to TKIs. The current study only included CCRCC patients treated with sunitinib. We found that lung lesions showed the greatest response to sunitinib and detected a modest correlation between initial tumor diameter and reduction in lesion size while even small lesions in other ans failed to respond. However the number of extra-pulmonary tumors assessed was too small to determine statistical significance and further studies with larger numbers of tumors are needed to obtain conclusive results. Only lesions with an initial diameter?<?20 mm achieved a CR in this study indicating that a lung-tumor reduction of?>?50% might be limited to smaller lesions. The cut-off value of 16.5 mm for a?>?50% reduction in diameter was calculated using ROC analysis with a sensitivity of 67.0% and a specificity of 77.8%. Some physicians may prefer conservative therapies without TKIs or a watchful waiting strategy in CCRCC patients with only small lung metastatic lesions [11]. Furthermore cytokine therapies are still employed in CCRCC patients especially in Japan because of their low toxicity and ability to achieve long-term stable disease [12]. However the present results suggest that smaller lung lesions are associated with a greater chance of response to TKIs and it is therefore important not to miss the opportunity for early initiation of TKI treatment in patients with PD during watchful waiting periods or cytokine therapy. Several studies have investigated the response of primary kidney lesions to TKIs [13-15]. Kroon et al. reported that smaller primary lesions were more responsive to treatment and that tumors of 5“7 cm may benefit from neoadjuvant treatment followed by nephron-sparing surgery. In contrast our results showed that the response of kidney lesions to sunitinib was independent of initial tumor size and many smaller lesions exhibited no response. A possible explanation for this difference may be the selection of patients; most of the kidney lesions were investigated in the neoadjuvant setting in Kroon et al.™s study while all the patients with kidney lesions in the current study had an extensive metastatic tumor burden. The different patient backgrounds may have led to different responses to TKIs particularly in small kidney lesions. CRP is an acute phase protein produced by the liver in response to various conditions such as inflammation infection and malignancy [16]. In the cytokine era elevated serum CRP level has been suggested as a biomarker for predicting poor survival in RCC patients [17-19]. Yasuda et al. recently demonstrated that CRP was a significant predictive marker for prognosis in metastatic RCC patients treated with TKIs [20]. In the current study the size reduction of lung lesions in patients with high serum CRP levels was lower than that in patients with low CRP levels irrespective of the initial size. This lower response to sunitinib in patients with higher serum CRP levels may be attributed to an aggressive disease status reflected by higher CRP levels the acquisition of resistance to therapeutic agents through an increase in inflammatory mediators in the cancer-cell microenvironment or compromised drug metabolism induced by such mediators associated with CRP [21]. Tumor response to treatment is currently assessed by imaging based on RECIST criteria [22]. However although marked central necrosis is often detected in lesions with a small size reduction after treatment with TKIs RECIST only considers one-dimensional lesional size changes suggesting that it may substantially underestimate the actual tumor response. Several studies recently reported novel criteria which may improve response assessment by evaluating changes in tumor attenuation and morphology on contrast-enhanced computed tomography scans in addition to size changes [522-26]. The results of this study therefore need to be interpreted carefully because lesions in different ans may exhibit distinct response patterns in imaging. Moreover the current study did not demonstrate an association between tumor response and patient survival and it is possible that percent change in tumor size might not correlate directly with survival. Further studies are needed to determine the influence of an-specific response patterns to TKI treatment on survival. Conclusions The results suggest that tumor-size reduction depends on initial tumor size and the ans involved as well as systemic reaction to the lung tumor as indicated by CRP levels. CCRCC patients with lung metastatic lesions?<?20 mm in diameter and lower CRP levels may achieve greater reductions in tumor size with sunitinib therapy than those with extra-pulmonary lesions lung lesions???20 mm in diameter and/or higher CRP levels. Abbreviations RCC: Renal cell carcinoma; TKI: Tyrosine kinase inhibitor; RECIST: Response evaluation criteria in solid tumors; CCRCC: Clear cell RCC; CTC-AE: Common Terminology criteria for Adverse Events; ROC: Receiver-operator curve; AUC: Area under the curve; CRP: C-reactive protein. Competing interests Norihiko Tsuchiya and Tomonori Habuchi received honoraria from Pfizer Japan Inc. Authors™ contributions NT TY JY and TH were involved in the conception and design of the study. TY KN MS and SM were involved in the provision of patients™ clinical data."
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continuously increasing with development of the economy and the environment [“] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [“] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [“] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrna™e1e2abcbasepairinge3dguriche4™ecrichpretrnarna bindingproteinsrbp™™™™™™™™gnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the ™ hydroxyl of the upstream exon reacts with the ™ phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the ™ hydroxyl of the ™ intron interacts with the ™ phosphate of the ™intron to form an ecircrna following an interaction between the ™ hydroxyl of the ™ exon and the ™ phosphate of the ™ exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a ™“™ phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [“] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin “ feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin “ li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci “ salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 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progression gene expr “ vandenbulcke h moreno c colle i alcohol intake increases the risk of hcc in hepatitis c virusrelated compensated cirrhosis a prospective study j hepatol “ fabris c toniutto p falleti e mthfr c677t polymorphism and risk of hcc in patients with liver cirrhosis role of male gender and alcohol consumption alcohol clin exp res “ vernon g baranova a younossi zm systematic review the epidemiology and natural history of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in adults aliment pharmacol ther “ bruix j reig m sherman m evidencebased diagnosis staging and treatment of patients with hepatocellular carcinoma gastroenterology “ zhang bh yang bh tang zy randomized controlled trial of screening for hepatocellular carcinoma j cancer res clin oncol “ verduci l ferraiuolo m sacconi a the oncogenic role of circpvt1 in head and neck squamous cell carcinoma is mediated through the mutant p53yaptead transcriptioncompetent complex genome biol yu j xu qg wang zg circular rna csmarca5 inhibits growth and metastasis in hepatocellular carcinoma j hepatol “ wang m yu f li p circular rnas characteristics function and clinical significance in hepatocellular carcinoma cancers basel guo w zhang j zhang d et al polymorphisms and expression pattern of circular rna circitch contributes to the carcinogenesis of hepatocellular carcinoma oncotarget “ han d li j wang h circular rna circmto1 acts as the sponge of microrna9 to suppress hepatocellular carcinoma progression hepatology “ qin m liu g huo x hsa_circ_0001649 a circular rna and potential novel biomarker for hepatocellular carcinoma cancer biomark “ yao z luo j hu k zkscan1 gene and its related circular rna circzkscan1 both inhibit hepatocellular carcinoma cell growth migration and invasion but through different signaling pathways mol oncol “ xu l zhang m zheng x the circular rna cirs7 cdr1as acts as a risk factor of hepatic microvascular invasion in hepatocellular carcinoma j cancer res clin oncol “ yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun “ liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun “ li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle “ “ shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c'
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despite great advances in recent decades in screening diagnosisand curative surgery hepatocellular carcinoma hcc remainsthe second leading cause of cancerrelated mortality worldwidegrandhi siegel epidemiologicalevidence has confirmed that the longterm outcomes of patientswith hcc have notimproved significantly with the rapiddevelopment of surgical techniques madduru moreimportantly because of the limitations of systematic statustumor position and the need to preserve liver function morethan of patients are not eligible for surgical treatment evenafter curative resection prognosis remains unsatisfactory becauseof a high incidence of postoperative recurrence colecchia the initiation and maintenance of hcc is a complexand regulated process involving the accumulation of numerousgenetic changes over decades niu erkekoglu these sequential alterations not only endow normal livercells with neoplastic ability enabling uncontrolled growth butalso provide potential therapeutic targets and biomarkers thusfurther understanding of the initiation and maintenance of hccat the molecular level is crucial to prolonging survival and makingindividual treatment decisionsthe exive development of highthroughput technologyhas provided powerful tools for the molecular study of cancerschmidt and hildebrandt rna sequencing rnaseq and microarraysthe most representative methods ofthis technology are mature enough for use in commercialapplications mantione zhang duringthe past decades the genomewide transcriptional analysis ofgene expression has become critically important to gain betterinsight into the biological processes of hcc and other typesof cancer jin xiong in additionto the aberrant expression of transcripts studies have focusedon diï¬erent molecular levels multilevel omicsincludingcopy number variation epigenetic modifications nucleotidepolymorphisms and dna methylation especially in hcclee lin evidence obtained from thesestudies clearly demonstrates that hcc is a disease causedby cumulative aberrations at diï¬erentlevels of molecularregulation thus only a highthroughput multiomics analysiscan decipher the complex biology of hcc many previousstudies despite promising results focused only on the aberrantregulation of expression and its biological eï¬ects howeverstructural transcript variation in hcc which is heavily shaped byalternative splicing as has until recently been less well studiedaccording to the manual genome annotation project harrow pruitt there are only about proteincoding genes this number is obviously inconsistent withthe overall cellular complexity which includes at least distinct proteincoding sequences harrow thisdiscrepancy between the numbers of transcripts and proteincoding genes in human cells indicates the existence of anadditional mechanism between the transcriptional and the posttranslational levels that increases the coding capacity of thegenome through the as process a single rna precursorcan be spliced via distinct arrangements to generate rnaslandscape of as in hccwith diï¬erent structures and functions biamonti song this may be one ofthe main causesof cellular complexity and proteome diversity experimentalstudies on the eï¬ects of individual as events suggest that asmay change the biological function of a protein by regulatingits stability controlling its location modifying the mutualinteractions of proteins and even adding or deleting activedomains brett yang these findingssuggest that as well as expression abundance the balance ofdiï¬erent as events that result from the same rna precursormust be considered howeverthe latter consideration hasoften been neglected in previous studies in fact emergingdata from genomewide analyses feng kahles indicate that as occurs in more than ofmultiexon genes suggesting thatthe widespread existenceof as is the product of physiological processes rather thantranscription errorsin recent years the diagnostic and the therapeutic role ofas in many human diseases has attracted increasing attentionlargescale screening of as events has been performed usingexpressed sequence tag libraries although this approach isprone to a high rate of false positives venables exonjunction probes provide a higher experimental validation ratelapuk however this method has the disadvantageof being limited to known splice junctions owing to thelimited available techniques complicated mechanisms and hugenumbers involved transcriptomewide as dysregulation and itspotential associations with biological behavior in hcc haveremained uncharacterizedrnaseq not only supports the quantitative measurementof novel as events but also provides deeper coverage higheraccuracy and better resolution li y thus it maybe the most suitable of the currently available approaches foras study in recent years the cancer genome atlas tcgatomczak wang has accumulated a richand publicly available source of rnaseq data and correspondingclinical information this enables the analysis of as dysregulationin hcc at a genomewide level tcga includes rnaseqdata samples obtained from hcc patients together withtheir corresponding clinical information thereby facilitating theclinical analysis of hccrelated as events in a large cohorthowever without reliable and efficient bioinformatical methodsthe advantages of rnaseq in as analysis cannot be fullyexploited spliceseq a recently developed bioinformatics toolcan exactly match rna reads with gene splice graphs and ishelpful for accurately calculating complex or lowfrequency asevents ryan there has been a lack of studies combining largescalernaseq data with the corresponding clinical information tocomprehensively analyze as at singleexon resolution howeverthis is very necessary especially in hcc thereforein thecurrent study we comprehensively analyzed wholegenome asin the tcga hcc cohort to screen out hccrelated asevents and further studied the relationships of these eventswith clinical outcomes our findings suggest that certain hccrelated as events including nek2at and troptat havecritical roles in the progression and maintenance of hcc morefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccimportantly these hccrelated as events represent potentialnew therapeutic targetsmaterials and methodstumorlocationinvasioninformation ofdata curationclinicopathologicalthe hcc cohort andcorresponding rnaseq data were retrieved and downloadedfrom tcga1 to ensure appropriate protection of patientprivacy the tcga data were stratified according to data typeand level conforming to the publishing guidelines formulatedby tcga wang then the rnaseq data andcorresponding clinicopathological information were mutuallypaired using the unique tcga barcodes only patients whomet the criteria listed below were included grandhi patients with corresponding rnaseq data siegel patients with complete clinicopathological informationincluding localsex age distalmetastasis pathological stage diï¬erentiation grade lymph nodemetastasis and survival information madduru histological diagnosis of hcc and colecchia survival for at least month after the primary pathologicaldiagnosis spliceseq was used to determine rna splicing patternsand produce as profiles for each hcc patient as previouslydescribed li y zhu each as eventwas quantified using the percent spliced in psi value rangingfrom to a commonly used method to reflect the abundanceof as events in order to remove the eï¬ects of splicing noiseand generate as reliable a set of as events as possible a seriesof strict filters average psi ‰¥ percentage of samples withpsi ‰¥ was applied to the detected as events the interactivesets between the seven types of as were quantitatively analyzedand the results were visualized in upset plots using upsetrversion conway circlize version wasused to generate circos plots to depict the parent genes and theiras events in chromosomes gu the details of thedesign of the present study are illustrated in supplementaryfigure s1 all the methods used in this study were in line withthe relevant guidelines and regulationsidentification of deas and enrichmentanalysisto screen the diï¬erentially expressed alternative splicing deasevents between hcc and corresponding normal tissues the psivalue of each as event was determined in the tcga hcccohort hcc tissue samples and paired adjacent normaltissues a generalized linear model was applied to remove thebatch eï¬ects the deas were determined based on adjustedp adj p and associated log2 fold change fc values withadj p ‰¤ and log2fc ‰¥ representing as events thatwere downregulated and upregulated respectively biologicalfunction enrichment analysis was performed based on the deasparent genes gene ontology go and kyoto encyclopediaof genes and genomes kegg terms with false discoveryrate less than were considered to be significantly enrichedand were selected for further analysis enrichment analysis wasperformed using the clusterprofiler package version yu the parent genes of deas events were importedinto the string database and used to determine protein“protein interactions ppis a relationship network was thengenerated using cytoscape version su clusteranalysis was conducted using the average linkage agglomerationalgorithm and correlation distance metricsestablishment of hccrelated splicingcorrelation networka total of splicing factors sfs supplementary table s1were identified by comprehensive and handcurated screening ofthe literature all the sfs included in the current study had beenexperimentally validated in previous research giulietti and included heterogeneous nuclear ribonucleoproteinsproteins serineargininerich proteins and other proteinsbelonging to the celf fox khdrbs nova and elav familiesthe expression of each sf was obtained from the broadinstitute2 correlations between the psi values of deas and theexpression of sfs were analyzed by weighted gene coexpressionnetwork analysis version langfelder and horvath benjamini and hochberg correlation was used to adjust thepvalues the adjusted pvalues less than were consideredto indicate statistically significant diï¬erences cytoscape version was used to generate the correlation plotssurvival analysisall the included hcc patients were divided into two groupsbased on the psi value of each deas median cut and thetwo artificial categories were modeled as continuous variables toderive more easily interpretable hazard ratios based on overallsurvival os and diseasefree survival dfs cox regression wasperformed to evaluate the prognostic value of each deas eventlogrank test and kaplan“meier analysis were used to comparepatient survival between subgroups p was consideredas statistically significant the overall survivalrelated deaswere further analyzed in lasso regression to identity the mostpowerful prognostic markers finally a prognostic model wasconstructed for predicting the os in order to quantify the risk ofos a standard form of risk score rs for each colorectal cancercrc patient was calculated combine the levels of the psiι1 psii × lito divide the patients into the high or lowrisk group kaplan“meier curves were used to estimate the survival for patients in thetraining the testing and the validation sets between the highriskand the lowrisk groupspsii and lasso coefficients li risk score pnfunctional experiment of cxcl12splicing variants in hccthe human hcc cell line hepg2 was obtained from the chineseacademy of sciences committee on type culture collectioncell bank shanghai china the cell line was cultured in 1httpsportalgdccancergov2httpgdacbroadinstitutefrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccprimers was used asgibco carlsbad ca united states supplemented with fetal bovine serum fbs bi beit haemek israel at —¦c with co2 total cdna from tissues was obtained as described abovethe pcr reaction was carried out using the forward primer5cid48tgcccttcagattgttgcac3cid48 common for allisoforms and theisoformspecific reverse primers 5cid48gctaactggttagggtaatac3cid48 and5cid48gctagcttacaaagcgccagagcagagcgcactgcg3cid48for np_9546371and np_0010290581 respectively bactin amplified usingthe forward 5cid48acactgtgcccatctagcagggg3cid48and reverse 5cid48atgatggagttgaaggtagtttcgtggat3cid48aloading control quantitative realtime pcr was performedin mx3005tm qpcr system with a mxpro qpcr software stratagene la jolla ca united states and sybr greendetection system the adherent hepg2 cells were transfectedwith the corresponding hiscxcl12 construct by the calciumphosphate method and cultured for h at h before collectingthem the cell supernatants were removed and when indicatedbrefeldin a was added to the fresh medium the collectedcells were left untreated or permeabilized with saponin andimmunolabeled with the his mab and a pegoat antiigsecondary antibody and analyzed by flow cytometry the cellinvasion assay was conducted using matrigelcoated chambers µm pore size corning costar corporation cambridgema united states in brief × cells were plated in theupper chamber coated with matrigel and supplemented withserumfree medium the lower chamber was filled with a culturemedium containing fbs incubation was carried out for h at —¦c following which the noninvasive cells were scrapedoï¬ with cotton swabs the cells that had successfully translocatedwere fixed with paraformaldehyde stained with crystalviolet and finally counted using an inverted microscope mttassay colony formation assay and soft agar growth assay wereperformed according to our previously described methods zhou protein structure homology modeling analysis wasperformed as previously described by using the online serverswissmodel waterhouse evaluation of the relationship of asclusters with clinicopathologicalfeaturesbased on the identified deas n the tcga hcc cohortin the current study was stratified by an unsupervised consensusapproach consensus cluster plus version wilkerson andhayes the optimal number of clusters was determinedby integrating the results of the elbow method and gap statisticthe relationship between clinical outcomes and as clusterswas evaluated using logrank test and kaplan“meier curves asdescribed by xiong 2018aresultslandscape of as event profiles in hccto systematically characterize the as events and their clinicalsignificance in hcc we collected rnaseq libraries andcorresponding clinicalinformation from hcc patientsthe tumor tissues and paired adjacent normal tissues from patients were sequenced simultaneously the includedpatients comprised males and femalesamong which patients developed recurrence and died of hcc the median followup period was months range “ months the general characteristicsof these hcc patients are fully detailed in supplementarytable s2 rnaseq data were associated with the clinicalthe corresponding patient via the tcgainformation ofbarcodes there were patients with rnaseq data bothfrom tumor tissue and adjacent normal tissue according tothe recommended analysis approach described in a previouslypublished study ryan we identified asevents from genes based on their splicing patterns theseas events could be roughly classified into seven types alternatepromoter ap mutually exclusive exons me retained intronri exon skipping es alternate acceptor site aa alternateterminator at and alternate donor site ad figure 1ato quantify the detected as events psi values were calculatedthese values measure the proportion of each detected splicingvariation in all of the expressed isoforms the expression ofcertain isoforms was fairly low psi and most of the asevents could not be stably detected in all of the given samplesafter screening average psi ‰¥ percentage of sampleswith psi ‰¥ a total of as events from geneswere obtained we further compared the variance in quantityof as events and the genes involved between tumor adjacentpaired normal and unpaired tumor tissues for diï¬erent splicingpatterns there were no significant diï¬erences in quantityvariations however on average one gene might have nearlythree as events figure 1b left panel moreover only annotated genes in this study stably underwent as figure 1bright panel notably diï¬erent as patterns may occur for a singlegene thus upset plots were used to depict the intersectionsbetween as types as demonstrated in figure 1c most of theparent genes only occurred in one type of as event whereascertain parent genes contained up to four types of as eventabout of the parent genes contained two or more asevents the arrangements of diï¬erent as types and as eventsbetween diï¬erent exonsintrons may be the major reason fortranscriptome diversity in order to comprehensively depict asevent profiles in hcc circos plots were used to visualize therelationships among as events and the corresponding parentgenes in chromosomes figure 1didentification of hccrelated deascomparing the variations in molecular components amongdiï¬erent pathological states using highthroughput techniquesis an eï¬ective way to screen key molecules this approachhas been widely used to identify diseaserelated molecules inprevious research xiong 2018ab it is reasonableto consider that significant diï¬erences in as events betweenprimary hcc tissues and adjacent normal tissues may be relevantto the initiation and maintenance of hcc in this study thetcga barcodes corresponding to tissue samples rnaseq data were analyzed from which as profiles of pairednormal and tumor tissues were finally extracted these pairedfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure landscape of alternative splicing as events in hepatocellular carcinoma hcc a diagrammatic sketch of the seven types of as events in the presentstudy alternate acceptor site aa alternate terminator at alternate promoter ap exon skipping es mutually exclusive exons me alternate donor site adand retained intron ri b number of as events and the corresponding parent genes illustrated according to as type left panel the color bar represents asevents filtered by criteria the black bar represents the corresponding genes involved in as each as type was divided into four groups based on the tissue sourcen normal tissue t tumor tissue pt paired tumor tissue npt unpaired tumor tissue number of detected as events asrelated genes filtered as events and thecorresponding genes right panel c intersection of parent genes between different as types n in hcc one gene may incur up to four types ofalternative splicing d circos plots depicting the distribution and the detailed alteration of as events and their parent genes in chromosomesas profiles were used to identify deas eventually deaswere identified from genes using a threshold of log2fc and adj p including aps ess ats risnine ads aas and one me figure 2a and supplementarytable s3 the top deas are listed in table notablythe proportion of as types between the filtered as and deaswas inconsistent the es events accounted for of filteredas but only of deas however the proportion of apevents rose from of filtered as to of deasfigure 2b these statistical findings suggest that as is notthe result of transcription errors but a tightly regulated processmoreover based on the identified deas the samples could beclearly separated into normal and tumor groups by unsupervisedhierarchical clustering figure 2c indicating that the deashad been reliably identified the psi values of deas eventsin diï¬erent hcc patients are illustrated in figure 2c as amatrix heat map the changes in color gradient intuitively revealthe heterogeneity of hcc a splice graph which representssplice junctions as edges and exons as rectangular nodes wasused to visualize some of the identified deas figure 2dfurthermore the diï¬erences in expression of these as eventsbetween primary hcc tissue and corresponding adjacent normaltissues are intuitively depicted in figure 2e taken together theseresults show that a significant variation of as occurred duringhcc initiation and maintenance indicating that the potentialrole of hccrelated as events requires further researchenrichment and interaction analysis ofdeasemerging evidence indicates that as could change a transcribedsequence directly with eï¬ects on expression abundance orprotein function thus the potential biological eï¬ects of deascould be determined by analyzing the corresponding proteinsas shown in supplementary figures s2a“c specific go termsclosely related to liver metabolism including negative regulationof hydrolase activity sterol homeostasis anic acid catabolicprocess and acidic amino acid transport were significantlyenriched by the parent genes of deas in addition certain keggpathways known to be involved in hcc were enriched includingthe cgmppkg signaling pathway the nfκb signaling pathwaythe mrna surveillance pathway and the phosphatidylinositolfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure identification of hepatocellular carcinoma hccrelated aberrant alternative splicing as a differences in as events between paired hcc tissue andparacancerous tissue volcano plot of the differentially expressed alternative splicing deas identified in hcc the blue and the red points represent the deas withstatistical significance logfc ‰¥ adj p b proportions of different as types among filtered as and deas c heat map of the deas the horizontal axisshows the clustering information of samples divided into two major clusters adjacent normal tissue n and paired tumor tissue n the left longitudinalaxis shows the clustering information of deas the gradual change of color from green to red represents the alteration of expression of deas from low to highd splice graph of some representative deas the thin exon sections represent untranslated regions and the thick exon sections represent coding regions theexons are drawn to scale and the connecting arcs represent splice paths e differences in percent spliced in values of as events between hcc and pairedadjacent normal tissuessignaling system supplementary figure s2d these resultssuggest that the parent genes of deas are critical in the biologicalregulation of hcc thus aberrant splicing of the transcribedsequences could influence their translation and change thecharacteristics of the resulting proteins therefore it is essentialto study as events from the perspective of ppi networks basedon the deasrelated genes a ppi network was establishedrepresenting not only normal interactions but also the potentialimpact of as events figure correlation network of sfs andhccrelated asas events are primarily regulated by sfs which attach to themrna precursor and aï¬ect the selection of exons and thechoice of splicing site aberrant as events in tumor tissuemay be orchestrated by a limited number of sfs for thisreason we conjecture that a few key sfs potentially regulatea large proportion of hccrelated as events to validate thisconjecture we first identified sfs supplementary table s1by comprehensive and handcurated screening of the literatureall of which had been previously experimentally validatedgiulietti then the copy number variation somaticmutations and expression abundance of these sfs in eachhcc patient were investigated using cbioportal figure 4avisualization using oncoprint revealed that each of the sfs harbored at least three molecular alterations figure 4aleft panel the most frequently aï¬ected sf was khdrbs3in which molecular alterations were detected in cases partly owing to the above changes the expressionabundance of the sfs showed a significant heterogeneity atan individual level figure 4a right panel the expressionprofiles of the sfs in diï¬erent cancer types also showedheterogeneous characteristics figure 4b more importantlythe expression of sfs also diï¬ered between paired normal andcancer tissues of the same hcc patient figure 4c nextcorrelation analyses were performed between the psi value ofeach deas event and the sfs according to the correlationcoefficient ttest p r a splicing regulatorynetwork was established as shown in figure 5a sfs weresignificantly correlated with deas events among which were downregulated and were upregulated several diï¬erentas events in the network were regulated by a single sf insome cases an sf had the opposite regulatory eï¬ect on diï¬erentas events figure 5a we also found that the same bindingsite as event could be competitively bound by diï¬erent sfsthese observations explain at least in part why one gene cangenerate several diï¬erent isoforms representative correlationsfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hcctable the top most different alternative splicing as eventssymbolas typeexonsfrom exonto exonmean nmean tlog fcadjusted pvaluedownregulatedmthfd2ligf2kif22gstz1serpinb5ppargfam3afip1l1tmem59tpm4cdh23mid1gnesamd12kif4afbxw7nek2padi4rnf115vti1aupregulatedrdm1nr1i3psphtmem145nr1i3gpr116piddnr1i3ano1cldn7sardhscp2znf331eno3pemtfn1tnfrsf10carhgef39igf2neil3apapadapatapaaatapapatapapatatapatatesatatatapatriesapriesapapesapatapesatapapatˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’1393eˆ’1593eˆ’4948eˆ’9941eˆ’2008eˆ’4387eˆ’8636eˆ’1393eˆ’4574eˆ’2519eˆ’1528eˆ’2202eˆ’6860eˆ’1522eˆ’2379eˆ’7699eˆ’1780eˆ’1843eˆ’4211eˆ’2609eˆ’1994eˆ’8164eˆ’1036eˆ’7765eˆ’1089eˆ’3569eˆ’1065eˆ’2135eˆ’5986eˆ’1038eˆ’1611eˆ’1110eˆ’2586eˆ’7736eˆ’2117eˆ’1072eˆ’4524eˆ’1151eˆ’1829eˆ’8136eˆ’between sfs and specific as events were illustrated using dotplots figures 5b“g for example the expression of esrp2 wassignificantly correlated with both es of ceacam1 figure 5cand at of epb41l5 figure 5fassociation of deas with prognosis ofhcc patientsa crossvalidation method was used to evaluate the accuracyof the survival data and the clinical information as shown insupplementary figure s3 stratifying patients according to thetnm stage resulted in separate kaplan“meier curves for bothos and dfs these results confirmed that the survival dataset forthe tcga hcc cohort although it contained censored data wasappropriate and informative for use in further survival analysistheeï¬ect of each deas on survival was determined by coxregression analysis the hcc patients were divided into twogroups according to their psi value median cut of eachdeas event according to univariate analyses a total of to investigate the prognostic significance of deasfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure protein“protein interaction ppi analysis of the identified differentially expressed alternative splicing deas interactions of the parent genes affectedby deas these genes were used to construct an intricate ppi network comprising nodes and edges the genes are denoted as nodes in the graph andthe interactions between them are represented as edges the shape size and color of the nodes respectively represent alternative splicing type value of logfcand change patterndeas events were significantly correlated with dfsand deas events were significantly correlatedwith os supplementary table s4 among these prognosisrelated deas events deas events were correlated withboth os and dfs p figure shows some of thedeas events for which the pvalue for both os and dfs waslower than to demonstrate the capability of as eventsfor prognostic prediction we randomly selected two prognosisrelated deas events and used them to draw survival curvesas shown in figure according to the psi value median ofnek2at and troptat the hcc patients could be stratifiedto form significant kaplan“meier curves by both os and dfssurvival analysis additionally the deas events that significantlycorrelated with survival in the univariate analysis were furtherassessed by multivariate analysis as shown in supplementaryfigure s4 there were five and six deas events that could berecognized as independent prognostic indicators for os and dfsrespectively these findings confirm that deas events possessnot only an important biological meaning but also a potentialclinical significanceconsidering the prognostic value of the aboveidentified asa prognostic model integrating multias was established so thatit can be easily applied to clinical practice based on the survivalrelated deas a relative regression coefficient was calculated bylasso analysis by forcing the sum of the absolute value ofthe regression coefficients to be less than a fixed value certaincoefficients were shrunk exactly to zero and the most powerfulprognostic marker of all the hcc survivalassociated deaswas selected including four as supplementary figure 5acombining the relative expression levels of the as in themodels and the corresponding lasso coefficients a riskscore was calculated for each patient obviously patientswith higher rs generally had a significantly worse overallsurvival than those with lower rs p supplementaryfigures 5bc as the majority of events occurred within years timedependent roc curves were used to assess theprognostic power based on os at and years respectivelysupplementary figure 5dclustering hcc patients using deasassociated with prognosisgiven our findings of significant heterogeneity among deas atan individual level which could reflect the diï¬erent outcomesfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure multiomics analysis of the splicing factors sfs in hepatocellular carcinoma hcc a cbioportal analysis of the sfs in the cancer genomeatlas hcc patients oncoprint was used to produce a landscape of genomic alterations legend in sfs rows at the individual level columns inframe deletionstruncated mutations and missense mutations with known or unknown significance are shown in orange blue green and gray respectively with onethird heightthe copy number variations are annotated with the full height amplification is shown in red and copy number loss is in blue heat map matrix shows the variation insfs at expression level the expression abundance from high to low is represented by color gradient from red to blue b expression of the sfs in tumortypes heat map color gradient depicts the normalized expression of sfs between different tumor types c differential expression analysis of representative sf tia1in hcc the expression of tia1 in hcc tissue was significantly higher than that in normal liver tissueof patients with hcc we conjectured that there might existdistinct patterns of as among diï¬erent hcc patients thishypothesis was verified using consensus unsupervised analysisbased on the deas the optimal number of clusters wasdetermined by combining the gap statistic and elbow method theoptimal balanced partition as suggested was k figure 8aaccordingly all the hcc patients were divided into four clustersas follows i n ii n iii n and iv n figures 8bc as illustrated bythe heat map the four clusters had a significant interconnectivityamong which cluster ii appeared as a wellindividualized clusterwhereas there was more classification overlap among clustersi iii and iv figures 8bc kaplan“meier survival analysisand logrank test were used to evaluate the associations betweenprognosis and the as clusters as illustrated in figures 8dethe stratification of hcc patients based on as clusters showeda significant correlation with distinct patterns of survival thevariation tendency that resulted in the as stratification betweenfrontiers in genetics wwwfrontiersinaugust volume 0cxiong landscape of as in hccfigure specific regulatory network of hepatocellular carcinomarelated alternative splicing as events a correlation network of splicing factors sfs anddifferentially expressed alternative splicing the shape size and color of nodes respectively represent type as event or sf value of logfc and change patternupregulated or downregulated the breadth of the line represents the interaction strength b“g representative dot plots of the correlations between theexpression of sfs and percent spliced in values of as eventsfigure prognostic value of differentially expressed alternative splicing deas in hepatocellular carcinoma part deas events simultaneously associated withoverall survival os and diseasefree survival dfs univariate analysis of deas for os and dfs respectively unadjusted hazard ratios boxes and confidenceintervals horizontal lines
0
"(B) E-cadherin was membrane positive and vimentin was negative in p120ctn membrane-positive lung cancer cells. (C) E-cadherin was negative and vimentin was positive in p120ctn cytoplasmic-positive lung cancer cells. .0088064.t001 Correlation between E-cadherin vimentin and lymph node metastasis and p120ctn. p120ctn N membrane cytolymph/nucleolus X2 p E-cadherin negative 56 9 47 30.166 <0.01 positive 22 18 4 Vimentin negative 53 23 30 5.633 0.022 positive 25 4 21 Lymph node metastasis No 41 19 22 5.251 0.032 Yes 37 8 29 Localization of p120ctn is consistent with E-cadherin in lung cancer cells We examined the protein expression levels of p120ctn and E-cadherin in normal HBE cells and nine lung cancer cell lines by Western blot and found that they all expressed mainly isoforms 1A (120 kDa) and 3A (100 kDa) of p120ctn (A). Although the protein expression levels of p120ctn were not related to E-cadherin the localization (membrane or cytoplasm) of p120ctn was always consistent with that of E-cadherin. We then screened cells expressing high levels of p120ctn and E-cadherin in the membrane (H460 cells) or cytoplasm (SPC cells) as well as those expressing low levels of p120ctn and E-cadherin in the membrane (H4299 cells) or cytoplasm (LK2 cells) for further study (B). .0088064.g002 Expression and localization of p120ctn and E-cadherin in H460 SPC H1299 and LK2 cells. (A) Western blot analyses showed expression of p120ctn and E-cadherin in nine lung cancer cell lines and HBE. (B) By immunofluorescence analysis the expression of E-cadherin and p120ctn were observed restricted to the cell membrane at cell-cell adherens junctions in H460 and H1299 cells whereas they both were confined to the cytoplasm in SPC and LK2 cells. Different functions of p120ctn isoform 1A in EMT are dependent on E-cadherin subcellular localization Knockdown of endogenous p120ctn isoform 1A by siRNA-p120ctn-1A resulted in decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells (A). However knockdown of endogenous p120ctn-1A by siRNA-p120ctn-1A showed opposite results in SPC cells where we found increased E-cadherin expression and decreased N-cadherin snail and vimentin expression (B). In comparison with the control the ablation of p120ctn isoform 1A also enhanced the H460 cells invasiveness (17.33±1.25 vs. 36.33±1.70 P<0.01) (C) whereas reduced the SPC cells invasiveness (23.0±0.82 vs. 13.0±0.82 P<0.01) (D). These results revealed that the p120ctn isoform 1A plays a different role in EMT and cell invasiveness in different E-cadherin subcellular locations. .0088064.g003 p120ctn isoform 1A plays a different role in regulating EMT in H460 and SPC cells. (A) Ablation of p120ctn isoform 1A decreased E-cadherin expression and increased N-cadherin snail and vimentin expression in H460 cells. (B) SPC cells were treated as in (A) and the opposite results were obtained. (C) Ablation of p120ctn isoform 1A enhanced the invasiveness of H460 cells (**P<0.01). (D) Ablation of p120ctn isoform 1A decreased the invasiveness of SPC cells (**P<0.01). Inhibitory function of p120ctn isoform 3A on EMT is not affected by differences in E-cadherin subcellular localization To verify whether p120ctn isoforms 1A and 3A play different roles in regulating EMT their expression plasmids were transiently transfected into lung cancer cells with low expression of p120ctn (H1299 with membrane E-cadherin expression and LK2 with cytoplasmic E-cadherin expression). The western-blot analysis demonstrated that overexpression of the p120ctn isoform 1A led to increased E-cadherin expression and decreased N-cadherin vimentin and snail expression (A); on the contrary the decreased E-cadherin expression and increased N-cadherin vimentin and snail expression were observed in LK2 cells (B). Overexpression of the p120ctn isoform 1A also reduced the H1299 cell invasiveness (52.0±2.65 vs. 33.33±2.64 P<0.01) (C) while enhanced the LK2 cell invasiveness (18.0±0.82 vs. 39.66±2.05 P<0.01) (D).. Overexpression of p120ctn isoform 3A led to increased E-cadherin expression decreased N-cadherin vimentin and snail expression (A 4B) and reduced cell invasiveness (52.0±2.65 vs. 29.66±1.53 P<0.01; 18.0±0.82 vs. 8.33±expression 0.47 P<0.01) (C 4D) in both of these cell lines. These results further confirmed that the p120ctn isoform 1A had a different effect on EMT depending on the subcellular localization of E-cadherin. They also revealed that the p120ctn isoform 3A maintained an inhibitory role in the EMT of lung cancer cells whether E-cadherin was localized to the membrane or the cytoplasm. .0088064.g004 p120ctn isoform 3A maintains the role of inhibitiing EMT independently of E-cadherin localization. (A B) Both H1299 (E-cadherin membrane localization) and LK2 cells (E-cadherin cytoplasmic localization) transiently transfected with the p120ctn isoform 3A plasmid showed increased E-cadherin expression and decreased N-cadherin vimentin and snail expression. (C D) Transient transfection of p120ctn isoform 3A plasmids into H1299 and LK2 cells resulted in decreased cell invasiveness (**P<0.01). (E) E-cadherin remained localized on the membrane in H1299 cells and in the cytoplasm of LK2 cells after transfection of the p120ctn isoform 3A plasmid. Discussion The phenomenon of EMT in tumor cells often leads to decreased cell adhesion and increased mobility and this transition is accompanied by decreased E-cadherin expression and increased expression of N-cadherin vimentin and other mesenchymal biomarkers [3] [4] [5] [6] [7]. As an important factor for stabilizing E-cadherin p120ctn plays a role in inhibiting or promoting tumor cell proliferation and invasion that is dependent on whether E-cadherin is expressed or not [16] [17]. Furthermore p120ctn isoforms 1A and 3A have shown different effects on E-cadherin expression and tumor cell invasiveness which are based on differences in the localization of E-cadherin [18]. These results strongly suggest that p120ctn most likely regulates the EMT of tumor cells by affecting E-cadherin expression and that p120ctn isoforms 1A and 3A play different roles in EMT expressing E-cadherin in different subcellular locations. We first found that the p120ctn membrane expression was positively correlated with E-cadherin expression and negatively correlated with vimentin expression and lymph node metastasis while the cytoplasmic expression of p120ctn was negatively correlated with E-cadherin expression and positively correlated with vimentin expression and lymph node metastasis by immunohistochemistry. Although these results were consistent with previous studies [13] [14] they further suggested that p120ctn likely affects the EMT by influencing the expression of E-cadherin and vimentin and thereby the cell invasion and metastasis in non-small cell lung cancer (NSCLC). To confirm the different impacts of p120ctn isoforms 1A and 3A on EMT in cells expressing E-cadherin in different locations we selected H460 and H1299 cells with E-cadherin membrane expression and SPC and LK2 cells with E-cadherin cytoplasmic expression for further analysis. Plasmids expressing the p120ctn isoforms 1A and 3A were constructed and the full-length p120ctn siRNA was synthesized for these experiments. Since the sequence beyond amino acids 1“101 of p120ctn isoform 1A is similar to that of p120ctn isoform 3A [24] [25] we could not design an interference sequence specifically for p120ctn isoform 3A. Therefore we had to further study the impact of the two isoforms on EMT and cell invasiveness in lung cancer cells with different E-cadherin locations specifically by knocking down p120ctn isoform 1A in H460 and SPC cells with high p120ctn expression and transfecting cDNA plasmids for exogenous p120ctn isoforms 1A and 3A into H1299 and LK2 cells with low expression of p120ctn. Knockdown of p120ctn isoform 1A in H460 cells destroyed the epithelial cell adhesion complexes. E-cadherin expression was also downregulated due to the loss of its important stabilizing factor p120ctn isoform 1A which was consistent with previous studies [20] [26]. Decreased E-cadherin expression and disrupted cell-cell adhesion may induce EMT [27] [28] [43] [44] which results in increased N-cadherin vimentin and snail expression and enhanced cell invasiveness. On the other hand overexpressed p120ctn isoforms 1A and 3A was shown to bind E-cadherin located on the membrane proactively in tumor cells [29] and then inhibit the degradation of E-cadherin and stabilize its expression contributing to the formation of effective epithelial cell adhesion complexes [30] [31] [32]. "
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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 includedorœhypothyroidism or œHT and œthyroid diseases orœbreast cancer or œBC or œbreast neoplasms or œmammarmy cancer and œrisk orœincidence At the sametime we manually screened out the relevant potentialliterature in the references extracteddysfunctionœthyroidtermsforInclusion 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 author™s 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 Cristofanilli™s research may causeheterogeneity [] Cristofanilli™s 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 Cristofanilli™s 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 Scand“Dargent 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 20121636e401“Kim 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 levels are positively associatedwith breast cancer risk in postmenopausal women Breast Cancer Res 123R33 httpsdoi101186bcr2587Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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expanding cancer predisposition genes with ultra‘rare cancer‘exclusive 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 individual™s 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 Meier“Kaplan survival rate estimate b Meier“Kaplan diseaseprogressionfree estimatesomatic mutations in any of these genes is much longer a0months Applying the Kaplan“Meier survival estimate yields a p value of 178eˆ’ in the Logrank test Fig a04a The Kaplan“Meier diseaseprogressionfree estimate was also worse for patients with somatic mutations in the novel CPGs with a p value of 603eˆ’ 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 a0S1“S24 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 ˜Caucasians™”individuals that were both genetically verified as Caucasians and declared themselves as ˜white™ ˜nonCaucasians™”individuals 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 cancer”insights 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 world™s 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
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lenvatinib inhibits tyrosine kinases including vascular endothelial growth factor vegf receptor fibroblast growth factor receptor platelet derived growth factor receptor alpha ret proto oncogene and kit proto oncogene receptor tyrosine kinase we assessed the efficacy and safety of lenvatinib in patients with metastatic colorectal cancer after failure of standard chemotherapiespatients and methods this was an open label single centre single arm phase study eligible patients had unresectable metastatic colorectal adenocarcinoma refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin trifluridinetipiracil anti vegf therapy and anti epidermal growth factor receptor therapy for tumours with wild type ras patients were treated with oral lenvatinib at mg one time a day in day cycles until disease progression or unacceptable toxicity the primary endpoint was centrally assessed disease control rate secondary endpoints included safety response rate progression free survival and overall survival the planned sample size was patients to expect a disease control rate of with a threshold disease control rate of one sided alpha of and power of results between october and january patients were enrolled and had received or ‰¥ lines of prior chemotherapy for metastatic disease respectively the median number of lenvatinib cycles was range “ the centrally assessed disease control rate was ci to one sided p00001 patients had a partial response and had a stable disease median progression free survival was months ci to median overall survival was months ci to the most common grade ‰¥ adverse events were hypertension thrombocytopenia increased alanine aminotransferase and anorexia eachs lenvatinib showed promising clinical activity and was tolerated in patients with metastatic colorectal cancer after failure of standard chemotherapiestrial registration number umin ctr umin000023446 and jamcct ctr jma iia00261introductionthe combination of cytotoxic chemotherapy with a molecular targeted agent has significantly key questionswhat is already known about this subject –º no studies have previously reported the efficacy and safety of lenvatinib monotherapy in patients with metastatic colorectal cancer refractory to standard chemotherapieswhat does this study add –º lenvatinib showed promising antitumour activity with acceptable toxicity for heavily pretreated patients with metastatic colorectal cancer refractory to standard chemotherapies –º no unexpected safety signals were observed and toxicities were manageable with dose modification interruptions and supportive medicationshow might this impact on clinical practice –º further prospective randomised studies are warranted to evaluate the efficacy of lenvatinib in patients with metastatic colorectal cancer refractory to standard chemotherapiesimproved the survival of patients with unresectable metastatic colorectal cancer1“ from results of recent clinical trials trifluridinetipiracil and regorafenib are recognised as new treatment options for patients with metastatic colorectal cancer refractory or intolerant to standard therapies6 nevertheless the prognosis of patients which are refractory or intolerant to standard chemotherapies is poor and there are still an unmet medical needs for these patients especially for those who are in a good performance status and eligible for further therapieslenvatinib is an oral multitargeted tyrosine kinase inhibitor of the vascular endothelial growth factor receptor vegfr “ fibroblast growth factor receptors “ platelet derived growth factor receptor alpha ret and kit8 preclinical studies have shown that iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesslenvatinib not only interferes the interaction between cancer cells and endothelial cells but also inhibits tumour growth10 several phase trials of patients with solid tumours in the usa11 europe12 and japan13 showed that the optimum dosage of lenvatinib was mg one time a day in a day cyclea total of patients were enrolled in four phase studies of lenvatinib monotherapy of whom had colorectal cancer disease control rate dcr was achieved in out of patients including one with a partial response which continued for weeks mg two times a day for weeks of a week cycle grade palmar plantar erythrodysesthesia was reportedly much lower in of patients treated with lenvatinib for thyroid cancer in a japanese population of the select trial than that of reported in a japanese population of correct trial using regorafenib for metastatic colorectal cancer15 these results suggested that lenvatinib may have a potential for improving the outcomes of patients with unresectable metastatic colorectal cancer who have already received conventional chemotherapy with a fluoropyrimidine irinotecan and oxaliplatinwe conducted a single centre phase study to evaluate efficacy and safety in patients with metastatic colorectal cancer failing to standard therapiespatients and methodsstudy design and patientsthis study was a single arm phase study conducted at national cancer center hospital tokyo japan the inclusion criteria were histological diagnosis of colorectal adenocarcinoma excluding carcinoma of the appendix and the anal canal unresectable metastatic disease an eastern cooperative oncology group performance status of or an age of “ years no previous treatment with regorafenib or lenvatinib sufficient oral intake adequate an and bone marrow function at least one measurable lesion in accordance with the response evaluation criteria in solid tumors recist version refractory or intolerant to fluoropyrimidine irinotecan oxaliplatin therapy and antiepidermal growth factor receptor therapy for tumours with wild type ras and no systemic therapy for at least weeks weeks if any investigational drug had been administered before study enrolment the exclusion criteria were provided in the online supplementary materialtrifluridinetipiracil anti vegf all patients provided written informed consentprocedurespatients received lenvatinib at mg one time a day in day cycles orally until disease progression or unacceptable toxicity the dose was reduced to mg mg mg mg and mg if a patient had an intolerable grade or grade adverse event treatment was discontinued if a dose interruption was required for more than consecutive daystumour response was assessed by the independent radiological review committee based on the ct or mri performed at baseline every weeks for weeks and every weeks thereafter until confirmed objective disease progression safety assessments including laboratory tests were done at screening days and of cycle and days and of the subsequent cycles urinalysis thyroid function prothrombin time international normalized ratio pt inr and tumour markers both carcinoembryonic antigen and carbohydrate antigen “ were measured at screening and on day of each treatment cycle adverse events were recorded from the first day of the protocol treatment to days after the last dose of study medication and graded using the national cancer institute common terminology criteria for adverse events version blood sampling for biomarker analyses was done at baseline on days and and at the end of treatment plasma levels of angiopoietin2 were measured by the human angiopoietin2 quantikine elisa kit rd systems minneapolis usaoutcomesthe primary endpoint was centrally assessed dcr which was defined as the proportion of patients with a complete response partial response or stable disease persisting for more than weeks from the initiation of study treatment according to recist version a complete response and partial response were needed to be confirmedthe secondary endpoints were the objective response rate orr proportion of patients who had a complete response or partial response progression free survival pfs time from the enrolment until investigator assessed disease progression or death overall survival os time from the enrolment until death due to any cause and adverse events the incidence of adverse events was calculated based on the information of the worst grade of each adverse event experienced in each patient relative dose intensity which is unprespecified outcome was calculated as the proportion of the actual cumulative dose divided by planned cumulative dose mg times treatment daysstatistical analysisfor this single arm study the required sample size of patients provided power to reject the null hypothesis of dcr ‰¤ with expectation that of patients would have a disease control one sided α of considering the possibility of a few ineligible patients we planned to recruit patientsthe final analysis was planned approximately months after enrolment of the last patient we included all eligible patients in the efficacy analysis and all patients receiving a least one dose of lenvatinib in the safety analyses for the primary analysis binomial test was performed and the centrally assessed dcr was estimated with ci using the clopper and pearson method which corresponds to one sided α of we also estimated the investigator assessed dcr a supplementary analysis of the primary iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0ctable baseline patient characteristicscharacteristicstable continuedoverall n characteristicsoverall n open access median range   continued intolerant wild type mutant ras mutational status braf mutational status wild type mutant unknownmsi status mss unkown there is an overlapping this number includes patients with the ras wild type and patient with mutant rasecog eastern cooperative oncology group egfr epidermal growth factor receptor msi microsatellite instability mss microsatellite stableendpoint and orr with cis using the same method we estimated the median time and month and year probability of os and pfs with the kaplan meier method the cis for the median time were calculated using brookmeyer and crowley method the cis of month and year survival probabilities were calculated based on the greenwood™s formula hrs and cis were estimated by cox regression we did subgroup analyses divided by prespecified baseline patient and disease characteristic variables including ras status for dcr pfs and os we also did a prespecified exploratory analysis of potential predictive biomarkers in blood samples we did all analyses with sas v94resultspatient characteristicsbetween october and january patients with unresectable metastatic colorectal cancer were enrolled all patients were eligible and received the study medication table summarises the baseline characteristics of all enrolled patients the median number of previous lines of palliative chemotherapy was range “ and patients had received or ‰¥ prior lines of chemotherapy for metastatic disease respectively the data cut off date was january with median follow up of months iqr “efficacythe centrally assessed dcr was ci to one sided p00001 two patients had a partial response and had a stable disease including unconfirmed pr table figure a total of patients had a reduction in target lesion size from baseline figure time on treatment for all patients is ‰¥ ‰¥ male female months ‰¥ months right sided colon left sided colorectum lung liver lymph node peritoneumage years sex ecog performance status primary site number of metastatic site metastatic an time from start of first line chemotherapy number of previous palliative chemotherapy previous chemotherapy and reason for discontinuation fluoropyrimidine refractory intolerantoxaliplatin irinotecan tas102 trifluridinetipiracil angiogenesis inhibitor anti egfr inhibitor refractory intolerant refractory intolerant refractory refractory intolerant refractory intolerantiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accesstable best response to treatmentcomplete responsepartial responsestable diseaseprogressive diseasenot evaluabledisease control rate ciresponse rate cicentral assessmentn30 to to investigator assessmentn30 to to shown in online supplementary figures and events for pfs were recorded in all patients and median pfs was months ci to figure all deaths were recorded median os was months ci to with a month and year os of ci to and ci to figure safetypatients received the study treatment for four cycles at median range “ the median relative dose intensity was iqr “ dose interruptions and reductions were required in and patients respectively the major treatment related adverse events ‰¥ for dose reduction were proteinuria patients palmar plantar erythrodysesthesia patients diarrhoea patients hypertension patients fatigue patients and thrombocytopenia patients the reasons for treatment discontinuation of all patients were disease progression in patients and adverse events in patients gastrointestinal perforation and grade proteinuria in of each after treatment with lenvatinib patients received a subsequent treatment online supplementary table most patients only had mild grades “ adverse events table the most common grade ‰¥ adverse events were hypertension patients thrombocytopenia patients increased alanine aminotransferase and anorexia patients each no clear relationship was found between the incidence of lenvatinib associated adverse event of any grade and baseline body surface area online supplementary table serious adverse events occurred in four patients including figure waterfall plot analysis of maximum percentage change from baseline in measurable target lesions response evaluation criteria in solid tumors version central reviewiwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen accessfigure kaplan meier curves of a progression free survival pfs by investigator assessment and b overall survival os in all patients n30five treatment associated events anorexia in two and gastrointestinal perforation central venous catheter related bloodstream infection caused by staphylococcus aureus and nausea in each one in each of four patients all patients recovered from these adverse eventssubgroup analysisin patients with wild type ras the median pfs was months ci to and that was months ci to in patients with mutant ras online supplementary figure in patients with wild type ras the median os was months ci ci to and months ci to in patients with mutant ras online supplementary figure plasma angiopoietin2 levels were decreased by lenvatinib treatment in almost all patients and increased at the iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776time of treatment discontinuation online supplementary table with a first quartile cut off point17 the eight patients with a first quartile or lower level of angiopoietin2 had a median os of months ci to months compared with months ci to in the patients with higher than a first quartile level of angiopoietin2 hr ci to online supplementary figure patients with a first quartile or less level of angiopoietin2 had a median pfs of months ci to compared with months ci to in the patients with more than a first quartile level of angiopoietin2 hr ci to online supplementary figure 0copen accesstable treatment related adverse events occurring in ‰¥ patients n30any gradegrade ‰¥treatment related adverse eventhypertensionproteinuriathrombocytopeniafatiguehypothyroidismweight losshoarsenesspalmar plantar erythrodysesthesia syndromeanorexiadiarrhoeamucositis oralserum ast increasedserum creatinine increasedast aspartate transaminase discussionpatients with metastatic colorectal cancer with disease progression after three or more lines of therapy have limited treatment options in this open label single arm phase study of patients with previously treated metastatic colorectal cancer lenvatinib demonstrated manageable toxic effects and promising antitumour activity a total of out of patients had disease control including with partial responses moreover patients experienced reduction in measurable tumour size the overall toxicity profiles were similar to that reported for lenvatinib across a spectrum of advanced malignant neoplasmstwo recent international phase studies reported that regorafenib or trifluridinetipiracil provided significant improvements in dcr pfs and os compared with placebo in patients with metastatic colorectal cancer after failure of standard chemotherapies dcr median pfs months median os months in the correct study and dcr median pfs months median os months in the recourse study6 interestingly the present single arm phase study of lenvatinib revealed favourable dcr and median pfs values in patients with metastatic colorectal cancer compared with those in the regorafenib or trifluridinetipiracil study moreover about half of the patients received post study treatment which led to a favourable osthe lenvatinib safety profile in this study was similar to the published safety profiles of lenvatinib for thyroid cancer and hepatocellular carcinoma in the japanese population18 moreover we found no unexpected or off target safety signals the most common adverse events were hypertension proteinuria thrombocytopenia and fatigue while the most case of grade or hypertension and proteinuria required treatment interruption and dose reduction while the target population for thyroid cancer or hepatocellular carcinoma that showed efficacy for lenvatinib was first line setting20 this study targeted patients receiving salvage line therapy most patients with metastatic colorectal cancer in the salvage line setting had grade or proteinuria and hypertension at baseline because of the long term prior treatment with anti vegfvegfr treatment whereas the occurrence of grade hypertension was significantly higher compared with that of regorafenib in a similar study population in the correct concur and consign trials7 it was manageable by dose reduction or interruption but it may be necessary to consider the starting dose in the future although palmar plantar erythrodysesthesia is a not life threatening toxicity these adverse events have a significant impact on treatment schedules and quality of life in treated patients grade ‰¥ palmar plantar erythrodysesthesia has been observed in and of patients treated with lenvatinib in this study and the select japanese population15 respectively while in patients treated with regorafenib in the correct japanese population16 to date the clear mechanism of palmar plantar erythrodysesthesia by vegf receptor tyrosine kinase inhibitors is not known but it has been reproduced that palmar plantar erythrodysesthesia by lenvatinib is well tolerated overall it is suggested that lenvatinib might be a favourable treatment option in terms of toxicitiesseveral preclinical studies demonstrated that vegf targeted treatment affects immune suppression by promoting the expansion of suppressive immune cell populations such as regulatory t cells and myeloid derived suppressor cells24 several clinical studies suggested that modulation of vegf mediated immune suppression via angiogenesis inhibition could potentially augment the immunotherapeutic activity of anti programmed cell death pd1 antibody26 regorafenib and nivolumab showed antitumour activity in patients with metastatic colorectal cancer including those with microsatellite stable tumours in a phase study28angiopoietin2 a relatively novel regulator of angiogenesis that acts through the tek tyrosine kinase endothelial tie2 receptor has been identified as a potential prognostic biomarker for some types of cancer although the baseline ang2 level was a predictive biomarker in patients with thyroid cancer in the select trial17 it did not become a reliable biomarker of lenvatinib response in this study prior treatment with anti vegfvegfr antibodies probably had an effect on baseline angiopoietin2 levels because the study population was refractory to standard treatment in this study the decrease in angiopoietin2 levels was observed after treatment therefore it may be an indicator of treatment responsethe limitations of our study include its small size which could limit the interpretation of the subgroup analyses iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0cand the absence of a comparison group however the level of clinical benefit in the form of confirmed responses observed in this study was remarkable in the historical context of other clinical trials done in heavily pretreated patients with metastatic colorectal cancer moreover most of the patients in our study had left sided tumours which were known to have a better prognosis compared with right sided tumoursin lenvatinib provided promising activity with prolonged survival relative to the anticipated median pfs in heavily pretreated patients with metastatic colorectal cancer the safety profile of lenvatinib was similar to that in other tumour types with no new safety signals recorded based on these findings further investigation of lenvatinib with anti pd1 antibody or other novel combinations with the potential to build on the benefit of lenvatinib is currently taking place nct03797326 and nct04008797acknowledgements the authors thank the patients and their families the members of the clinical research support office for their support with data collection and running the study and nai incorporated for editing a draft of this manuscriptcontributors all authors conceived and designed the study and drafted and revised the manuscript for publication si no hs yh at kk th nb and yy collected data ak go mk and kn analysed the data and managed data and study progress all authors interpreted the data and approved the final version of the manuscriptfunding the study was supported by the project promoting clinical trials for development of new drugs and medical devices japan medical association from the japan agency for medical research and development grant number jp18lk0201037 and by eisai cocompeting interests si has received research grants from eisai and merck biopharma th has received research grants from eisai and honoraria from merck serono yy has received honoraria from eisaipatient consent for publication not requiredethics approval the study was conducted in accordance with the declaration of helsinki and good clinical practice guidelines the study protocol was approved by the national cancer center institutional review board t4329provenance and peer review not commissioned externally peer revieweddata availability statement data are available upon reasonable request proposals should be directed to siwasa ncc go jp the data will be available for achieving aims in the approved proposalopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited any changes made are indicated and the use is non commercial see a0http creativecommons licenses by nc orcid idssatoru a0iwasa http orcid yasuhide a0yamada http orcid references saltz lb clarke s díaz rubio e et a0al bevacizumab in combination with oxaliplatin based chemotherapy as first line therapy in metastatic colorectal cancer a randomized phase iii study j clin oncol “ tabernero j yoshino t cohn al et a0al ramucirumab versus placebo in combination with second line folfiri in patients with metastatic colorectal carcinoma that progressed during or after first line therapy with bevacizumab oxaliplatin and a fluoropyrimidine raise a randomised double blind multicentre phase study lancet oncol “open access van cutsem e tabernero j lakomy r et a0al addition of aflibercept to fluorouracil leucovorin and irinotecan improves survival in a phase iii randomized trial in patients with metastatic colorectal cancer previously treated with an oxaliplatin based regimen j clin oncol “ yamazaki k nagase m tamagawa h et a0al randomized phase iii study of bevacizumab plus folfiri and bevacizumab plus mfolfox6 as first line treatment for patients with metastatic colorectal cancer wjog4407g ann oncol “ price tj peeters m kim tw et a0al panitumumab versus cetuximab in patients with chemotherapy refractory wild type kras exon metastatic colorectal cancer aspecct a randomised multicentre open label non inferiority phase study lancet oncol “ mayer rj van cutsem e falcone a et a0al randomized trial of tas102 for refractory metastatic colorectal cancer n engl j med “ grothey a van cutsem e sobrero a et a0al regorafenib monotherapy for previously treated metastatic colorectal cancer correct an international multicentre randomised placebo controlled phase trial lancet “ matsui j yamamoto y funahashi y et a0al e7080 a novel inhibitor that targets multiple kinases has potent antitumor activities against stem cell factor producing human small cell lung cancer h146 based on angiogenesis inhibition int j cancer “ matsui j funahashi y uenaka t et a0al multi kinase inhibitor e7080 suppresses lymph node and lung metastases of human mammary breast tumor mda mb231 via inhibition of vascular endothelial growth factor receptor vegf r and vegf r3 kinase clin cancer res “ wiegering a korb d thalheimer a et a0al e7080 lenvatinib a multi targeted tyrosine kinase inhibitor demonstrates antitumor activities against colorectal cancer xenografts neoplasia “ hong ds kurzrock r wheler jj et a0al phase i dose escalation study of the multikinase inhibitor lenvatinib in patients with advanced solid tumors and in an expanded cohort of patients with melanoma clin cancer res “ boss ds glen h beijnen jh et a0al a phase i study of e7080 a multitargeted tyrosine kinase inhibitor in patients with advanced solid tumours br j cancer “ yamada k yamamoto n yamada y et a0al phase i dose escalation study and biomarker analysis of e7080 in patients with advanced solid tumors clin cancer res “ nakamichi s nokihara h yamamoto n et a0al a phase study of lenvatinib multiple receptor tyrosine kinase inhibitor in japanese patients with advanced solid tumors cancer chemother pharmacol “ kiyota n schlumberger m muro k et a0al subgroup analysis of japanese patients in a phase study of lenvatinib in radioiodine refractory differentiated thyroid cancer cancer sci “ yoshino t komatsu y yamada y et a0al randomized phase iii trial of regorafenib in metastatic colorectal cancer analysis of the correct japanese and non japanese subpopulations invest new drugs “ tahara m schlumberger m elisei r et a0al exploratory analysis of biomarkers associated with clinical outcomes from the study of lenvatinib in differentiated cancer of the thyroid eur j cancer “ takahashi s kiyota n yamazaki t et a0al a phase ii study of the safety and efficacy of a0lenvatinib in patients with advanced thyroid a0cancer future oncol “ yamashita t kudo m ikeda k et a0al reflect a phase trial comparing efficacy and safety of lenvatinib to sorafenib for the treatment of unresectable hepatocellular carcinoma an analysis of japanese subset j gastroenterol “ kudo m finn rs qin s et a0al lenvatinib versus sorafenib in first line treatment of patients with unresectable hepatocellular carcinoma a randomised phase non inferiority trial lancet “ schlumberger m tahara m wirth lj et a0al lenvatinib versus placebo in radioiodine refractory thyroid cancer n engl j med “ li j qin s xu r et a0al regorafenib plus best supportive care versus placebo plus best supportive care in asian patients with previously treated metastatic colorectal cancer concur a randomised double blind placebo controlled phase trial lancet oncol “ van cutsem e martinelli e cascinu s et a0al regorafenib for patients with metastatic colorectal cancer who progressed after standard therapy results of the large single arm open label phase iiib consign study oncologist “iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0copen access ott pa hodi fs buchbinder ei inhibition of immune checkpoints and vascular endothelial growth factor as combination therapy for metastatic melanoma an overview of rationale preclinical evidence and initial clinical data front oncol kato y tabata k kimura t et a0al lenvatinib plus anti pd1 antibody combination treatment activates cd8 t cells through reduction of tumor associated macrophage and activation of the interferon pathway plos one 201914e0212513 taylor mh lee c h makker v et a0al phase ibii trial of lenvatinib plus pembrolizumab in patients with advanced renal cell carcinoma endometrial cancer and other selected advanced solid tumors j clin oncol “ makker v rasco d vogelzang nj et a0al lenvatinib plus pembrolizumab in patients with advanced endometrial cancer an interim analysis of a multicentre open label single arm phase trial lancet oncol “ fukuoka s hara h takahashi n et a0al regorafenib plus nivolumab in patients with advanced gastric gc or colorectal cancer crc an open label dose finding and dose expansion phase 1b trial regonivo epoc1603 jco iwasa a0s et a0al esmo open 20205e000776 101136esmoopen2020000776 0c'
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"The work cannot be changed in any way or used commercially. Introduction: In non“small-cell lung cancer an exon 19 deletion and an L858R point mutation in the epidermal growth factor receptor (EGFR) are predictors of a response to EGFR-tyrosine kinase inhibitors. However it is uncertain whether other uncommon EGFR mutations are associated with sensitivity to EGFR-tyrosine kinase inhibitors. Methods: A post-hoc analysis to assess prognostic factors was performed with the use of patients with EGFR mutations (exon 19 deletion L858R G719X and L861Q) who were treated with gefitinib in the NEJ002 study which compared gefitinib with carboplatin-paclitaxel as the first-line therapy. Results: In the NEJ002 study 225 patients with EGFR mutations received gefitinib at any treatment line. The Cox proportional hazards model indicated that performance status response to chemotherapy response to gefitinib and mutation types were significant prognostic factors. Overall survival (OS) was significantly shorter among patients with uncommon EGFR mutations (G719X or L861Q) compared with OS of those with common EGFR mutations (12 versus 28.4 months; p = 0.002). In the gefitinib group (n = 114) patients with uncommon EGFR mutations had a significantly shorter OS (11.9 versus 29.3 months; p < 0.001). By contrast OS was similar between patients with uncommon mutations and those with common mutations in the carboplatin-paclitaxel group (n = 111; 22.8 versus 28 months; p = 0.358). Conclusions: The post-hoc analyses clearly demonstrated shorter survival for gefitinib-treated patients with uncommon EGFR mutations compared with the survival of those with common mutations and suggest that the first-line chemotherapy may be relatively effective for non“small-cell lung cancer with uncommon EGFR mutations. Gefitinib G719X L861Q NEJ002 Uncommon epidermal growth factor receptor mutations OPEN-ACCESS TRUE The clinical efficacy of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) such as gefitinib and erlotinib has been demonstrated in non“small-cell lung cancer (NSCLC) patients in whom standard chemotherapy has failed.12 Further studies have revealed that the presence of activating mutations in the EGFR kinase domain is strongly associated with the therapeutic efficacy of EGFR-TKIs.34 Randomized phase 3 trials have demonstrated that EGFR-TKIs significantly improve median progression-free survival (PFS) compared with platinum-doublet therapy in EGFR-mutated patients.5“8 However not all mutations in the EGFR kinase domain are responsive to EGFR-TKI treatment. These phase 3 trials have shown that EGFR-TKIs are effective for patients with common EGFR mutations such as an exon 19 deletion or the L858R point mutation which account for more than 90% of EGFR mutations. Retrospective studies and case reports suggest that some uncommon mutations are associated with sensitivity to EGFR-TKIs.9“20 These mutations include G719X in exon 18 which accounts for approximately 3% of EGFR mutations and L861Q in exon 21 which represents approximately 2% of EGFR mutations. However these uncommon EGFR mutations have not been clearly shown to be predictive markers for the efficacy of EGFR-TKIs because of their low frequency. To investigate the efficacy of gefitinib in patients with uncommon mutations we conducted a post-hoc analysis of the NEJ002 which compared gefitinib and carboplatin-paclitaxel as first-line therapies for advanced NSCLC with activating EGFR mutations. PATIENTS AND METHODS Patient Population We retrospectively analyzed the data of 225 patients who received gefitinib treatment at any point in the NEJ002 study.6 The eligibility criteria of the NEJ002 study included the presence of advanced NSCLC harboring an EGFR mutation (exon 19 deletion or L858R G719X or L861Q point mutation) without the resistant EGFR mutation T790M (identified using the peptide nucleic acid“locked nucleic acid polymerase chain reaction clamp method) no history of chemotherapy an age of 75 years or younger a performance status of 0 to 1 and appropriate an function.2122 Patients provided a written informed consent. The study was conducted in accordance with the Helsinki Declaration of the World Medical Association. The protocol was approved by the institutional review board of each participating institution. Treatment Eligible patients were randomly assigned to receive either gefitinib (250 mg/day) or paclitaxel (200 mg/m2)/carboplatin (area under the curve 6.0) on day 1 every 3 weeks. Chemotherapy was continued for at least three cycles. Gefitinib was administered until the disease progressed intolerable toxicities developed or consent was withdrawn. The protocol recommended that the crossover regimen be used as a second-line treatment. Clinical Assessments The antitumor response to treatment was assessed using computed tomography every 2 months. Unidirectional measurements were adopted on the basis of the Response Evaluation Criteria in Solid Tumors (version 1.0).23 PFS was evaluated from the date of randomization to the date when disease progression was first observed or death occurred. The treatment response and PFS were determined by an external review of computed tomography scans by experts who were not aware of the treatment assignments. Overall survival (OS) was evaluated from the date of randomization to the date of death. Statistical Analysis To assess prognostic factors for OS we used univariate and multivariate Cox proportional hazards models. Kaplan“Meier survival curves were constructed for PFS and OS and differences between groups were identified using the log-rank test. Differences in response rates were identified using Fisher™s exact test. Each analysis was two sided with a 5% significance level and a 95% confidence interval. All analyses were performed using SAS for Windows software (release 9.1; SAS Institute Cary NC). RESULTS Patient Population A total of 230 chemonaive patients were enrolled in the NEJ002 study: 115 patients were assigned to receive gefitinib and 115 were assigned to receive carboplatin-paclitaxel (Fig. 1). To evaluate the efficacy of gefitinib in NSCLC patients with uncommon EGFR mutations we analyzed the data of 114 patients in the gefitinib group and 111 patients in the carboplatin-paclitaxel group. We identified five patients who had uncommon EGFR mutations in each group. Two patients who had common mutations and were treated with first-line chemotherapy consisting of carboplatin-paclitaxel were excluded from the PFS analysis in the NEJ002 study. However both were treated with gefitinib and were included in this post-hoc analysis. The demographic and disease characteristics of the patients with uncommon EGFR mutations were similar to those of patients with common EGFR mutations (). The characteristics of each patient with uncommon EGFR mutations are shown in supplementary Table S1 (Supplemental Digital Content 1 http://links.lww.com/JTO/A494). FIGURE 1. Enrollment randomization and follow-up of the study patients. TABLE 1. Patient Characteristics Survival Factors In the univariate analysis of 225 patients who received gefitinib at any point uncommon EGFR mutations had a significant detrimental effect on survival (). We also identified performance statuses 1 and 2 distant metastasis brain metastasis stable disease and progressive disease as significant predictors of worse prognosis for standard chemotherapy and stable disease and progressive disease as significant predictors of worse prognosis for gefitinib. When these variables were included in the Cox proportional hazards model we found that uncommon EGFR mutations performance statuses 1 and 2 stable disease and progressive disease for standard chemotherapy and stable disease and progressive disease for gefitinib had significant hazard ratios (). TABLE 2. Univariate and Multivariate Analysis by Cox Proportional Hazards Model Uncommon EGFR Mutations and Survival The Kaplan“Meier curve for OS for uncommon versus common EGFR mutations is shown in A. The OS was significantly shorter among patients with uncommon EGFR mutations compared with OS of those with common EGFR mutations in the overall population (12 versus 28.4 months; p = 0.002). A significantly shorter survival time was observed in patients with uncommon EGFR mutations compared with survival time in those with common EGFR mutations in the gefitinib group (11.9 versus 29.3 months; p < 0.001) (Fig. 2B). However a similar survival time was observed between the subgroups of uncommon and common EGFR mutations in the carboplatin-paclitaxel group (22.8 versus 28 months; p= 0.358) (Fig. 2C). FIGURE 2. The overall survival curves of patients with common mutations and uncommon mutations in the entire population (A) the gefitinib group (B) and the carboplatin-paclitaxel group (C). To examine whether the sequence of platinum doublet and gefitinib affected OS we performed a further subgroup analysis. The survival time tended to be shorter among patients receiving first-line gefitinib compared with the survival time among those receiving first-line carboplatin-paclitaxel in the uncommon EGFR mutation group (11.9 versus 22.8 months; p = 0.102). Consistent with previous publications a similar survival time was observed between patients receiving first-line gefitinib and those receiving first-line carboplatin-paclitaxel in the common EGFR mutation group (29.3 versus 28 months; p = 0.378). Uncommon EGFR Mutations PFS and Response In the gefitinib group the median PFS was significantly shorter for patients with uncommon EGFR mutations compared with median PFS of those with common EGFR mutations (2.2 versus 11.4 months; p < 0.001) (Fig. 3A). By contrast the median PFS did not differ significantly between patients with uncommon EGFR mutations and those with common EGFR mutations in the carboplatin-paclitaxel group (5.9 versus 5.4 "
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high throughput methods in biological and biomedical fields acquire alarge number of molecular parameters or omics data by a single experimentcombining these omics data can significantly increase the capability for recoveringfinetuned structures or reducing the effects of experimental and biological noise indataresultsfhclust for identifying patient subgroups from different omics information eg geneexpression mirna expression methylation in particular hierarchical structures of patientdata are obtained in each omic or view and finally their topologies are merged byconsensus matrix one of the main aspects of this methodology is the use of ameasure of dissimilarity between sets of observations by using an appropriate metricfor each view a dendrogram is obtained by using a hierarchical clustering based on afuzzy equivalence relation with łukasiewicz valued fuzzy similarity finally a consensusmatrix that is a representative information of all dendrograms is formed by combiningmultiple hierarchical agglomerations by an approach based on transitive consensusmatrix construction several experiments and comparisons are made on real data egglioblastoma prostate cancer to assess the proposed approachs fuzzy logic allows us to introduce more flexible data agglomerationtechniques from the analysis of scientific literature it appears to be the first time that amodel based on fuzzy logic is used for the agglomeration of multiomic data theresults suggest that fhclust provides better prognostic value and clinical significancecompared to the analysis of singleomic data alone and it is very competitive withrespect to other techniques from literaturekeywords multiomics data data integration hierarchical clustering fuzzy similarityfuzzy aggregation the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriatecredit to the original authors and the source provide a link to the creative commons licence and indicate if changes weremade the images or other third party material in this are included in the ™s creative commons licence unlessindicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and yourintended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creativecommons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data madeavailable in this unless otherwise stated in a credit line to the data 0cciaramella bmc bioinformatics 21suppl page of nowadays high throughput methods in biological and biomedical fields acquire a largenumber of molecular parameters by a single experiment in particular such measuredparameters are collected in œomics datasets eg genomics transcriptomics methylomics among multiple measured parameters dna genome sequence rna expressionand dna methylation are representative instances for individually analysing suchdata several methodologies have been introduced in literature even though recentlya number of studies pointed out the best performance coming from the integration ofmultiomics data for instance analysing each omic or view in the machine learningjargon set separately fundamental patterns can be detected from data however somefinetuned structures such as cancer subtypes can be highlighted by both gene expression and dna methylation information so that multiomics analysis can reduce theeffects of experimental and biological noise in data from literature three kinds ofintegration methodologies emerge¢ early integration builds a single featurebased matrix by concatenating each omic¢ intermediate integration builds a joint representation of data given the views¢ late integration each omic is analysed separately and the solutions are integratedin general late integration methods and in particular clustering are preferred whenthe analysis combines continuous and discrete data together for a review of integrationapproaches and their comparisons the reader may refer to in this work a multiviewclustering methodology named fhclust is introduced see fig for its general schemafor identifying patient subgroups from different omics information or datasets eg geneexpression mirna expression methylation specifically for each omic dataset a fuzzybased hierarchical clustering approach is adopted and finally the results are mergedby consensus matrix the idea behind the proposed approach comes from observingthat a hierarchical clustering dendrogram can be associated with a łukasiewicz fuzzydataset ie view and applies a singleomic analysisfig proposed approach a data preparation b data normalization and feature selection c multiomicshierarchical agglomerations d data integration e clustering and visualization 0cciaramella bmc bioinformatics 21suppl page of similaritybased equivalence relation so that a consensus matrix that is the representative information of all dendrograms is derived by combining multiple hierarchicalagglomerations following an approach based on transitive consensus matrix constructionmethodscluster analysis or clustering is an unsupervised technique that aims at agglomerating aset of patterns in homogeneous groups or clusters [ ] hierarchical clustering hc isone of several different available techniques for clustering which seeks to build a hierarchyof clusters and it can be of two types namely agglomerative where each sample starts inits own cluster and pairs of clusters are merged as one moves up the hierarchy or divisivewhere all samples start in one cluster and splits are performed recursively as one movesdown the hierarchy thus hc aims at grouping similar objects into a cluster and werethe endpoint is a set of clusters where each cluster is distinct from each other and theobjects within each cluster are broadly similar to each other hc can be performed eitheron a distance matrix or raw data agglomerative hc starts by treating each observationas a separate cluster and it repeatedly executes the following two steps identifies thetwo clusters that are closest together and merges the two most similar clusters thisprocess continues until all the clusters are merged togetherthe main output of hc is a dendrogram which shows the hierarchical relationshipbetween the clusters distances many distance metrics have been developed and thechoice should be made based on theoretical concerns from the domain of studylater on it is necessary to determine how the distance is computed eg singlelinkagecompletelinkage averagelinkage as with distance metrics the choice of linkage criteria should be based on theoretical considerations from the application domainin nonfuzzy clustering or hard clustering data is divided into distinct clusters andeach data point can only belong to exactly one cluster in fuzzy clustering data pointscan potentially belong to multiple clusters for example in hard clustering given someparameters a œsymptom can be in a mutually exclusive way present or absent red orblue whereas in fuzzy clustering that œsymptom could simultaneously be of somegrade red and some other grade blue in fig a comparison between hard and fuzzycategorisation is shown the reader can refer to for a recent comparison betweenhard and fuzzy clustering in this work we introduce a data integration methodologybased on fuzzy concepts in particular we associate a dendrogram to a fuzzy equivalencerelation ie łukasiewicz valued fuzzy similarity so that a consensus matrix in a multiview clustering that is the representative information of all dendrograms can be obtainedfrom multiple hierarchical agglomerations [ ] the main steps of fuzzy agglomerationcan be summarised as follows characterisation of membership functions computation of a fuzzy similarity matrix or dendrogram for all models at a giventime construction of a consensus matrix for all hierarchical agglomerationsmembership functionswhen dealing with clustering tasks fuzzy logic fl permits to obtain a soft clusteringinstead of an hard clustering of data specifically data points can belong to more 0cciaramella bmc bioinformatics 21suppl page of fig hard vs fuzzy in symptom risk example a hard categorization b fuzzy categorizationthan one cluster simultaneously the fundamental concept in fl upon which all thesubsequent theory is constructed is the notion of fuzzy set a generalisation of a crisp setfrom classical set theorya fuzzy set generalises a crisp set by allowing its characteristic function ie itsmembership function assuming values in the interval [ ] rather than in the set in this way a given item belongs to the fuzzy set with a degree of truthranging from do not belong at all ie its membership function assumes value to completely belong ie the membership function assumes value in fl applications fuzzy sets make it possible to represent qualitative nonnumeric values ielinguistic variables such as high medium low for approximate reasoninginference or fuzzy control systems linguistic variables can be represented by fuzzy setsthrough a transformation step called fuzzification and it is achieved by using different types of membership functions representing the degree of truth to whicha given input sample belongs to a fuzzy set see œmembership functions sectionin supplementary material 0cciaramella bmc bioinformatics 21suppl page of fuzzy similarity matrixa measure of similarity or dissimilarity defines the resemblance between two samples orobjects similarity measure is a significant means for measuring uncertain informationfuzzy similarity measure is a measure that depicts the closeness among fuzzy sets and hasbeen used for dealing issues of pattern recognition and clustering analysisa binary fuzzy relation that is reflexive symmetric and transitive is known as a similarity relation fuzzy similarity relations are the generalisation of equivalence relationsin binary crisp relations to binary fuzzy relations in details a fuzzy similarity relationcan be considered to effectively group elements into crisp sets whose members are similar to each other to some specified grade and it is a generalization of classical equivalencerelation as described in œfuzzy similarity section in supplementary material in orderto introduce the fuzzy similarity in the following we focus on the properties of thełukasiewicz tnorm tl and the biresiduum in this way we obtain a fuzzy equivalencerelation that can be used for building dendrogram for more details in the derivation ofthese results see œfuzzy similarity section in supplementary materialdendrogram and consensus matrixif a similarity relation is mintransitive ie t min then it implies the existence ofthe dendrogram see œdendrogram and consensus matrix section in supplementarymaterial for details the mintransitive closure of a relation matrix r can be easilycomputed and the overall process is described in algorithm the last ingredient to accomplish an agglomerative clustering is a dissimilarity relationhere we considered the following result lemma letting r be a similarity relation with the elements rcid2x ycid3 ˆˆ[ ] and lettingd be a dissimilarity relation which is obtained from r bydx y ˆ’ rcid2x ycid3then d is ultrametric iif r is mintransitivein other words we have a onetoone correspondence between mintransitive similaritymatrices and dendrogram and between ultrametric dissimilarity matrices and dendrograms finally after the dendrograms have been obtained each time a consensus matrixie the representative information of all dendrograms is obtained by combining thetransitive closures ie maxmin operation the overall approach is described inalgorithm the overall workflow of the proposed approach is summarised in fig in particular for each omic data set xi a fuzzification step is adopted for obtaining thenew data set yi see supplementary material successively adopting a fuzzy similaritymeasure the similarity matrix si is computed and to guarantee the transitive closure ofthe matrix a new matrix ci is computed see algorithm finally all the ci matricesare collected for obtaining the consensus matrix a and the overall final dendrogram seealgorithm in fig we show an example that summarize a realistic agglomeration result we plotin figs 4abc three input hierarchies obtained on datasets that should be combinedin this case four sequences of patients are considered namely s1 s2 s3 and s4 respectively in fig 4d we show the final result by agglomerating dendrograms we observe that 0cciaramella bmc bioinformatics 21suppl page of fig workflow of the fuzzy based hierarchical clusteringthe output hierarchy contains clusters s1 s2 s3 and s1 s2 s3 s4 at different levels andeach of these clusters eg s1 s2 s3 are repeated at least in two out of the three inputdendrograms moreover it is worth stressing that the proposed approach based on theagglomeration of dendrograms can also be applied with commonly used metrics egeuclidean distance in fig we show a comparison between the dendrograms obtainedby using an euclidean metric and a similarity based approach ie łukasiewicz tnormrespectively in this realistic example we simulate three omic data sets with rows ienumber of patients and columns ie features we split the single datasets in twopartitions or clusters such that the first rows are random samples from a standard normal distribution with variance and the other rows have the same distribution withfig combination algorithm abc input dendrograms d combined hierarchy 0cciaramella bmc bioinformatics 21suppl page of fig crisp hierarchical clustering vs fuzzy based hierarchical clustering a dendrogram of euclidean basedhierchical clustering b dendrogram of similarity based hierachical clusteringalgorithm mintransitive closure input relation si output transitive relation ci sti elaborate compute sˆ— if sˆ—ielse ci sti si ˆª si —¦ sicid8 si replace si with sˆ—i sˆ—i and go to step i and the algorithm terminatesvariance obtaining a sort of an overlap we observe that both methods find two separated clusters but the similarity based approach in fig 5b permits to obtain a perfectseparation of the source partitionsresults and discussionin the following we describe the behaviour of the proposed methodology on multiomicsbenchmark datasetsalgorithm combination of dendrograms input ci ‰¤ i ‰¤ l l input similarity matrices dendrograms output similarity matrix dendrogram a aggregate the similarity matrices to a final similarity matrixa aggregate c1 c2 cla let aˆ— be the identity matrixb for each ci calculate e aˆ— aˆ— ˆª aˆ— —¦ cic if aˆ— is not changed a aˆ— and goto step else goto step 1b create the final dendrogram from aomics datasetswe consider multiomics cancer datasets available from the cancer genome atlastcga tcga is a large multiomic repository of data on thousands of cancerpatients all datasets contain three omics gene expression mirna expression and 0cciaramella bmc bioinformatics 21suppl page of table datasets description three omics are provided for each dataset respectively dna geneexpression mirna and methylationcases dnaoridatasetamlbiccoadgbmkirclihcluscskcmovsarclnrfmirnaorilnmethyorilnmultiomicsorilnrfrfrfthe number of features at each variable selection method is shown ori original variable dimension ln logarithm andnormalisation and rf random forest based on mean decrease gini indexdna methylation1 in table are summarised the main properties of the datasetsnamely acute myeloid leukemia aml breast invasive carcinoma bic colon adenocarcinoma coad glioblastoma multiforme gbm kidney renal clear cell carcinoma kirc liver hepatocellular carcinoma lihc lung squamous cell carcinomalusc skim cutaneous melanoma skcm ovarian serous cystadenocarcinoma ovsarcoma sarc the number of patients ranges from for aml to for bicmultiview clustering algorithmsfor validating the effectiveness of our model we compared it against several categories ofmultiview clustering algorithms2¢ kmeans and spectral clustering techniques ¢ lracluster it is a lowrank approximation based integrative probabilistic model¢ pins perturbation clustering for data integration and disease subtyping pinsis able to address subtype discovery as well as integration of multiple data types thealgorithm is built upon the resilience of patient connectivity and cluster ensembles toensure robustness against noise and biasto fast find the shared principal subspace across multiple data types¢ snf similarity network fusion snf allows for discovery of disease subtypesthrough integration of several types of highthroughput data on a genomic scale snfcreates a fused network of patients using a metric fusion technique and thenpartitions the data using spectral clustering snf appears to be the state of the art inthis area and has proven to be very powerful however the unstable nature ofkernelbased clustering makes the algorithm sensitive to small changes in molecularmeasurements or in its parameter settings¢ mcca multi canonical correlation analysis mcca which extends theapplication of canonical correlation analysis cca to more than two views is oneof the most widely used dimension reduction method for finding linear relationsbetween two or more multidimensional random variables1row data are available at httpacgtcstauacilmulti_omic_benchmarkdownloadhtml2httpsgithubcomshamirlabmultiomicscancerbenchmark 0cciaramella bmc bioinformatics 21suppl page of evaluation metricsin order to assess the performance of each method we adopt three evaluation metricsthat are the logrank test the enrichment of clinical labels in the clusters and the methods runtime the logrank test assumes that if clusters of patients have significantlydifferent survival they are different in a biologically meaningful way for the enrichmentof clinical labels in clusters six clinical labels are considered gender age at diagnosispathologic tumor pathologic metastases pathologic lymph nodes and pathologic stagethe four latter parameters are discrete pathological parameters measuring the progression of the tumor metastases and cancer in lymph nodes and the total progressionpathologic stage enrichment for discrete parameters was calculated using the χ2 testfor independence and for numeric parameters using kruskalwallis test not all clinicalparameters were available for all cancer types so a total of clinical parameters wereavailable for testing to derive a pvalue for the logrank test the χ2 test for independence the kruskalwallis test and the statistic for these three tests is assumed to have χ2distribution preprocessingtcga datasets were preprocessed as follows patients and features with more than missing values were removed and missing values were imputed using knearest neighborimputation the sequence features were logtransformed the features with highestvariance from geneexpression and methylation omics were selected in the mirna omicfeatures with zero variance were filtered all features were then normalized to have zeromean and standard deviation for methylation we selected the features with maximal variance in each dataset and also adopted the standard pipeline proposed in whose procedure filters out the probes from the x and y chromosomes or probes that areknown to have common snps at the cpg sitea further unsupervised variable selection step has been performed by using the meandecrease gini based on random forest the mean decrease in gini is the average of a variable total decrease in node impurity weighted by the proportion of samplesfig mean performance of the algorithms on ten multiomics cancer datasets the xaxis measures thedifferential survival between clusters mean log10 of logrank™s test pvalue and the yaxis is the meannumber of clinical parameters enriched in the clusters 0cciaramella bmc bioinformatics 21suppl page of fig performance of the algorithms on ten multiomics cancer datasets for each plot the xaxis measuresthe differential survival between clusterslog10 of logrank™s test pvalue and the yaxis is the number ofclinical parameters enriched in the clusters red vertical lines indicate the threshold for significantly differentsurvival pvalue cid2 reaching that node in each individual decision tree in the forest this is effectively a measure of how important a variable is for estimating the value of the target variable acrossall of the trees that make up the forest a higher mean decrease in gini indicates highervariable importance therefore the most important variables to the model is the highestin the plot with the largest mean decrease in gini values conversely the least importantvariable is the lowest in the plot with the smallest mean decrease in gini values by following this strategy we cutoff all those variables whose importance is zero the numberof variable cutoff at each step is summarised in table experimental resultsin the experiments for all methods the number of searched clusters is selected in therange [ ˆ’ ] to determine the number of clusters for a method we used the œelbowmethod to automatically pick out the optimal elbow rather than choose it manually weused as approximation the second derivative of a vector vv [i ] v [i ˆ’ ] ˆ’ 2v[ i] in particular we consider the index i that brings this expression to a maximum or minimum depending on whether v increases or decreases for all methods we adhered tothe guidelines for usage and parameter selection given by the developers in some casestable performance on ten multiomics number of clinical parameters enriched in the clustersfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeans 0cciaramella bmc bioinformatics 21suppl page of table performance on ten multiomics differential survival between clusters log10 of logrank™stest pvaluefhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeanswhere no information was provided by the authors we devised parameter selection methods we performed the same process pipeline used in for evaluating the performanceof our method all methods were run on a multicore intelr xeonr cpu e52620v3 240ghz with gb ram in the following the obtained experimental results aredescribedfigure shows the average performance for multiomics data and for each singleomicseparately across all cancer types and fig shows the performance on the different cancer datasets all algorithms show quite similar performance in either differential survivalor enriched clinical parameters with respect to survival our fhclust method achievedthe overall best prognostic value sum of ˆ’log10 pvalues while pins and mcca ranked second and third respectivelyin table the differential survival between clusters mean ˆ’log10 of logrank™s testpvalue are reported spectral achieved the highest total number of significant clinical parameters with parameters fhclust along with lracluster and kmeansplaced themselves second with parameters snf achieved the third position with parameterswith respect to survival table fhclust outperformed its competitors achieving parameters mcca pins and snf have achieved good results with and enriched parameters respectivelywe also counted the number of datasets for which a method solution obtains significantly different survival these results are reported in table all methods that weredeveloped for multiomics data had at least four cancer types with significantly differentsurvival in this case fhclust and pins had different cancer subtypes for which itsclustering had significantly different prognosis fhclust spectral clustering and mccahad enrichment in cancer typeson average fhclust pins and mcca had better prognostic value but found lessenriched clinical labels as compared to spectral clustering methodtable for each benchmarked algorithm the number of cancer subtypes for which its clusteringhad significantly different prognosis first row and had at least one enriched clinical label secondrow are shownsignificant different survivalsignificant clinical enrichmentfhclustkmeansspectrallraclusterpinssnfmcca 0cciaramella bmc bioinformatics 21suppl page of table number of clusters chosen by the benchmarked algorithms on ten multiomics cancerdatasetsfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccaamlbiccoadgbmkirclihcluscskcmovsarcmeansthe number of clusters found for each dataset are presented in table ranging from to because of the good methods performance in the previous analysis partitioning thedata into a relatively high number of clusters could indicate that clustering cancer patientsinto more clusters improves prognostic value and clinical significanceconcerning with methods computational burden their run times are reported intable fhclust takes on average seconds per dataset while spectral and snf gotlower timing the worst method takes roughly minutes per dataset see fig finally fig shows the benchmarked methods performance for singleomic datamoreover for each dataset and method the single omic that gave the best results forsurvival and clinical enrichment are also shown these results suggest that fhclust provides better prognostic value and clinical significance on multiomics data compared tothe analysis of singleomic data used separately nevertheless the interested reader mayrefer to the supplementary material for details on additional results concerning singleomics we also stress that the proposed method differently from other methods suchas snf does not need any hyperparameter tuning moreover clustering is embeddedin the data integration and vice versa and the use of fuzzy concepts ie tnormsfrom one hand permits to obtain a generalisation of the clustering approaches whereason the other hand gives the possibility to apply an inference system eg mamdanifor a quantitative and qualitative measure eg œhigh œmedium œlow in cancer riskassessmentsin this work we proposed a multiview clustering methodology for identifying patientsubgroups from different omics data in biological and biomedical fields combining theseomics data can significantly increase data mining capabilities one of the main aspects oftable runtime in seconds of the algorithms on ten multiomics cancer datasetsovfhclustcrisp hclustkmeansspectrallraclusterpinssnfmccacoadskcmbicamlgbmkirclihcluscsarcmeans 0cciaramella bmc bioinformatics 21suppl page of fig computational time comparisonsthis methodology is the use of a measure of dissimilarity between sets of observations byusing an appropriate metric and a consensus matrix that is a representative agglomerateinformation of all the dendrograms as emerged from the analysis of the scientific literature to the best of our knowledge our work concerns for the first time a model based onfuzzy logic used for the agglomeration of multiomic data the use of fuzzy logic allowsus to introduce more flexible data mining features also related to approximate reasoningseveral experiments and comparisons have been made on real data eg glioblastomaprostate cancer to assess the proposed methodology the results suggest that fhclustprovides better prognostic value and clinical significance compared to analysis of singleomic data alone fuzzy logic concepts and in particular membership functions permitsfig summarized performance of the algorithms across ten cancer datasets for each plot the xaxismeasures the total differential prognosis between clusters sum across all datasets of “log10 of logrank™s testpvalue and the yaxis is the total number of clinical parameters enriched in the clusters across all cancertypes a“c results for singleomic datasets d results when each method uses the single omic that achievesthe highest significance in survival e same with respect to enrichment of clinical labels 0cciaramella bmc bioinformatics 21suppl page of to develop a fuzzy inference model ie mamdani fuzzy cognitive maps for easilyobtaining a model for a quantitative and qualitative risk assessment of the cancer themodel based on approximate reasoning can be particularly useful for embedded devicesin future work it could be possible to improve results for multiomics analysis ina number of ways for instance more accurate feature selection algorithms couldbe adopted for improving the overall performance on one hand the integration oflabelled data could improve the feature selection step on the other hand some specific feature extraction strategies could be adopted indeed approaches based on thesignal analysis of gene expression data eg nonlinear principal component analysis compressive sensing could possibly further improve the performance [ ]in future it is possible to foresee a different weight for each omic data in order toobtain a more robust similarity and parametric similarity measures can be adoptedeg uninorm for generalizing the concept of and and or connections betweenclusterssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12859020035676additional file supplementary materialabbreviationsfhclust fuzzyhierarchical clustering dna deoxyribonucleic acid rna ribonucleic acid hierarchical clustering hccrisp hc crisp hierarchical clustering fl fuzzy logic tcga the cancer genome atlas aml acute myeloid leukemia bicbreast invasive carcinoma coad colon adenocarcinoma gbm glioblastoma multiforme kirc kidney renal clear cellcarcinoma lihc liver hepatocellular carcinoma lusc lung squamous cell carcinoma skcm skim cutaneousmelanoma ov ovarian serous cystadenocarcinoma sarc sarcoma pins perturbation clustering for data integrationand disease subtyping lracluster low rank approximation based multiomics data clustering snf similarity networkfusion mcca multi canonical correlation analysisabout this supplementthis has been published as part of bmc bioinformatics volume supplement proceedings from the 13thbioinformatics and computational biology international conference bbcc2018 the full contents of the supplement areavailable online at httpsbmcbioinformaticsbiomedcentralcomssupplementsvolume21supplement10authors™ contributionsac originally designed the methodology ac and dn worked on the developing of the method and the design of theexperiments ac dn and as contributed for interpreting and for analysing the results all authors contributed forwriting the manuscript read and approved the final manuscriptfundingpublication costs are funded by a grant from the dipartimento di scienze e tecnologie università degli studi di napoliœparthenope tecniche di machine learning e soft computing per l™elaborazione di dati multivariati softmulan piciaramellaavailability of data and materialscode and data of the proposed approach are available on multiomicscancerbenchmark github repositoryethics approval and consent to participateno ethics approval was required for the studyconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsauthor details1dipartimento di scienze e tecnologie università degli studi di napoli œparthenope centro direzionale c4 island naples italy 2hitachi rail sts via argine naples italypublished august 0cciaramella bmc bioinformatics 21suppl page of referencescamastra f di taranto md staiano a statistical and computational methods for genetic diseases an overviewcomput math meth med 20152015 id “serra a fratello m fortino v raiconi g tagliaferri r greco d mvda a multiview genomic data integrationmethodology bmc bioinformatics “rappoport n shamir r multiomic and multiview clustering algorithms review and cancer benchmark nucleicacids res “reddy ck aggarwal cc data clustering boca raton chapman and hallcrc camastra f ciaramella a son lh riccio a staiano a fuzzy similaritybased hierarchical clustering for atmosphericpollutants prediction lncs “ciaramella a staiano a on the role of clustering and visualization techniques in gene microarray data algorithmsbora dj gupta ak int j emerg trends technol comput sci “napolitano f pinelli m raiconi g tagliaferri r ciaramella a staiano a miele g clustering and visualizationapproaches for human cell cycle gene expression data analysis int j approx reason “ciaramella a cocozza sand clustering of genomic data neural netw “iorio f miele g napolitano f pinelli m raiconi g tagliafer
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"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 œposed 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 cells”which 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 œsex or œgender 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 women™s 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 women™s 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 charit”universittsmedizin berlin berlin germany prof v regitzzagrosek md department of cardiology university hospital z¼rich university of z¼rich switzerland prof v regitzzagrosek center for women™s 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 ·influenza and pneumonia ·suicide ·alzheimer™s disease ·type diabetes ·stroke ·cpd ·injuries ·female individualsother·septicaemia ·chronic kidney disease ·influenza and pneumonia ·type diabetes ·injuries ·alzheimer™s 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 women™s hearts develop less inflammation resulting in less fibrosis than men™s 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 women™s 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
breast cancer patients especially those with triple‘negative breast cancer is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancer Although collagen type VIII alpha chain COL8A1 has been shown to be downregulated in BRIP1‘knockdown breast cancer cells its clinical role in breast cancer remains unknownMethods Gene microarrays and mRNA sequencing data were downloaded and integrated into larger matrices based on various platforms Therefore this is a multi‘centered study which contains breast cancer patients and controls COL8A1 mRNA expression in breast cancer was compared between molecular subtypes In‘house immunohistochemistry staining was used to evaluate the protein expression of COL8A1 in breast cancer A diagnostic test was performed to assess its clinical value Furthermore based on differentially expressed genes DEGs and co‘expressed genes CEGs positively related to COL8A1 functional enrichment analyses were performed to explore the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerResults COL8A1 expression was higher in breast cancer patients than in control samples standardized mean differ‘ence confidence interval [CI] “ Elevated expression was detected in various molecular subtypes of breast cancer An area under a summary receiver operating characteristic curve of CI “ with sensitivity of CI “ and specificity of CI “ showed moderate capacity of COL8A1 in distinguishing breast cancer patients from control samples Worse overall survival was found in the higher than in the lower COL8A1 expression groups Intersected DEGs and CEGs positively related to COL8A1 were significantly clustered in the proteoglycans in cancer and ECM‘receptor interaction pathwaysConclusions Elevated COL8A1 may promote the migration of breast cancer by mediating the ECM‘receptor interac‘tion and synergistically interplaying with DEGs and its positively related CEGs independently of molecular subtypes Several genes clustered in the proteoglycans in cancer pathway are potential targets for developing effective agents for triple‘negative breast cancerKeywords COL8A1 Breast cancer Immunohistochemistry staining MechanismCorrespondence fengzhenbo_gxmu163com chenganggxmueducn Wei Peng and Jian‘Di Li contributed equally as first authors Department of Pathology The First Affiliated Hospital of Guangxi Medical University NO6 Shuangyong Road Nanning Guangxi People™s Republic of ChinaFull list of author information is available at the end of the BackgroundBreast cancer poses a grave threat to female health According to the latest American cancer statistics breast cancer is estimated to be the most common cancer and the second most common cause of cancerassociated 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 0cPeng a0et a0al Cancer Cell Int Page of deaths in women [] Owing to higher distal metastatic and recurrent rates triplenegative breast cancer TNBC patients exhibit worse overall and diseasefree survival than any other type of breast cancer [“] Etiology investigations suggest that hereditary factors account for nearly onetenth of breast cancer cases Other risk factors such as early or delayed menstruation nulliparity hormone replacement therapy and alcohol consumption also contribute to the prevalence of breast cancer [] Clinical practice guidelines recommend that females aged “ or “ who are at higher risk screen for breast cancer [] Imaging examinations such as bilateral breast Xray imaging positron emission tomography“computed tomography and ultrasound histological findings and especially molecular pathology are the predominant methods of breast cancer diagnosis and assessment [ ] Based on the tumor burden optimal treatments namely breastconserving surgery radiotherapy chemotherapy and endocrine therapy are individually designed for breast cancer patients [] For TNBC patients anthracyclines and taxanes are preferred in the initial treatment and neoadjuvant therapy has been recognized as a standard strategy [“] Unfortunately neither endocrine therapy nor trastuzumab treatment is effective for TNBC Targeted drugs for example vascular endothelial growth factor [VEGF] antibodies epidermal growth factor receptor [EGFR] inhibitors and mammalian target of rapamycin [mTOR] inhibitors are gradually being employed in TNBC treatment even though their therapeutic effects are unsatisfactory [“] Therefore the situation faced by breast cancer”especially TNBC”patients is still grave More effective therapeutic targets are needed to optimize the clinical management of breast cancerMolecular events occurring in breast cancer help us better understand the onset and progression of breast cancer It is generally agreed that chromosome 1q amplification chromosome 16q deletion and PIK3CA mutations are the most common pathways leading to luminal breast cancer [ ] Moreover breast cancer gene BRCA1 and P53 mutations EGFR upregulation and cytokeratin downregulation have been associated with TNBC [ ] It has also been reported that circSEPT9 promotes tumor formation and TNBC progression [] Recently ˆ†Np63 has been found to participate in breast cancer metastasis and dissemination [] On the other hand several genes protect patients from breast cancer progression For example ZNF750 miR5745p and circKDM4C can inhibit breast cancer progression by mediating the epigenetic regulation of prometastatic genes indirectly suppressing SKILTAZCTGF and miR548pPBLD axis regulation [“] Based on these discovered molecular mechanisms some progress has been made in breast cancer treatment Delivering dual microRNA using CD44targeted mesoporous silica nanops proved to be effective in TNBC treatment [] Although many studies provided in a0vitro and in a0vivo a detailed molecular picture of TNBC cancers [“] the underlying cause of breast cancer has not been fully understood Further research is required to elucidate the breast cancer mechanisms and discover effective therapeutic targets for TNBCCollagen type VIII alpha chain COL8A1 also named C3orf7 is located at chromosome and encodes alpha chain in collagen type VIII which is an essential component of extracellular matrix ECM [] Previous studies mainly addressed the relevance between COL8A1 and agerelated macular degeneration ADM as well as cell proliferation [“] Recently limited studies demonstrate the deregulation of COL8A1 in various cancers Elevated COL8A1 expression was found in gastric cancer patients and higher COL8A1 correlated with advanced tumor stages and worse overall survival condition and COL8A1 was selected as a candidate diagnostic biomarker in gastric cancer [“] Additionally upregulation of COL8A1 was also reported in adamantinomatous craniopharyngioma [] Furthermore COL8A1 proved to participate in the progression of colon adenocarcinoma possibly by mediating focal adhesionrelated pathways [] COL8A1 upregulation induced by TGFβ1 was found in renal cell carcinoma carcinogenesis and also correlated with poor prognosis [] Moreover elevated COL8A1 in hepatocellular carcinoma promoted tumor cells proliferation invasion and in a0vivo tumorigenicity [] Thus far only few studies mentioned COL8A1 in breast cancer COL8A1 was one of the key genes restored by epigallocatechin3gallate in a murine breast cancer model [] COL8A1 proved downregulated in both BRIP1knockdown breast cancer cells and MCF10A a0CDH1 noncancer breast cells [ ] However the role of COL8A1 in breast cancer remains unknownConsidering this knowledge gap our study aimed to explore the role of COL8A1 in breast cancer We were focused on investigating the expression of COL8A1 messenger RNA mRNA in various molecular subtypes of breast cancer by analyzing gene microarray and RNA sequencing data sets The COL8A1 protein expression level was validated by immunohistochemistry staining We also aimed to determine prognostic value of COL8A1 in breast cancer to pave the way for future clinical applications Moreover we explored the molecular mechanisms of COL8A1 underlying breast cancer to improve our knowledge of breast cancer carcinogenesis and progression 0cPeng a0et a0al Cancer Cell Int Page of MethodsExpression of a0COL8A1 mRNA in a0breast cancerWe integrated gene microarrays and mRNA sequencing data downloaded from Gene Expression Omnibus The Cancer Genome Atlas TCGA the GenotypeTissue Expression the Sequence Read Archive ArrayExpress and Oncomine The search formula based on MESH terms was as follows Breast OR mammary AND neoplasm OR cancer OR adenoma OR carcinoma OR tumor OR BRCA OR neoplasia OR malignant OR malignancy Studies were screened according to the following criteria i the studied species should be Homo sapiens ii the studied specimens should be tissue dissected from patients or healthy individuals rather than cell lines In the case of duplicated studies or samples the most recent version was retained The exclusion criteria were as follows i expression profiles not including COL8A1 ii breast cancer patients receiving hormone therapy or chemotherapy iii stromal rather than epithelial tumors and iv metastatic rather than primary tumors The included data sets were carefully checked and a log2 transformation was performed if any matrices had not been normalized Additionally the data sets were integrated into larger matrices according to various platforms and batch effects between studies were removed using the limmavoom package in R v361 Subsequently COL8A1 expression values were extracted and grouped according to specimen types Standardized mean difference SMD were calculated to compare the expression of COL8A1 mRNA between breast cancer patients and control samples using STATA v120 Heterogeneity between the included studies was assessed with the I2 statistic Statistical significance was set to an I2 value greater than with a Pvalue less than A random effects model was used in the case of significant heterogeneity Sensitivity analysis was performed to probe the potential source of heterogeneity and a publication bias test was used to evaluate the stability of the SMD results Subgroup analysis was performed to compare the COL8A1 expression levels between molecular subtypes luminal A luminal B human epidermal growth factor receptor 2positive [HER2 ] and TNBCDiagnostic value of a0COL8A1 in a0breast cancerA diagnostic test was performed to assess the clinical significance of COL8A1 in breast cancer Based on the expression value of COL8A1 a receiver operating characteristic ROC curve was plotted to compute the area under the curve AUC using IBM SPSS Statistics v190 AUC values of less than between and and greater than represented weak moderate and strong discriminatory capability of COL8A1 respectively between breast cancer patients and control samples The true positives false positives true negatives and false negatives rates were calculated and the cutoff values were identified A summary receiver operating characteristic sROC curve was drawn using STATA v120 to assess the general discriminatory capability of COL8A1 between breast cancer patients and control samples The significance of the area under the sROC curve was consistent with that of the ROC curve The diagnostic odds ratio DOR sensitivity specificity positive diagnostic likelihood ratio DLR P and negative diagnostic likelihood ratio DLR N were also calculated to precisely determine the accuracy and validity of COL8A1 in distinguishing breast cancer patients from control samplesPrognostic value of a0COL8A1 in a0breast cancerTo explore the relation between COL8A1 mRNA expression and prognosis of breast cancer patients information on clinicopathological parameters was collected The independent samples ttest or oneway analysis of variance was used to identify statistically significant differences in COL8A1 expression between two or more groups A Pvalue of was considered statistically significant Kaplan“Meier curves were used to compare high and low COL8A1 expression groups in terms of survival The logrank test was used to determine whether the prognostic difference was statistically significantInvestigation of a0COL8A1 protein expression in a0breast cancer by a0immunohistochemistry stainingA total of nonspecific invasive breast carcinoma and normal breast tissue specimens were obtained from the First Affiliated Hospital of Guangxi Medical University PRCHINA All patients had previously signed informed consent forms and our research was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University The breast cancer and normal breast tissue specimens were fixed with formalin The two steps immunohistochemistry method was used to determine the protein expression of COL8A1 The primary antibody was polyclonal Antibody to COL8A1 concentrated dilution purchased from Wuhan Pujian COLTD Supervision TM MouseRabbitHRP Broad Spectrum Detection System Product No D300415 was purchased from Shanghai Long Island The experimental procedure conformed to the manufacturer™s instructions The patients™ clinicopathological information was analyzed to determine the relationship between COL8A1 protein expression and prognosisEvaluation of a0genetic alteration and a0mutation landscapesThe cBioPortal for Cancer Genomics httpcbiop ortal proved to be a powerful tool and facilitated our online search and cancer genomics data analysis Using 0cPeng a0et a0al Cancer Cell Int Page of cBioPortal we gained insight into the genetic alterations of COL8A1 in breast cancer patients and obtained information on the association between COL8A1 alterations and breast cancer patient survival We selected the Breast Invasive Carcinoma TCGA Firehose Legacy cohort which contains patients and used a method of mRNA expression zscores relative to diploid samples RNASeqV2 RSEM We also considered the mutation types of COL8A1 in breast cancer patients using the Catalogue of Somatic Mutations in Cancer COSMIC which has been recognized as the most detailed resource for somatic mutations in cancerIdentification of a0differentially expressed genes and a0COL8A1 co‘expressed genes in a0breast cancerTo gain insight into the role of COL8A1 in breast cancer we identified DEGs and COL8A1 CEGs using the limmavoom package The DEG criteria were as follows ilog2FoldChange and ii adjusted Pvalue The CEG criteria were as follows irelation coefficient and ii Pvalue Upregulated DEGs and CEGs positively related to COL8A1 were intersected Similarly downregulated DEGs and CEGs negatively related to COL8A1 were intersectedMolecular mechanisms of a0COL8A1 underlying breast cancerOverlapping genes were used to perform function enrichment to shed light on the potential mechanisms of COL8A1 underlying breast cancer The R clusterProfiler package was used to conduct Gene Ontology GO Kyoto Encyclopedia of Genes and Genomes KEGG Disease Ontology DO and Reactome pathway analyses Proteintoprotein interaction PPI network was constructed using STRING https strin gdb to investigate protein interactions Hub genes and functional modules were identified using Cytoscape v361 Based on breast cancer patients the mutation landscapes of genes clustered in essential pathways were visualized using the TCGAmutations package in R v361ResultsUpregulation of a0COL8A1 mRNA in a0breast cancerAdditional file a0 Figure S1 shows the flow diagram of the study inclusion process A total of studies were included and integrated into larger platform matrices covering breast cancer patients and controls Table a0 COL8A1 was generally upregulated in breast cancer compared to normal breast tissue Thirteen of the twenty platforms showed much higher COL8A1 expression in breast cancer patients than in control samples Additional file a0 Figure S2 Because of significant heterogeneity I2 P a random effects model was used An SMD value of confidence interval [CI] “ showed that COL8A1 expression was significantly higher in breast cancer than in nonbreast cancer tissue Fig a0 Sensitivity analysis indicated that the included studies could not explain the source of heterogeneity Additional file a0 Figure S3a No publication bias existed Additional file a0 Figure S3b Subsequently we compared COL8A1 expression levels between different subtypes of breast cancer COL8A1 expression was universally higher in luminal A luminal B HER2 and TNBC patients than in control samples Additional file a0 Figure S4 Additional file a0 Figure S5 and Additional file a0 Figure S6a Furthermore three platforms showed significantly higher COL8A1 expression in nonTNBC than TNBC while only one showed lower expression in nonTNBC than TNBC Additional file a0 Figure S6b However an SMD of ˆ’ CI ˆ’“ showed no difference in COL8A1 expression between TNBC and nonTNBC patients Additional file a0 Figure S7 Subgroup analysis of four molecular subtypes of breast cancer showed no significant differences in COL8A1 expression levels between them Fig a0Diagnostic and a0prognostic value of a0COL8A1 mRNA in a0breast cancerThe clinical value of COL8A1 in breast cancer was found to be promising Among the thirteen platforms showing high COL8A1 expression twelve platforms indicated the ability of COL8A1 in differentiating breast cancer patients and control samples where four platforms showed strong discriminatory capability of COL8A1 between breast cancer patients and control samples Additional file a0 Figure S8 An area under the sROC curve of CI “ with sensitivity of CI “ and specificity of CI “ displayed moderate capacity in distinguishing breast cancer patients from control samples Fig a03a A DOR of CI “ also highlighted the discriminatory ability of COL8A1 in breast cancer Fig a03b The DLR P and DLR N were CI “ and CI “ respectively Additional file a0 Figure S9 As shown in Additional file a0 Figure S10 and Additional file a0 Table a0S1 elevated COL8A1 expression correlated with race white black molecular subtypes of breast cancer luminal B luminal A TNBC ER PR and HER2 status Moreover Kaplan“Meier curves indicated worse overall survival in high compared to low COL8A1 expression groups Fig a0Expression levels and a0clinical significance of a0COL8A1 protein in a0breast cancerClinical data of breast cancer samples used to perform in immunohistochemistry was summarized 0cPeng a0et a0al Cancer Cell Int Page of ldnaoPESGi abarA iduaSESG ecnarFESG ASUESG inapSESG ecnarFESG ecnarFESG adanaCESGkramneDESGi eropagnSESGl yatIESG ASUESG ynamreGESG inapSESG ASUESGASUESG cil bupeRhcezCESG ldnaerIESGi eropagnSESGASUESG ailartsuAESG iocxeMESGi abarA iduaSESGASUESG ASULPG‘ESGadanaCESG ASUESG adanaCESGASULPG‘ESGASULPG‘ESGadanaCESGianhCESG anhCi ESG ASUESG ASUESGianhCESGl aisyaaMESGi anhCESGadanaCESGi anhCESGASUESGi anhCESGASUESGi eropagnSESGl aisyaaMESGASUESG ESG ESG ESG ESG ESGASUESG ESGASU ESGASU ESGanhCi ESGynamreG ESGASU ESGˆ’‘ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’LPGLPGLPGLPGLPGLPGLPGˆ’LPGLPGLPGLPGLPGLPGLPGxETG‘AGCTESGESGESGESGESGsrebmun latoTseirtnuoC seireSOEG NTNFPFPTleuav‘PfdtlortnoC ACRBnoisseccADSMNDSMN stesataddell a0 orneeht a0fonoitamrofni cisaB elbaT 0cPeng a0et a0al Cancer Cell Int Page of Fig General expression status of COL8A1 in breast cancer BRCA compared to non‘BRCA tissues A standardized mean difference SMD value and 95CI with no overlap of zero indicated COL8A1 was significantly upregulated in BRCA compared to non‘BRCA tissuesAdditional file a0 Table a0 S2 Protein expression confirmed the upregulation of COL8A1 in breast cancer The breast cancer patients whose specimens were analyzed in this study were aged between and mean and their followup durations ranged from to a0 days Immunohistochemistry staining showed varying coloration intensity of COL8A1 in breast cancer and normal breast tissue COL8A1 was negatively weakly moderately or strongly stained in normal breast epithelium Fig a05a“d and breast cancer tissue Fig a0 5e“h According to the staining intensity and color range percentages of breast cancer tissue specimens exhibited low and exhibited high COL8A1 expression whereas of normal breast tissue specimens exhibited low and exhibited high COL8A1 expression A Chi square test confirmed the significantly higher expression of COL8A1 in breast cancer than normal breast tissue χ2 P Moreover elevated COL8A1 expression correlated with estrogennegative ER breast cancer P Genetic alterations and a0mutation kinds of a0COL8A1 in a0breast cancerAlterations and mutations of COL8A1 in breast cancer were relatively frequent Based on cBioPortal COL8A1 was altered in of breast cancer patients Additional file a0 Figure S11 Amplification and high and low mRNA were the main alterations No statistically significant difference in overall and diseasefree survival was found between high and low COL8A1 expression breast cancer groups P Furthermore according to COSMIC substitution missense mutations were the most frequent types Additional file a0 Table a0S3DEGs and a0COL8A1 CEGs in a0breast cancerA total of platform matrices were collected to determine the DEGs The approach has been aforementioned Initially upregulated DEGs downregulated DEGs CEGs positively related to COL8A1 and CEGs negatively related to COL8A1 were identified Additional file a0 Figure S12 shows partial DEGs and CEGs After being intersected 0cPeng a0et a0al Cancer Cell Int Page of Fig Subgroup analysis based on the subtypes of breast cancer The result indicated that the elevated COL8A1 expression shared no significant difference among Luminal A Luminal B HER‘ and Three Negative Breast Cancer TNBC subgroupsthe DEGs and CEGs were divided into two gene sets overlapping upregulated DEGs and CEGs positively related to COL8A1 all genes appeared in no fewer than three data sets and overlapping downregulated DEGs and CEGs negatively related to COL8A1Potential mechanisms of a0COL8A1 underlying breast cancerThe GO KEGG DO and Reactome pathway analyses based on the intersected genes are shown in Additional file a0 Table a0S4 Regarding the overlapping upregulated DEGs and CEGs positively related to COL8A1 the following KEGG pathways were significantly aggregated 0cPeng a0et a0al Cancer Cell Int Page of Fig Diagnostic value of COL8A1 in breast cancer BRCA a Summary receiver operating characteristic sROC curve b Forest plot of diagnostic odd ratio DOR An AUC value and a DOR signified COL8A1 possessed moderate capability in distinguishing BRCA from non‘BRCA patients AUC area under the curve 0cPeng a0et a0al Cancer Cell Int Page of Fig The prognostic value of COL8A1 mRNA in breast cancer tissues a GSE25307 b GSE35629‘GPL1390 c TCGA In the GSE25307 cohort high COL8A1 group possessed poor overall survival condition compared to low COL8A1 group in breast cancer patients 0cPeng a0et a0al Cancer Cell Int Page of See figure on next pageFig Protein expression levels of COL8A1 in breast cancer BRCA and normal breast tissues a‘d normal breast tissues e“h BRCA tissues Magnification × COL8A1 was negatively stained in normal breast epithelium a and BRCA e COL8A1 was weakly stained in normal breast epithelium b and BRCA f COL8A1 was moderately stained in normal breast epithelium c and BRCA g COL8A1 was strongly stained in normal breast epithelium d and BRCA h According to staining intensity and percentage of color range BRCA tissues exhibited low COL8A1 expression and BRCA tissues exhibited high COL8A1 expression While normal breast tissues exhibited low COL8A1 expression and normal breast tissues exhibited high COL8A1 expressionFig a06a proteoglycans in cancer Additional file a0 Figure S13 ECMreceptor interaction Additional file a0 Figure S14 and several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly genes WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B BAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though is not in the top KEGG pathways Moreover DO analysis indicated that these genes are closely associated with myeloma and bone marrow cancer Fig a06b as well as renal cell carcinoma ovarian cancer renal carcinoma and other types of cancer Furthermore Reactome pathway analysis revealed extracellular matrix anization ECM proteoglycans integrin cell surface interactions and degradation of the extracellular matrix as the top four metabolic pathways Fig a0 6c Regarding GO enrichment extracellular matrix anization extracellular matrix and extracellular matrix structural constituent were the most clustered Biological Process BP Cellular Component CC and Molecular Function MF terms respectively Fig a07a The proteoglycans in cancer and ECMreceptor interaction pathways were selected to construct PPI networks Fig a07b c FN1 and ITGB1 were identified as the hub genes in the two networks respectively The mutation landscapes of the genes in these two important pathways are shown in Fig a0 In particular FN1 was altered in of breast cancer samples where missense mutations accounted for The regulatory networks of COL8A1 and enriched genes in the proteoglycans in cancer and ECMreceptor interaction pathways as well as the top two functional modules are displayed in Additional file a0 Figure S15 On the other hand the enrichment results regarding the overlapping CEGs negatively related to COL8A1 and downregulated DEGs showed no statistical significance these data are therefore not shown Additional file a0 Table a0S5DiscussionThe highlight of this study is that it comprehensively explored the upregulation of COL8A1 mRNA in breast cancer from multiple aspects based on breast cancer patients and controls Our study is multicentered because we collected breast cancer patients from Asia American Europe and Oceania covering different countries This is the first study to investigate the protein expression of COL8A1 in breast cancer using immunohistochemistry staining Moreover this study is the first to assess the clinical prognostic value of COL8A1 in breast cancer Furthermore our study sheds light on the biological function and potential molecular mechanisms of COL8A1 underlying breast cancerThis study demonstrates the upregulation of COL8A1 in breast cancer We found higher COL8A1 expression in breast cancer than normal breast tissue based on large platform matrices integrated from data sets Subgroup analysis based on four molecular subtypes of breast cancer showed that elevated COL8A1 expression is independent of subtypes Further analysis also revealed universally higher COL8A1 expression levels in luminal A luminal B HER2 and TNBC patients than in control samples A comparison of COL8A1 expression between nonTNBC and TNBC patients showed no statistically significant difference Immunohistochemistry staining confirmed the upregulation of COL8A1 protein in breast cancer We thus concluded that COL8A1 expression is higher in breast cancer patients than in control samples and that upregulation is independent of molecular subtypes of breast cancer Though the expression of COL8A1 in tissue cannot reflect the early diagnostic value in breast cancer we assume that if COL8A1 is also differentially expressed in the bodily fluid of patients it will be possible to serve as a potential diagnostic marker for breast cancer Nevertheless no previous studies have demonstrated the expression level of COL8A1 in the bodily fluid of breast cancer patients until nowWe determined the clinical value of COL8A1 in breast cancer for the first time Our diagnostic test showed a moderate discriminatory capability of COL8A1 between breast cancer and normal breast tissue Higher COL8A1 expression levels in breast cancer patients correlated with worse survival outcomes Additionally COL8A1 expression was much higher in patients of the white than of the black race Our TCGA cohort analysis showed lower COL8A1 upregulation levels in TNBC than in the luminal A and B subtypes Furthermore high COL8A1 protein levels were related to ER breast cancer Thus COL8A1 might serve as a prognostic marker for breast cancer 0cPeng a0et a0al Cancer Cell Int Page of 0cPeng a0et a0al Cancer Cell Int Page of Fig Functional enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a Kyoto Encyclopedia of Genes and Genomes b Disease Ontology c Reactcome DEGs differentially expressed genes CEGs co‘expressed genes 0cPeng a0et a0al Cancer Cell Int Page of Fig GO enrichment based on overlapping genes of upregulated DEGs and COL8A1 positively related CEGs a GO analysis b Protein‘to‘protein internet based on KEGG pathway proteoglycans in cancer ID hsa05205 c Protein‘to‘protein internet based on KEGG pathway ECM‘receptor interaction ID hsa04512 GO Gene Ontology DEGs differentially expressed genes CEGs co‘expressed genes KEGG Kyoto Encyclopedia of Genes and GenomesMore importantly our study provides important clues about the role of COL8A1 in breast cancer for the first time The intersected CEGs positively related to COL8A1 and upregulated DEGs were significantly aggregated in several cancer pathways such as thyroid cancer colorectal cancer and hepatocellular carcinoma Interestingly we noticed that genes related to COL8A1 WNT2 GADD45B FZD2 CDKN1A KRAS LEF1 WNT7B 0cPeng a0et a0al Cancer Cell Int Page of Fig Mutation landscapes of COL8A1 and co‘expressed genes clustered in two Kyoto Encyclopedia of Genes and Genomes pathways proteoglycans in cancer and ECM‘receptor interactionBAK1 BRCA2 CDK4 GRB2 HRAS PIK3R3 and POLK were associated with the breast cancer pathway ID hsa05224 even though this pathway is not in
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" 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 pearson™s 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 cancercenter™s 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 × bp the data wereprocessed according to the manufacturer™s protocol the mean coverage was × and × 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 ×candidate 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 pearson™s 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 695eˆ’ to 452eˆ’ 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 3k“80k 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 ia“iiia 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 a“f 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 iiib“iv 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 3k“80k 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 “× 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 œaccompanying 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 lyu™s 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 people™s republic of china for their contribution tothe st
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"Previous evidence has suggested that lower gestational vitamin D levels might increase the risks ofadverse pregnancy and birth outcomes The results remain inconsistent and require further explorationMethods A total of Chinese motherinfant pairs were included in this retrospective cohort study Serumconcentrations of 25OHD were reviewed in early pregnancy ± weeks Outcomes of maternal gestationaldiabetes mellitus GDM cesarean section fetal distress preterm birth low birth weight LBW and macrosomiawere extracted from the medical records Cox regression analysis was used to explore these associationsResults In total of mothers were pregnant at an advanced age ‰¥ years and of pregnant womenhad vitamin D deficiency nmolL After adjusting for potential covariates the hazard ratio HR CI perstandard deviation SD increase of serum 25OHD concentrations was for GDM for preterm birth and for LBW Similar protective associations were found for GDMcesarean section and preterm birth for a better vitamin D status when compared with vitamin D deficiencyConclusion Higher early pregnancy vitamin D was associated with a lower risk of GDM cesarean section pretermbirth and LBWKeywords 25ohd Vitamin D Pregnancy Maternal outcome Infant outcomeBackgroundVitamin D is a secosteroid hormone that is well knownfor its physiological function in maintaining bone metabolism and health A high prevalence of vitamin D deficiencyusually defined as serum 25OHD levels nmolL hasbeen found in pregnant women globally especially in developing countries [] including China [ ] Increasing evidence has suggested that vitamin D sufficiency is important Correspondence liuzphlk81outlookcom Gengdong Chen and Tingting Pang contributed equally to this work1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong ChinaFull list of author information is available at the end of the for the prevention of pregnancy complications in mothersand adverse fetal birth outcomes [ ] although divergentresults have been reported in other studies [ “] Severalsystematic reviews based on randomized clinical trials orobservational studies have suggested that lower vitamin Dstatus contribute to adverse outcomes such as preeclampsia[] gestational diabetes mellitus GDM [ ] low birthweight LBW [] and preterm birth [] However increasing evidence shows different associations [ ]and no definitive has yet been made [] Severalproblems remain to be solved by further studies the heterogeneity of associations from diverse areas and populations with different vitamin D status serve as evidencethewhen makingguidelinesregionsforspecific 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 Pregnancy and Childbirth Page of observation or supplementation of vitamin D in the thirdtrimester in many studies might present the problem ofcausal inference or miss the practical period for the intervention In addition the results from early trimesters mightbe helpful and more important for the early prevention ofadverse outcomesIn additionit has been suggested that the risk ofadverse complications or birth outcomes increases asmaternal age increases [“] With the change ofpopulation policy in China the percentage of womenpregnant at an advanced age years has increasedand proper strategies are urgently needed for the prevention of adverse complications [] However whetherthe influence of vitamin D on maternal and infant outcomes remained the same for women pregnant at young years and advanced ages remains unclear andmore studies are needed to better illustrate the problemWe investigated the relationship between early gestational serum 25OHD concentrations and several adverse maternal and infant outcomes in a retrospectivecohort study including Chinese motherinfantpairs Our results provide further evidence for clinicalrecommendations on the early prevention of related adverse outcomes in this fieldMethodsThe study used data that were gathered from a largecenter Affiliated Foshan Maternity Child HealthcareHospital Southern Medical University Foshan CityGuangdong Province China from September toJuly The hospital is the largest gynecology andobstetrics center in Foshan City and covers a large population of million people The included subjects werewomen who underwent early pregnancy serum vitaminD measurement ‰ gestational weeks and deliveredtheir infants at the hospital The exclusion criteria included twin or higherorder multiple pregnancies seriousdiseases such as type diabetes mellitus cardiovasculardiseases thyroid disorder and cancer that occurred before pregnancy Ultimately a total motherinfantpairs were included in this study The study was approvedby the ethics committee of Affiliated Foshan Maternity Child Healthcare Hospital Southern Medical UniversityData collectionVitamin D data from the clinicallaboratory werereviewed Blood samples were collected during the regular obstetric checkups and immediately measured by aclinical laboratory without being frozen Serum concentrations of 25OHD 25OHD2 and 25OHD3 weredetected using colloidal gold immunochromatographyCommercial kits were obtained from Mei Ning KangCheng Bio Tec Inc The intraassay and interassay coefficients of variation were less than Outcomes including GDM cesarean section fetal distress preterm birth low birth weight and macrosomiawere extracted from medical records and reexamined bytwo independent staff members The disease diagnoseswere made by professional doctors with the samestandardization criteria and were extracted from themedical records Gestational hypertension preeclampsiaor eclampsia was not included because of the lack ofavailable cases An oral glucose tolerance test was performed from to gestational weeks and GDM wasdiagnosed if the subjects met any of the following criteria fasting blood glucose ‰¥ mmolL onehour bloodglucose postoral sugar ‰¥ mmolL or twohour bloodglucose postoral sugar ‰¥ mmolL Preterm birth wasdefined as delivery at ‰¥ but gestational weeksLBW was diagnosed if the neonatal birth weight g while a neonatal birth weight ‰¥ g was defined asmacrosomia Other variablesincluding the maternalage body mass index BMI gestational age parity season of blood collection and time of delivery were alsoextracted from the medical recordsStatistical analysesContinuous variables were represented by the mean ±standard deviation SD or median interquartile rangeand tested by Student™s ttest Categorical variables wererepresented by frequencies percentage and tested bythe chisquare test Cox regression analysis was performed to explore the associations between vitamin Dand maternal or infant outcomes Serum concentrationsof 25OHD 25OHD2 and 25OHD3 were first Zstandardized before being included in the regressionVitamin D deficiency was defined as serum 25OHDlevels of nmolL Two different models were testedwith Model as a univariate model without adjustmentand Model adjusted for maternal age BMI parity andseason the blood was collected and measured Stratifiedanalyses were performed according to the gestationalage young years advanced ‰¥ years All the analyses were performed using SPSS software version SPSS Inc Chicago IL USA A twosided P value ofless than was considered statistically significantResultsA total motherinfant pairs were included in thisstudy A high prevalence of gestational vitamin D deficiency was discovered in the mothers Even insubjects without vitamin D deficiency the highest serum25OHD concentration was only nmolL Compared with women pregnant at a younger age the subjects with advanced age of pregnancy ‰¥ years tendedto have a higher BMI parity higher percentage of vitamin D deficiency higher incidence of GDM cesareansection preterm birth and LBW but a lower gestational 0cChen BMC Pregnancy and Childbirth Page of age lower serum 25OHD and OHD3 concentrations lower incidence of fetal distress and lower incidence of macrosomia Table As shown in Table although the 25OHD concentrations were statistically higher in spring and summerthan those in autumn and winter only small differenceof 25OHD values to nmolL was observedbetween different seasons especially for 25OHD2 Significant protective associations were found between thevitamin D levels and GDM and preterm birth Afteradjusting for potential covariatesTable higher25OHD concentrations per one SD increase were associated with a HR CI decrease in the GDM risk a HR CI decrease in the preterm birth risk and a HR CI decrease in theLBW risk Similar protective results were also found for25OHD2 and 25OHD3 but the associations tended tobe more pronounced for 25OHD2 Null associationsbetween vitamin D and cesarean section fetal distressand macrosomia were observed in all the subjects Maternal serum 25OHD levels ‰¥ nmolL were associated with a decrease in the GDM risk a decrease in the cesarean section risk and a decrease in the preterm birth risk but the trend did nothold with the other outcomes compared with those withvitamin D deficiency Table After stratification by gestational age Table higherlevels of vitamin D were associated with a lowerrisk of GDM in those years In contrast a protectiveassociation of vitamin D with low birth weight wasfound for women pregnant at ‰¥ years but not years However no significant interactions were discovered Pinteraction Table Characteristic of subjectsAge yearsBMI kgcm2Gestational age weeksParity timesNeonatal birth weight kg25OHD nmolL25OHD2 nmolL25OHD3 nmolLVitamin D deficiency N YesNoGestational diabetes mellitus N YesNoCaesarean section N YesNoFetal distress in uterus N YesNoPreterm birth N YesNoLow birth weight N YesNoMacrosomia N YesNoTotal N ± years N ± ‰¥ years N ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± P 0cChen BMC Pregnancy and Childbirth Page of Table Seasonal difference between serum vitamin D indicatorsDetected Seasonspring summer ± ± ± autumn winter ± ± ± P 25OHD nmolL25OHD2 nmolL25OHD3 nmolLDiscussionIn this retrospective cohort study including Chinese motherinfant pairs protective associations werefound for higher serum 25OHD concentrations withGDM cesarean section postpartum hemorrhage preterm birth and low birth weight but not for the otheroutcomes The results for 25OHD2 tended to be morepronounced than those for 25OHD3Interestinglyhigher serum vitamin D contributes to a higher risk ofpostpartum anemia in women pregnant at a young agebut not in advanced yearsIn this population with a high prevalence of vitamin Ddeficiency better serum vitamin D status contributed toa lower risk of GDM This result was consistent withseveral other studies A to higher risk of GDMwas observed for pregnant women with insufficient ordeficient vitamin D status in three systematic reviewsand metaanalyses based on observational studies orclinical trials [ ] In a large randomized controltrial RCT based on Iranian women the intervention of 25OHD3 was associated with lower risk ofGDM [] The results were further supported by severalother prospective cohort studies [ ] However nullassociations were found between the vitamin D status orsupplements and GDM in a systematic review based onfive randomized trials including subjects [] anested casecontrol study of women [] and alarge prospective study including motherchildpairs [] Additionally two studies found detrimentalassociations of higher vitamin D levels with GDM butthe effects were tiny [] or restricted to specificethnicities Hispanic []Consistent with our results the protective associationof maternal vitamin D status with a lower risk of preterm or low birth weight has also been reported in severalstudies An inverse doseresponse relation ofvitamin D status was found for preterm birth in a systematic review and metaanalysis based on longitudinal studies[] Rostami reported that a 25OHD3 interventionof monthly IU could be beneficial for the preventionof preterm delivery in an RCT of Iranian women []In a large prospective cohort including motheroffspring pairs the risk of low birth weight was CI and CI among subjects with vitamin D deficiency and insufficiency respectively [] Higher maternal serum 25OHD was positivelyTable Associations between early pregnant serum vitamin D concentrations and maternal infant outcomes25OHD aHR95CIPa25OHD2HR95CIPa25OHD3HR95CIPGestational diabetes mellitusModel Model Caesarean sectionModel Model Fetal distressModel Model Preterm birthModel Model Low birth weightModel Model MacrosomiaModel Model Cox regression analysis were operated for exploration of associations Model without adjustment Model adjusted for age BMI parity season of bloodcollected a Per one SD increase 0cChen BMC Pregnancy and Childbirth Page of Table Associations between vitamin D status and maternal infant outcomesGestational diabetes mellitusCaesarean sectionFetal distressPreterm birthLow birth weightMacrosomia25OHD nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolL nmolL‰¥ nmolLHR95CI PCox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collectedassociated with higher birth weight in a study of UnitedArab Emirates [] and supplementation of vitamin D at adose of IUd was optimal and safe for mothers andtheir infants in United Arab Emirates [] However nullor detrimental associations were also reported in otherstudies Although vitamin D increased the mean birthweight it did not significantly reduce the risk of low birthweight or preterm birth in a metaanalysis based on randomized trials [] these results were further supported byanother update study [] In addition higher 25OHDconcentrations were found to increase the riskof preterm delivery in a prospective study of pregnant Chinese women []Gestational vitamin D deficiency was found to be associated with a 2fold increased risk of cesarean section ina cohort of lowincome pregnant women []Within a cohort of women from the United Statesfor women with 25OHD concentrations lower than nmolL the risk of a primary cesarean section wasalmost times higher [] These results were consistent with ours However a metaanalysis of trials subjects found null associations of vitamin D supplements with cesarean section []Much heterogeneity exists in the previous evidence inthis field and there may be several factors that partly explain these differences First most of the randomized trials included had a small sample size and were of lowquality Most subjects included in the trials had sufficient vitamin D status ‰¥ nmolL and a further doseof a or IUd supplement might have attenuatedTable Subclass analysis of relationship between serum vitamin D concentrations and related outcomes stratified by gestational ageP bP bP baa25OHD aHR95CIP25OHD2HR95CIP25OHD3HR95CIPGestational diabetesmellitusCaesarean sectionFetal distressPreterm birth y ‰¥ y y ‰¥ y y ‰¥ y y ‰¥ y Low birth weight y ‰¥ y Macrosomia y ‰¥ y Cox regression analysis were operated for exploration of associations and adjusted for covariates including age BMI parity season of blood collecteda Per one SD increase b P for interaction 0cChen BMC Pregnancy and Childbirth Page of the detrimental influence of vitamin D deficiency or insufficiency in the reference group [] Increasing benefitsmight not exist for higher doses Second the vitamin Dreceptor is important for the utilization of vitamin D[ ] The difference in vitamin D receptor gene polymorphism might help explain the ethnic heterogeneity[] This requires further confirmation in the futureThird the studies were conducted during different trimesters of gestation which might increase the difficultyof making comparisons those studies conducted duringan early gestational period might be advantageous forcausal inference and early preventionBecause 25OHD3 contributes most to the 25OHDconcentrations it has been used to represent 25OHDin many studies and only a few studies have focused on25OHD2 In our study the results were generally consistent between these two subcomponents The associations tend to be more pronounced in 25OHD2 than in25OHD3 and mightindicate the prominence of25OHD2 over 25OHD3 for the prevention of relateddiseases This theory merits further confirmation Nevertheless our results and others provide further evidencefor the importance of a better early vitamin D status forthe prevention of GDM cesarean section preterm birthand low birth weight in a population with a high prevalence of vitamin D deficiency Our results indicated thatmore consideration should be given to distinguishing between the vitamin D subcomponents women pregnantat different ages and women with more pregnancy complications in this field Considering the high prevalenceof vitamin D deficiency during pregnancy observed inChina [ ] it is a matter of urgency to call for the supplementation of vitamin D and more efforts should bemade to improve the gestational vitamin D status ofChinese populationVitamin D deficiency was found to decrease vasculardiameter within the labyrinth region and dysregulateplacental development during early pregnancy in an animal experiment [] Vitamin D supplementation duringearly pregnancy might rescue this situation while furthersupplementation might be futile after missing this timepoint [] Moreover vitamin D supplementation duringearly pregnancy is likely to affect genetic information ofsystemic inflammation and immune responses involvedin the development of gestational comorbidities egGDM preeclampsia and infection as reviewed by Hollis [] These mechanisms help to explain the beneficialinfluences of vitamin D for maternal and infanthealth and emphasize the importance of improving vitamin D status during early pregnancyOurstudy had several advantages Firstserum25OHD concentrations were measured during an earlygestational period and with the retrospective cohort design we assured the temporal sequence and avoided thepossibility of causal inversion Second we studied multiple outcomes and conducted further analysis of thesubcomponent of vitamin D and a stratified analysis ofgestational age which provided a more comprehensiveunderstanding of this field There are also several limitations of our study First our study was limited by thelack of information on dietary vitamin D intake including by supplement and sunlight exposure during thepregnancy period The obtained data were difficult touse for a retrospective study while the use of blood indicators might be more precise than a dietary survey sowe adjusted the association for different seasons to attenuate their influence Second the 25OHD concentration was measured only once we could not monitor thedynamic changes afterward or determine whether theywere normalized after receiving vitamin D supplementation However the associations tended to be underestimated instead of overestimated Third Wagner et al[] and Mirzakhani [] indicated a 25OHDconcentration ‰¥ nmolL in early pregnancy was optimal for the prevention of preterm birth and preeclampsia however the highest 25OHD concentration in ourstudy is only nmolL Therefore we were unable toperform the analyses using a threshold of nmolLand assess the influences of higher 25OHD concentrations Finally residual confounding might still existthough we tried to control several potential covariatesConclusionsThis retrospective cohort study showed the beneficial associations of early gestational vitamin D with outcomesof GDM cesarean section preterm birth and low birthweight More welldesigned randomized clinical trialsare needed for further exploration and confirmation ofthese resultsAbbreviationsGDM Gestational diabetes mellitus LBW Low birth weight BMI Body massindex SD Standard deviationAcknowledgementsWe would like to thank Ye Shaoxin Yang Xiaoming Liu Haojing Wangdong and Huang Shaobing for their generous help in this studyAuthors™ contributionsGDC and ZPL devised the idea and designed the study GDC TTP PSLZXZ DXL DZF contributed to the primary data collection GDC and TTP reexamined the data GDC TT P PS L ZX Z DXL DZF contributedto the analysis of the data GDC and TTP wrote the original draft whichwas revised by XLG and ZPL XLG and ZPL supervised the study andZPL administered the project All authors have read and approved themanuscriptFundingThis work was supported by the Basic and Applied Basic ResearchFoundation of Guangdong Province No 2019A1515110163 GDC and theFoundation of Bureau of Science and Technology of Foshan City No GDC The funding sponsors had no role in the design ofthe study in the collection analyses or interpretation of data in the writingof the manuscript and in the decision to publish the results 0cChen BMC Pregnancy and Childbirth Page of Availability of data and materialsThe datasets used andor analyzed during the current study are availablefrom the corresponding author upon reasonable requestEthics approval and consent to participateThe study was approved by the ethics committee of Affiliated FoshanMaternity Child Healthcare Hospital Southern Medical University TheAffiliated Foshan Maternity Child Healthcare Hospital providedadministrative permissions for the research team to access and use the dataincluded in this research Data were extracted from medical records and theconsent to participate was unavailable due to the retrospective design of thestudy and difficulty in reconnection however the private information waswell protectedConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Foshan Institute of Fetal Medicine Department of Obstetrics AffiliatedFoshan Maternity Child Healthcare Hospital Southern Medical UniversityFoshan Guangdong China 2Department of Medical RecordsAffiliated Foshan Maternity Child Healthcare Hospital Southern MedicalUniversity Foshan Guangdong ChinaReceived February Accepted August ReferencesSaraf R Morton SM Camargo CA Jr Grant CC Global summary of maternaland newborn vitamin D status a systematic review Matern Child Nutr“Yun C Chen J He Y Mao D Wang R Zhang Y Yang C Piao J Yang XVitamin D deficiency prevalence and risk factors among pregnant Chinesewomen Public Health Nutr “Zhou J Su L Liu M Liu Y Cao X Wang Z Xiao H Associations between hydroxyvitamin D levels and pregnancy outcomes a prospectiveobservational study in southern China Eur J Clin Nutr “Agarwal S Kovilam O Agrawal DK Vitamin D and its impact on maternalfetal outcomes in pregnancy a critical review Crit Rev Food Sci Nutr “von Websky K Hasan AA Reichetzeder C Tsuprykov O Hocher B Impact ofvitamin D on pregnancyrelated disorders and on offspring outcome JSteroid Biochem Mol Biol “Nobles CJ Markenson G ChasanTaber L Early pregnancy vitamin D statusand risk for adverse maternal and infant outcomes in a biethnic cohort thebehaviors affecting baby and you BaBY study Br J Nutr “Roth DE Leung M Mesfin E Qamar H Watterworth J Papp E Vitamin Dsupplementation during pregnancy state of the evidence from a systematicreview of randomised trials BMJ 2017359j5237HautaAlus HH Viljakainen HT HolmlundSuila EM EnlundCerullo MRosendahl J Valkama SM Helve OM Hytinantti TK Makitie OM AnderssonS Maternal vitamin D status gestational diabetes and infant birth size BMCPregnancy Childbirth Khaing W Vallibhakara SA Tantrakul V Vallibhakara O Rattanasiri S McEvoyM Attia J Thakkinstian A Calcium and vitamin D supplementation forprevention of preeclampsia a systematic review and network metaanalysisNutrients 2017910E1141 Zhang Y Gong Y Xue H Xiong J Cheng G Vitamin D and gestationaldiabetes mellitus a systematic review based on data free of Hawthorneeffect BJOG “ Zhang MX Pan GT Guo JF Li BY Qin LQ Zhang ZL Vitamin D deficiencyincreases the risk of gestational diabetes mellitus a metaanalysis ofobservational studies Nutrients “ Aghajafari F Nagulesapillai T Ronksley PE Tough SC O'Beirne M Rabi DMAssociation between maternal serum 25hydroxyvitamin D level andpregnancy and neonatal outcomes systematic review and metaanalysis ofobservational studies BMJ 2013346f1169 Qin LL Lu FG Yang SH Xu HL Luo BA Does Maternal Vitamin D DeficiencyIncrease the Risk of Preterm Birth A MetaAnalysis of Observational StudiesNutrients 201685E301 Rodriguez A GarciaEsteban R Basterretxea M Lertxundi A RodriguezBernalC Iniguez C RodriguezDehli C Tardon A Espada M Sunyer J et alAssociations of maternal circulating 25hydroxyvitamin D3 concentrationwith pregnancy and birth outcomes BJOG “ Ogawa K Urayama KY Tanigaki S Sago H Sato S Saito S Morisaki NAssociation between very advanced maternal age and adverse pregnancyoutcomes a cross sectional Japanese study BMC Pregnancy ChildbirthKhalil A Syngelaki A Maiz N Zinevich Y Nicolaides KH Maternal age andadverse pregnancy outcome a cohort study Ultrasound Obstet Gynecol“Jolly M Sebire N Harris J Robinson S Regan L The risks associated withpregnancy in women aged years or older Hum Reprod “Li Q Deng D New medical risks affecting obstetrics after implementation ofthe twochild policy in China Front Med “ Poel YH Hummel P Lips P Stam F van der Ploeg T Simsek S Vitamin Dand gestational diabetes a systematic review and metaanalysis Eur J InternMed “ Rostami M Tehrani FR Simbar M Bidhendi Yarandi R Minooee S Hollis BWHosseinpanah F Effectiveness of prenatal vitamin D deficiency screeningand treatment program a stratified randomized field trial J Clin EndocrinolMetab “ Xu C Ma HH Wang Y Maternal early pregnancy plasma concentration of25Hydroxyvitamin D and risk of gestational diabetes mellitus Calcif TissueInt “ Boyle VT Thorstensen EB Mourath D Jones MB McCowan LM Kenny LCBaker PN The relationship between 25hydroxyvitamin D concentration inearly pregnancy and pregnancy outcomes in a large prospective cohort BrJ Nutr “Schneuer FJ Roberts CL Guilbert C Simpson JM Algert CS Khambalia AZTasevski V Ashton AW Morris JM Nassar N Effects of maternal serum hydroxyvitamin D concentrations in the first trimester on subsequentpregnancy outcomes in an Australian population Am J Clin Nutr “ Amegah AK Klevor MK Wagner CL Maternal vitamin D insufficiency andrisk of adverse pregnancy and birth outcomes a systematic review andmetaanalysis of longitudinal studies PLoS One 2017123e0173605 Chen YH Fu L Hao JH Yu Z Zhu P Wang H Xu YY Zhang C Tao FB XuDX Maternal vitamin D deficiency during pregnancy elevates the risks ofsmall for gestational age and low birth weight infants in Chinesepopulation J Clin Endocrinol Metab “ Amirlak I Ezimokhai M Dawodu A Dawson KP Kochiyil J Thomas LAbdulle AM Current maternalinfant micronutrient status and the effects onbirth weight in the United Arab Emirates East Mediterr Health J “ Dawodu A Saadi HF Bekdache G Javed Y Altaye M Hollis BW Randomizedcontrolled trial RCT of vitamin D supplementation in pregnancy in apopulation with endemic vitamin D deficiency J Clin Endocrinol Metab“ PerezLopez FR Pasupuleti V MezonesHolguin E BenitesZapata VA Thota PDeshpande A Hernandez AV Effect of vitamin D supplementation duringpregnancy on maternal and neonatal outcomes a systematic review and metaanalysis of randomized controlled trials Fertil Steril “1288e1274Scholl TO Chen X Stein P Maternal vitamin D status and delivery bycesarean Nutrients “ Merewood A Mehta SD Chen TC Bauchner H Holick MF Associationbetween vitamin D deficiency and primary cesarean section J ClinEndocrinol Metab “ Chun RF Peercy BE Orwoll ES Nielson CM Adams JS Hewison M VitaminD and DBP the free hormone hypothesis revisited J Steroid Biochem MolBiol Pt A132“ Bikle DD Gee E Halloran B Kowalski MA Ryzen E Haddad JG Assessmentof the free fraction of 25hydroxyvitamin D in serum and its regulation byalbumin and the vitamin Dbinding protein J Clin Endocrinol Metab “ Powe CE Evans MK Wenger J Zonderman AB Berg AH Nalls M Tamez HZhang D Bhan I Karumanchi SA Vitamin Dbinding protein and 0cChen BMC Pregnancy and Childbirth Page of vitamin D status of black Americans and white Americans N Engl J Med“Liu NQ Ouyang Y Bulut Y Lagishetty V Chan SY Hollis BW Wagner CEquils O Hewison M Dietary vitamin D restriction in pregnant female miceis associated with maternal hypertension and altered placental and fetaldevelopment Endocrinology “ Mirzakhani H Litonjua AA McElrath TF O'Connor G LeeParritz A Iverson RMacones G Strunk RC Bacharier LB Zeiger R Early pregnancy vitaminD status and risk of preeclampsia J Clin Invest “ Hollis BW Wagner CL Vitamin D supplementation during pregnancyimprovements in birth outcomes and complications through directgenomic alteration Mol Cell Endocrinol “ Wagner CL Baggerly C McDonnell SL Baggerly L Hamilton SA Winkler JWarner G Rodriguez C Shary JR Smith PG Posthoc comparison ofvitamin D status at three timepoints during pregnancy demonstrates lowerrisk of preterm birth with higher vitamin D closer to delivery J SteroidBiochem Mol Biol “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"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 DDP‘resistant cells A549DDP and paclitaxel PTX‘resistant 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 cancer‘associated 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 ChinaE‘mail xyhuangrm163comKey words lung cancer close homolog of cisplatin paclitaxel chemosensitivitycharacteristics of cancer cells lung cancer can be roughly clas‘sified into non‘small‘cell lung cancer NSCLC and small‘cell lung cancer Among patients with lung cancer nearly are diagnosed as NSCLC which manifests with earlier diffu‘sion 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 chemo‘therapy is a remarkably important treatment Cisplatin DDP is widely applied in the treatment of several malignan‘cies 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 subse‘quent 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 anti‘proliferation and anti‘metastasis 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 DDP‘resistant 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 PTX‘resistant 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 DDP‘resistant H460 cells sample and each cell has three repeats samples The online tool GEO2R httpwwwncbinlmnihgovgeogeo2r was used to determine the differen‘tially expressed genes in H460 and DDP‘resistant H460 cells P005 and log2fold‘change‰¥ were set as cut‘off standardsCell culture The human NSCLC cell line A549 the PTX‘resistant cell line A549PTX and the DDP‘resistant cells A549DDP were purchased from Procell Life Science Technology Co Ltd The cells were cultured in Ham's F‘12K medium supplemented with fetal bovine serum both purchased from Thermo Fisher Scientific Inc Uml penicillin and Uml strep‘tomycin cat no Thermo Fisher Scientific Inc in a ˚C humidified incubator with CO2Cell transfection The resistant cells A549PTX and A549DDP cells were transfected with µg CHL1 recombinant expres‘sion plasmid cat no HG10143‘NY Sino Biological Inc Empty vector pCMV3‘SP‘N‘HA 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 ˚CRNA extraction and reverse transcription‘quantitative PCR RT‘qPCR assay Total RNAs were isolated using TRIzol reagent Thermo Fisher Scientific Inc according to the manufacturer's instructions and the mixed DNAs were elim‘inated by DNase I New England Biolabs Inc First‘strand cDNA synthesis was conducted using the GoScriptTM kit Promega Corporation according to the manufacturer's instructions The reaction conditions for reverse transcription were ˚C for min ˚C for min and ˚C for min The SYBR Green Real‘Time PCR Master mix Thermo Fisher Scientific Inc was used to perform RT‘qPCR 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 ˚C followed by cycles at ˚C for sec sec at ˚C and a final extension at ˚C 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 ˚C 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 ˚C µl MTT mgml solution was added to each well and incubated for h at ˚C 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 F‘12K medium with or without DDP or PTX at ˚C for h A total of weeks later cells were fixed in paraformalde‘hyde 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 V‘FITC 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 SDS‘PAGE gel Next the proteins were transferred onto a polyvinylidene membrane Thermo Fisher Scientific Inc blocked with BSA Thermo Fisher Scientific Inc for h at ˚C and incubated overnight at ˚C with primary antibodies against CHL1 cat no ‘‘AP ProteinTech Inc multi‘drug resistance gene MDR1 cat no ‘‘AP ProteinTech Inc multidrug resistance‘associated protein MRP cat no ‘‘Ig ProteinTech Inc low‘density lipoprotein receptor‘related protein LRP cat no ‘‘AP ProteinTech Inc phosphorylated p‘Akt cat no ab38449 Abcam and Akt cat no ab227385 Abcam After washing three times with PBS the membrane was incubated with horseradish peroxide‘conjugated goat anti‘rabbit cat no ab6271 Abcam_or rabbit anti‘mouse 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 Image‘pro 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 BALBc‘nu mice ‘week‘old ± 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 ‘ËšC 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 subcu‘taneously 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 vector‘DDP group A549DDP cells trans‘fected with empty vector and treated with mgkg DDP iii CHL1 group A549DDP cells transfected with CHL1 overexpression vector and treated with µl saline solu‘tion and iv CHL1‘DDP 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 post‘injection mice were euthanized with CO2 at volume displacement rate VDR per min using a programmable logic controller Barry‘Wehmiller 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 tripli‘cate and data are presented as the mean ± standard deviation All experiments were performed at least three times Paired Student's t‘test was performed for comparisons between two groups and one‘way analysis of variance followed by Tukey's multiple comparison post‘hoc 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 A549PTX‘resistant cells In order to investigate the mechanism of chemore‘sistance in lung cancer the lung adenocarcinoma cell line A549 the DDP‘resistant cells A549DDP and PTX‘resistant 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 drug‘resistant 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 DDP‘resistant 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 expres‘sion 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 CHL1‘knockdown enhanced the resistance to DDP and PTX in A549 cells Fig 2B and C Colony formation assay revealed that compared with the control group CHL1‘knockdown 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 demon‘strated significantly reduced apoptosis in CHL1‘knockdown cells after DDP and PTX treatment compared with that of the control group Fig 2E\x0cCAI CHL1 ENHANCES THE CHEMOSENSITIVITY OF LUNG CANCER CELLSFigure CHL1 is downregulated in DDP and PTX‘resistant A549 cells A Cell survival of A549 and A549‘resistant 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 resistance‘related proteins MDR1 MRP and LRP in A549 cells and A549‘resistant cells A549DDP and A549PTX P005 vs A549 cells The protein and mRNA expression levels of CHL1 in A549 cells and A549‘resistant cells A549DDP and A549PTX were analysed by D western blotting and E reverse transcription‘quantitative 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 multi‘drug resistance gene MRP multidrug resistance‘associated protein LRP low‘density lipoprotein receptor‘related protein IC50 half maximal inhibitory concentration CHL1 overexpression enhances the sensitivity of A549 resistant cells to DDP and PTX As CHL1 is downregu‘lated 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 resis‘tance 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 treat‘ment Fig 3ETo further validate the effects of CHL1 overexpression on DDP or PTX sensitivity xenograft mice model experi‘ments 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 aggra‘vated DDP‘mediated 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 CHL1‘knockdown elevated the expression of p‘Aktser473 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 CHL1‘silenced A549 cells were treated with the Akt inhibitor SC66 and it was demon‘strated that inhibiting Akt activity significantly reduced the promotive effects on cell survival Fig 4B and clone forma‘tion Fig 4C and the inhibitory effects on apoptosis Fig 4D induced by CHL1‘depletion These results confirmed that CHL1 mediates chemosensitivity in NSCLC by inhibiting the Akt pathway\x0cONCOLOGY LETTERS Figure CHL1‘knockdown 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 CHL1‘knockdown 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 cell‘cell interactions and mediating cell‘cell and cell‘matrix 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 expres‘sion 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\x0cCAI 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 demon‘strated 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 DDP‘resistant cells in a GEO dataset CHL1 was demonstrated to be upregulated in DDP‘resistant 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 chemotherapy‘sensitive tumor tissues is higher compared with that in drug‘resistant 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 ‘fluro‘uracil 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 down‘regulated 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 p‘Akt and total Akt in CHL‘silenced 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 si‘CHL1 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 si‘CHL1 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 p‘Akt regulates cell survival cell growth cell motility and angiogenesis and prevents apoptosis Additionally Akt activation is associated with tumor chemore‘sistance The results of the present study demonstrated that compared with the control groups the expression of p‘Akt was increased in CHL1‘knockdown A549 cells and its expres‘sion 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 CHL1‘knockdown 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 CHL1‘based 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 S21‘S30 Chen W Cancer statistics Updated cancer burden in China Chin J Cancer Res Oser MG Niederst MJ Sequist LV and Engelman JA Transformation from non‘small‘cell lung cancer to small‘cell lung cancer Molecular drivers and cells of origin Lancet Oncol e165‘e172 Thatcher N Faivre‘Finn C Blackhall F Anderson H and Lorigan P Sequential platinum‘based chemotherapy‘thoracic radiotherapy in early stage non‘small cell lung cancer Clin Cancer Res Suppl S5051‘S5056 Yano T Okamoto T Fukuyama S and Maehara Y Therapeutic strategy for postoperative recurrence in patients with non‘small cell lung cancer World J Clin Oncol ‘ Fang Z Chen W Yuan Z Liu X and Jiang H LncRNA‘MALAT1 contributes to the cisplatin‘resistance of lung cancer by upregulating MRP1 and MDR1 via STAT3 activation Biomed Pharmacother ‘ Cai Y Dong ZY and Wang JY LncRNA NNT‘AS1 is a major mediator of cisplatin chemoresistance in non‘small 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 upregulation‘induced 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 Niimori‘Kita 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 array‘CGH 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 progres‘sion Biochem Biophys Res Commun ‘ Martín‘Sánchez E Mendaza S Ulazia‘Garmendia A Monreal‘Santesteban I Blanco‘Luquin I Córdoba A Vicente‘García F Pérez‘Janices N Escors D Megías D CHL1 hypermethylation as a potential biomarker of poor prog‘nosis 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 miR‘‘5p promotes proliferation and invasion of colon adeno‘carcinoma cells through targeting CHL1 Mol Med Hötzel J Melling N Müller J Polonski A Wolters‘Eisfeld G Izbicki JR Karstens KF and Tachezy M Protein expression of close homologue of L1 CHL1 is a marker for overall survival in non‘small cell lung cancer NSCLC J Canc
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prevalence of pathogenic variants in DnA damage response and repair genes in patients undergoing cancer risk assessment and reporting a personal history of early‘onset renal cancerTiffiney a0R a0Hartman12 a0Elena a0V a0Demidova345 a0Randy a0W a0Lesh6 a0Lily a0Hoang7 a0Marcy Richardson7 a0Andrea a0Forman8 a0Lisa a0Kessler1 a0Virginia a0Speare7 a0Erica a0A a0Golemis4 a0Michael a0J a0Hall38 a0Mary a0B a0Daly38 Sanjeevani Arora3Pathogenic a0variants a0PVs a0in a0multiple a0genes a0are a0known a0to a0increase a0the a0risk a0of a0earlyonset a0renal a0cancer a0eoRC a0However a0many a0eoRC a0patients a0lack a0PVs a0in a0RCspecific a0genes a0thus a0their a0genetic a0risk a0remains a0undefined a0Here a0we a0determine a0if a0PVs a0in a0DNA a0damage a0response a0and a0repair a0DDRR a0genes a0are a0enriched a0in a0eoRC a0patients a0undergoing a0cancer a0risk a0assessment a0Retrospective a0review a0of a0deidentified a0results a0from a0 a0eoRC a0patients a0undergoing a0testing a0with a0a a0multigene a0panel a0for a0a a0variety a0of a0indications a0by a0Ambry a0Genetics a0PVs a0in a0cancerrisk a0genes a0were a0identified a0in a0 a0of a0patients”with a0 a0in a0RCspecific a0and a0 a0in a0DDRR a0genes a0DDRR a0gene a0PVs a0were a0most a0commonly a0identified a0in a0CHEK2 a0BRCA1 BRCA2 and ATM a0Among a0the a0 a0of a0patients a0with a0a a0BRCA1 or BRCA2 a0PV a0 a0 a0reported a0a a0personal a0history a0of a0hereditary a0breast a0or a0ovarianassociated a0cancer a0No a0association a0between a0age a0of a0RC a0diagnosis a0and a0prevalence a0of a0PVs a0in a0RCspecific a0or a0DDRR a0genes a0was a0observed a0Additionally a0 a0patients a0reported a0at a0least a0one a0additional a0cancer a0breast a0cancer a0being a0the a0most a0common a0 a0of a0females a0 a0of a0males a0Multigene a0testing a0including a0DDRR a0genes a0may a0provide a0a a0more a0comprehensive a0risk a0assessment a0in a0eoRC a0patients a0Further a0validation a0is a0needed a0to a0characterize a0the a0association a0with a0eoRCRenal cancer RC often develops with no signs or symptoms and is referred to as the œsilent disease While factors including smoking environment obesity and race have been linked to increased risk of RC inherited factors are the most wellvalidated source of increased risk2“ Hereditary RC syndromes typically associated with earlyonset disease and a clinically significant family history of cancer result from germline pathogenic variants PV in highpenetrance ˜RCspecific™ genes including VHL MET FLCN TSC1 TSC2 FH SDH PTEN and BAP15“ A previous report of an earlyonset RC eoRC cohort screened with an RCspecific panel found of individuals had a PV in an RCspecific gene7 However for most eoRC patients a PV in an RCspecific gene is not identified leaving many eoRC genetically undefined Thus there is a need to identify additional genes related to eoRC risk Currently there are no National Comprehensive Cancer Network NCCN guidelines for detection prevention or risk reduction in individuals who present with an eoRC but lack a PV in a defined RCspecific gene81Arcadia University Glenside PA USA 2Cancer Biology Program Fox Chase Cancer Center Philadelphia PA USA 3Cancer Prevention and Control Program Fox Chase Cancer Center Cottman Avenue Philadelphia PA USA 4Molecular Therapeutics Program Fox Chase Cancer Center Philadelphia PA USA 5Kazan Federal University Kazan Russian Federation 6Geisinger Commonwealth School of Medicine Scranton PA USA 7Ambry Genetics Konica Minolta Aliso Viejo CA USA 8Department of Clinical Genetics Fox Chase Cancer Center Philadelphia PA USA email SanjeevaniArorafccceduScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cDNA damage response and repair genes DDRR play an important role in maintaining genome integrity and when mutated in the germline can increase cancer risk for several types of cancers including breast colorectal ovarian and others9 Although PVs in DDRR genes are associated with increased risk of a variety of cancer types they are not typically considered risk factors for RC However germline PVs in some DDRR genes have been observed in RC including PVs in the DNA mismatch repair Lynch syndrome genes MSH2 and MLH1 in renal urothelial carcinoma and PVs in CHEK2 in advanced renal cell carcinoma10“ To address the hypothesis that PVs in additional DDRR genes may contribute to the missing heritability of eoRC we analyzed germline sequencing data from a cohort of individuals with RCMaterials and methodsAmbry a0Genetics a0eoRC a0study a0cohort a0and a0variant a0determination a0 Deidentified data were requested from RC patients that were tested by Ambry Genetics Konica Minolta Aliso Viejo California using germline cancer testing panels Ambry samples were selected for patients with RC and deidentified data was obtained for all RC patients tested with multigene cancer panels n ‰ a0years at diagnosis specimens collected between July “December All genetic test results from germline testing of individuals diagnosed with RC at ‰ during this time period were used in this studyThere is currently no standard definition specifying the age when RC is considered earlyonset Different models have been used to determine a specific age as a trigger for germline testing in patients with RC who lack family history of RC including ages or a0years For this study we selected individuals a0years or younger as the cutoff for our cohort which is substantially below the median age of RC diagnosis of a0years in the general population as reported in SEER2223 but considerably older than other suggested cutoffs We did so because the main hypothesis of the study was that PVs in DDRR genes might be responsible for increased risk of RC Variants in multiple DDRR genes are associated with earlyonset colorectal cancer2425 which typically manifests in patients at a0years or younger We considered that PVs in DDRR genes were most likely to impact repair of DNA damage induced during cell replication leading to genetic instability and cancer given renal cells turn over much less frequently than colon cells we hypothesized that it may take longer for cancers associated with PVs in DDRR genes to manifest in RC causing us to select a cutoff of ‰ a0years old for assessmentDeidentified data included family history of cancer genetic test results personal history of cancers apart from RC presence of multifocal tumors and RCsubtypestage The RC patients had been tested with CancerNext versions “ and CancerNextExpanded versions and Table a0S1 Deidentified patient information was analyzed for genetic test results and personal and family medical histories Classification of variants by Ambry Genetics is based on ACMG recommendations for standards for interpretation and reporting of sequence variations These variants are also regularly deposited in ClinVar by Ambry Genetics Variant classification was updated through March for all data Gene variants were classified as pathogenic variant PV”see below for criteria variant of uncertain significance VUS or inconclusive or negativeindeterminate Ambry Genetics follows strict criteria when classifying variants as PV Variant Likely Pathogenic VLP VUS Variant Likely Benign VLB and Benign for details see wwwambry gencomclini cianourscien tific excel lence varia ntclass ifica tion Variants reported as PV and VLPs were grouped as PVs All test results were used for this study The analysis of VUS which currently lack clinical significance was beyond the scope of this study Given updating of test panels by Ambry Genetics not all patients were tested for all genes Individuals were provided different versions of the panel over the course of the study see below and also see Table a0S1Any deidentified personal or family history information including sex ethnicityrace age of cancer diagnosis tumor histology history of additional personal cancer and history of family cancer and types was reported first as summarized data and later as deidentified individual case reports For analysis comparing outcomes for RCspecific genes versus genes not typically associated with RC we focused our statistical comparison on only those individuals who had CancerNext Expanded panel version testing which analyzes all genes including the RCspecific genes individuals who had the CancerNext Expanded version test were used for this statistical comparison For additional statistical test comparisons that analyzed the correlations between specific genes and categories such as tumor pathology or age any individual who had been tested for that specific gene was includedThe Western IRB issued a regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus the requirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB provided study oversight and approval protocol number Ambry Genetics provided deidentified patient medical and family history where available and genetic results for the patients All methods were performed in accordance with the relevant guidelines and regulation of the approved studyGenetic a0analysis a0with a0Ambry a0CancerNext a0and a0CancerNext a0Expanded a0panels a0Individuals were provided different versions of the panel by their healthcare provider see Table a0S1 The number of genes in the panels ranged from the smallest CancerNext panel Version which include genes APC ATM BARD1 BRIP1 BMPR1A CDH1 CHEK2 EPCAM MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 PTEN RAD50 RAD51C SMAD4 STK11 TP53 to the largest CancerNext Expanded Version panel which contained genes APC ATM BAP1 BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM FH FLCN GREM1 MAX MEN1 MET MITF MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE PTEN RAD50 RAD51C RAD51D RET SDHA SDHAF2 SDHB SDHC SDHD SMAD4 SMARCA4 STK11 TMEM127 TP53 TSC1 TSC2 VHL The DDRRs identified in germline testing of this cohort are bolded26Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAmbry Genetics sequenced genomic DNA that was obtained from patient blood or saliva samples DNA was evaluated by next generation sequencing NGS of all coding sequences and ± bases into the ² and ² ends of flanking introns and untranslated regions In addition sequencing of the promoter region was performed for the following genes PTEN cˆ’ to cˆ’ MLH1 cˆ’ to cˆ’ and MSH2 cˆ’ to cˆ’ Additional Sanger sequencing was performed for any regions missing or with insufficient depth of coverage for reliable heterozygous variant detection and on potentially homozygous variants variants in regions with complicated pseudogene interference and when variant calls did not meet allele frequency quality thresholds Additional details on specific testing methods are available at wwwambry gencomclini ciangenet ictesti ng28oncol ogycance rnext expan dedControl a0population a0in a0ExAc a0and a0gnomAD a0 To compare the frequency of DDRR gene PVs found in the study to that in the general population our results were compared to the Exome Aggregation Consortium ExAc dataset of largely unrelated whole exome sequencing results and to the Genome Aggregation database gnomAD dataset consisting of exomes and genomes2728 These datasets are the most commonly used genomic data at the populationlevelClinVar a0analysis a0 ClinVar wwwncbinlmnihgovclinv ar a database of medically relevant gene variants was used to investigate all PVs in this study retrieved on February PVs that were not reported in ClinVar were noted as ˜not reported™ Associated conditions for each PV were categorized into hereditary cancer predisposing syndromes conditions related to renal cancer and any other conditions To further elucidate any PVs related to renal cancer the search term œrenal cancer was queried and the results were noted as œassociated with ClinVar search term ˜Renal Cancer™Statistical a0 analysis a0 To identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis genes were combined into pathwaysgroups of interest histology™s were grouped and cancer types were grouped Each individual was categorized as having a variant in one of the genes within the group or no variant in the group Gene categories were used for comparison of RC diagnosis with a DDRR or an RCspecific geneWe also tested the hypothesis that different gene groups are associated with age at RC diagnosis We used the median age of RC diagnosis in the study cohort a0years and studied PVs in patients a0years or ‰¥ a0years of age To test the association between the presence of PVs and age of RC diagnosis twosided Fisher™s exact tests were used and a0pvalues ‰ were considered significant Odds ratios OR were calculated to determine the odds that an outcome had occurred given a particular variant compared to the odds of the outcome occurring in the absence of that variant in the population tested Finally we queried the SEER database for patients under a0years old with RC to compare the distribution of their clinical characteristics where available to those in our study cohort22Due to the evolving nature of the panels during the course of this study each version included a different total number of genes and analysis of each gene is based on the number of individuals whose test included that gene Table a0S1 Only data from individuals was considered for comparison of individuals with RCspecific genes compared to those with variants in genes not typically associated with RC as the other individuals did not have all genes analyzed For statistical comparisons analyzing correlations between specific genes with various characteristics all individuals who had been tested for that specific gene were includedTo identify potential correlations between PVs and characteristics such as tumor pathology additional primary tumor type and age of diagnosis RCspecific genes other cancerassociated genes and DDRR genes were combined into groups and histologies were grouped The categories for comparison of PVs and patient characteristics are as follows Known RC genes BAP1 FH FLCN MEN1 MET MITF PTEN SDHA SDHAF2 SDHB SDHC SDHD TSC1 TSC2 and VHL versus genes not typically associated with RC APC ATM BARD1 BRCA1 BRCA2 BRIP1 BMPR1A CDH1 CDK4 CDKN2A CHEK2 EPCAM GREM1 MAX MLH1 MRE11A MSH2 MSH6 MUTYH NBN NF1 PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D RET SMAD4 SMARCA4 STK11 TEMEM127 TP53 versus DDRR genes alone ATM BARD1 BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A MSH2 MSH6 MUTYH NBN PALB2 PMS2 POLD1 POLE RAD50 RAD51C RAD51D Histology categories combined from the original categories Chromophobe Papillary renal Clear cell Wilms Renal cell likely clear cell but cannot be confirmed Unknown Mixed papillary [clear cell papillary type papillary renalchromophobe renal and sarcomatoidpapillaryclear cell] Mixed chromophobe [chromophobeoncocytoma chromophoberenal cell clear cellchromophobe and clear celloncocytomachromophobe] Oncocytoma Mixed oncocytoma [clear celloncocytoma oncocytomacollecting duct and renal celloncocytoma] and Others [included clear cellsarcomatoid collecting duct mixed epithelial and stromal mucinous tubular and spindle cell multilocular cystic renal neuroendocrine renal cellWilms renal cortical sarcomatoid transitional urothelial and urothelial transitional] Transitional urothelial urothelial and papillary transitional cases were not included in the analysis for counts of pathogenic variants Renal oncocytomas mixed epithelial and stromal tumors are considered benign tumors and were not included in the analysis for counts of pathogenic variants Study a0approval a0 The Western IRB issued a a0regulatory opinion that the Genomic Data Sharing Policy for Ambry Genetics does not involve human subjects based on CFR46102f and associated guidance thus a0the Scientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0crequirement to obtain written patient informed consent was waived A Data Use Agreement and Materials Transfer Agreement was established between Ambry Genetics and Fox Chase Cancer Center The FCCC Institutional Review Board IRB a0provided study oversight and approval protocol number Ambry Genetics a0provided deidentified results for the study All methods were performed in accordance with the relevant guidelines and regulation of the approved studyResultsPatient a0characteristics a0 We first benchmarked the eoRC study cohort to the reported incidence of RC in SEER data for the general US population to provide context In the study cohort of cases were between “ a0years of age and median age of diagnosis was a0years As expected a higher percentage of RC cases were diagnosed between “ a0years of age as compared to patients ‰ diagnosed with RC in the general US population SEER versus Fig a01A The study cohort was female and male Fig a01B Table a0 versus female and male for the general US population prevalence of RC diagnosed ‰ Fig a01B Raceethnicities in study cohort were Caucasian African AmericanBlack Ashkenazi Jewish Hispanic other and unknown Table a0The tumor pathologies reported varied Fig a01C and Table a0 Clear cell constitutes of all RCs in SEER and was the most commonly reported histology in the eoRC cohort Renal cell not defined but likely to predominantly reflect clear cell was also common Fig a01C and Table a0 Papillary and chromophobe histology were each identified in “ of the individuals and respectively Other histologies were identified rarely but included Wilms tumor and oncocytoma For of patients the RC subtype was unknownHigh a0incidence a0of a0other a0cancers a0in a0study a0cohort a0 n of the cases in the study cohort reported at least one additional primary cancer Fig a01D Table a0 Table a0S2 Each of the primary cancer types is also represented at a higher level in the study cohort than in the general US population as reported by the SEER database Fig a01D For femalespecific cancers of females also had breast cancer in comparison to the breast cancer rate in women ‰ in the general population SEER Fig a01D and Table a0S2 The rate of additional primary cancer in the study cohort is much higher than the rate of each cancer type observed in SEER cases with eoRC Fig a01E Finally patients out of reported a family history of cancer and of these patients specifically reported at least one family member with RC Table a0Multigene a0cancer a0panel a0testing a0identifies a0PVs a0in a0DDRR a0genes a0in a0the a0study a0cohort a0 The most common gene with PVs identified in the eoRC patients was the DDRR gene CHEK2 Fig a02A Table a0S3 and S4 consistent with a recent report by Carlo et a0al16 Of patients with CHEK2 PVs n had a common highly damaging variant c1100delC pThr367Metfs that is known to be associated with an increased risk for breast prostate colorectal and thyroid cancers Table a0S434“After CHEK2 PVs were most frequently observed in the DDRR genes BRCA2 ATM and BRCA1 Table a0S3 We compared the overall frequency of PVs in CHEK2 BRCA1 BRCA2 and ATM to the control population in ExAc and gnomAD representing individuals sequenced for diseasespecific and population genetic studies2728 Overall PVs in each of these genes were more common in the study cohort versus the control populations Fig a02BC Table a0S5A An outlier was the moderate risk CHEK2 c470TC p I157T PV38 identified in individuals in the study cohort which was higher in the controls gnomAD OR CI “ ExAc OR CI “ We compared the prevalence of all PVs in DDRR genes presented in Table a0S4 from cases to controls from gnomAD23 We found 48fold enrichment of PVs in DDRR genes in the study cohort versus the controls in gnomAD vs respectively Table a0S5B each DDRR gene was corrected for number of patients assessedCancer risk with MUTYH DDRR gene has only been defined for individuals with homozygous or compound heterozygous PVs but not for heterozygous carriers39 We identified individuals with MUTYH PVs out of which were heterozygous carriers and only was compound heterozygous Only the individual with compound heterozygous MUTYH PVs was counted in the full study cohort n Table a0S3 and Fig a02A Similar to MUTYH cancer risk from the FH RCspecific gene c1431_1433dupAAA pK477DUP variant is currently considered to be pathogenic only in the compound heterozygous or homozygous state40 We identified RC patients who were heterozygous carriers of this specific FH variant Tables a0S3 and S4The overall gene variation rate in the full study cohort n is presented in Table a0S3 The full study cohort was not tested for all genes The largest panel was tested in the subcohort of cases and consisted of genes which included RCspecific genes and othercancer associated genes including DDRR genes Table a0S1 Here of cases had PVs PVs were identified in one or more of the genes not typically associated with RC in cases n Table a0S6 versus n with a PV in RCspecific genes Fig a02D Table a0S6 Of the genes not typically associated with RC were in DDRR genes n or n Among the patients patients were found to have PVs in two genes One patient had PVs in two DDRR genes BRCA1 and MUTYH het and the other patient in a RCspecific gene and a DDRR gene SDHB and MUTYH het Table a0S4 There was no MUTYH or FH compound heterozygous or homozygous PV in the subcohort of casesDDRR a0genes a0PVs a0are a0similarly a0enriched a0in a0patients a0diagnosed a0with a0eoRC a0alone a0or a0with a0eoRC a0and a0other a0cancers a0Individuals who were tested for all genes n could be further separated into two subcohorts those with eoRC as their only diagnosis n and those with eoRC and one or more additional types of cancer n To test the hypothesis that DDRR gene PVs might be Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cAiega yb ssongad iCsesac AgenrnrWilmsersothal cellnrenrnwonknunramebohpomchroebod hmixepomchroar cellncocytocleodncocytomixeoamalnpillarypillary read pmixeapKey A C D E FSEER cohort n97805Ambry cohort n844Bsesac FemaleMaleSEER cohortAmbry cohortDtear recnac brainstabrectalolorecmiaekuleamonelamnariavoaticcrenapstateproamoarcsyroidthetrialuterinemodneEsesac number of primary cancers reportedFigure a0 Patient characteristics A Age range of individuals diagnosed with RC ‰ a0years in SEER cohort compared to the study cohort n of the remaining individuals in the study were diagnosed a0years were diagnosed at a0years and were excluded from the calculations as their age was reported as a wide range of years B Percentage of males and females diagnosed with RC ‰ a0years in SEER compared to the study cohort n C The percentages of reported RC histology up to age a0years in the SEER data n compared to the study cohort n not all histological subtypes reported in SEER were reported in the study cohort D The percentage of cancer incidence at ‰ a0years in the general SEER population versus the study cohort The SEER data reflect individuals reporting the indicated cancer type not individuals with RC in addition to the indicated cancer type E Percentage of different primary cancers reported ‰ a0years in SEER n versus the study cohort n Less than not reported for figure clarityScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cCharacteristicSexMaleFemaleEthnicityAfrican AmericanAshkenazi JewishAsianCaucasianHispanicMiddle EasternMixed EthnicityNative AmericanOtherUnknownMedian age of testingHistologyChromophobeMixed chromophobeClear cellOncocytomaMixed oncocytomaPapillary renalMixed papillaryRenal cellWilmsOthersUnknownPersonal cancer historyRenal cancer onlyRenal cancer plus additional cancer typeFamily history of cancerYesNoNot reportedunknownFamily history of renal cancerYesNoTotalNumber of patients in Ambry study cohort Rate in general population from birth to age SEER of renal cancers of renal cancersnrnrnrnrnrnr a0years Table Demographics and clinical characteristics of RC patients in the Ambry Genetics study cohort Demographics and clinical characteristics of the RC cases in the study cohort were compared to those of RC from birth to age in the SEER data Personal and family history of cancer were reported for the cases in the study cohort For family history of renal cancers numbers include only those who reported on cancer history n nr not reported SEER data included types of renal cancer histologies not all were represented in dataset œother based on other category from Ambry cohort Family histories as selfreported on the intake formmedical records and have not been validatedassociated with eoRC we first analyzed PVs in eoRC cases with no additional primary cancer diagnosis Among the patients who only presented with eoRC PVs were identified in of cases n Fig a02E which is approximately twice the reported frequency of PVs in RCspecific genes7 Among this n of PVs were in one of DDRR genes ATM BRCA1 BRCA2 BRIP1 CHEK2 MLH1 MRE11A NBN PALB2 RAD51C n were in one of RCspecific genes BAP1 FLCN SDHB VHL and the remaining cases bore PVs in nonDDRR genes associated with cancers other than RC Fig a02ENext we performed similar analysis as described above for patients who presented with eoRC plus one or more additional cancers Among the patients who presented with eoRC and at least one additional cancer Scientific RepoRtS 101038s41598020704495Vol1234567890wwwnaturecomscientificreports 0cPVs were identified in cases Fig a02F Among these of cases PVs in othercancer associated genes including DDRR genes were found in of cases n versus n of cases with PVs in RCspecific genes This population was also enriched for PVs in DDRR genes n ATM BRCA1 BRCA2 CHEK2 MSH6 PALB2 PMS2 versus PVs in RCspecific genes BAP1 FLCN MET MITF PTEN SDHB VHLOverall these data suggest that DDRR gene PVs are enriched similarly in individuals diagnosed with eoRC alone or eoRC plus at least one additional primary cancer but that the frequency of PVs in DDRR genes in either group exceeded that in the control populations tested gnomADExAc Fig a0 Table a0S5A The specific PVs identified were similar in frequency to those identified in the full patient cohort n with CHEK2 the most represented DDRR genes Fig a0 To gain additional insight into the prevalence of these PVs in cancer patients we surveyed ClinVar wwwncbinlmnihgovclinv ar and found that multiple PVs from this study Table a0S4 have been reported in hereditary cancer predisposing syndromes HCPS summarized in Table a0S7 HCPS reflects a pattern of cancers in a family characterized by earlier onset with individuals not necessarily having the same tumor andor having more than one primary tumor and having tumors that are more likely to be multicentricRC a0patients a0with a0BRCA1 or BRCA2 a0PVs a0 Notably of the eoRC cases had a BRCA2 PV and RC cases had a BRCA1 PV Table a0 Table a0S3 This included n Table a0 of the cases who presented with only eoRC Interestingly despite the fact that the cohort was female of the detected BRCA1 and BRCA2 PVs were in males Table a0 Of the RC cases with a BRCA1 or BRCA2 PV had an additional cancer associated with hereditary breast and ovarian cancer HBOC syndrome breast ovarian prostate pancreatic or melanoma had an additional nonHBOC cancer and presented with only eoRC Table a0 Family history was reported for cases and of those indicated that at least one family member had an HBOCassociated cancer Of those with a BRCA2 PV reported that at least one family member had RC Table a0No a0correlation a0between a0age a0of a0RC a0diagnosis a0and a0type a0of a0PV a0in a0RC a0 To determine if identification of specific classes of germline PV correlated with age of diagnosis in this cohort genes were divided into four broad overlapping categories all genes in the panel RCspecific genes nonRC genes including DDRR genes and DDRR genes see œMethods The groups were compared to median age at first RC diagnosis of or ‰¥ a0years Given the invariable earlyonset of Wilms tumor the individuals with this diagnosis were excluded from the analysis Within this eoRC cohort there was no significant association between age at diagnosis of RC and the type of PV for any of the four broad categories above Fig a03ACorrelation a0of a0renal a0histologies a0with a0PVs a0in a0specific a0genes a0 Of the clear cell cases in this cohort had a PV of which were RCassociated PVs Similar findings were observed for the cases described as renal cell carcinoma had a PV of which were RCassociated DDRR gene PVs were found in of clear cell cases and in of renal cell cases Figure a03BC contrast the findings in clear cell and renal cell histology with the other nonclear cell histologiesDiscussionThis study for the first time demonstrates that PVs in multiple DDRR genes occur in patients with eoRC Importantly this study found that DDRR gene PVs were represented both in cases diagnosed with eoRC and additional cancers and also cases diagnosed with eoRC alone Comparison with a large control population indicated that germline PVs in DDRR genes were more common in this study cohort than in the control population although further studies are required to confirm this finding and estimate the penetrance of PVs in DDRR genes for eoRC We also found that germline testing using an RCspecific panel would have identified only of the RC cases with actionable PVs according to the NCCN recommended screening or management guidelines compared to the additional cases identified with the expanded panelsThe most common gene with PVs identified in the patients in this study was the DDRR gene CHEK2 This is consistent with recent reports by Carlo et a0al and Huszno et a0al1516 While evidence is mounting that CHEK2 PVs may increase risk for RC in this study we did not consider CHEK2 as a gene typically associated with RC as it is not currently included on RC panels and would fail to be included in testing in many cases In addition limitations of the previous studies and the analysis reported here together indicate that larger studies with appropriate controls are needed before confirming that CHEK2 indeed confers a risk for RCIdentification of germline DDRR gene PVs can have specific implications for the proband and the family For example of cases diagnosed with eoRC alone had PVs in BRCA1 or BRCA2 but not all of these cases had a family history strongly indicative of HBOC syndrome This is important because identification of a BRCA PV can potentially change medical management for instance PARP inhibitor therapy is effective in tumors with BRCA PVs including nonbreast tumors4142 Also screening and prevention of HBOCsyndrome cancers would likely be increased significantly in the proband and in family members found to have the same PV Further many of the specific PVs identified in this study have been annotated as relevant to various HCPS emphasizing their role in the development of multiple cancer types Our results support broader panel testing as a way to identify unexpected highpenetrant PVs in eoRC patients when there is a personal or family history of additional cancers especially an HBOCsyndrome cancerScientific RepoRtS 101038s41598020704495Vol0123456789wwwnaturecomscientificreports 0cA stinairav cnegohapt lan deifitneditot BKey A D E FDDRR genesother cancer associated genesrenal cancer associated genesMTADRABACRBACRBPIRBKEHCHLMHSMHSMAERMHYTUMNBNBLAPSMPCDARCPAARPMBANKDCFNPTPABNCLFTEMFTIMNETPAHDSBHDSLHVPathogenic DDRR variants in Ambry cohort vs ExAc populationC Pathogenic DDRR variants in Ambry cohort vs GnomAD populationKey B CATM BRCA1BRCA2CHEK229211GA3576GA8655dupT5712dupA68_69delAG2475delC7558CT9294CG7069_7070delCT3847_3848delGT2339CG4284dupT518delG4441GA4441CT470TC1
2
" bridge to surgery bts using a selfexpandable metallic stent sems for the treatment of obstructivecolorectal cancer improves the patient™s quality of life this study aimed to examine prognostic factors ofobstructive colorectal cancermethods we analyzed stage iiiii resectable colon cancer cases cur a retrospectively registered between january and december overall patients with cur a obstructive colorectal cancer were evaluated ofthem underwent emergency surgery es group and of them after bts with sems placement bts group wecompared surgical results and prognoses between the two groupsresults a total of patients underwent endoscopic sems placement which technical success of andmorbidity rate of primary anastomosis rates were in es and in bts p postoperativecomplication in es and in bts p pathological findings of lymphatic invasion in es and in bts p venous invasion were in es and in bts p and recurrence of in esand in bts the 3year overall survival was significantly different between two groups es 868bts bts is worse than es logrank test p venous invasion independently predicted worsened recurrencefreeand overall survivals the vascular invasiveness was correlated with tumor progression after sems placement and thesurvival rate was lower in bts sems potentially worsens prognostic outcomes in stage ii“iii obstructive colorectalcancerkeywords bowel obstruction colorectal cancer selfexpandable metallic stent colorectal cancer crc remains the leading cause ofcancerrelated deaths worldwide because several patientsare initially diagnosed during advanced stages correspondence ohtakmedkindaiacjp1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japanfull list of author information is available at the end of the approximately “ of patients with crc were diagnosed with acute colonic obstruction [“] severe malignancy with bowel obstruction needs urgent surgicalintervention which includes primary lesion resectionand stoma creation leading to increased morbidity andmortality and a potential failure to achieve completeoncological resection [ ]an endoscopic procedure with selfexpandable metallic stent sems is an acceptable bridge to surgery bts the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc surgery page of treatment for acute colonic obstruction [“] preoperative sems placement provides an opportunity to perform medical resuscitation comorbidity optimizationbowel preparation tumor staging and observation ofproximal lesions the procedure prevents highriskemergency surgeries and increase oncological resectionand primary anastomosis rates [ ] after the inclusion of colonic sems placement as bts in the coverageof the national health insurance in japan several physicians joined the colonic stent safety procedure researchgroup and developed skills to provide safe treatmentthe largest multicenter prospective study demonstratedthe feasibility and safety of sems placement as bts inpatients with malignant colorectal obstruction the oncological safety and minimalinvasiveness ofthis procedure have confirmed that sems placement asa bridge to elective surgery is not recommended as astandard treatment for symptomatic leftsided malignantcolonic obstruction [ ] several studies reportedthat prognostic factors of malignant colonic obstructionin sems placement had oncological disadvantages compared with those in emergency surgery es [ ] incontrast several trials showed that sems placement as abridge to elective surgery did not improve the survivalrates [“] how sems placement worsens prognosticoutcomes remains unclear [ ]this study aimed to evaluate the induction of curativesurgery in patients with malignant colorectal obstructionafter a sems placement and its longterm results andprognostic factors postoperatively compared to patientswithout sems placement we demonstrated prognosticfactors and overall survival os and recurrencefreesurvival rfs rates for curative surgery after a semsplacementmethodspatientsmedical records of patients who underwent primarycolorectal resection at higashiosaka city medical centerbetween january and december werereviewed all participants provided written informedconsent oralintake and symptoms before and aftersems placement were assessed in table using thecolorectal obstruction scoring system cross from to we recruited patients with all class oftable the colorectal obstruction scoring system crosspatient™s symptom and their condition of an oral intakesolid meal low residue and full diet without symptomcrosscross as stent insertion candidate from we excluded patients with cross and based on updatedstent insertion guideline malignant colorectal obstruction was diagnosed through clinical examinationcross radiography and computed tomography surgery was performed using three approaches es comprised laparotomylymph node dissection as possibleand primary anastomosis on the same day between and bts after sems placement comprised standbylaparoscopy d3 lymph node dissection and primaryanastomosis since january overall patientswith stage iiiii cur a obstructive colorectal cancerwere evaluated of them underwent emergency surgery as es group and of them after bts with semsplacement as bts group we compared surgical resultsand prognoses between the two groupssems devices and the procedurepatients were endoscopically treated with placement ofan uncovered wallflex enteral colonic stent boston scientific corporation natick ma usa or nitis enteralcolonic uncovered stent taewoong inc gimpo southkorea placements were performed as presented in thepreintroduction publicity announcement placement details were mentioned on the website as a brief guideline obstruction structures were determined using aguide wire and a contrast tube was inserted into theproximal colorectal lumen obstructions were measuredusing contrast agents and then the endoscopist determined the number size and type of stent pathologicalbiopsies were recommended after sems locations andintraluminal or extraluminal marking using an endoscopic clip were recommended via visual recognition ofthe endoscopist dilatation of the colonic obstructionbefore sems placement was generally not allowedhistological findingsparaffinembedded specimens were obtained from a cohort of patients diagnosed by the union for international cancer control stage ii“iiisurvival definitionsos was defined as the duration from surgery to anydeath or last followup diagnosis of recurrence was calculated based on recist according to the chemotherapy criteria rfs was defined as the durationfrom surgery to any recurrence includes local recurrenceor distant metastasissolid meal low residue and full diet with symptomliquid or enteral nutrient intakeno oral intakerequiring continuous decompressionstatistical analysisstudent™s ttest and wilcoxon test for continuous variables and the χ2 and fisher™s exact tests for categoricalvariables were conducted survival curves were generated using the kaplan“meier method and compared 0cohta bmc surgery page of table baseline characteristics and outcomes of endoscopicsems placementtable comparison of baseline characteristics in patientsundergoing emergency surgery and bridge to surgerybts n es n pmalefemalemedian range “ “genderagelocationmalefemalemedian rangececumascendingtransversedescendingsigmoidrectumlength of obstructionmedian range cmtechnical successprocedurestentingmorbiditymortalityclinical successthrough the scopethrough the wirewall flex cmnitis cmoverall cda iiicda vcrossbts n “ “ a clavien“dindo classificationusing a logrank test univariate and multivariate survival analyses were performed using the cox proportional hazards regression model all statistical analysesused jmp version sas institute cary nc or statistical scripting language r httpwwwrprojectpvalues of ‰¤ twosided were considered statistically significant this prognostic study complied withthe reporting recommendationsfor tumor markerprognostic studies resultsa total of patients underwent endoscopic semsplacement which was technically safe for malignantcolorectal obstruction with the technical success rate of the clinical success rate was and the patient™ssymptoms and oral intake dramatically improved afterthe sems placement shown in table a total of patients were reviewed and patients underwentes and bts respectively as shown in table baselineclinical characteristics were balanced between the twogroups moreover cases of patients underwent es on the same day as in open surgery the median waiting period for surgery was days for bts thegenderagelocationtype of operationcecumascendingtransversedescendingsigmoidrectumstandbyemergency “ “duration tooperationmedian range dayssurgical procedurelaparotomy laparoscopy timemedian range minblood lossmedian range ml“ ““ “ before afterstoma creation “morbidity30day complication cda iii anastomosticleakagehospital stayaclavien“dindo classificationmedian range days “ “primary anastomosis ratios were in es and in bts p postoperative complication rateswere in es and in bts p postoperative hospital stay was shorter in bts days comp patients withpared to esobstructive crc showed significantinpostoperative complication rate and hospital stay withsems placement operative procedures were dramatically changed and the primary anastomosis rate improved after the sems placementimprovement daysthe pathological tissue type accounted for of differentiated types shown in table tumordepth was similar between the two groups lymphatic vesselinvasion ratios were in es and in bts p and venous invasion ratioswere in es and in bts p recurrence rates were cases in es and cases in bts nodenegative patients stage iimorewhereas nodepositive patients stage iii more frequently had liver metastasis in the kaplan“meier survival analysis in fig 1a the 3year rfs waslung metastasisfrequentlyhad 0cohta bmc surgery page of table comparison of pathological characteristics of emergency surgery and bridge to surgerypt factortotal lymph nodespn factorhistologicallymphatic invasionvenous invasionsurgical clearancet4b t4a t3median rangen0 n1 n2 n3tub1 tub2 othersly v cur a b csignificantly different between the two groups es bts which was significantly low inbts than that in es logrank test p the3year os rate was also significantly different between thetwo groups es bts p shown in fig 1b the relationship between lymph node metastasis and sems placementwas also evaluated the pathological nodenegativestage ii 3year rfs rate was not different betweenthe two groups es bts as shown infig 2athe pathological nodepositivestage iii 3year rfs rate was different between thetwo groups es bts as shown in fig 2bthe stage ii 3year os rate was not different between thetwo groups es bts as shown in fig 2cwhereas stage iii 3year os rate was different between thetwo groups es bts as shown in fig 2dthese results suggestthat vascular invasiveness andpathological nodepositive status were correlated withtumor progression after sems placementthein contrastthuses n “ bts n “ p “survival rate was affected by poor prognosis in the btsgroupresults of adjusted multiple cox proportional hazard regression for rfs and os in all stages and stage iii diseaseare presented in table after adjusting for possible confounders venous invasion and bts independently predicted poor rfs in all stages and venous invasionindependently predicted poor rfs in stage iii disease venous invasion and bts were also significantly associatedwith os in stage iii diseasediscussionacute colonic obstruction requires emergent surgicalintervention a mandatory conventional treatment skillemergent surgicalis associated with highmorbidity mortality and stoma creation rates affectingthe quality of life of patients malignant colorectal obstruction is not only an intestinal obstruction but alsoan advanced stage crc their prognosis was poorerthan that in patients with nonocclusive disease becausetreatmentfig kaplan“meier survival curves in patients undergoing emergency surgery vs bridge to surgery a recurrencefree survival b overall survival 0cohta bmc surgery page of fig kaplan“meier survival curves in patients undergoing emergency surgery vs bridge to surgery a rfs nodenegative patients b rfs nodepositive patients c os nodenegative patients d os nodepositive patientsof highly invasiveness and distant metastasis [ ]chen revealed that the prognosis in patients withperforation associated with obstruction was poor early intervention in the clinical setting before the colonic perforation has been established endoscopic placement of colonic stents improves the high decompressioneffect and reduces clinical symptoms high postoperative complication rates were correlatedwith poor prognosis in patients with cancer in severalans [“] reducing complication rates can improve the prognosis our results showed high clinicalsuccess rate after sems placement and high primarysurgicalinterventions howeveranastomosis rate stentrelated complications requiredemergentthe stentplacement is safe and feasible in this study moreoverthe laparoscopic rate was high and postoperative complication rate was clinical results including shortterm outcomesin bts after sems were verifiedthrough a metaanalysis [ ]the prognosis was poor in patients with stent perforation and increased local recurrence rate after the colonic stent placementthe longtermprognosis in patients with colorectal obstruction afterbts was not different compared with that in patients however 0cohta bmc surgery page of table multivariate analysis of recurrencefree survival at all stages and stage iiipvaluevariablesrecurrence free suvivalall stages hazard ratio ± sd cistage iii hazard ratio ± sd cipvaluees vs semspt factor t3t4pn factor n0n1n2“verous invasion v0“1v2“lymphatic invasion ly0“1ly2“hr ± hr ˆ’ ± ˆ’ ˆ’hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ overall suvivales vs semspt factor t3t4pn factor n0n1n2“verous invasion v0“1v2“lymphatic invasion ly0“1ly2“hr ± hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ without obstruction [“] according to the europeansociety of gastrointestinal endoscopy clinical guidelinethat considers the risk of perforation due to colorectalstents only limited uses are allowed therefore colorectalstent placement is not a standard treatment [“]the prognostic outcomes of bts in this study were significantly worse than those of es particularly in lymphnodepositive patientslymphatic and venous invasionseemed to be a significant prognostic factor althoughreduced postoperative complication rate would improve the prognosis our results were contradictoryafter the stent replacement these results suggestedthat stent placement leads to poor prognosis a concern that colonic stents may be associated with adverse effects of mechanical expansion also exists mechanical expansion may be associated with thegrowth of solid tumors particularly lymphatic andvenous invasion [ ]we found that recurrence and os were associatedwith high vascular invasion after a colonic stent placement venous invasion was an independent factor for recurrence and prognosis the ck20 mrna level anepithelial marker is significantly increased in peripheralblood serum suggesting stent deployment into the vasculature alliteratively ki67 level associated withcellular proliferation and p27 gene assisting cell cycleprogression were measured using specimens obtainedbefore and after sems insertion next the ki67 leveldecreased in the specimen after an sems placementcompared with that before and cell proliferation wassuppressed the prognostic nutritionalindex andserum albumin levels were significantly decreased afterstenting suggesting its disadvantage as bts theduration from stent placement to surgery was daysoncological and nutritional factors might change in theblood and contribute to poor prognosis during the waiting period mechanical expansion of the replacement hr ± ˆ’ hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ hr ± hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ should be minimized to prevent perforation and molecular cytological factors to improve the materials expansion and establishment of new mechanism are necessaryin colorectal obstruction [ ]thisisafirstretrospectivethese findings should be considered in light of severallimitationsnonrandomized small sample sized study from a single institution thereby the heterogeneity of the surgical strategy may have affected the prognostic factors secondalthough validated endoscopic procedures were validated stent devices used in this study had differentlengths types and thickness and obtained from differentvendors lastly we performed stent placement in the patients with cross and who are not indicated forstent insertion until to investigate the oncological longterm prognosis ofcolonic sems placement as a bridge to elective surgerylarge sample size and prospective randomized controlledstudies are warranted to develop a treatment strategy forcrc with obstructionvascular invasiveness was correlated with tumor progression after a sems placement and os and rfs rateswere lower in bts sems placement potentially worsensprognostic outcomes in stage ii“iii malignant colorectalobstructionabbreviationsbts bridge to surgery crc colorectal cancer cross colorectal obstructionscoring system es emergency surgery esge european society ofgastrointestinal endoscopy jsccr japanese society for cancer of the colonand rectum recist response evaluation criteria in solid tumors version os overall survival rfs recurrencefree survival sems selfexpandablemetallic stent wses world society of emergency surgeryacknowledgementsthe authors thank for contribution as endoscopic technical adviser kenkonishi md phd from department of surgery hyogo prefecturenishinomiya hospital 0cohta bmc surgery page of authors™ contributionsall authors have read and approved the manuscript ko protocolproject development data collection and management andmanuscript writingediting mi protocolproject developmentmanagement and manuscript writingediting mu protocolprojectdevelopment and data collection and management jm se jm and ikdata collection and management yt and sn data collection ki andtn data analysis and manuscript writingediting mt and ty dataanalysis and managementfundingauthors have no grant support and no financial relationship for this studyavailability of data and materialsthe datasets used and analyzed during this study are available from thecorresponding author upon reasonable requestethics approval and consent to participateall procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional researchcommittee and with the helsinki declaration and its later amendmentsor with comparable ethical standards all participants or their guardians haveprovided their written informed consent and that the study protocol wasapproved by higashiosaka city medical center ethical committee on humanresearch assignment number “consent for publicationno applicablecompeting intereststhe authors declare that they have no conflict of interest to discloseauthor details1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japan 3thoracic surgeryhigashiosaka city medical center osaka japan 4digestive surgery kawasakimedical school okayama japan 5gastroenterology higashiosaka citymedical center osaka japanreceived june accepted august referencesjemal a bray f center mm ferlay j ward e forman d global cancerstatistics ca cancer j clin “ pubmed pmid epub eng winner m mooney sj hershman dl feingold dl allendorf jd wright jd incidence and predictors of bowel obstruction in elderly patients withstage iv colon cancer a populationbased cohort study jama surg “ pubmed pmid pubmed central pmcid pmc45 epub engjullumstro e wibe a lydersen s edna th colon cancer incidencepresentation treatment and outcomes over years color dis “ pubmed pmid epub engcheynel n cortet m lepage c benoit l faivre j bouvier am trends infrequency and management of obstructing colorectal cancers in a welldefined population dis colon rectum “ pubmed pmid epub engcuffy m abir f audisio ra longo we colorectal cancer presenting assurgical emergencies surg oncol ““ pubmed pmid epub eng mcardle cs hole dj emergency presentation of colorectal cancer isassociated with poor 5year survival br j surg “ pubmedpmid epub eng mainar a tejero e maynar m ferral h castanedazuniga w colorectalobstruction treatment with metallic stents radiology “pubmed pmid epub engzhang y shi j shi b song cy xie wf chen yx comparison of efficacybetween uncovered and covered selfexpanding metallic stents inmalignant large bowel obstruction a systematic review and metaanalysiscolor dis 2012147e367“ pubmed pmid epub engtilney hs lovegrove re purkayastha s sains ps westonpetrides gk darziaw comparison of 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pmid pubmed central pmcid pmc5737828 epub eng mcshane lm altman dg sauerbrei w taube se gion m clark gm reportingrecommendations for tumour marker prognostic studies remark eur jcancer “ pubmed pmid epub eng hashiguchi y muro k saito y ito y ajioka y hamaguchi t japanesesociety for cancer of the colon and rectum jsccr guidelines for thetreatment of colorectal cancer int j clin oncol pubmed pmid epub eng cortet m grimault a cheynel n lepage c bouvier am faivre j patterns ofrecurrence of obstructing colon cancers after surgery for cure a population 0cohta bmc surgery page of based study color dis “ pubmed pmid epub eng nojiri t maeda h takeuchi y funakoshi y kimura t maekura r predictive value of btype natriuretic peptide for postoperative atrialfibrillation following pulmonary resection for lung cancer eur jcardiothorac surg “ pubmed pmid epub engpubmed pmid pubmed central pmcid pmc4128744 epub engkaragiannis gs poutahidis t erdman se kirsch r riddell rh diamandis epcancerassociated 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improvingthe success rate of selfexpandable metal stent insertion front oncol pubmed pmid pubmed central pmcid pmc7237730epub eng miyasako y kuwai t ishaq s tao k konishi h miura r newlydeveloped selfexpandable nitis md colonic metal stent for malignantcolonic obstruction world j gastrointest surg “ pubmedpmid pubmed central pmcid pmc7215972 epub engpublisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationstype natriuretic peptide as a predictor of postoperative cardiopulmonarycomplications in elderly patients undergoing pulmonary resection for lungcancer ann thorac surg “ pubmed pmid epub eng cowie mr struthers ad wood da coats aj thompson sg poolewilsonpa value of natriuretic peptides in assessment of patients withpossible new heart failure in primary care lancet “pubmed pmid epub eng cuthbertson bh card g croal bl mcneilly j hillis gs the utility of btypenatriuretic peptide in predicting postoperative cardiac events and mortalityin patients undergoing major emergency noncardiac surgery anaesthesia“ pubmed pmid epub engsabbagh c browet f diouf m cosse c brehant o bartoli e isstenting as a bridge to surgery an oncologically safe strategy for themanagement of acute leftsided malignant colonic obstruction acomparative study with a propensity score analysis ann surg “ pubmed pmid epub engtung kl cheung hy ng lw chung cc li mk endolaparoscopic approachversus conventional open surgery in the treatment of obstructing leftsidedcolon cancer longterm followup of a randomized trial asian j endoscsurg “ pubmed pmid epub eng gianotti l tamini n nespoli l rota m bolzonaro e frego r aprospective evaluation of shortterm and longterm results from colonicstenting for palliation or as a bridge to elective operation versus immediatesurgery for largebowel obstruction surg endosc “pubmed pmid epub eng yang sy park yy han yd cho ms hur h min bs oncologicoutcomes of selfexpandable metallic stent as a bridge to surgery andsafety and feasibility of minimally invasive surgery for acute malignantcolonic obstruction ann surg oncol “ pubmed pmid epub engvan hooft je van halsema ee vanbiervliet g beetstan rg dewitt jmdonnellan f selfexpandable metal stents for obstructing colonic andextracolonic cancer european society of gastrointestinal endoscopy esgeclinical guideline endoscopy “ pubmed pmid epub eng ansaloni l andersson re bazzoli f catena f cennamo v di saverio s guidelenines in the management of obstructing cancer of the leftcolon consensus conference of the world society of emergency surgerywses and peritoneum and surgery pns society world j emerg surg pubmed pmid pubmed central pmcid pmc3022691epub eng arezzo a balague c targarona e bhi f giraudo g ghezzo l colonic stenting as a bridge to surgery versus emergency surgery formalignant colonic obstruction results of a multicentre randomisedcontrolled trial esco trial surg endosc “ pubmedpmid epub eng amelung fj burghgraef ta tanis pj van hooft je ter b f siersema pd critical appraisal of oncological safety of stent as bridge to surgery inleftsided obstructing colon cancer a systematic review and metaanalysiscrit rev oncol hematol “ pubmed pmid epub eng domingo s puertolas s graciavilla l puertolas ja mechanical comparativeanalysis of stents for colorectal obstruction minim invasive ther alliedtechnol “ pubmed pmid epub engsuzuki n saunders bp thomasgibson s akle c marshall m halligan scolorectal stenting for malignant and benign disease outcomes incolorectal stenting dis colon rectum “ pubmed pmid epub eng voutouri c mpekris f papageis p odysseos ad stylianopoulos t roleof constitutive behavior and tumorhost mechanical interactions in thestate of stress and growth of solid tumors plos one 201498e104717 0c"
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"Despite the biological link between thyroid hormones and breast cancer cell proliferation shown inexperimental studies little is known about the association between hyperthyroidism and breast cancer as well asits association with the most common mammographic and genetic risk predictors for breast cancerMethods This study estimates the incidence rate ratios IRRs of breast cancer among women diagnosed withhyperthyroidism compared to those who are not using two cohorts a Swedish national cohort of the generalfemale population n “ and the Karolinska Mammography Project for Risk Prediction of BreastCancer KARMA n “ We used logistic regression to estimate the odds ratios ORs ofhyperthyroidism according to the mammographic and genetic risk predictors for breast cancerResults An increased risk of breast cancer was observed in patients in the national cohort with hyperthyroidismIRR CI “ particularly for toxic nodular goiter IRR CI “ Hyperthyroidismwas associated with higher body mass index early age at first birth and lower breastfeeding duration Highermammographic density was observed in women with toxic nodular goiter compared to women withouthyperthyroidism Additionally among genotyped women without breast cancer in the KARMA cohort N hyperthyroidism was associated with a high polygenic risk score PRS for breast cancer overall OR CI “ and for estrogen receptorpositive specific PRS OR CI “Conclusion Hyperthyroidism is associated with an increased risk of breast cancer particularly for patients withtoxic nodular goiter The association could be explained by higher mammographic density among these womenas well as pleiotropic genetic variants determining shared hormonalendocrine factors leading to the pathology ofboth diseasesKeywords Breast cancer Hyperthyroidism Mammographic density Genetic pleiotropy Correspondence haominyangkise1Department of Epidemiology and Health Statistics School of Public HealthFujian Medical University Xuefu North Road University Town Fuzhou China2Department of Medical Epidemiology and Biostatistics Karolinska InstitutetSE17177 Stockholm SwedenFull 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 0cYang BMC Medicine Page of BackgroundBreast cancer is the most common cancer diagnosed amongwomen worldwide and the leading cause of cancer deathsamong women [] Breast cancer is usually regarded as ahormonerelated cancer with approximately “ ofcases being estrogen receptorpositive [] Experimental studies show that thyroid hormones can stimulate cell proliferation in breast tissue [ ] At high serum concentrationsthyroid hormones can have estrogenlike effects [“] whichinduce the expression of progesterone receptors [] and canenhance estradiolmediated effects on cell proliferation [ ]Several populationbased studies have reported hyperthyroidism excessive production of thyroid hormones tobe associated with breast cancer [“] while others havefound no association [“] The disparity of findingsmay be explained by differences in study design smallsample sizes or using a specific subgroup of womenMoreover no study to date has examined the impact ofdifferent subtypes of hyperthyroidism on breast cancerrisk which may differ in their pathological processAlthough it is shown that thyroid hormones are associated with breast tissue proliferation and a subsequent increase in breast cancer risk [ ] the association betweenhyperthyroidism and mammographic features of breasttissue is less studied [] Mammographic density refers tothe percentage of radiologically dense epithelium stromaand connective tissues identified in a mammogram Mammographic density is a widely used mammographic featurefor breast cancer risk prediction and is considered an intermediate phenotype for breast cancer [ ] It can therefore be a powerful proxy when investigating the associationbetween hyperthyroidism and breast cancerBesides the direct effect of thyroid hormones on breastcancer risk the association between hyperthyroidism andbreast cancer could also be explained by genetic pleiotropicpathways leading to both hyperthyroidism and breastcancer [] A recent study has shown an associationbetween thyroidrelated single nucleotide polymorphismsSNPs and breast cancer risk [] Howeverit is stillunclear whether a genetic predisposition to breast cancer isassociated with hyperthyroidism which is important forexamining a potential pleiotropic genetic effectIn this study we investigated mechanisms behind the association between hyperthyroidism and breast cancer Specifically whether a genetic susceptibility to breast cancermeasured using polygenic risk score and mammographicdensity could explain some of this association while beingable to account for important factors such as menopausalstatus body mass index BMI and reproductive healthMethodsStudy populationsTo study the association between hyperthyroidism andbreast cancer risk we used two cohorts a Swedishnational cohort of the general female population and a mammographic screeningbased cohort KarolinskaMammography Projectfor Risk Prediction of BreastCancer KARMA Additional file Fig S1The national cohort included all women above age years who lived in Sweden between and N Having a main diagnosis of hyperthyroidismwas retrieved from the Swedish Patient Register andclassified according to the International Classification ofDiseases ICD code E05 Types of hyperthyroidism weredefined by the ICD codes E050 Graves™ disease E051“E052 toxic nodular goiter and E053“E059 other orunspecified types Followup of the cohort ended on thedate of breast cancer diagnosis date of death date ofemigration or end of followup December whichever came first Information on breast cancer diagnosis death and emigration was obtained by usingunique personal identification numbers to link the cohort to the Swedish Cancer Register Swedish Cause ofDeath Register and the Swedish Migration Register TheSwedish Cancer Register was founded in The completeness and accuracy of this register are estimated at and respectively [] The ICD7 code wasused to identify breast cancer diagnoses in the cancerregister and to exclude breast cancer cases before includingquestionnaireKARMA is a screeningbased cohort formed throughan invitation to all women participating in a mammographic screening or clinical mammography in one offour hospitals in Sweden between January andMarch [] During the recruitment period women consented to join the study with a participationrate of Aside from mammographic imaging andblood sample collection participants also answered awebbaseddemographicanthropometric reproductive lifestyle and familial riskfactors related to breast cancer This cohort was linkedto the Swedish Patient Register to retrieve diagnoses ofhyperthyroidism Breast cancer cases in the cohort wereidentified through linkage to the Swedish Cancer Register For consistency with the national cohort followupof the KARMA cohort also started from and endedwith the same criteria as the national cohort except foran extension of the followup time until Dec We therefore excluded women with hyperthyroidism orbreast cancer before ending with the final studypopulation of women Characteristics of womenin the national and the KARMA cohort are shown inAdditional file Table S1Polygenic risk scoreBlood samples from a subset of women who didnot have breast cancer when they joined the KARMAcohort were genotyped These women were part of theBreast Cancer Association Consortium and were 0cYang BMC Medicine Page of iSelectcustom Illuminarandomly selected as controls for the genomewideassociation studies GWAS [ ] Genotyping wasperformed using aarrayiCOGS comprising SNPs [] or OncoArraycomprising of SNPs [] Details of the arraydesign sample handling quality control processes andimputation of nongenotyped variants are described elsewhere [ ] Hyperthyroidism cases were defined aswomen who were diagnosed with hyperthyroidism from to while the controls were women without adiagnosis of hyperthyroidism To assess whether hyperthyroidism is associated with a genetic predisposition ofbreast cancer we selected genomewide significantSNPs reported in a recent metaanalysis of breast cancerGWAS for constructing polygenic risk scores for breastcancer overall and by estrogen receptor ER status []For all individuals a weighted polygenic risk score PRSwas calculated using the following formulaPRS ¼ βx1 þ βx2 þ ¦Î²kxk þ βnxnwhere β is the perallele log odds ratio OR of breastcancerassociated risk allele for SNP k xk is the numberof alleles for the same SNP and n is the totalnumber of the breast cancer SNPs included in the profile The SNPs and corresponding betas used to derivethe PRS are summarized in Additional file Table S2For the analyses PRS was categorized into quartilesMammographic densityFullfield digital mammograms from mediolateral oblique views of the left and right breasts in the most recent screening before were used For the KARMAcohort we used the areabased STRATUS method tomeasure mammographic density [] Percent densitywas calculated by dividing the dense area by the totalbreast area in the mammogram and further categorizedinto quartiles For this part of the study we excludedwomen with any cancer diagnosis as well as those whohad a breast enlargement reduction or surgery resulting in a final study population of Only womendiagnosed with hyperthyroidism before the latest screening were considered as casesThe study was approved by the Regional Ethical Review Board in Stockholm Sweden Dnr and Statistical analysisFor both cohorts the incidence rate ratio IRR of breastcancer among hyperthyroidism patients was calculatedusing Poisson regression using attained age as the timescale and adjusting for calendar period 3year categories For these analyses hyperthyroidism was treated as atimevarying exposurein which the exposed persontime was counted from months after the hyperthyroidism diagnosis index date and the unexposed persontimewas counted from Jan and ended on the date ofbreast cancer diagnosis date of death date of emigrationindex date or end of followup whichever came first Theanalysis was further stratified by menopausal status ageand time since hyperthyroidism diagnosis and type ofhyperthyroidism In the analyses Model adjusted forcalendar period and Model further adjusted for BMIage at menarche number of births family history of breastcancer oral contraceptive use hormone replacementtreatment and having had a benign breast diseaseThe association between hyperthyroidism and variouslifestyle risk factors for breast cancer were assessed amongKARMA women using logistic regression models Theserisk factors included having had a benign breast diseasenumber of births age at first birth breastfeeding durationBMI family history of breast cancer oral contraceptiveuse hormone replacement therapy use age at menarcheand menopausal status Two models were conducted forthis analysis a univariable model and a multivariablemodel adjusting for all these risk factors simultaneouslyGiven that mammographic density is causally relatedto breast cancer risk we also tested the associationbetween having a prior diagnosis of hyperthyroidism andmammographic density among KARMA women usinglogistic regression models Model was the univariablemodel Model adjusted for age at mammogram andBMI and Model further adjusted for age at menarchenumber of births family history of breast cancer oralcontraceptive use hormone replacement treatment andhaving had a benign breast disease Linear regressionmodels were also used to test the effect of hyperthyroidism on mammographic density square roottransformed as a continuous variableTo test the association between hyperthyroidism andhaving a genetic predisposition to breast cancer we usedlogistic regression models to calculate the ORs of hyperthyroidism among KARMA women who had beengenotyped by quartiles of PRS for breast cancer overallfor ERpositive cancers and for ERnegative cancersThese associations were adjusted for age the first threeprinciple components and for genotyping array We further adjusted for age at menarche number of birthsfamily history of breast cancer BMI menopausal statusoral contraceptive use hormone replacement treatmentand having had a benign breast diseaseStatistical analyses were performed using SAS version SAS Institute Inc Cary NC USA and Stata softwareversion Stata Corporation College Station TXResultsIn the national cohort cases of breast cancerpatients were observed during person years 0cYang BMC Medicine Page of following a diagnosis of hyperthyroidism correspondingto an incidence rate of person years Patientswith hyperthyroidism experienced a increased riskof breast cancer IRR CI “ In theKARMA cohort there were cases of breast cancerafter hyperthyroidism diagnosis during a followup timeof person years corresponding to an incidence rateof person years KARMA women with adiagnosis of hyperthyroidism also had a increasedrisk of breast cancer although the association was notstatistically significantin the multivariable adjustedmodel IRR CI “ In both cohortswomen diagnosed with hyperthyroidism aged youngerthan years had a significantly increased risk of breastcancer while there was no strong difference for the riskof breaststatusTable Analyses by type of hyperthyroidism in thenational cohort indicated a stronger association betweenbreast cancer and toxic nodular goiter IRR CI “according to menopausalcancerAmong the major risk factors for breast cancer a diagnosis of hyperthyroidism was significantly associatedwith obesity BMI OR CI “which was predominantly driven by toxic nodular goitersAdditional file Table S3 Other reproductive factorsincluding early age at first birth breastfeeding for lessthan a month and menopausal status were also associated with hyperthyroidism Table When investigating the association between hyperthyroidism and mammographic density no overall statistically significant association was observed Table However a prior diagnosis of toxic nodular goiter wassignificantly associated with high mammographic densityOR for the highest quartile CI “ p for continuous even after adjustingfor all other potential risk factors for breast cancerAdditional file Table S4Among KARMA women with genotyped data a higherPRS for breast cancer was associated with hyperthyroidism OR for the highest quartile CI “ This association was mainly driven by the association between hyperthyroidism and ERpositive breastcancer OR for the highest quartile CI “ Table There was no association betweenhyperthyroidism and PRS for ERnegative breast cancerDiscussionWomen diagnosed with hyperthyroidism had an increased risk of breast cancer compared to the generalpopulation This finding was more pronounced amongTable Risk of breast cancer in women in the national and KARMA cohorts with a diagnosis of hyperthyroidism compared towomen without hyperthyroidismSwedish national cohort N “IRR CI Model No HT “ “ “No BCKARMA cohort N “No HTNo BCIRR CI Model “ “ “IRR CI Model “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “OverallPremenopausalPostmenopausalBy age years “ By years sincehyperthyroidismdiagnosis““By types of hyperthyroidismGraves™ diseaseToxic nodular goiterOthers or unspecified “ “ “ “ “ “ “ “ “Hyperthyroidism patients were identified by the main diagnosis given in the inpatient and outpatient registers IRRs were calculated by comparinghyperthyroidism patients to women without hyperthyroidism using Poisson regression using age as the underlying time scale In the analyses stratified bymenopausal status women with age younger than in the national cohort were considered as premenopausal women while in the KARMA cohort the exactage at menopause was used Model adjusted for calendar period and Model further adjusted for BMI age at menarche number of births family history ofbreast cancer hormone replacement therapy use oral contraceptive use and benign breast disease Statistically significant results are boldedHT hyperthyroidism BC breast cancer IRR incidence rate ratio CI confidence interval 0cYang BMC Medicine Page of Table The association between hyperthyroidism and major breast cancer risk predictors in KARMA cohort N Variable namesBenign breast diseaseNo of nonhyperthyroidismOR2 CINo of hyperthyroidismOR1 CINoYesNumber of births Age at first birthyears “‰¥ Breastfeeding duration months “ BMI kgcm2 category ““ Family history of breast cancerNoYesOral contraceptive useNoYesHormone replacement therapy useNoYesAge at menarche years “ Menopausal statusPremenopausalPerimenopausalPostmenopausalHistory of irregular menstrual periodsNoYes REF “ REF “ “ “ REF “ “ REF “ “ “ REF “ “ REF “ REF “ REF “ REF “ “ REF “ REF “ “ “ REF “ “ REF “ “ “ REF “ “ REF “ REF “ REF “ REF “ “ REF “ “ REF “ “ REF “ REF “OR1 was calculated using a univariate model while OR2 was calculated with a multivariate model including all risk predictors simultaneously The analysis wasrestricted to KARMA women who had not had a breast cancer diagnosis when they entered the studyOR odds radio BMI body mass indexAnalyses for age at first birth and breastfeeding duration were limited to parous women 0cYang BMC Medicine Page of Table The association between hyperthyroidism and mammographic density among KARMA women without a cancer diagnosisn Mammographic densityQ1Q2Q3Q4P for square root continuousNoYesOR CIModel REF “ “ “Model Model REF “ “ “ REF “ “ “Model is the univariate model Model adjusted for age and BMI Model further adjusted for age at menarche number of births family history of breastcancer hormone replacement therapy use oral contraceptive use and benign breast diseasethose diagnosed at a younger age and with a toxic nodular goiter Hyperthyroidism was also associated withbreast cancer risk predictors such as BMI age at firstbirth and duration of breastfeeding The association between mammographic density and hyperthyroidism wasonly observed in women with a diagnosis of toxic nodular goiter Additionally having hyperthyroidism wasassociated with a high PRS for breast cancer particularlyfor ERpositive breast cancerOur estimates for an increased risk of breast canceramong women diagnosed with hyperthyroidism were thesame when analyzing data from the national andKARMA cohorts which is also consistent with previousin Denmark and Taiwan [ ] ThisestimatesTable The association between hyperthyroidism and breast cancer polygenic risk scores PRS among women in the KARMAcohort N PRS for BC overallQ1Q2Q3Q4P for trendStandardized continuousPRS for ER BCQ1Q2Q3Q4P for trendStandardized continuousPRS for ER ˆ’ BCQ1Q2Q3Q4P for trendStandardized continuousNoNo caseOR1 CIOR2 CI REF “ “ “ “ REF “ “ “ “ REF “ “ “ “ REF “ “ “ “ REF “ “ “ “ REF “ “ “ “Analysis was performed among KARMA women without breast cancer and who had genotyped data OR1s were adjusted for age array of genotyping andprinciple components OR2s were additionally adjusted for BMI menopausal status age at menarche number of births family history of breast cancer HRT useoral contraceptive use and benign breast disease Statistically significant results are boldedNo case the number of hyperthyroidism patients No the number of women without hyperthyroidism OR odds ratio CI confidence interval ER estrogen receptor 0cYang BMC Medicine Page of association however was not statistically significant inthe KARMA cohort probably due to a limited numberN The associationof breast cancer patientsbetween hyperthyroidism and breast cancer was furthersupported by an increased risk of breast cancer amongwomen with high serum thyroxine T4 [ ] Despitethis the effect of serum triiodothyronine T3 on breastcancer risk is still conflicting in different studies [ ]which might be influenced by nonthyroidalillnesssyndrome in cancer patients []Several biological pathways may exist between hyperthyroidism and breast carcinogenesis T3 could activatethe PI3K pathways mediated by integrin αvβ3 andstimulate breast cancer cell invasion [] In additionT4 induces MAPKmediated nuclear ERα phosphorylation and promotes cell proliferation to an extent comparable to the effect of estradiol [] which could beinhibited by the T4 analog tetrac []In addition to the known association between thyroidhormones and breast cancer risk it has been hypothesized that I131 radioactive iodine treatment for hyperthyroidism may also increase the risk of breast cancerHowever majority of the studies did not find a significant association between I131 treatment and breastcancer [“] After surgery or radioactive iodine treatment hyperthyroidism patients may subsequently reacha hypothyroid state and thus be prescribed thyroidreplacement medication eg levothyroxine [] Despite this a recent metaanalysis did not find an increasedrisk of breast cancer after subsequent hypothyroidism[] nor following treatment with levothyroxine []Moreover considering the potential late effect of treatment on breast cancer the observed association betweenhyperthyroidism and breast cancer in our study particularly for short term risk cannot be explained by theeffect of treatmentIn this study hyperthyroidism was associated withseveral risk factors for breast cancer Both Graves™disease and toxic nodular goiter are strongly influencedby pregnancy [ ] which could result in early cessation of breastfeeding and supports our finding of anassociation between reduced breastfeeding duration andhyperthyroidism The strong association between hyperthyroidism and breast cancer observed among womenaged below years in both the national and KARMAcohortsfurther suggests that closer surveillance forbreast cancer may be useful among women diagnosedwith hyperthyroidism during their reproductive yearsWe found a significantly higher BMI among patientswith hyperthyroidism despite previous knowledge that reduced weight is associated with hyperthyroidism [] Thisdisparity could be explained by treatments for hyperthyroidism which might eventually result in excess weightgain for patients [ ] Moreoverthe associationbetween hyperthyroidism and BMI was mainly driven bytoxic nodular goiters This finding is consistent withprevious studies indicating that obesity is associated withthyroid nodules [ ]Independent of BMI toxic nodular goiters were associated with higher mammographic density While oneprevious study did not find a significant association between thyroid disorders including hyperthyroidism andthyroid nodules and mammographic densitythis islikely due to having a limited sample size [] Thisfinding in the KARMA cohort was consistent with thepronounced association between breast cancer and toxicnodular goiter in the national cohort Unlike Graves™disease which is an autoimmune disease toxic nodulargoiters are likely to be hormonerelated and thereforemore closely linked to breast cancer Nodular thyroiddiseases are associated with benign breast disease andbreast cancer [ ] and some shared pathways are involved in the proliferation of thyroid and breast tissues[ ] Hence the association between hyperthyroidism and breast cancer could result from both a hormonal effect and tissue proliferation and therefore possiblymediated through mammographic densityAdditionally we found a significant association between breast cancer PRS and hyperthyroidism Amongthose GWAS significant SNPs for breast cancer previous studies show that one SNP in the ABO gene is associated with thyroid function [ ] Another geneoverlapping the GWAS for thyroidstimulating hormoneand breast cancer is IGFBP5 [ ]indicating theshared growth hormoneinsulinlike growth factor GHIGF pathways between thyroid function and breast cancer Although insignificant at the GWAS level one SNPin the DIO1 gene has been associated with both free T4and breast cancer [] suggesting the role of deiodinaseactivity in the association between thyroid function andbreast cancer [] Overall given the genetic associationwas only significant for the ERpositive breast cancerPRS and not for ERnegative PRS we hypothesize thatgenetic pleiotropy between hyperthyroidism and breastcancer is involved in the shared hormonalendocrinepathways for both diseasesConsidering the phenotypic and genetic associations between hyperthyroidism and breast cancer as well as thebeneficial effect of introduced euthyroid hypothyroxinemia in the setting of breast cancer [] hyperthyroidismshould be considered in the evaluation of women™s risk ofbreast cancer Despite concerns of possible overdiagnosisand anxiety caused by mammographic screening [ ]better adherence to the scheduled breast cancer screeningprogram is recommended for patients with hyperthyroidism in order to detect breast cancer at an early stageThe main strength of our study is having a large sample size and a populationbased cohort design including 0cYang BMC Medicine Page of both Swedish health registers and a mammographicscreening cohort Other strengths include the KARMAcohort containing rich lifestyle mammographic andgenetic data which allowed us to explore underlyingmechanisms for the associations investigated We alsoacknowledge severallimitations The validity of ICDcodes in the Swedish patient register is about “[] which indicates potential misclassification Therefore we tried to minimize any potential misclassificationby using main diagnoses only Due to increased medicalsurveillance of patients with hyperthyroidismthesewomen may be diagnosed earlier with breast cancer thanwomen without hyperthyroidism While this possiblebias might slightly shift the temporal risk pattern amongwomen with hyperthyroidism it would not strongly influence the overall association An older age at breastcancer diagnosis among patients with hyperthyroidismsee Additional file Table S1 further argues againstthis surveillance bias In the national cohort we werenot able to adjust for breast cancer risk factors Nevertheless in the KARMA cohort which contains detailedinformation on these confounders multivariable adjustment did not change the point estimates indicating aweak confounding effect ofthese breast cancer riskfactors In the analysis of breast cancer risk using theKARMA cohort we started the followup before womenentered the KARMA study which could have introducedsurvivor bias However we believe the effect of any suchbias would be minimal given that hyperthyroidism isnot a deadly disease and the same estimates were foundusing the national cohortConclusionIn conclusion we found that women diagnosed withhyperthyroidism were associated with an increasedrisk of breast cancer and major predictors for breastcancerincluding mammographic density and polygenic risk score These findings may partly be explained byfactorsbetween these two diseases and may contribute to amorecomprehensive understanding of hormonalendocrinal factors contributing to breast cancer riskshared geneticand hormonalSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1291602001690yAdditional file Table S1 Characteristics of women in the national andKARMA cohorts by hyperthyroidism status Table S2 Single nucleotidepolymorphisms SNPs used for constructing the polygenic risk score PRS forbreast cancer Table S3 The association between hyperthyroidism and majorbreast cancer risk predictors among the KARMA cohort by type ofhyperthyroidism N Table S4 The association betweenhyperthyroidism and mammographic density among KARMA women withoutcancer by type of hyperthyroidism n Figure S1 Sample attritionfor the analyses using the KARMA cohortAbbreviationsBMI Body mass index CI Confidence interval ER Estrogen receptWAS Genomewide association studies ICD International Classification ofDiseases IRR Incidence rate ratio KARMA Karolinska Mammography Projectfor Risk Prediction of Breast Cancer OR Odds ratio PRS Polygenic risk scoreSNPs Single nucleotide polymorphisms T3 Triiodothyronine T4 ThyroxineAcknowledgementsWe thank all the participants in the KARMA studyAuthors™ contributionsHY had full access to all of the data in the study and takes responsibility forthe integrity of the data and the accuracy of the data analysis KC NH andHY conceived and designed the study All authors acquired analyzed orinterpreted the data HY and NH drafted the manuscript All authors criticallyrevised the manuscript for important intellectual content HY performed thestatistical analysis KC and HY obtained the funding All authors read andapproved the final manuscriptAuthors™ informationNot applicableFundingThis work was supported by the Swedish Research Council [grant no ] Swedish Cancer Society [grant no CAN190266] and FORTE [grant no] We would like to also acknowledge Märit and Hans Rausing™s Initiative against Breast Cancer Haomin Yang is supported by Startup Fund forhighlevel talents of Fujian Medical University [grant no XRCZX2020007] andStartup Fund for scientific research Fujian Medical University [grant no2019QH1002] access funding provided by Karolinska InstituteAvailability of data and materialsThe KARMA data used in the present study are available from thecorresponding author upon reasonable request The registerbasednationwide cohort datasets linked and analyzed in the current study are notpublicly available due to Swedish law but are available by applying throughthe Swedish National Board of Health and Welfare and Statistics SwedenDetailed information on data application can be found using the followinglinks httpsbestalladatasocialstyrelsensedataforforskning and httpswwwscbsevaratjansterbestallamikrodataEthics approval and consent to participateThe study was approved by the Regional Ethical Review Board in StockholmDnr and KARMA participantsconsented to participate in the study with written informed consentConsent for publicationAll authors approved the manuscript and consented to its publicationCompeting interestsThe authors declare no competing interestAuthor details1Department of Epidemiology and Health Statistics School of Public HealthFujian Medical University Xuefu North Road University Town Fuzhou China 2Department of Medical Epidemiology and BiostatisticsKarolinska Institutet SE17177 Stockholm Sweden 3Department of OncologySouth General Hospital SE11883 Stockholm SwedenReceived February Accepted June ReferencesGlobal Burden of Disease Collaboration Global regional and nationalcancer incidence mortality years of life lost years lived with disability anddisabilityadjusted lifeyears for cancer groups to a systematicanalysis for the global burden of disease study JAMA Oncology “Allred DC Brown P Medina D The origins of estrogen receptor alphapositive and estrogen receptor alphanegative human breast cancer BreastCancer Res “ 0cYang BMC Medicine Page of Hall LC Salazar EP Kane SR Liu N Effects of thyroid hormones onhuman breast cancer cell proliferation J Steroid Biochem Mol Biol““Conde I Paniagua R Zamora J Blanquez MJ Fraile B Ruiz A Arenas MIInfluence of thyroid hormone receptors on breast cancer cell proliferationAnn Oncol “Dinda S Sanchez A Moudgil V Estrogenlike effects of thyroid hormone onthe regulation of tumor suppressor proteins p53 and retinoblastoma inbreast cancer cells Oncogene “Nogueira CR Brentani MM Triiodothyronine mimics the effects of estrogenin breast canc
2
] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0“ in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global moran™s index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of pm25 and pm10 in “ respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range “ “ “ pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0“ was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0“ was associated with a increase of daily confirmed new cases wuhan rr ci1032“ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p χ2 p a μgm3 increase in pm25 was associated with a “ increase in fatality rate χ2 p a μgm3 increase in pm10 was associated with a “ increase in fatality rate no2 a 10μgm3 no2 increase lag0“ was associated with a increase in daily confirmed new cases wuhan rr ci “ xiaogan rr ci “ huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci “ estimate on deaths value cid0 ci “ kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearman™s one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci “ and deaths cid0 ci “ low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years “ and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0“ in no2 is associated with a ci “ increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053“ xiaogan rr ci “ but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations “ pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol “ httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res “ httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health “ httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med “ httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health “ https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res “ https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 “ conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop
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the incidence and death rate of nonsmall cell lung cancer nsclc in china ranks the first among the malignant tumors circular rna circrna was reported to be involved in the progression of nsclc our study aimed to investigate the underlying mechanism of circ_0020123 in nsclc progressionmethods quantitative realtime polymerase chain reaction qrtpcr was used to detect the expression of circ_0020123 mir5905p and thrombospondin thbs2 in nsclc tissues and cells cell proliferation and migration were examined by cell counting kit8 cck8 assay and transwell assay respectively flow cytometry assay was used to detect the apoptosis of nsclc cells the protein levels of ki67 matrix metalloprotein9 mmp9 cleavedcaspase9 cleavedcasp9 and thbs2 were detected by western blot the targets of circ_0020123 and mir5905p were predicted by starbase and targetscan and then confirmed by dualluciferase reporter assay and rna immunoprecipitation rip assay the animal experiment showed the effect of circ_0020123 on tumor growth in vivoresults the expression of circ_0020123 was upregulated in nsclc tissues and cells functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of nsclc cells interestingly circ_0020123 directly targeted mir5905p and inhibition of mir5905p reversed the knockdown effects of circ_0020123 on nsclc cells more importantly thbs2 was a target of mir5905p and thbs2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in nsclc cells finally suppression of circ_0020123 inhibited tumor growth in vivo through mir5905pthbs2 axis circular rna circ_0020123 regulated thbs2 by sponging mir5905p to promote cell proliferation and migration and inhibit cell apoptosis in nsclc cellskeywords nsclc circ_0020123 mir5905p thbs2highlights circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in nsclc cellscorrespondence bskrju163comdepartment of thoracic surgery lianyungang second people™s hospital no hailian east road haizhou district lianyungang jiangsu china circ_0020123 directly targeted mir5905p and mir5905p downregulation reversed the knockdown effects of circ_0020123 on nsclc progression thbs2 acted as a target of mir5905p and overthe effects of expression of thbs2 reversed circ_0020123 knockdown on nsclc progression downregulation of circ_0020123 suppressed tumor growth in vivo through mir5905pthbs2 axis the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cwang a0et a0al cancer cell int page of lung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis small cell lung cancer sclc accounts for about of all lung cancer cases however nonsmall cell lung cancer nsclc accounts for of lung cancer and the a0years overall survival rate os is only about therefore it is important to find the effective treatment and potential molecular targets of nsclc progressioncircular rna circrna is a single stranded rna molecule with a closed circular structure recently amounts of circular dna have been discovered and most of which were thought to be the byproducts of typical splicing [ ] previous reports indicated that the expression of circrna was tissuespecific and the change of its expression intensity was associated with some diseases [“] furthermore circrna was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] for example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging mir34a5p to regulate cc chemokine ligand ccl22 meanwhile hsa_circ_0043256 participated in the progression of nsclc cells by mediating the cinnamaldehyde treatment a previous report suggested that circ_0020123 acted as an oncogene in nsclc and circ_0020123 regulated zincfingerenhancer binding protein zeb1 and enhancer of zeste homolog ezh2 by competitively binding with mir144 to induce cell progression and migration these reports suggested that circ_0020123 was a vital factor in the pathogenesis of nsclc and its function and molecular mechanism need to be further studiedas a small endogenous rna microrna mirna is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers recently some aggregated mirnas have been found in prostate cancer such as mir221222 mir143145 mir23b27b241 and mir1133a which were downregulated and had tumor inhibiting functions a previous study found that circulating mir5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy besides overexpression of mir5905p reduced the development of nsclc cells and regulated the expression of epithelialmesenchymal transformation emtrelated proteins by targeting the signal transducers and activators of transcription stat3 however the precise mechanism by which mir5905p affects nsclc needs further investigationthrombospondin thbs2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer colorectal cancer and nsclc a previous report suggested that thbs2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by mir20a tian et a0al found the expression and clinicopathological features of thbs2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis however the molecular function of thbs2 in nsclc remains poorly definedin this study the targeting relationship between circ_0020123 and mir5905p was firstly detected the effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesmaterials and a0methodspatients and a0specimensnsclc tissues and the adjacent healthy lung tissues were taken from nsclc patients in the lianyungang second people™s hospital all volunteers signed written informed consents nsclc tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °c for further experiments this research was approved by the ethics committee of lianyungang second people™s hospitalcell culture and a0cell transfectiontwo nsclc cell lines a549 and h1299 and one normal lung cell line imr90 were obtained from the beijing concorde cell library beijing china a549 h1299 and imr90 cells were cultivated in dulbecco™s modified eagle medium dmem hyclone logan ut usa supplementing with fetal bovine serum fbs hyclone and cultured in an incubator at a0„ƒ with co2small interfering rna sirna targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin rna shrna targeting circ_0020123 shcirc_0020123 mir5905pinhibitors sirna negative control sinc shnc and ncinhibitors were all obtained from biomics biotech jiangsu china full length of thbs2 cdna sangon biotech shanghai china was subcloned into pcdna31 plasmid ekbioscience shanghai china then cell transfection was performed by lipofectamine thermo fisher scientific waltham ma usa 0cwang a0et a0al cancer cell int page of rna isolation and a0quantitative real‘time polymerase chain reaction qrt‘pcrthe trizol reagent invitrogen carlsbad ca usa was used for extracting the total rnas next the reversed transcription was carried out by rtpcr kit invitrogen the qrtpcr was performed using the abi sybr green master mix invitrogen the primers in our study were as follows f5²ttc gga cga ccg tca aac at3² and r5²agg atc cct gca cca caa tg3² for circ_0020123 f5²tga aag acg tga tgg cac ac3² and r5²ctt cca ttt tgg for mir5905p f5²aga agg ggt ttt tgg3 ² ctg ggg ctc att tg3² r5²agg ggc cat cca cag tct tc3² for glyceraldehyde3phosphate dehydrogenase gapdh f5²gcg gct ggg tct att tgt c3² and r5²gca gga ggt gaa gaa cca tc3² for thbs2 f5²att gga acg ata cag aga agatt3² and r5²gga acg ctt cac gaa ttt g3² for u6 gapdh and u6 were the internal parameterscell counting kit‘ cck‘ assaythe proliferation viability of a549 and h1299 cells were detected by the cellcounting kit8 msk wuhan china a549 and h1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°c for or a0h then a0μl fresh medium and cck8 solution was added after incubation at a0°c for a0h the od values were detected by the multiskan ascent microplate reader abcam cambridge ma usatranswell assaytranswell chamber corning life sciences corning ny usa was used to detect cell migration firstly the serumfree dmem thermo fisher scientific was fixed with cell suspension cells and seeded into the upper chamber and the dmem containing serum was put into the lower chamber after incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed finally the migrated cells were stained with crystal violet corning life sciences and observed by using a microscopeflow cytometryfirstly a549 and h1299 cells were cultured and pbs was used for washing cells then the binding buffer was used to resuspend cells and the annexin vfluorescein isothiocyanate vfitcpropidium iodide pi apoptosis detection kit thermo fisher scientific was used to stain cells finally cell apoptosis was detected by flow cytometry thermo fisher scientificwestern blot analysisthe total proteins of nsclc tumors or cells were collected by ripa lysis buffer sangon biotech then the proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis sdspage and transferred to polyvinylidene fluoride pvdf membranes thermo fisher scientific the skimmed milk was added and incubated with primary antigapdh antibody invitrogen carlsbad ca usa antiβactin antibody invitrogen antiki67 antibody invitrogen antimatrix metalloprotein9 mmp9 antibody invitrogen anticleavedcaspase9 cleavedcasp9 antibody invitrogen or antithbs2 antibody invitrogen at a0°c overnight finally the membranes were incubated with the secondary antibody for a0 h at room temperature the results were viewed using kodak film developer fujifilm japandual‘luciferase reporter assaysthe wild type circ_0020123 sequences circ_0020123wt mutant circ_0020123 sequences circ_0020123mut wild type thbs2 ²utr sequences thbs2wt mutant thbs2 ²utr sequences thbs2mut were cloned into pgl3 luciferase reporter plasmid promega madison wi usa then the plasmid and mir5905p or mirnc were cotransfected into a549 and h1299 cells by lipofectamine thermo fisher scientific after transfection for a0h the dualluciferase reporter assay system promega was performed to detect the luciferase activityrna immunoprecipitation ripfirstly the magna rip rnabinding protein immunoprecipitation kit gzscbio guangzhou china was performed to verify the relationship between circ_0020123 and mir5905p in brief the magnetic beads and antiago2 antibody abcam were added into cells and incubated for a0h then the proteinase k and the phenol“chloroformisoamyl alcohol reagent were added for purifying rnas finally qrtpcr was used to measure circ_0020123 enrichmentanimal experimentsthe 4weekold balbc male nude mice vitalriver beijing china were raised in a sterile environment for 0cwang a0et a0al cancer cell int page of experiments then pbs was used to suspend a549 cells × transfected with shcirc_0020123 or shnc next the nude mice were divided into two groups n a549 cells transfected with shcirc_0020123 or shnc were shcirc_0020123 or shnc inoculated into the nude mice the tumor volume was detected every a0 days after a0days the nude mice were euthanatized and the tumor weight was detected besides the tumor tissues from each group were collected to detect the expression of circ_0020123 mir5905p and thbs2 the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalstatistical analysisthe software graphpad prism was performed for statistical analysis the data was displayed as mean ± standard deviation sd the significant difference was calculated by student™s t test and oneway analysis of variance anova p was considered as statistically significantresultscirc_0020123 was a0upregulated in a0nsclc tissues and a0cellsto begin with qrtpcr was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in nsclc tissues compared with the adjacent healthy tissues fig a0 1a similarly the expression of circ_0020123 in nsclc cells a549 and h1299 was markedly higher than that in normal cells imr90 fig a01b from these data it is speculated that circ_0020123 might be acted as an oncogene in nsclcfig circ_0020123 was upregulated in nsclc tissues and cells a qrtpcr was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b the expression of circ_0020123 in normal cell line imr90 and nsclc cell lines a549 and h1299 was detected by qrtpcr p downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0nsclc cellsto investigate the functional effects of circ_0020123 on nsclc cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into a549 and h1299 cells firstly the transfection efficiency was detected by qrtpcr fig a02a next cck8 was used to detect the proliferation and the results showed that the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced fig a0 2b moreover the migration of a549 and h1299 cells was significantly downregulated by circ_0020123 knockdown fig a02c in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with sinc fig a02d finally the protein levels of cell proliferationrelated protein ki67 and cell migrationrelated protein mmp9 were inhibited while cell apoptosisrelated protein cleavedcasp9 was upregulated in nsclc cells transfected with sicirc_00201231 or sicirc_00201232 fig a02e these data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in nsclc cellscirc_0020123 directly targeted mir‘‘5pby searching in the online software starbase the potential binding sites between circ_0020123 and mir5905p were detected fig a0 3a to confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123wt reporter plasmid was reduced by mir5905p mimic while the circ_0020123mut reporter plasmid activity was not changed in a549 and h1299 cells fig a03b furthermore the expression of mir5905p was lower in a549 and h1299 cells compared with that in imr90 cells fig a0 3c in contrast mir5905p expression was elevated in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a0 3d finally the rip assay was also used to confirm the targeting relationship between circ_0020123 and mir5905p and the results showed that circ_0020123 and mir5905p were enriched in antiago2 group fig a03emir‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0nsclc cellsto further explore the functional effects between circ_0020123 and mir5905p mir5905pinhibitor was established qrtpcr was used to detect the transfection efficiency fig a0 4a interestingly mir5905p was upregulated in a549 and h1299 cells transfected with sicirc_00201231 while the expression of mir5905p was recovered in cells transfected with 0cwang a0et a0al cancer cell int page of fig downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of nsclc cells a the transfection efficiency of sicirc_00201231 and sicirc_00201232 in a549 and h1299 cells was detected by qrtpcr b cck8 assay was used to detect the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 c the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by transwell assay d flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 e the protein levels of cell proliferation related protein ki67 cell migration related protein mmp9 and cell apoptosis related protein cleavedcasp9 were detected by western blot p fig a0sicirc_00201231 mir5905pinhibitors 4b moreover circ_00201231 knockdown inhibited cell proliferation and migration while the mir5905p inhibitor reversed these effects fig a0 4c d in addition the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 was increased which was abolished by mir5905pinhibitor fig a0 4e similarly mir5905p inhibitors reversed the effects on the protein levels of ki67 mmp9 and cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 fig a0 4f these results that mir5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of nsclc cellsindicated mir‘‘5p targeted thbs2 in a0nsclc cellsthe thbs2 ²utr was predicted to contain the binding sites of mir5905p through the online software targetscan fig a05a then the dualluciferase reporter assay was used to confirm the targeting relationship the results showed that cotransfection of mir5905p and thbs2wt significantly limited the luciferase activity in both a549 and h1299 cells the luciferase activity was not altered in cells cotransfected with mir5905p and thbs2mut fig a05b importantly the mrna and protein level of thbs2 was enahnced in nsclc cells fig a05c d we further explored whether circ_0020123 affected the functions of thbs2 in nsclc cells the mrna and protein expression of thbs2 were repressed in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 fig a05e f 0cwang a0et a0al cancer cell int page of fig circ_0020123 directly targeted mir5905p a the binding site between circ_0020123 and mir5905p was detected by the online software starbase b the luciferase activity of circ_0020123wt or circ_0020123mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p was detected by dualluciferase reporter assay c qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells d the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr e rip assay was used to confirm the relationship between circ_0020123 and mir5905p p thbs2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0nsclc cellsbased on the work ahead of us the pcdna31thbs2 was constructed then the qrtpcr and western blot were used to detect the transfection efficiency and the thbs2 expression was increased in a549 and h1299 cells transfected with pcdna31thbs2 fig a0 6a b in addition the proliferation and migration rates of a549 and h1299 cells transfected with sicirc_00201231 pcdna31thbs2 were higher than that transfected with sicirc_00201231 fig a0 6c d meanwhile a similarly phenomenon was also observed in cell apoptosis the pcdna31thbs2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis fig a0 6e furthermore the effects of circ_0020123 deletion on ki67 mmp9 and cleavedcasp9 protein levels were also reversed by thbs2 overexpression fig a0 6f these data suggested that overexpression of thbs2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisreduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123mir‘‘5pthbs2 axisto further explore the function of circ_0020123 in nsclc cells the shcirc_0020123 was constructed and the xenograft tumor was established then a549 cells transfected with shcirc_0020123 or shnc were injected into the nude mice the xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shnc group fig a07a moreover tumor weight was inhibited by circ_0020123 knockdown fig a0 7b furthermore the expression circ_0020123 and thbs2 was decreased while the mir5905p was increased in xenograft tumor transfected with shcirc_0020123 fig a0 7c western blot assay also revealed that the protein level of thbs2 was repressed by circ_0020123 knockdown fig a07d finally the digital tomosynthesis dts was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group fig a07e the results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivodiscussionclinically only a few nsclc patients were diagnosed at an early stage and treated by surgical resection more than of nsclc patients were diagnosed with the advanced stage or metastatic tumors thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of nsclcrecently circrna was no longer considered as a random product in the rna shearing process and its biological significance and function in malignant tumors 0cwang a0et a0al cancer cell int page of fig mir5905p downregulation reversed circ_0020123 knockdown effects in nsclc cells a qrtpcr was used to detect the expression of mir5905p in a549 and h1299 cells transfected with mir5905pinhibitors b the expression of mir5905p in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was detected by qrtpcr c the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors was tested by cck8 assay d transwell assay was used to measure the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors e flow cytolysis assay was used to detect the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 mir5905pinhibitors were detected by western blot p had received more and more attention previous reports revealed that circ_0020123 was involved in the development of nsclc moreover the level of circ_0020123 was elevated in nsclc cells consistently we found that the expression of circ_0020123 was markedly higher in nsclc tissues and cells moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of nsclc cells in a0 vitro besides circ_0020123 promoted tumor growth in a0vivoendogenous circrnas could act as microrna sponges to inhibit their function and some studies linked mirna sponges to human diseases including cancer a previous study indicated that circrna ctransferrin receptor ctfrc regulated tfrc by sponging mir107 to facilitate bladder carcinoma development mir5905p was studied in different cells such as airway smooth muscle cells colon epithelial cells and nsclc cells however the potential relationship between mir5905p and circrna has not been researched in this study circ_0020123 directly targeted mir5905p and mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc progression these data provided a clue to the therapeutic strategy for nsclc 0cwang a0et a0al cancer cell int page of fig mir5905p targeted thbs2 in nsclc cells a the potential binding site between thbs2 ²utr and mir5905p was predicted by the online software targetscan b dualluciferase reporter assay was used to measure the luciferase activity of thbs2wt or thbs2mut reporter plasmid in a549 and h1299 cells transfected with mirnc or mir5905p c qrtpcr was used to detect the mrna expression of thbs2 in nsclc cells d the protein level of thbs2 in nsclc cells was tested by western blot e the mrna expression of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qrtpcr f western blot was used to measure the protein level of thbs2 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201232 p our study also confirmed that mir5905p could target thbs2 directly in nsclc cells thbs2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase mmp genes involved in tissue formation and repair [ ] a previous document suggested that thbs2 acted as a target of mir2213p and participated in lymph node metastasis in cervical cancer the data in this research showed that the expression of thbs2 in nsclc cells was markedly higher than normal healthy cells furthermore overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells suggesting that circ_0020123 promoted the progression of nsclc cells through mir5905pthbs2 axisin our research showed that the expression of circ_0020123 was higher in nsclc tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of nsclc cells and also suppressed tumor growth in a0 vivo moreover circ_0020123 directly targeted mir5905p while mir5905p inhibition reversed the effects of circ_0020123 knockdown on nsclc cells more importantly circ_0020123 regulated the expression of thbs2 by sponging mir5905p and upregulation of thbs2 reversed the effects of circ_0020123 knockdown on nsclc cells therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cwang a0et a0al cancer cell int page of fig overexpression of thbs2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of nsclc cells a b the mrna and protein expression of thbs2 in a549 and h1299 cells transfected with pcdna31thbs2 was detected by qrtpcr and western blot c cck8 assay indicated the proliferation of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 d the migration of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was measured by transwell assay e the apoptosis of a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 was detected by flow cytolysis assay f the protein levels of ki67 mmp9 cleavedcasp9 in a549 and h1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcdna31thbs2 were detected by western blot p apoptosis of nsclc cells by sponging mir5905p to regulate thbs2results and develop the manuscript all authors read and approved the final manuscriptabbreviationsnsclc nonsmall cell lung cancer circrna circular rna qrtpcr quantitative realtime polymerase chain reaction cck8 cell counting kit8 mmp9 matrix metalloprotein9 cleavedcasp9 cleavedcaspase9 cleavedcasp9 cleavedcaspase9 rip rna immunoprecipitation zeb1 zincfingerenhancer binding protein ezh2 zeste homolog stat3 signal transducers and activators of transcription thbs2 thrombospondin acknowledgementsnot applicableauthors™ contributionslw collaborated to design the study lz were responsible for experiments analyzed the data yw wrote the paper all authors collaborated to interpret fundingnoneavailability of data and materialsplease contact corresponding author for data requestsethics approval and consent to participatethis research was approved by the ethics committee of lianyungang second people™s hospital the animal experiment was approved by the animal experimentation ethics committee of lianyungang second people™s hospitalconsent for publicationall listed authors have actively participated in the study and have read and approved the submitted manuscript 0cwang a0et a0al cancer cell int page of fig reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123mir5905pthbs2 axis a a total of × a549 cells transfected with shcirc_0020123 or shnc were injected into nude mice to establish the xenograft tumor tumor volume was measured every d after injection b tumor weight was measured on d c the expression of circ_0020123 mir5905p and thbs2 in xenograft tumor was measured by qrtpcr d the protein level of thbs2 in xenograft tumor was evaluated by western blot e the number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis dts p competing intereststhe authors declare that they have no competing interestsreceived april accepted july references bray f ferlay j soerjomataram i siegel rl torre la jemal a global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries ca cancer j clin “ abe h takase y sadashima e fukumitsu c murata k ito t kawahara a naito y akiba j insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value cancer cytopathol “li c zhang l meng g wang q lv x zhang j li j circular rnas pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer j cancer res clin oncol “ belousova ea filipenko ml kushlinskii ne circular rna new regulatory molecules bull exp biol med “ zhang z xie q he d ling y li y li j zhang h circular rna new star new hope in cancer bmc cancer li l chen y nie l ding x zhang x zhao w xu x kyei b dai d zhan s guo j zhong t wang l zhang h myodinduced circular rna cdr1as promotes myogenic differentiation of skeletal muscle satellite cells biochim biophys acta gene regul mech “ greco s cardinali b falcone g martelli f circular rnas in muscle function and disease int j mol sci weng xd yan t liu cl circular rna_larp4 inhibits cell migration and invasion of prostate cancer by targeting foxo3a eur rev med pharmacol sci “ deng n lei d huang j yang z fan c wang s circular rna expression profiling identifies hsa_circ_0011460 as a novel molecule in severe preeclampsi
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among synchronous colorectal cancers scrcs reported previously the incidence of quadruple advanced scrcs is very rarewe present the case who underwent laparoscopic twosegment resection of the colon requiring two anastomoses that wasperformed for quadruple advanced cancers and four tumors were curatively removed there were no signs of recurrence at months after surgery laparoscopic surgery provided less invasiveness even for quadruple advanced scrcs in terms of earlyrecovery with an acceptable longterm outcomeintroductionsynchronous colorectal cancers scrcs are characterized bythe simultaneous occurrence of multiple primary tumors inthe same patient synchronous malignancies most commonlyoccur in the colon among other ans [“] the occurrence ofadvanced scrcs is rare and may be identified at any locationwithin the large intestine the prevalence of scrcs is reportedto range from to among these however the incidence of quadruple advanced scrcs is extremely rare accounting for of all scrcs surgical resection is considered thestandard treatment for scrcs as a surgical approach laparoscopic surgery has significant advantages in terms of shortterm outcomes including early recovery and no disadvantageouslongterm outcomes according to recent reports laparoscopicsurgery has been used in scrcs but these reports noted thatcontroversy remains concerning operative procedures for multiple segmental resections and for total or subtotal colectomywe report the case who presented with quadruple synchronousadvanced cancers arising from the colon which were successfully treated with laparoscopic twosegment colectomyreceived may accepted june published by oxford university press and jscr publishing ltd all rights reserved the authors this is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly citedfor commercial reuse please contact spermissionsoupcom 0cj ma figure colonoscopy images showing four tumors a one cauliflowerlike tumor with lumen stenosis is located in the ascending colon b another cauliflowerliketumor is located in the descending colon the third c and fourth d tumors are located in the sigmoid coloncase presentationa 70yearold man who was positive for a blood stool testvisited our hospital colonoscopy computed tomography ctand barium enema indicated quadruple concurrent locallyadvanced cancers the firsttumor with observed lumenstenosis was located in the ascending colon the secondtumor was located in the descending colon and the third andfourth tumors were located in the sigmoid colon fig ctrevealed marked intestinal wall thickness in the ascendingdescending and sigmoid colon fig preoperative precisesimulation using 3d angiography was performed to determineadequate lymph node dissection along the arteries feedingthe tumors and appropriate resection to avoid anastomoticleakageswe planned the appropriate placement of trocars as shownin figure because we wanted to create a single minilaparotomy for specimen retrieval and extracorporeal reconstruction after lymph node dissection and mobilization of thecolonduring the operation laparoscopic exploration confirmed thepresence of known four tumors with no invasion of the serosasubsequently a right hemicolectomy and sigmoid colectomywere performed laparoscopically the right half of the colon wasseparated and a sidetoside anastomosis between the jejunumand transverse colon was performed followed by the sigmoidcolon and a colorectal anastomosis between the descendingcolon and rectum was performed the resected tissue specimensrevealed four tumors fig histological examination showedthat the first tumor in the ascending colon the second tumor inthe descending colon and the third tumor in the sigmoid colonhad invaded up to the subserosa whereas the fourth tumor inthe sigmoid colon had invaded up to the muscularis propriafig according to the american joint committee on cancertumornodemetastasis staging system the pstage was iiia t3n1m0the patient was discharged days after surgery for adjuvantchemotherapy the patient chose to take an oral fluoropyrimidine agent for months fortunately there have been no signsof metastasis or recurrence after the operation at months offollowupdiscussionwe reported a rare case of quadruple scrcs all four tumorswere removed curatively by laparoscopic surgery with d3 lymphnode dissection we planned a strategy for quadruple scrcsbased on preserving the remnant large intestine and sufficientd3 lymph node dissection through a laparoscopic approachwe believe that laparoscopic surgery can be a safe even forquadruple scrcs this is the first case report of laparoscopicsurgery with d3 lymph node dissection for quadruple advancedscrcsthe incidence of malignant scrc with four or five synchronous lesions is extremely rare with a rate of beingreported this is a quite rare case of quadruple synchronous 0cquadruple advanced synchronous colorectal cancersfigure placement of trocars and miniincision in the present casethan the index cancer however all of the scrcs in our patienthad the same histological grade and t staging pstage iiiawith the tumor locations being in the ascending descending andsigmoid colonsurgical management of scrcs needs to be tailored tothe individual based on tumor location invasion status andthe patient™s health condition some studies have suggestedtotal or subtotal colectomy to remove any potential existingsynchronous tumors or polyps that have not been detected however other studies recommend a more conservativesurgical approach it is thought that the removal of the entirecolon will prevent the development of metachronous tumorsand a previous study indicated that subtotal colectomy mayincrease defecation frequency as the normal colon cannot bepreserved we successfully performed laparoscopic surgerycombining twosegment resection of a right hemicolectomyand sigmoid colectomy with no intra or postoperative adverseevents in our patient we tried preserving as much colonas possible considering the patient™s quality oflife aftersurgery in addition to performing sufficient d3 dissection ofcourse the meaning of preserving colon in terms of patientpostoperative quality of life needs to be more clearly assessed infuturewe encountered a rare case of advanced quadruple scrcsfor which we achieved a curative resection that required twoanastomoses through a laparoscopic approach we suggest thatlaparoscopic surgery that requires multiple anastomoses foradvanced scrcs can be a safe procedure even if the number ofcolorectal cancers is multiplefigure abdominal ct scan revealing a tumor of the ascending colon aarrowhead a tumor in the descending colon b arrowhead and two tumorsin the sigmoid colon are also visible c d arrowheadadvanced cancer arising from the ascending descending andthe sigmoid colon it was reported that scrcs often occur inthe same or adjacent segment of the large intestine and thatother smaller colorectal cancers in the patients with scrcs wereusually smaller and of lower pathological grade and t stagingconflict of interest statementnone declared 0cj ma figure the surgical specimens of the ascending colon cancer a descending colon cancer b and the two sigmoid colon cancers c and dfigure histopathological examination of the tissue specimens revealed four tumors showing cancerous cells arranged in a tubular pattern 0cfundingnonereferencesjiang x xu c tang d wang d laparoscopic subtotal colectomy for synchronous triple colorectal cancer a case reportoncol lett “ yang j peng jy chen w synchronous colorectal cancersa review of clinical features diagnosis treatment and prognosis dig surg “ aky l synchronous colorectal cancer clinical pathological and molecular implications world j gastroenterol“ fukatsu h kato j nasu ji kawamoto h okada h yamamotoh clinical characteristics of synchronous colorectalcancer are different according to tumour location dig liverdis “ holubar sd wolff bg poola vp soop m multiple synchronous colonic anastomoses are they safe colorectal dis“quadruple advanced synchronous colorectal cancers li z wang d wei y liu p xu j clinical outcomes oflaparoscopicassisted synchronous bowel anastomoses forsynchronous colorectal cancer initial clinical experienceoncotarget “ nosho k kure sirahara n shima k baba yspiegleman d a prospective cohort study showsunique epigenetic genetic and prognostic features ofsynchronous colorectal cancers gastroenterology “20e1“ lam ak carmichael r gertraud buettner p gopalan dho yh siu s clinicopathological significance of synchronous carcinoma in colorectal cancer am j surg “ easson am cotterchio m crosby ja sutherland h dale daronson m a populationbased study of the extent ofsurgical resection of potentially curable colon cancer annsurg oncol “ tsantilas d ntinas apetrasp zambas n aimogrambi s frangandreas g metachronouscolorectals202“adenocarcinomastech coloproctol 0c'
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"Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunityNatasha D Sheybani1 Alexandra R Witter2 Eric A Thim1 Hideo Yagita3 Timothy N J Bullock Richard J Price To cite Sheybani a0ND Witter a0AR Thim a0EA et a0al Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity Journal for ImmunoTherapy of Cancer 20208e001008 101136jitc2020001008 –º Additional material is published online only To view please visit the journal online http dx jitc NDS and ARW contributed equallyAccepted July Authors or their employers Re use permitted under CC BY NC No commercial re use See rights and permissions Published by BMJ1Biomedical Engineering University of Virginia Charlottesville Virginia USA2Pathology University of Virginia Charlottesville Virginia USA3Department of Immunology Juntendo University Graduate School of Medicine Bunkyo ku Tokyo Japan4Radiology Medical Imaging University of Virginia Charlottesville Virginia USACorrespondence toDr Richard J Price rprice virginia eduDr Timothy N J Bullock tb5v virginia eduBackground Triple negative breast cancer TNBC remains recalcitrant to most targeted therapy approaches However recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy We therefore tested a strategy for immune sensitization of murine TNBC 4T1 tumors through combination of focused ultrasound FUS thermal ablation and a chemotherapy gemcitabine GEM known to attenuate myeloid derived suppressor cells MDSCsMethods We applied a sparse scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity We also tested this combination in Rag1ˆ’ˆ’ mice or T cell depleted wild type mice to determine the essentiality of adaptive immunity Further we layered Programmed cell death protein PD1 blockade onto this combination to evaluate its impact on tumor outgrowth and survivalResults The immune modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1 consistent with the dominant MDSC driven immunosuppression evident in this model The combination of FUSGEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice Tumor control correlated with increased circulating antigen experienced T cells and was entirely dependent on T cell mediated immunity The ability of FUSGEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti PD1Conclusion Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth improves survival and enhances immunogenicity in a murine metastatic TNBC model This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluationTrial registration numbers NCT03237572 and NCT04116320BACKGROUNDMetastatic breast cancer BrCa particularly the triple negative breast cancer TNBC phenotype is resistant to most chemical and molecularly targeted therapeutic approaches Interestingly TNBC is often infiltrated with immune cells and the presence of these cells has been shown to have a favorable prognosis in patients treated with neoadjuvant chemotherapy1 Early studies in the use of immunotherapies targeting the PD1Programmed death ligand PD L1 checkpoint inhibitory axis showed some efficacy2“ in TNBC compared with other BrCa subtypes which are generally recalcitrant to checkpoint blockade Activity in the TNBC subtype may be related to the relatively high immune infiltration and correlated with the higher mutational burden observed in TNBC Greater immunotherapy efficacy in TNBC has been recently observed with the use of antibodies targeting the PD1PD L1 checkpoint inhibitory axis in combination with Nab paclitaxel5 This outcome suggests that inducing tumor damage augments antitumor immunity either by promoting antigen availability or disrupting the immunosuppressive tumor microenvironment TME found in TNBCAmong the potential networks in TNBC that could constrain the activity of antitumor immunity is the presence of immunosuppressive myeloid cell subsets These have the capacity to impair adaptive immunity and promote tumor growth and metastasis Among these cell types myeloid derived suppressor cells MDSCs prevail as a heterogeneous population of immature myeloid cells which serve the eponymous role of suppressing the antitumor immune response limiting both T cell activation and effector functions6 Increased levels of this cell type have been demonstrated in tumor tissues of patients with primary BrCa while those with metastatic disease bear the highest abundance of circulating MDSCs8 Studies have Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access shown that approaches that either stimulate myeloid cells with inflammatory mediators or eliminate MDSC can improve antitumor immunity9“To this end the central premise put forth in this study is that focused ultrasound FUS”a safe noninvasive and nonionizing strategy for localized acoustic energy deposition into tissues”can synergize with immunotherapy in a murine model of metastatic TNBC FUS is capable of rapidly heating tumors to thermally ablative temperatures Its extracorporeal application obviates the need for catheterization injection or implantation FUS can be targeted with millimeter precision under MRI or ultrasound guidance thereby allowing for thermal damage and destruction of tumor tissue without compromising healthy intervening or peripheral tissues The bioeffects of FUS hold distinct implications for tumor antigenicity immune cell activation and trafficking13 Thermally active FUS regimes have elicited antitumor immune responses in implantable models of melanoma15 pancreatic16 prostate17“ colon20 kidney21 and BrCa23 Pertaining to the challenge of myeloid cell immunosuppression in TNBC thermally ablative FUS has been shown to induce the expression of heat shock proteins24 and proinflammatory cytokines including interleukin IL12 interferonÎ IFNÎ and tumor necrosis factorα TNFα from a variety of cancer cell lines and after in vivo treatment of tumors26 Whether the ability of FUS to induce these inflammatory mediators is sufficient to overcome myeloid suppression in the context of BrCa is currently under debate with some studies showing activation of antigen presenting cells and T cell recruitment in patients with BrCa treated with thermally ablative FUS28 while others show that additional innate stimuli are needed to support antitumor immunity23 Notably some studies have suggested that a sparse scan thermal ablation regimen more effectively recruits and activates dendritic cells DCs and antitumor immunity than total thermal ablation perhaps by limiting thermal denaturation of tumor antigens and innate stimuli31Based on the improved myeloid cell maturation that occurs with sparse scan regimens we herein tested the ability of a sparse scan partial thermal ablation FUS regimen as a monotherapy to promote antitumor immunity in an aggressive syngeneic model of metastatic murine TNBC with extensive granulocytic MDSC involvement that is recalcitrant to anti PD1 While some activity is evident with the partial ablation approach significantly greater control was achieved by targeting MDSC inhibition in combination with thermally ablative FUS This control was completely dependent on the adaptive immune responseMoreover we demonstrate that layering anti PD1 immune checkpoint blockade onto this combinatorial regimen moderately improves tumor growth restriction These data suggest that in disease settings where myeloid allied approaches to attenuate myeloid immunosuppression may be employed to reveal the full immunotherapeutic immunosuppression predominates potential of thermally ablative FUS Once immunosuppressive myeloid cells are accounted for FUS treatment can promote adaptive immunity that in turn potentiates immune checkpoint blockadeMETHODSCell line maintenance4T1 and E0771 cell lines were maintained in RPMI L glut or Dulbecco™s Modified Eagle™s Medium DMEM gL D glucose L glutamine respectively supplemented with Fetal Bovine Serum FBS at °C and CO2 Thawed cells were cultured for up to three passages and maintained in logarithmic growth phase for all experiments Cells tested negative for mycoplasmaEight week old to week old female BALBc or C57Bl6 mice were obtained from NCI Charles River NCI CRL or The Jackson Laboratory Female BALBc Rag1ˆ’ˆ’ mice were obtained from The Jackson Laboratory 4T1 or E0771 cells × were subcutaneously implanted into the right flank of mice Mice were housed on a hour12 hour lightdark cycle and supplied food ad libitum Tumor outgrowth was monitored via digital caliper measurements Tumor volume was calculated as follows volume length×width22 Approximately days 4T1 or days E0771 following tumor implantation mice were randomized into groups in a manner that ensured matching mean starting tumor volume across experimental groupsIn vivo ultrasoundguided FUS partial thermal ablationMice were treated with FUS either days 4T1 cohorts or days E0771 postimplantation On treatment day mice were anesthetized with intraperitoneal injection of ketamine mgkg Zoetis and dexdomitor mgkg Pfizer in sterilized saline Mouse flanks were shaved and depilated following which ultrasound guided FUS thermal ablation was performed using one of the two systems System and treatment details are provided in online supplementary materials and methods Mice that did not receive FUS treatment consistently underwent anesthesia and depilation of the flank Additionally these mice underwent a ˜sham™ treatment consisting of exposure to the °C degassed water bath exposure for min Following ˜sham™ or FUS treatment all mice were moved to a heating pad and given Antisedan for anesthesia reversal and recoveryGemcitabine therapyGemcitabine GEM mgmouse in µL volume Mylan diluted in saline and filter sterilized through a µm syringe filter was administered intraperitoneally once a week on the day of FUS treatment following which administration was repeated for an additional weeks Administration of GEM doses was based on existing literature demonstrating the use of GEM for inhibition of MDSCs in 4T112 The initial dose of GEM was administered immediately prior to ˜sham™ or FUS treatment Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cMice that did not receive GEM received an intraperitoneal injection of ˜vehicle™ treatment µL of sterile saline at the time points specifiedPD1 blockade therapyFor checkpoint inhibitor therapy the rat anti mouse PD1 antibody αPD1 RMP114 diluted in sterilized saline was administered intraperitoneally every days for a total of five doses µg per mouse Treatment was initiated on day ˜early αPD1™ or day ˜delayed αPD1™T cell depletionsT cell depletion antibodies”anti CD8 clone Bio X Cell and anti CD4 GK15 clone Bio X Cell”were diluted in sterilized saline and administered intraperitoneally every to days starting at day days post FUS for a total of seven doses µg of each antibody for a total µg per mouseImmunohistochemistryOn day sham or FUS exposed tumors were excised and fixed in neutral buffered formalin Sigma Fixed tumors were paraffin embedded sectioned and stained for hematoxylin and eosin Digital images of stained slides were acquired using the Vectra Automated Quantitative Pathology Imaging System Akoya Biosciences Whole slide screening and image capture were subsequently performed using Phenochart Akoya BiosciencesFlow cytometryMice were bled at days and via tail vein and samples were RBC lysed Hybri Max Sigma and stained for flow cytometry analysis At days post tumor implantation tissues were obtained from euthanized tumor bearing animals for immune response assessment In order to gain resolution into tissue resident versus vascular immune cell populations mice were injected intravenously with rat anti mouse CD45 FITC clone F11 BD Biosciences min prior to euthanasia 4T1 tumors spleens cardiac blood axillary and brachial tumor draining lymph nodes tumor DLNs pooled and nondraining inguinal lymph nodes were harvested processed and stained for flow cytometry analysis Additional details are provided in online supplementary materials and methodsSamples were acquired on an Attune NxT flow cytometer ThermoFisher Scientific and data were analyzed with FlowJo TreeStar or FCS Express De Novo Software A representative gating strategy for granulocytic myeloid derived suppressor cell G MDSC and CD44 T cells is provided in online supplementary figure Statistical analysisAll statistical analyses were performed in GraphPad Prism GraphPad Software A detailed description of statistical methods for each experiment is provided in the corresponding figure legend accessAnimal study approvalAll animal work was performed under a protocol approved by the Animal Care and Use Committee at the University of Virginia and conformed to the National Institutes of Health guidelines for the use of animals in researchRESULTSPartial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsetsTo achieve partial thermal ablation of 4T1 tumors we used an ultrasound guided FUS system equipped with a single element therapeutic transducer driven at MHz figure 1A online supplementary figure A grid of sonications was overlaid on the ultrasound visible tumor and ablated in a raster pattern under B mode ultrasound guidance figure 1B“C The exceptionally small focus of this system rendered a low ablation fraction “ of total tumor volume Immediately following ablation tumors displayed evidence of coagulative necrosis in the ablated zone with surrounding periablative margins figure 1D One week following FUS partial thermal ablation tumors and secondary lymphoid ans were excised for immunological characterization by flow cytometry figure 1B FUS partial thermal ablation of 4T1 tumors conferred a significant increase fold in the absolute number of CD11c hi DCs within the axillary tumor draining lymph node aDLN of mice figure 1E While this was accompanied by a nearly threefold elevation in the absolute number of CD86 DCs within the aDLN figure 1F the percentage of DCs expressing CD86 did not change figure 1G Increased numbers of DCs”and CD86 DCs in particular”suggest FUS is promoting the maturation or trafficking of these cells in the DLNs where they could encounter and activate T cells However this did not translate to tumor growth restriction data not shown We also did not observe significant differences in the absolute number of activated T cells in 4T1 tumors figure 1H or DLNs data not shown following FUS exposure suggesting limitations in the ability of FUS activated DC to further drive an antitumor T cell responseImmune profiling by flow cytometry revealed that irrespective of FUS exposure of the intratumoral CD45 immune cell population is comprised of CD11b myeloid cells figure 1I Similarly approximately of the circulating immune cell population in 4T1 tumor bearing mice is comprised of myeloid cells a striking fold elevation in circulating myeloid burden compared with naive mice online supplementary figure Notably Ly6G granulocytic myeloid derived suppressor cells G MDSCs significantly dominated the immune cell repertoire within 4T1 tumors relative to other myeloid including F480 macrophages Ly6C cell subsets monocytic myeloid derived suppressor cells M MDSCs and CD11c hi DCs figure 1J FUS partial thermal ablation did not significantly alter the absolute number per Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Partial thermal ablation of established TNBC tumors promotes peripheral DC activation but has limited impact on the presence of T cells and other myeloid cell subsets A Design overview of a custom ultrasound guided FUS system consisting of a MHz single element transducer orthogonally co registered to an MHz linear ultrasound imaging array The tumor bearing flank of each anesthetized mouse was acoustically coupled to ultrasound transducers via degassed water bath maintained at °C ˜Sham™ mice were similarly positioned but did not undergo sonications B Schematic illustration of FUS partial thermal ablation scheme and study layout for evaluation of immune sequelae in 4T1 tumor bearing mice A grid of sonications was applied in a raster pattern onto the B mode ultrasound visible tumor In total two planes of sonication spaced mm apart were applied to each tumor Grid points were spaced mm apart within a single plane One week following thermal ablation tumors and secondary lymphoid ans were excised for sham n6 or FUS treated n5 mice and processed for flow cytometry C Representative B mode ultrasound images of ectopic 4T1 tumors either before top or during bottom FUS exposure Sonication grid depicting targets red points is superimposed on B mode image during treatment Subsequent to thermal ablation hyperechoic signatures yellow arrow are occasionally observed D Representative HE staining of either sham 4T1 tumors or those resected immediately following FUS partial thermal ablation Zoomed insets depict the transition from necrotic to intact tumor tissue within the periablative zone scale bars400 µm and µm on left and right inset respectively E Absolute number of CD11c hi DCs in the axillary tumor draining lymph node aDLN of 4T1 tumor bearing mice p00136 vs sham F Absolute number of CD86 CD11c hi DCs in the aDLN p00063 vs sham G Percentage of CD86 subset out of total CD11c hi DCs within aDLN H Absolute number of intratumoral CD44 CD8 and CD44 CD4 T cells and regulatory T cells Tregs per gram tumor I Percentage of CD11b myeloid cells out of total CD45 immune cells across tumor spleen aDLN inguinal DLN iDLN and nontumor draining axillary and inguinal LNs nDLNs p005 vs all other groups irrespective of FUS exposure specifically tumor vs spleen p00226 tumor spleen vs all other ans p00001 J Absolute number of intratumoral myeloid cells CD11c hi DCs F480 macrophages Ly6C monocytic myeloid derived suppressor cells M MDSCs Ly6G granulocytic myeloid derived suppressor cells G MDSCs per gram 4T1 tumor p00001 vs all other cell types irrespective of FUS exposure All data represented as mean±SEM Significance assessed by unpaired t test F“H or two way analysis of variance followed by Tukey multiple comparison correction I“K ˜ns™not significant DCs dendritic cells FUS focused ultrasound HIFU high intensityfocused ultrasoundSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0cgram tumor of these myeloid cell subsets These observations led us to formulate the hypothesis that widespread immunosuppressive mechanisms associated with the 4T1 TME must be addressed in order to facilitate the T cell response to FUSFUS partial thermal ablation in combination with GEM constrains primary TNBC tumor outgrowth and extends overall survivalOur observation of the overwhelming MDSC burden following 4T1 tumor implantation warranted implementation of an allied therapeutic strategy in order to counter this immunosuppressive barrier To this end we tested a combinatorial paradigm incorporating GEM a myelosuppressive chemotherapy demonstrated to inhibit MDSCs transiently in the 4T1 model without consequence to T cell phenotype or function12To evaluate the efficacy of FUS and GEM in combination we used a preclinical ultrasound guided FUS system to achieve partial thermal ablation of established 4T1 tumors 14d after tumor implantation average tumor volume of mm3 In combination with the single session of FUS thermal ablation we initiated GEM therapy mgmouse which was then readministered weekly for a total of three GEM doses figure 2A Combinatorial therapy synergized to produce significant constraint of 4T1 tumor outgrowth compared with sham and monotherapy groups figure 2B“CBy termination of treatments at day 4T1 tumors exposed to FUSGEM combination saw nearly × and × reductions in average volume compared with sham or GEM exposed tumors respectively figure 2B Two dimensional tumor projections at day postimplantation saw a nearly fold reduction in area from sham to combinatorial therapy setting figure 2D“E In a fraction of mice treated with FUSGEM we observed complete regression of 4T1 tumors although transient figure 2C tumor outgrowth eventually rebounded after termination of treatments 4T1 tumor bearing mice receiving FUSGEM treatment additionally saw the greatest extension in overall survival with and increases in median survival time compared with sham and GEM groups respectively HRs and for FUSGEM relative to sham and GEM groups respectively figure 2F We additionally observed that FUSGEM significantly constrained outgrowth in a separate C57Bl6 metastatic mammary carcinoma model E0771 online supplementary figure To further the clinical relevancy of these findings we applied this combinatorial strategy with the research grade analog of a clinical ultrasound guided FUS system Theraclion Echopulse that is already CE marked for applications in breast fibroadenoma thyroidparathyroid gland and varicose vein ablation and currently in use for multiple clinical trials leveraging FUS thermal ablation in combination with cancer immunotherapy We observed that partial thermal ablation using the Theraclion visualization and treatment unit MHz in combination accesswith GEM controlled 4T1 tumor outgrowth to a degree comparable with that observed with the custom in house system online supplementary figure These findings lend credence to the notion that the impact of combining GEM with FUS may be conserved across partial thermal ablation regimens Moreover they demonstrate that the efficacy of FUS partial thermal ablation in combination with GEM can be recapitulated on a system with a larger focus and in line image guidance that is currently in use clinicallyCombination of FUS partial thermal ablation with GEM increases the levels of circulating T cellsLymphocytes”in particular CD8 and CD4 T cells”play an important role in responding to tumor antigen and generating a durable antitumor response Based on the extended protective effect observed in mice treated with FUSGEM flow cytometry analysis was performed to evaluate the contribution of T cells in generating systemic and local tumor control We sampled the circulating immune cell repertoire in 4T1 tumor bearing mice via serial tail bleeds days and prior to readministration of GEM and a terminal cardiac bleed at the time of spleen harvest day figure 3A Combinatorial therapy significantly elevated absolute number of CD8 and CD4 T cells in the circulation at days and figure 3B“C and E“F Moreover a trend threefold to fivefold increase in circulating T cells was noted in the FUS group relative to sham figure 3B“C and E“F From days to systemic CD44 expressing antigen experienced T cell populations both CD8 and CD4 saw a steady significant increase after combinatorial therapy figure 3D and G A similar modest trend was noted for the FUS monotherapy group relative to sham and GEM figure 3D and G These changes were concordant with a decrease in circulating myeloid CD11b cells in GEM recipient groups demonstrating the ability of GEM to partially alleviate circulating myeloid burden figure 3HSplenomegaly is a common signature that arises in parallel with the leukemoid reaction to 4T1 tumors that is the expansion of immunosuppressive myeloid cells during tumor progression We observed that combinatorial therapy most significantly reverses splenomegaly online supplementary figure 6A“B Consistent with this observation immunological characterization of spleens revealed a significant decrease in CD11b myeloid cells”a “ reduction in FUSGEM spleens relative to sham or monotherapy figure 3I While there appeared to be a trend toward more CD11b cells in the monotherapy groups compared with the sham this difference was not significant and there was no difference between these groups in terms of absolute CD11b cell numbers within the spleen data not shown The decrease in myeloid cells in the combination treatment group was accompanied by a significant corresponding elevation in lymphocytes in the spleen following FUSGEM treatment Relative to these sham and GEM groups combination therapy elevated splenic CD8 T lymphocytes by fold and Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access Figure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM constrains primary triple negative breast cancer outgrowth and extends overall survival A Overview of experimental design for evaluation combination of FUS with serial GEM treatment in murine mammary carcinoma B Average 4T1 tumor outgrowth in sham n7 FUS monotherapy n5 GEM monotherapy n10 and combinatorial FUSGEM therapy groups n10 Data are represented up to select time points corresponding with mouse dropout due to humane endpoints All data represented as mean±SEM Significance assessed on outgrowth up to day by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Tukey multiple comparison correction p005 vs all other groups specifically sham vs FUSGEM p00001 FUS vs FUSGEM p00001 shamGEM vs FUSGEM p00026 C 4T1 tumor outgrowth from individual mice in sham FUS shamGEM or FUSGEM groups Data represent outgrowth from initiation of treatments at day up to removal of mouse from study for meeting a humane endpoint D Representative images of 4T1 tumors excised at day Scale bar1 cm E Quantification of 2D tumor areas from images in previous panel F Kaplan Meier curve depicting overall survival of sham treatment n9 FUS monotherapy n6 GEM monotherapy n10 and combinatorial FUSGEM therapy n10 recipient mice Significance assessed by log rank Mantel Cox test p005 vs all other groups specifically sham vs FUS p02154 sham vs FUSGEM p00001 sham vs shamGEM p00050 FUS vs FUSGEM p00021 FUS vs shamGEM p00312 FUSGEM vs shamGEM p00041Sheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c accessFigure Combination of focused ultrasound FUS partial thermal ablation with gemcitabine GEM increases the levels of circulating T cells A Overview of experimental design to understand the impact of FUS andor GEM treatment on circulating immune cells B“C Absolute number of circulating CD8 T cells at day B and day C D Percentage of circulating CD8 T cells expressing CD44 from days to E“F Absolute number of circulating CD4 T cells at day E and day F G Percentage of circulating CD4 T cells expressing CD44 from days to H Percentage of CD11b myeloid cells out of total CD45 immune cell in circulation from days to I“K Percentage of myeloid cells I CD8 T cells J and CD4 T cells K out of total CD452 immune cells All data represented as mean±SEM All data representative of sham n6“ FUS monotherapy n4“ GEM monotherapy n9 and combinatorial FUSGEM therapy n6“ groups Significance assessed by analysis of variance followed by Tukey multiple comparison correction for B C E F or Fisher™s least significant difference LSD without multiple comparisons correction for I“K Significance for D G and H assessed by repeated measures mixed effects model implementing restricted maximum likelihood method followed by Fisher™s LSD without multiple comparisons correction p005 vs all other groups unless otherwise indicated p001 p0001 vs groups indicatedSheybani a0ND et a0al J Immunother Cancer 20208e001008 101136jitc2020001008 0c access fold figure 3J and CD4 T lymphocytes by fold and fold figure 3K These elevations were accompanied by a modest increase in percentage of Foxp3 regulatory T cells Tregs online supplementary figure 6E Additionally increases in percentage of NK and B cells were noted twofold to fivefold online supplementary figure 6C“D These findings indicate that combinatorial therapy with FUSGEM promotes a systemic lymphocyte response that is discrete from the effects of either intervention alone which may account for reduced mortality associated with pulmonary metastasesCombinatorial FUSGEM therapy does not promote robust local antitumor T cell responsesGiven the robust systemic immune signatures within the blood and spleen following FUSGEM we assayed 4T1 tumors at a time point within the window of tumor growth restriction and subsequent to termination of treatments ie day to interrogate whether tumor control correlates with an increase in the effector functions of the intratumoral T cell response figure 4A Approximately hours prior to euthanasia mice received intravenous brefeldin A injection to inhibit cytokine secretion for subsequent intracellular cytokine staining by flow cytometry Immune characterization of tumors at days postimplantation”that is days subsequent to final GEM administration”revealed no significant changes in absolute number of antigen experienced CD44 CD8 or CD4 T lymphocytes figure 4B“C Moreover the polyfunctionality of these T cells as denoted by IFNÎ and granzyme B expression was not significantly altered figure 4D“E However intratumoral functional changes were noted in the myeloid compartment GEM monotherapy modestly increased IL 12p40 production by DCs fold but this was not conserved in the combinatorial therapy group figure 4F Moreover while FUS monotherapy generated a trend in elevated TNFα production by intratumoral G MDSCs GEM recipient groups saw a significant increase threefold relative to sham figure 4G These findings indicate that changes in the myeloid compartment in response to monotherapy and combination therapy may contribute to tumor control but are unlikely to drive the protective response entirely Interestingly intratumoral T cell representation correlates poorly with circulating lymphocytes suggesting a transitory immune response that either cannot be fully characterized at this time point or is hampered by additional modes of immunosuppressionProtection conferred by combination of FUS and GEM is dependent on adaptive immunitySince our findings revealed no obvious advantage or function of adaptive immunity in the local TME we next investigated the overarching role of the adaptive immune system in protection offered by combinatorial therapy with FUSGEM To this end we utilized an Rag1ˆ’ˆ’ model that is deficient in T and B cells to address the hypothesis that mature T andor B cells play a role in the observed response Wild type WT or Rag1ˆ’ˆ’ mice bearing 4T1 tumors were randomized into groups in a manner that preserved similarity in average initial tumor volumes Mice were subsequently treated with either GEM monotherapy or the combination of FUSGEM The tumor growth inhibition offered by FUSGEM was entirely lost in Rag1ˆ’ˆ’ mice relative to their WT counterparts with average 4T1 tumor volume in Rag1ˆ’ˆ’ mice being over fivefold higher than that of WT mice on terminati
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Rheumatoid arthritis RA is a systemic chronic inflammatory disease that affects synovial joints and has various extraarticular manifestations including atherosclerotic cardiovascular disease CVD Patients with RA experience a higher risk of CVD leading to increased morbidity and mortality Inflammation is a common phenomenon in RA and CVD The pathophysiological association between these diseases is still not clear and thus the risk assessment and detection of CVD in such patients is of clinical importance Recently artificial intelligence AI has gained prominence in advancing healthcare and therefore may further help to investigate the RACVD association There are three aims of this review to summarize the three pathophysiological pathways that link RA to CVD to identify several traditional and carotid ultrasound imagebased CVD risk calculators useful for RA patients and to understand the role of artificial intelligence in CVD risk assessment in RA patients Our search strategy involves extensively searches in PubMed and Web of Science databases using search terms associated with CVD risk assessment in RA patients A total of peerreviewed s were screened for this review We conclude that a two of the three pathways directly affect the atherosclerotic process leading to heart injury b carotid ultrasound imagebased calculators have shown superior performance compared with conventional calculators and c AIbased technologies in CVD risk assessment in RA patients are aggressively being adapted for routine practice of RA patientsKeywords Arthritis a0· Rheumatoid a0· Atherosclerosis a0· Cardiovascular disease a0· Inflammation a0· Carotid artery diseases a0· Carotid intimamedia thickness a0· Risk assessmentIntroductionRheumatoid arthritis RA is a chronic inflammatory disease that not affects only synovial joints but also has several extraarticular involvements including those related to the skin eyes heart lungs kidneys and other ans [ ] It affects of the global population with a higher prevalence in females when compared with males [ ] Cardiovascular disease CVD is a common manifestation in RA patients with a two to threefold higher risk of cardiovascular events and mortality compared with a normal population [] However this increased risk is not entirely jasjitsuriatheropointcom Jasjit S Suri Extended author information available on the last page of the explained by conventional risk factors [] Current statistically derived CVD risk calculators use conventional risk factors alone [“] are not suitable for RA patients and they either underestimate or overestimate the risk [“] This may be because of the paradoxical behavior of some of the conventional risk factors such as body mass index lowdensity lipoprotein highdensity lipoprotein and total cholesterol in RA [ ] Despite this lack of clarity the guidelines by the European League Against Rheumatism EULAR recommend aggressive control of these conventional risk factors [ ] Recent attempts were made to improve the CVD risk assessment in the RA population including the development of œRAspecific risk factors in the CVD risk calculators [“] However such calculators could not provide adequate improvement in risk Vol01234567891 0c Rheumatology Internationalprediction and reportedly still underestimated or overestimated CVD risk in RA patients [ ]To provide a better CVD risk assessment in RA a pathophysiological association between these diseases should be understood as this would help in refining CVD risk predictors in RA patients [] Atherosclerosis a common phenomenon in RA [ ] can be adequately monitored using imaging modalities such as magnetic resonance imaging [] computed tomography [] optical coherence tomography [] and ultrasound [] Each of these imaging modalities offers unique information about morphological variations in atherosclerotic plaque Ultrasound imaging specifically in carotid arteries is a comparatively lowcost noninvasive radiationfree and easytouse imaging modality that is widely adopted in preventive cardiovascular and clinical vascular practices [ ] The imagebased phenotypes of carotid ultrasound such as carotid intimamedia thickness cIMT and carotid plaque are considered surrogate markers of coronary artery disease and have been used for preventive CVD risk assessments in several studies [“] These imagebased phenotypes indicate the morphological variations in the atherosclerotic plaque and are associated with the inflammatory markers of RA [“] Patients with RA have elevated cIMT and have more plaque area PA when compared with nonRA patients [“] Thus the inclusion of these imagebased phenotypes in risk prediction models may improve the CVD risk assessments of RA patients Recent studies have combined the effect of these imagebased phenotypes with conventional risk factors including proinflammatory markers like erythrocyte sedimentation rate ESR to perform CVD risk assessment [“] Such integrated risk calculators have demonstrated better CVD risk stratification when compared to traditional CVD risk calculators in nonRA patients [ ]Besides these statistically derived CVD risk calculators artificial intelligence AIbased techniques are also penetrating several medical imaging and risk assessment applications [“] AIbased algorithms such as machine learning ML methods provide a better CVD risk assessment when compared with statistically derived conventional risk calculators [ ] So far AI algorithms have been used for CVD risk assessment in the nonRA population and their potential still needs to be evaluated in RA cohorts However AI is well adapted for RA screening and diagnosis [“] This review provides an insight into how the AIbased algorithms can be used for CVD risk assessment in RA patients There are three aims of this review to summarize the pathophysiological pathways that link RA with CVD to identify several traditional and carotid ultrasound imagebased CVD risk calculators for RA patients and to provide an understanding of the role of artificial intelligence in CVD risk assessment in RA patientsSearch strategyFigure a0 shows a flow diagram indicating the search strategy for this narrative review To write a comprehensive narrative review it is essential to select at least two credible databases that provide highquality peerreviewed s [] This review is the outcome of several searches in the PubMed and Web of Science databases using keywords such as œcardiovascular diseases AND œrisk assessment AND œrheumatoid arthritis œcarotid atherosclerosis AND œrheumatoid arthritis œnoninvasive imaging AND œrheumatoid arthritis œcarotid ultrasound AND œrheumatoid arthritis œcarotid intimamedia thickness OR œcarotid plaque AND œinflammatory markers œcarotid atherosclerosis AND œerythrocyte sedimentation rate OR œC reactive protein œmachine learning AND œrheumatoid arthritis and œmachine learning AND œcardiovascular risk assessment AND œrheumatoid arthritis The availability of all these keywords in the and the full text was investigated to select the relevant s Peerreviewed s published in the last a0years were then given priority Citations from the published s were also shortlisted for this review All these s were subsequently filtered by the expert coauthors to select only those that met the objectives of this review leading to sPathophysiology of a0RA leading to a0CVDThe pathophysiological association between RA and CVD can be explained in two stages the role of traditional risk factors and direct vascular damage Inflammation plays a pivotal role in both of these stages []The role of a0traditional risk factors in a0the a0pathophysiology of a0RA‘driven atherosclerotic CVDThe righthand panel of Fig a0 explains the pathophysiological association between RA and CVD via four pathways [Ia“Id] governed by traditional risk factors such as hypertension proatherogenic dyslipidemia insulin resistance and obesity Patients with RA are generally found with proinflammatory cytokines such as interleukin IL IL6 and tumor necrosis factor α TNFα [] These proinflammatory cytokines are found in the synovium which triggers a systemic inflammatory response and may result in damage to the vascular endothelial cells [] Nitric oxide NO and cyclooxygenase1 are two essential components required 0cRheumatology International Fig Flow diagram for the search strategyto maintain the healthy endothelium which is inhibited by TNFα and IL6 thereby resulting in endothelial cell damage [ ] Inhibiting endothelial NO leads to arterial stiffness [] and is further associated with an increase in peripheral vascular resistance PVR [] thus leading to hypertension in RA patients Additionally several medications used to treat RA such as diseasemodifying antirheumatic drugs DMARDs leflunomide and cyclosporine glucocorticoids nonsteroidal antiinflammatory drugs NSAIDs and cyclooxygenase II inhibitors Cox IBs may also be involved in the development of hypertension in RA patients [ ]Another pathophysiological link between RA and CVD is proatherogenic dyslipidemia [] Nearly “ of RA patients have proatherogenic dyslipidemia [] In nonRA patients increased CVD risk is associated with elevated levels of lowdensity cholesterol LDLc total cholesterol and reduced highdensity lipoprotein cholesterol HDLc However in RA patients low levels of total cholesterol TC low levels of LDLc and suppressed levels of HDLc have been reported This condition is known as œthe lipid paradox [] Highly suppressed HDL levels in RA patients are œproatherogenic [] Furthermore RA patients show high atherogenic index levels despite low lipid levels The atherogenic index is calculated as a ratio of TC HDLc and it may vary according to their levels [] Apolipoprotein B Apo B is a major apolipoprotein in LDL and several studies have indicated an increase in the ratio of Apo B Apo A in RA patients [] A combination of low TC LDLc and suppressed HDLc levels with a high atherogenic index and a high ApoBApoA ratio behaves as proatherogenic dyslipidemia [ ] Longstanding proatherogenic dyslipidemia causes atherosclerosis and eventually CVDRheumatoid cachexia is another important RAspecific characteristic that increases CVD risk [] It is characterized by significantly increased adiposity and reduced muscle mass while one maintains their bodyweight [] The pathophysiology [shown in œpathwayI c] behind R cachexia can be explained in two ways It is characterized by the reduction of muscle mass that is largely due to increased inflammatory cytokines particularly TNFα by activating the transcriptional nuclear factorkappa B cells NFkB pathway and promoting the ubiquitin pathway which causes catabolismproteolysis muscle protein breakdown [ ] Central obesity or abdominal obesity is present in “ of women and “ of men This causes visceral adiposity in RA which has an additional adverse impact on CVD [] On the other hand increased adiposity also induces the production of inflammatory cytokines in RA which further worsens this 0c Rheumatology InternationalFig Pathophysiological association between rheumatoid arthritis and cardiovascular disease IL1 interleukin IL6 interleukin TNFα tumor necrosis factor α EC endothelial cells SMC smooth muscle cells MCP1 monocyte chemoattractant protein MCSF macrophage colonystimulating factor VCAM vascular cell adhesion molecule ICAM intercellular adhesion molecule NSAIDs nonsteroidal antiinflammatory drugs CoxIBs cyclooxygenase inhibitors HTN hypertension PVR peripheral vascular resistance TC total cholesterol HDL highdensity lipoprotein LDL lowdensity lipoprotein APOB apolipoprotein B APOA apolipoprotein A NFkB nuclear factorkappa B cellsscenario [] This syndrome may be explained in the triad of increased adiposity reduced muscle mass and low body mass index BMIEpidemiological studies have suggested a strong association between insulin resistance IR metabolic syndrome and RA [ ] [shown in œpathwayI d in the dark greendotted box] Inflammation plays a crucial role in these three conditions [] In patients with RA IR serves as an independent prognostic risk factor that signifies the presence of subclinical atherosclerosis it is determined by carotid intimal thickness cIMT and is measured by carotid ultrasonography [] Longstanding inflammation due to RA promotes oxidative stress endothelial dysfunction and atherosclerosis in this population []Progression of a0atherosclerosis and a0direct vessel damage in a0RAIn RA the activation of Tcells and mast cells increases the production of proinflammatory cytokines such as IL1 IL6 and TNFα These proinflammatory cytokines stimulate endothelial cells ECs and smooth muscle cells SMCs in subendothelium [] by expressing cell adhesion molecules such as vascular cell adhesion molecule VCAM and the œintercellular adhesion molecule ICAM [] and by producing chemokines including monocyte chemoattractant protein MCP and macrophage colonystimulating factor MCSF The activation of endothelial cells allows the migration of LDLc into the subendothelial layer where 0cRheumatology International it becomes oxidized and triggers the inflammatory response for the recruitment of immune cells such as T lymphocytes and monocytes in the intimal layer Once they enter the intimal layer monocytes are transformed into macrophages and they then take up the oxidized LDLc to become foam cells The completion of this complex process then leads to the formation of atherosclerotic plaque Macrophages also trigger the migration of smooth muscle cells from tunica media to tunica intima and initiate their proliferation The SMCs form a thin fibrous cap to prevent the encroachment of atherosclerotic plaque towards the lumen However over time proinflammatory cytokines enzymes and free radicals cause fibrous cap erosion and make the plaque vulnerable for rupture The amplification of the inflammatory response results in the acceleration of plaque formation eventually leading to plaque rupture and thrombotic events which damage the blood vessels Pathway II of Fig a0 represents this processCurrent conventional CVD risk prediction models for a0RAOver the last decade several CVD risk assessment calculators have been developed very few of which are recommended by the cardiovascular risk management guidelines [ ] Some standard cardiovascular risk prediction algorithms are the Framingham risk score FRS [] Systematic Coronary Risk Evaluation SCORE [] American College of CardiologyAmerican Heart Association ACCAHA risk score [] World Health anization WHO risk charts [] and Reynolds™s risk score RRS [] These risk calculators use traditional risk factors such as patient demographics age gender ethnicity blood biomarkers lowdensity lipoprotein cholesterol highdensity lipoprotein cholesterol and total cholesterol behavioral markers smoking and alcohol consumption and physiological markers height weight body mass index All these risk calculators were initially developed for nonRA populations therefore when used in RA cohorts CVD risk is substantially underestimated [“] The use of traditional risk factors alone while not considering RAspecific inflammatory markers could be another reason for such underestimation However RRS included an RAspecific inflammatory marker called œhigh sensitivity Creactive protein hsCRP [] for CVD risk prediction but did not report any significant improvement in the CVD risk assessment [] Rajagopalan et a0al [] also reported a small improvement in area under the curve in females and in males when C reactive protein CRP or erythrocyte sedimentation rate ESR was added to the FRSOver the past few years several efforts have been made to improve the cardiovascular risk assessment in RA patients The EULAR guidelines recommended the use of a modified SCORE mSCORE in RA patients positive with rheumatoid factor RF or anticitrullinated protein antibodies ACPA and RA duration of more than a0years [ ] Cox et a0al [ ] developed the QRISK2 and QRISK3 algorithms which use the presence of RA as a CVD risk predictor hazard ratio confidence interval “ Solomon et a0al [] also developed an RAspecific CVD risk calculator called expanded risk score or ERS by including RAspecific biomarkers [such as disease activity disease duration a modified health assessment questionnaire HAQ disability index and daily prednisone use] with the traditional biomarkers used in the Coxbased model The authors reported an improvement of in cindex when validating the risk score on the reserved dataset Recently Curtis et a0al [] also proposed a CVD risk prediction tool for RA patients by combining conventional and RAspecific risk factors The authors predicted the risk of composite CVD events such as MI stroke and death during the followup period of a0years The area under the curve AUC for cardiovascular risk stratification for this model was All these RAspecific CVD risk scores reported a better risk assessment on the proprietary databases Still when compared with other risk calculators in different RA cohorts these calculators have demonstrated mixed results [“] Crowson et a0al [] reported an underestimation of CVD risk by FRS and RRS in RA patients The observed risk was twice the predicted risk Furthermore the authors did not report any improvement in cardiovascular risk prediction when CRP was added to their model Arts et a0al [] investigated the roles of SCORE FRS RRS and QRISK2 in RA patients Out of these four models SCORE FRS and RRS underestimated CVD risk in RA patients whereas the QRISK2 reported an overestimation The AUC ranged between and for the four risk models A similar study by Arts et a0al [] investigated the performance of the original recalibrated and improved version of SCORE calculators to predict the CVD risk in RA patients The AUC values for these scores were CI “ “ and “ All these three scores underestimated the CVD risk in RA patients In short even after the SCORE was redesigned using the RAbased risk factors it did not result in an adequate CVD risk assessment In another study by Crowson et a0al [] of RA patients a CVDrisk prediction model was developed that reported better performance AUC compared with conventional risk calculators such as FRS AUC ACCAHA AUC SCORE AUC and QRISK2 AUC Furthermore conventional risk calculators either overestimated or underestimated CVD risk in RA patients Wahlin et a0al [] compared the expanded risk score ACCAHA risk score and a modified version of ACCAHA with a multiplier of for a CVD risk assessment of 0c Rheumatology International RA patients The authors also reported an underestimation of CVD risk by all calculators However the discrimination ability was slightly better since AUC for ERSRA risk was compared to AUC of for two variants of ACCAHAThe overall trend of all these risk prediction algorithms developed for general and RA cohorts indicates a œpoor CVD risk assessment in patients with RA One possible reason for such poor performance is the paradoxical behavior of some of the risk factors such as lipids and body mass index Another potential reason for this outcome is the inclusion of risk factors that do not provide complete information about the CVD risk profile in RA patients [] Corrales et a0al [] indicated a high prevalence of carotid atherosclerosis plaque in patients with lowCVD risk This observation demonstrated the limited ability of conventional risk factorbased algorithms to improve the CVD risk assessment process which may be improved using imaging modalities Therefore there is still room to develop more accurate automated and reliable risk calculators for RA patients by exploring and including nontraditional risk factors such as genetic biomarkers inflammatory biomarkers or imagebased atherosclerotic plaque phenotypes in the risk prediction algorithmCarotid ultrasound atherosclerosis imaging for a0CVD risk assessment in a0RA patientsImaging modalities are becoming essential for the visualization of atherosclerotic plaque and CVD risk assessment in RA patients [] Noninvasive imaging modalities such as computed tomography magnetic resonance imaging ultrasound and positron emission tomography are currently used to assess carotid atherosclerosis in RA patients [] MRI is used to measure the plaque composition including calcification lipidrich necrotic core and fibrous cap thickness [] Computed tomography is generally used to determine carotid artery stenosis [] Ffludeoxyglucose“positron emission tomography FDGPET is a nuclear imaging modality that quantifies the inflammation in carotid atherosclerotic plaque [] Noninvasive carotid ultrasound is a commonly adopted imaging modality that can capture morphological variations in the atherosclerotic plaque quantified using carotid intimamedia thickness cIMT carotid intimamedia thickness variability IMTV and plaque area [] When compared with other noninvasive counterparts carotid ultrasound is less expensive and easier to use [ ] Therefore the scope of this review is restricted to the use of carotid ultrasound for CVD risk assessment in RA patients The automated cIMT and carotid PA are considered surrogate markers of coronary artery disease and widely used for CVDstroke risk assessment [“]Several studies have shown a high prevalence of increased cIMT and carotid plaque in RA patients [“] Studies have also demonstrated the significant association between these carotid atherosclerosis biomarkers and RAspecific markers of inflammation such as ESR CRP and IL6 [“] Table a0 provides some of such studies that link both carotid atherosclerosis and RA using two sets of biomarkers ie imagebased phenotypes and inflammatory biomarkers One common observation from these studies is that patients with RA show an elevated cIMT and carotid plaque area compared with nonRA cohorts row R2“R4 of Table a0 [ ] This association between carotid atherosclerosis and RA also seems independent of the three carotid artery segments common carotid artery carotid bulb and internal carotid artery from where the cIMT or plaque was measured [ ] However several studies have reported more aggressive atherosclerotic plaque formation in the carotid bulb segment when compared to other arterial segments [] The higher plaque prevalence in the carotid bulb is a consequence of turbulent blood flow and reduced shear stress which leads to endothelial dysfunction [ ] This observation of higher plaque in a bulb has also been confirmed in RA patients [] Figure a0 shows carotid ultrasound scans for RA Fig a03a b and nonRA patients Fig a03c d The lefthand side panel of Fig a03a c shows the raw carotid ultrasound scans measured using a Bmode ultrasound scanner The broad usage of carotid ultrasoundbased phenotypes and their significant association with RAspecific Similarly the righthand side panels of Fig a03b d show the processed scans tracking morphological variations in the carotid atherosclerotic plaque for the quantification of cIMT and plaque area The cIMT and plaque area are both greater in RA patients than in nonRA patientsAnother important observation from Table a0 is the significant association between carotid atherosclerotic biomarkers and RAspecific inflammatory markers such as ESR CRP and IL6 [ ] ESR is a relatively inexpensive measure of inflammation in RA patients”therefore several studies have used ESR for CVD risk assessment [“] Some of such studies are listed in Table a0 All these studies indicated a substantially higher CVD event rate in patients with elevated ESR levels Besides ESR studies have also suggested the use of other popular RAspecific inflammatory markers such as CRP or hsCRP and IL6 for the improvement in the CVD risk assessment [ ] Furthermore these RAspecific inflammatory markers are also associated with the annual progression of cIMT [ “] which is a prominent surrogate marker of cardiovascular events In a study with RA patients Kaseem et a0al [] demonstrated the association of ESR CRP and IL6 with carotid atherosclerosis with significant odds ratios p of and respectively 0c hti foTM wdetaicossa era PCIc PRCdna RSE ecneserp dna TM mm a0 yb tinueno sesaercniyreve rof RSEni esaercniIc sepytonehp desabegami sisorelcsorehta ditoraC ni rehgih naht stneitapARniyltnacfiingis era noitalupopARnon eht hti wdetaicossa sawAR orehta fo AC I ditorac ACChti ytireves hgihni sisorelcs wnaht blub ni rehgih stnemges saw stneitapyretra eerht nehw ACI FIB ACC slortnoc ot derapmoc ylevitagen si tnemtaert ARhti wdetalerrocnoitammaflniTMIc oslA era srekramyrotammaflnI detaicossa elcsorehta ditorac yltnacfiingis ARni TMI ditoraChtiwsisorfiingis erew smret rotcaf ksir DVC×RSEdna RSE hti wdetaicossa yltnacnoissergorpTM Ic ti w fo ecnelaverP euqalp dna sraey a0 ehT llomm a0 ARni euqalp ega CTditorac llew ew eb dna ega gnisu nac stneitapdetciderpCTRheumatology International yrammuSCstluseRCsrotcaf ksir egaminoNCepytonehp desabegamICsraey ega naeMC PRCdna rof p p r r RSEhti w detaicossa TMIc PRCdna RSEPC ecneserp dna TMIc dna AR roF 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Cytivitisnesnietorpopil ytisnedhgih oitar sddoRO erusserp cRheumatology Internationalinflammatory markers has also enabled their inclusion in the CVD risk prediction calculators [ ] Recently several CVDstroke risk prediction models have been developed that have combined the effect of conventional risk factors and the automated carotid atherosclerosis biomarkers [ ] These risk prediction models reported a better performance in identifying high CVD risk patients compared with current standardofcare risk calculators However such socalled integrated risk prediction models were developed for the general population They were based on the annual progression rates of carotid atherosclerotic biomarkers and conventional risk factors [“] Therefore given the progression rates of cIMT and PA due to the RAspecific inflammatory markers such models can be updated and might be useful for CVD risk assessment in RA patientsArtificial intelligence in a0CVDstroke risk assessmentArtificial intelligence AI is expeditiously changing the landscape of the global healthcare system and assisting the healthcare workforce in clinical decisionmaking [] Machine learning ML and deep learning DL are the two common branches of AI that have broad ranges of applications in almost every medical imaging sector eg classification and plaque characterization for stroke risk assessment [] thyroid cancer characterization [] liver cancer diagnosis [] prostate cancer diagnosis ovarian cancer diagnosis [] lung cancer detection [] brain tumor classification [] and heart disease prediction and disease classification [ ] During the recent global pandemic of coronavirus diseases AI is providing promising results in the diagnosis of patients with the help of several imaging techniques such as computed tomography [] and Xrays []Since this review is on CVDstroke risk assessment we have summarized several studies that have used MLbased algorithms for CVDstroke risk assessment Table a0 All of these studies follow a supervised learning approach in which the MLbased classifier is trained to identify the correct output labels using input risk factors or features and predefined gold standards or labels Figure a0 shows the generalized framework of supervised MLbased CVD risk assessment In the case of CVD risk assessment the gold standard can be the primary endpoints such as presence or absence of cardiovascular events or surrogate endpoints such as cIMT PA and CAC score or a combination of these risk factors [ ] Several types of input features can be used to train the AIbased algorithms They can be traditional risk factors imagebased phenotypes grayscale image features or statistically derived features Once the offline ML classifier is trained using these features and gold DAC rof RO roF gnikomS MD NTH PBD PBS IMB egAaimedipilrepyH thgiehPCdna TM Ic ± ][ nosetnavSR 0cRheumatology International Fig Carotid ultrasound image of the common carotid artery for control patientsstandard its coefficients are then used in the online ML system to predict the out risk labels Online ML systems do not require a gold standard to make the final risk classification All the studies provided in Table a0 used this approach for CVD risk assessment Unlike MLbased algorithms DLbased models such as convolutional neural networks do not require input features beforehand Instead such algorithms automatically learn their offline coefficients from the input image datasets [] Currently AIbased techniques are used in the diagnosis of RA [] the identification of RA disease severity [] the classification of several RA synovial tissues [] and mortality prediction due to RA [] Although MLbased algorithms are used in the RA field no efforts have been made to assess the CVD risk in RA patients using such automated intelligencebased paradigms MLbased algorithms have been used to perform CVD risk assessments in nonRA populations and reported a better performance in identifying highrisk CVD patients when compared with the current standard of care conventional risk calculators [ ] Patients with RA experience more atherosclerotic plaque in the carotid artery which might lead to cardiovascular events [“] In recent years several studies have demonstrated a better stroke risk assessment using MLbased strategies [] and DLbased strategies [] Besides all these studies attempts can be made to develop more accurate CVD risk prediction tools for RA patients using AI techniques Figure a0 conceptualizes several components required for CVD risk assessment in RA patients The AIbased CVD risk assessment for RA patients can be made possible by combining several types of risk factors such as patients™ demographics physiological parameters behavioral risk factors imagebased phenotypes and most importantly RAspecific inflammatory markers This combined set of features can be used as inputs along with the gold standard to identify what CVD risk category RA patients belong to As such both ML and DLbased systems can be employed to performed CVD risk assessment in patients Because of the significant association between carotid atherosclerosis and RA researchers can conduct a pilot study with cIMT and plaque areas as the surrogate markers for CVD risk assessmentSummaryIn this review we provided several
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"Gastric neoplasms containing neuroendocrine carcinoma NEC components are rare malignancieswith highly aggressive behavior and a poor prognosis and include pure NEC and mixed tumors containing NECcomponents We aimed to investigate whether there is a distinct difference in overall survival OS between gastricneoplasms containing NEC components and gastric adenocarcinomaMethods Surgically resected gastric neoplasms containing NEC components n and gastricadenocarcinomas n from January to December at Peking University Cancer Hospital wereretrospectively analysed Patients were categorized into a surgical group and a neoadjuvant group and adjustedusing 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 Kim™sfrom 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 Kim™s 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 Kim™s 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 Kim™s 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 CI“Multivariate 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 Park™s 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 Van™s 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 Pathol“Rayhan N Sano T Qian ZR Obari AK Hirokawa M Histological andimmunohistochemical study of composite neuroendocrineexocrinecarc
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" atopic dermatitis ad is a worldwide chronic skin disease which burden public health sea buckthornsbt hippophae rhamnoides l elaeagnaceae oil as a traditional herbal medicine has been used for diseasetreatment for many years the effects of sbt oil on ad mouse model induced by repeated administration of dinitrochlorobenzene dncb in balbc mice was evaluated in this studymethods mice were divided into four groups including the normal control group ad model group ad modelgroup treated with sbt oil mlkg and ad model group treated with sbt oil mlkg same volume at differentconcentrations of sbt oil was applied daily on the latter two groups by gavage for days following ad modelinduction the function of skin barrier and the production of il4 ifnÎ tnfα and tslp were examined afteranimal sacrifice the migration and mature of langerhans cell lcs in lymph node was further assessed by flowcytometryresults sbt oil alleviated dermatitis scores decreased ear thickness prevented infiltration of mast cell reducedlymph node weight and depressed activity of th2 cells sbt oil also reduced the expression of il4 ifnÎ tnfα andtslp in ear tissue ige level in serum and mrna relative expression of il4 ifnÎ tnfα in lymph node moreoversbt oil inhibited the migration of lcs cells from local lesions to lymph node and it™s mature in lymph nodes these results suggest sbt oil had a beneficial effect either systemic or regional on dncbinduced admice via maintain the balance of th1th2 and may be a potential complementary candidate for ad treatmentkeywords atopic dermatitis sea buckthorn oil 24dinitrochlorobenzene cytokine ad is a chronic inflammatory skin disease characterizedwith eczematous pruritic rash which has high morbidityin children and could be recurrent in adulthood [ ]as a general public health disease the prevalence of adhas increased in recent years [ ] ad affects nearly correspondence hongquanguansinacom houdiandong163com xinxin wang and sijia li contributed equally to this work5college of integrated traditional chinese and western medicine liaoninguniversity of traditional chinese medicine chongshan road no79shenyang liaoning pr chinafull list of author information is available at the end of the of children and of adults worldwide and the incidents become higher and higher although thepathogenesis of ad is not explicit utterly genetic riskenvironmental factors skin barrier dysfunction and immune dysregulation are thought to play important rolesduring the pathogenesis of ad [“] as for immunedysregulation th2 skewing seems to be the key point ofad pathogenesis [ ] immunological disorder ofth1th2 balance due to strong type immune responses characterized by over infiltration of mast cellincreased production of th2 cytokines and ige level in the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc complementary medicine and therapies page of serum plays crucial role in the onset and process of adthese th2 cytokines subsequently induce the release ofother proinflammatory cytokines such as ifnÎ throughactivating of th1 cells in clinical practicedue to the heavy burden ad placed on society and patients [ ] treatment approaches are needed imperativelyregional emollient andsystemic corticosteroids were generally used to cure ad[ ] however experts ofinternational eczemacouncil reached on a that although the useof corticosteroids for ad is widespread it is also discouraged due to the side effects and the risk of reboundin consideration of potential fearful side effect of topicalsteroid and immunosuppressive application [ ]there is a strong enthusiasm in seeking alternative andcomplementary medication to treat ad recently seeking new potential candidate from natural materials forad management attracted greatly attention [ “]sbt is a wild deciduous shrub or dwarf tree belongingto the elaeagnaceae family which has been used in tibetan mongolian and chinese traditional medicine extensively for disease management [“] according tomany researchers sbt has various medicinal effects suchas antitumor antistress antiinflammatory antiulcerantioxidant healing regulation of cardiovascular andimmune system [“] sbt oil which contains richfatty acids tocopherols ω3 and ω6 etc is a main bioactive part of sbt and it has been proved to have beneficial effect on skin inflammation conditions and have theability to improve the composition of fatty acid in skin[ ] therefore this study carried out to explore thebeneficial effects of sbt oil on dncbinduced admouse model and its possible mechanismmethodssbt oilsbt oil was provided by liaoning dongning pharmceutical co ltd the oil was extracted from thedried press residue including berry flesh seeds andpeel of sbt juice processing by aseptic supercriticalcarbon dioxide process analysis of samples was performed using a hp5ms capillary column m — mm μm agilentinc santaclara ca usa in a gcms 5975c agilent technologies inc sample was injected into the columnand ran using split mode split ratio the helium carrier gas was programmed to maintain atechnologiesconstantflow rate of mlmin oven was initially °c for min then finally raised to °c at °cmin fatty acids were identified by a reference standard mixture fame supelco bellefonte pa usa analyzed under the same operating conditions as thoseemployed for fame of the samples the componentsin sbt oil are exhibited in table animals and animal treatmentfemale healthy specific pathogenfree balbc miceaged weeks weighted ± g were provided byliaoning changsheng biotechnology co ltd benxichina all mice were housed in groups of mice percage waiting to be grouped in a specific pathogenfreeenvironment in h lightdark cycle and allowed free towater and food mice were acclimatized for week before ad model induction mice were randomly dividedinto groups the normal control group ad modelgroup ad model group treated with sbt oil mlkgand ad model group treated with sbt oil mlkgeach group with mice the dorsal skin of each mousewas shaved following dncb μl1 sensitizationthree times in total from day to day and the skin ofleft ear was challenged by dncb μl05 seventimes in total from day to day ad model grouptreated with sbt oil mlkg was given ml sbt oilplus ml olive oil per mice ad model group treatedwith sbt oil mlkg was given ml sbt oil permice oil was applied by intragastric administration oncea day from day15 to day olive oil ml was givenfor the normal control group and ad model group atthe same time at the same time the thickness of left earwas measured every days all animals were sacrificedat day and samples including blood left ear tissuesand submaxillary lymph nodes were collected the micewere anesthetized with avertin solution g tribromoethanol powder dissolved into ml 2methyl2butanol and ml pbs at °c which was filtered usinga nalgene μm filter thermo fisher scientific incwaltham ma usa and sacrificed via exsanguination the fullscale procedures of ad model inductionand treatment are shown in fig all experimental procedures performed were approved by the ethical committee of experimental animal careat liaoninguniversity of traditional chinese medicine shenyangpr chinatable major fatty acids and contents of sitosterol and βcarotene in sbt oilfatty acids myristic acidc140palmitic acidc160palmitoleicacidc161stearic acidc180oleic acidc181linoleic acidc182linolenic acidc183sitosterolmggβcarotenemgg 0cwang bmc complementary medicine and therapies page of fig general schematic diagram for ad model induction and sbt oil treatmentevaluation of ad severitydermatitis severity was assessed by ear thickness anddermatitis scores through the method of blind ear thickness was measured every days since day with a digitalthickness gauge mitutoyo co kanagawa japan dermatitis scores was calculated according to main characteristics drynesscrusting hemorrhageerythema erosionexcoriation and edema each one was marked on ascale from none mild moderate to severethe overall dermatitis score was consist of the sum of individual scores which range from to μm thick werehistopathological analysisthe left ear samples of mice were collected on day then fixed in formalin and embeded in paraffin thesectionseither withhematoxylin and eosin he for visualizing dermal andepidermal thickness or with toluidine blue tb for visualizing mast cell numbers the mast cells were countedin sections of power fields at — magnificationstainedimmunohistochemistryin short after deparaffinization and rehydration the eartissue slides were treated with hydrogen peroxidemethanol for inhibiting endogenous peroxidase and withhigh pressure for antigen retrieval then the slides wereincubated with sheep serum for min at °c withprimary antibodies abcam for overnight at °c withsecondary antibodies provided by zhongshanjinqiaobeijing china for h at °c at last the slides werestained with diaminobenzidine dab provided byzhongshanjinqiao beijing china for coloration resultwas analyzed by imagejrealtime polymerase chain reaction rtpcrmouse submaxillary lymph nodes were collected andweighted while sacrifice and total rna was extractedfrom lymph node tissues using trizol reagent invitrogen thermo fisher scientificfollowing theincmanufacturer™s protocols and reverse transcribed withprime scripttmrt reagent kit takara biotechnologyco ltd dalian china realtime polymerase chain reaction analyses were performed under the protocols ofsybr®premix ex taqtm ii takara biotech co ltddalian china and the primers used in this study weredesigned as shown in table relative quantities of alltargets in test samples were normalized to their corresponding gapdh levels the 2δδct method was usedto calculate relative expression quantifyflow cytometrythe antibodies used for flow cytometry were providedby bd usa and the scheme was performed follow induction about — “ — cells of submaxillarylymph node was collected in ep tube centrifuged at rpm and °c for min one hundred μl pbs wasmixed with cells after discarding the supernatant thenanother μl pbs containing fluorescent antibodieswas added for staining at °c for min and the processwas kept out of the sun five hundred μl pbs was addedand blended repeatedly for washing then centrifuged at rpm and °c for min the supernatant was discarded carefully and another μl pbs was added andmixed finally the mixture was transferred to flow tubefor flow cytometrytable primers used for rtpcrgeneil4ifnÎtnfαgapdhprimer sequenceforward ²acaggagaagggacgccat3²reverse ²gaagccctacagacgagctca3²forward ² tgagctcattgaatgcttgg ˆ’²reverse ² ggccatcagcaacaacataa ˆ’²forward ²ggaaaggacggactggtgta3²reverse ² tgccactggtctgtaatcca ˆ’²forward ²tggtgaaggtcggtgtgaac3²reverse ²actgtgccgttgaatttgcc3² 0cwang bmc complementary medicine and therapies page of statistical analysisthe data is presented as mean ± sd the significance ofdifferences of different groups was evaluated by oneway analysis of variance anova followed by the dunnett t test p005 was considered statistically significantresultssbt oil has a beneficial effect on skin against thedevelopment of dncbinduced ad models in balbcmiceto investigate the effect of sbt oil on adlike skinlesions in our model sbt oil mlkg10 mlkg wasapplied by gastric administration once a day followingthe ad model induction by dncb showed in fig topical application of dncb including sensitizationand challenge induced adlike skin lesions presenting as erythemaitching and hemorrhage companiedby abnormal scratching marks and dryness dermatitisseverity was assessed by ear thickness and dermatitisscores ear thickness was measured every days sinceday and the dermatitis scores was evaluated according to main characteristics as described previously after sbt oil administration for days theear thickness and dermatitis scores were significantlydecreased in a dosedependent manner compared tothe ad model group fig fig effects of sbt oil on ad model skin induced by dncb a examples of characteristic of adlike skin lesions b the ear thickness of the micec the dermatitis scores were summarized by the sum of scores according to various ad symtoms p p 005vs the control group p p vs the ad model group 0cwang bmc complementary medicine and therapies page of sbt oil contributed to the skin barrier repair anddecreased infiltration of mast cell in ad model miceinduced by dncbto evaluate the effect of sbt oil on adlike skin lesions histologically he and tb staining were performed on tissue slides repetitive application ofdncb induced dermal thickening epidermal hyperplasia and increased mast cellinfiltration in admodel group while according to he staining slidesthe dermal and epidermalthickness were both decreased and the epidermal hyperplasia was suppressedafter sbt oil administration for days which relatedto dosage fig 3a b according to tb staining slidesthe infiltration numbers of mast cell were also decreased in mice treated with sbt oil fig 3b csbt oil decreased the lymph node weights in ad modelmice induced by dncbthe submaxillary lymph nodes were collected andweighted after mice sacrifice to estimate whether sbtoil play a part in the process of ad induction the results indicated an increase in submaxillary lymph nodeweights in ad model group which was decreased bysbt oil administration fig sbt oil inhibited the expression of il4 ifnÎ tnfα andtslp in ear tissue in ad model mice induced by dncbin order to evaluate the effects of sbt oil on regulationof th1th2 cytokines the expression of il4 ifnÎtnfα and tslp in ad model mice was examined byimmunohistochemistry staining on ear tissue slides results demonstrated an upregulation expression of il4fig effects of sbt oil on histological ear skin tissue a he staining — thickness in hestained tissue mast cell number in tb stained tissue c tb staining — epidermis mast cell dermis b dermal and epidermal 0cwang bmc complementary medicine and therapies page of fig weights of submaxillary lymph node a submaxillary lymph node b lymph node weight a normal control group b ad model groupc treated with sbt oil mlkg d treated with sbt oil mlkg p p vs the control group p p vs the admodel groupifnÎ tnfα and tslp in ad model group which wasinhibited dosedependent by application of sbt oilfig sbt oil downregulated ige level in serum and mrnarelative expression of il4 ifnÎ and tnfα in lymphnodewe next measured ige levelin serum by elisa andmrna relative expression of il4 ifnÎ and tnfα inlymph node by rtpcr we found that ige levelinserum was increased in ad model mice induced bydncb the increase was suppressed significantly ingroups treated with sbt oil in a dosedependent manner the same as above mrna relative expression ofil4 ifnÎ and tnfα in lymph node was increased inad model group and decreased in groups treated withsbt oil fig sbt oil decreased numbers of lcs in draining lymph nodeand the expressions of cd86 ox40l and mhcii on lcsin order to assess the effect of sbt oil on the maturityand migration of lcs cell in submaxillary lymph nodewe detected cell numbers expressing cd207cd326cd86 cd80 ox40l and mhc ii by flow cytometryresults as shown in fig indicated that the expressionsof cd207cd326 cd86 ox40l and mhciion lcs cellin submaxillary lymph node were all increased in admodel groups induced by dncb and decreased ingroups treated with sbt oildiscussionad is a skin inflammatory disease induced by haptenand mediated by t cells clinically the main characteristics of ad are erythema edema papule blisterbleb bullous reaction and even necrosis the pathological changes of ad were infiltration of inflammatorystudy weand tissuecellsedemain thisemployed dncbinduced ad model using balbcmice which has been proposed as an appropriate representative of human ad because of similar symptoms including skin erosion hemorrhage epidermalhyperplasia mast cellinfiltration and increased igelevel in serum etc sbt oil as a traditional herbal extracts have been proved diversified pharmacologicalactions such as antiinflammatory relieve the pressure protecting vascular endothelial cell and immunomodulatory effects [ ] the main constituents ofsbt oilinclude fatty acids such as myristic acidpalmitic acid palmitoleic acid oleic acid etc sitosterol and βcarotene fatty acids are crucial components of cell membranes and play important role inbiologicalfunction of cells some of the fattyacids are required for innate immune activation andpathogen defense sitosterol is the main constituent of many plants and vegetables and has the abilityto modulate the functions of macrophages and antiinflammation [ ] βcarotenealso has beenshown to suppress the cellular and tissue response toinflammation [ ] in view of the immunoregulation and antiinflammatory actions of sbt oil weassessed the antiad effects of sbt oil in vivotopical application of dncb followed schedule including sensitization and challenge induced adlikeskin lesions presenting as erythemaitching andhemorrhage companied by abnormal scratching marksand dryness the ear thickness and dermatitis scoreswere all significantly increased in ad model groupcompared to control group after sbt oil administration for daysthickness and dermatitisscores in groups treated with sbt oil were significantly decreased in a dosedependent manner compared to the ad model group which indicate thatsbt oil administration suppressed the development ofadlike skin lesionsthe ear 0cwang bmc complementary medicine and therapies page of fig effects of sbt oil on expression of il4 ifnÎ tnfα and tslp in ear tissue a immunohistochemical staining of il4 ifnÎ tnfα and tslpin ear tissue b aod analysis of il4 ifnÎ tnfα and tslp p vs the control group p vs the ad model groupad is recognized as a th2midiated allergic disease withover expression of th2 cytokines and increased serum igelevel being the antibody synthesized by plasma cellsige plays an essential role in some hypersensitivity suchas ad allergic asthma and allergic rhinitis ige has thecapability of elevating the production of th2 cytokinesth2 cytokines il4 induced immunoglobulin switching inplasma cells and resulting in upregulation of serum igelevel mast cell as one of granular leukocytes can releasemany cytokines to mediate inflammatory reaction and immune regulation these cytokines also participate inpathological manifestations of many allergic disorders including ad the infiltration of mast cell which wasactivated by ige is one of the key features of adlike skin 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on ige level in serum and mrna relative expression of il4 ifnÎ and tnfαin lymph node a ige level in serum b mrnarelative expression of il4 ifnÎ and tnfα in lymph node p p vs the control group p p vs the ad model grouplesions [ ] cytokines released from activated mastcells attract eosinophils into the skin which give rise toskin tissue damage histologically according to tb staining slides the numbers of mast cell infiltration in ear skintissue of ad model mice were increased by application ofdncb and were inhibited remarkably by sbt oil the results indicated that sbt oil has beneficial effects on suppression of skin tissue mast cell accumulation in dncbinduced ad mice our tb staining results indicated thatmast cells in skin tissue were scarce in control group whileabundance in ad model group which highly conform tothe pathological changes of ad the mast cell numberwas reduced remarkably after sbt oil administration insbt oil treated group compared with ad model groupthese results suggest that sbt oil has inhibiting effect ofmast cell infiltrationaccording to studies published the over expression ofth2 cytokines go hand in hand with tslp tslp whichcan strongly promote the differentiation of th0 cells toth2 phenotype through activation of dendritic celldcs was determined as a crucial factor in the induction of th2 skewing in ad the expression of tslphas been shown to be enhanced markedly in keratinocytes of ad lesions both in ad patients and in mousemodels il4 can in turn induce the synthesis oftslp by keratinocytes importantly the migration ofdcs to draining lymph node was triggered by tslpmore interesting th1 cytokines ifnÎ which can activate keratinocytes was found also elevated in ad ifnÎand tnfα can synergistically stimulate the release ofcytokines and chemokines in chronic stage of ad inour study sbt oiltreatment reduced the increasedserum ige level which was induced by dncb application moreover sbt oil treatment also reduced dncbinduced increases in expression of il4 ifnÎ tnfαand tslp in ear tissue and mrna relative expression ofil4 ifnÎ and tnfα in lymph node these resultssuggest that sbt oil ameliorated ad symptoms partlythrough the activity suppression of th1th2 cells according to the downregulated effects sbt oil did to thetslp expression in ear tissue we speculated that sbtoil may have the ability to suppress both the activationand migration of dcs cell in order to clarify our speculationflow cytometry was used to do further studyabout lcsdraining lymph node plays an important role in thepathogenesis of ad the weight and volume of lymphnode will increase company with strengthened functionwhen it is active we investigated the local lymph nodesthrough different means first of all the submaxillarylymph node weights of dncbinduced ad model micewere increased significantly which were markedly reducedafter intragastric application of sbt oil for days in adosedependent manner we further assessed the expressions of cd207cd326 mhc class ii cd80 cd86 onlcs and ox40l on cd4 t cells in lymph node by flowcytometry because the complex immune reaction of adwas mainly taken place in lymph node langerin cd207a type ii transmembrane protein is a ctype lection oflcs lcs are virtual mediators of immune surveillance and tolerance which resided at epidermis as dc subpopulation antigens both external and internal werecaptured by lcs and presented to th0 cells within theskin draining lymph node cd207 is the only surface antigen just restricted to lcs epithelial cell adhesionmolecule epcamcd326 a cell surface protein is highlyexpressed on lcs and appears to stimulate lcs migration since cd207cd326 as the main symbol of lcs 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on the maturity and migration of lc cell a the scatter diagram which indicate the proportion of cells in lymph nodeexpressing cd207cd326 mhc ii cd86 cd80 and ox40l b the histogram of abovementioned results p p 005vs the controlgroup p p vs the ad model groupmigrated into lymph nodethe proportion changesshowed that sbt oil suppressed the migration of lcs cellfrom skin lesion to draining lymph node after degradingproteins derive from extracellular environment were takenup by endocytosis or phagocytosis and captured by mhcclass ii molecules then result in peptideloaded mhc iiand migrate to the surface of antigen presenting cell waiting for recognition by cd4 t cells finally activate adaptive immune response the increased cell proportionof mhc class ii indicated the uptrend tendency of mature 0cwang bmc complementary medicine and therapies page of lcs in lymph node in dncb induced ad model micecompared with normal control mice while this uptrendtendency was inhibited by sbt oil application in micetreated with sbt oil constant epidermal lcs are immature normally and barely express costimulatory moleculessuch as cd80 and cd86 while upon lcsmaturation the expression of these costimulatory molecules was enhanced in this study sbt oil down regulatedthe expression of cd86 on lcs in lymph node which wasenhanced by dncb in ad model mice it suggested theeffect of sbt oil on inhibiting lcs maturationox40cd134 is a transmembrane protein of tumor necrosis factor receptor superfamily member which mainlyexpressed on activated cd4 t cells and upregulatedwithin inflammatory lesions on the antigenactivated tcells [ ] tslp can stimulate the expression of ox40ligand ox40l on lcs lcs expressing ox40l migratefrom skin lesion to local lymph node and induce the transformation of th0 cells to th2 cells on one hand the increased proportion of ox40l cells in lymph node ofdncb induced ad mice was suppressed by sbt oil administration which confirmed the effect of sbt oil on activation of cd4 t cells on the other hand sbt oilconduced to the normal function of lcs through renovating the keratinocyte and suppressing tslp release as aresultthe abnormal th2 skewing inflammation wasinhibited by sbt oil administrationall in all our results suggest that sbt oil inhibitedboth the migration of lcs to lymph node and its maturation in lymph node thereby inhibited the transformation of th0 cells to th2 cells and finally limited theoccurrence of th2 type inflammatory responsein summary our study results attested that sbt oil application suppressed dncbinduced adlike symptomsby downregulating serum ige level and the productionof cytokines and chemokines and regulated th1th2balance in addition our results also indicated that sbtoil treatment inhibited the migration of lcs to draininglymph node and its maturation taken together sbt oilhas excellent therapeutic effect on inflammatory skindiseases and might be a potential complementary candidate for ad treatment in further studiesit will beworthwhile to explore the mechanism of sbt oil and itsactive constituent in the treatment of adabbreviationsad atopic dermatitis dab diaminobenzidine dcs dendritic cell dncb dinitrochlorobenzene he hematoxylin and eosin ifnÎ interferonÎige immunoglobulin e il4 interleukin4 lcs langerhans cell mhcii majorhistocompatibility complex ii rtpcr realtime polymerase chain reactionsbt sea buckthorn tb toluidine blue th1 thelper th2 thelper tnfα tumor necrosis factorα tslp thymic stromal lymphopoietinacknowledgementsnot applicableauthors™ contributionsxxw carried out the experiment and drafting of the manuscript sjl and jplassisted to accomplish the experiment dnk and xwh carried out statisticalanalysis pl and mx did the interpretation work hqg and ddh revised theresearch and manuscript ddh designed the experiment and submitted themanuscript all the authors read and approved the final manuscriptfundingthis project was supported by the national natural science foundation ofchina grant no which was granted to diandong hou the resultsindicated in this manuscript were main achievements of the projectavailability of data and materialsall data and materials are contained and described within the manuscriptethics approval and consent to participateall experimental procedures were conducted according to the guidelinesprovided by the ethical committee of experimental animal care at liaoninguniversity of traditional chinese medicine shenyang pr chinaconsent for publicationnot applicablecompeting intereststhe authors state no potential conflict of interestauthor details1liaoning university of traditional chinese medicine shenyang liaoning prchina 2basic medical and forensic medicine baotou medical collegebaotou inner mongolia pr china 3neurosurgery department northernhospital of inner mongolia baotou inner mongolia pr china 4liaoningdongning pharmceutical co ltd fuxin liaoning pr china 5college ofintegrated traditional chinese and western medicine liaoning university oftraditional chinese medicine chongshan road no79 shenyang liaoning pr chinareceived december accepted june referencesklonowska j new cytokines in the pathogenesis of atopic dermatitisnew therapeutic targets int j mol sci choopani r treatment of atopic dermatitis from the perspective oftraditional persian medicine presentation of a novel therapeutic approach jevid based complementary altern med “brunner pm guttmanyassky e leung dy the immunology of atopicdermatitis and its reversibility with broadspectrum and targeted therapiesj allergy clin immunol 20171394ss65“leung dym new insights into atopic dermatitis j clin investig “kim je molecular mechanisms of cutaneous inflammatory disorderatopic dermatitis int j mol sci kim j molecular mechanism of atopic dermatitis induction followingsensitization and challenge with 24dinitrochlorobenzene in mouse skintissue toxicol res “hou dd sea buckthorn hippophae rhamnoides l oil improvesatopic dermatitislike skin lesions via inhibition of nfkappab and stat1activation skin pharmacol physiol “campana r molecular aspects of allergens in atopic dermatitis curropin allergy clin immunol “brandt eb sivaprasad u th2 cytokines and atopic dermatitis j clin cellimmunol de vuyst e atopic dermatitis studies through in vitro models frontmed lausanne park jg tabetri tabebuia avellanedae ethanol extract amelioratesatopic dermatitis symptoms in mice mediat inflamm 0cwang bmc complementary medicine and therapies page of liu r βsitosterol modulates macrophage polarization and attenuatesrheumatoid inflammation in mice pharm biol “ vollmer d west v lephart e enhancing skin health by oral administrationof natural compounds and minerals with implications to the dermalmicrobiome int j mol sci cicero afg colletti a effects of carotenoids on health are all the sameresults from clinical trials curr pharm des “ito t il33 promotes mhc class ii expression in murine mast cellsimmun inflamm dis “ dubois a regulation of th2 responses and allergic inflammationthrough bystander activation of cd8 t lymphocytes in early life jimmunol “ girtsman t natural foxp3 regulatory t cells inhibit th2 polarizationbut are biased toward suppression of th17driven lung inflammation jleukoc biol “ wallmeyer l tslp is a direct trigger for t cell migration in filaggrindeficient skin equivalents sci rep leyvacastillo jm tslp produced by keratinocytes promotes allergensensitization through skin and thereby triggers atopic march in mice jinvest dermatol “feinberg h trimeric structure of langerin j biol chem “ zhang x tim4 is differentially expressed in the distinct subsets ofdendritic cells in skin and skindraining lymph nodes and controls skinlangerhans cell homeostasis oncotarget “kumkate s cd207 langerhans cells constitute a minor population ofskinderived antigenpresenting cells in the draining lymph node followingexposure to schistosoma mansoni int j parasitol “ gaiser mr cancerassociated epithelial cell adhesion moleculeepcam cd326 enables epidermal langerhans cell motility and migrationin vivo proc natl acad sci u s a 201210915e889“ natarajan k the role of molecular flexibility in antigen presentationand t cell receptormediated signaling front immunol weinberg ad science gone translational the ox40 agonist storyimmunol rev “kinnear g a diametric role for ox40 in the response of effectormemory cd4 t cells and regulatory t cells to alloantigen j immunol“publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsshrestha s burden of atopic dermatitis in the united states analysis ofhealthcare claims data in the commercial medicare and medicaldatabases adv ther “ arima k burden of atopic dermatitis in japanese adults analysis ofdata from the national health and wellness survey j dermatol “ megna m systemic treatment of adult atopic dermatitis a reviewdermatol ther heidelb “ glatz m emollient use alters skin barrier and microbes in infants at riskfor developing atopic dermatitis plos one 2018132e0192443 drucker a
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edited byfang zhoucas lamvac biotech co ltd chinareviewed bybart evertsleiden university medicalcenter netherlandsmaria rosa bonouniversity of chile chileandrew john staggqueen mary university of londonunited kingdomcorrespondencedipayan rudradipayanrudragmailcomrudradpostechackrsinhyeog imiimshpostechackr present addresshyunja kokobiolabs inc seoul south koreasungwook hongdepartment of microbiology andimmunology centre for immunologyuniversity of minnesota medicalschool minneapolis mnunited states¡these authors have contributedequally to this work§deceasedspecialty sectionthis was submitted toimmunological tolerance andregulationa section of the frontiers in immunologyreceived april accepted july published august citationko hj hong sw verma r jung jlee m kim n kim d surh cdkim ks rudra d and im sh dietary glucose consumptionpromotes raldh activity in smallintestinal cd103cd11b dendriticcells front immunol 103389fimmu202001897intestinal dendritic cells dcs are critical for the initiation and regulation of innate and adaptiveimmunity by delivering self or foreign antigens to t cells “ the intestine is spontaneouslyexposed to innumerable antigens comprising of intestinal microbes as well as dietarycomponents to maintain immune homeostasis intestinal dcs regulate the balance betweenthe tolerogenic immune response by inducing cd4foxp3 regulatory t cells treg cells “and the protective immune responses by inducing eï¬ector t cells dysregulation of thisbalance by harmful pathogens or dietary intake results in ‚ammatory disorders such as‚ammatory bowel disease ibd celiac disease and food allergy frontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityintestinal dcs are located in the peyer™s patches ppsmesenteric lymph nodes mlns and lamina propria lp andcomprise cellular subsets that have diï¬erent origins and functions “ among these dc subtypes intestinal cd103 dcshave the unique function that metabolizes vitamin a to retinoicacid ra through the activation of aldehyde dehydrogenase member a2 [aldh1a2 also called retinaldehyde dehydrogenaseraldh2] enzyme the ra produced by intestinaldcs play an important role in orchestrating immune responsesimprinting guthoming specificity on t cells b cells andinnate lymphoid cells ilcs inducing igaproducing b cellspromoting tgfdependent diï¬erentiation of induced tregcells suppressing the diï¬erentiation of th17 cells enhancing il production by Îδ t cells and ilcs as well as inducing eï¬ectorfunctions in t cells “while vitamin a derived from dietary intake can induceraldh enzymatic activity the ra produced from intestinalepithelial cells iecs by raldh1 and stroma cells in lp andmln by raldh2 in a trans activating mechanism is alsocapable of inducing raldh expression in intestinal dcs “ furthermore recent data suggest that ra is also involvedin the development of a gut homing precursor for intestinaldcs in the bone marrow as well as is required for theirtranscriptional programming and maturation severalendogenous factors that regulate raldh expression in lpdcs are also reported cytokines such as il4 and granulocytemacrophage colonystimulating factor gmcsf induce orenhance the expression of raldh enzymes in lpdcs whileprostaglandin e2 pge2 negatively regulates raldh activitythrough the induction of inducible cyclic amp early repressoricer “ despite these findings whether additionalcomponents in diet can induce raldh activity in the intestineand promote immune tolerance remains unknown in this studywe uncover a hitherto unknown role of dietary glucose in shapingup intestinal immunological tolerance by facilitating raldhexpression specifically in intestinal lpdcsresultsmice administered antigen free diet havedefects in development and raldhactivity in cd103cd11b silpdcsto investigate the ‚uence of commensal microbiota and foodcomponents on intestinal immunity we utilized the previouslyestablished œantigen free af mice model where germfreegf mice are raised on welldefined elemental diet [termedœantigen free diet afd] devoid of macromolecules such asproteins and starches when dcs in small intestine wereassessed we observed comparable frequencies of cd11cmhcii silpdcs in specific pathogen free spf gf and af micefigures 1ab however indepth analyses revealed alterationin the frequencies of tolerogenic dc subtypes the frequencyof cd103cd11b silpdcs a subset known to be a majortolerogenic dc population was slightly but significantly lowerin af when compared to spf and gf mice figure 1c acompensatory increase on the other hand was observed inthe cd103cd11bˆ’ silpdc compartment more interestinglywhile the expression of the characteristic dc surface markerslargely remained comparable in all three groups figure s1athe expression of all three representative genes tested namelyaldh1a2 indoleaminepyrrole 23dioxygenase ido1 andtransforming growth factor beta tgfb1 that are functionallyimplicated in tolerogenic phenotype of cd103cd11b dcswere dramatically reduced in af mice figure 1d interestinglythe expression of aldh1a2 was found to be specifically reduced inmice raised under afd a phenomenon that was not observedin gf mice figure 1d left panel on the other hand theabsence of gut microbiota appeared to partially ‚uence theexpression of ido1 and tgfb1 which was further enhanced byafd figure 1d middle and right panel these results indicatedthat certain dietary components otherwise absent or underrepresented in afd have most specific and the largest ‚uenceon the expression of aldh1a2 for this study we therefore focusedon the ‚uence of normal diet on raldh activity in silpdcsraldh is an enzyme that irreversibly metabolizes vitamin ato ra which in turn acts as a key modulator of mucosal immuneresponses “ to determine whether in concert to itsreduced expression the function of raldh in lpdcs fromaf mice was also negatively aï¬ected we next examined raldhenzyme activity in lpdcs from spf gf and af mice usingthe aldefluor assay in this assay which has been previouslyemployed in the context of cd103 lpdcs and mlndcs the raldh enzyme activity is measured in individual cellsby flow cytometry with a fluorescent substrate based assay system in agreement with the results obtained by realtime pcranalysis cd103 silpdcs both cd11b and cd11bˆ’ subsetsfrom af mice displayed significantly lower enzyme activity whencompared with spf and gf mice figures 1ef figure s1b ofnote the characteristic frequencies of the aforementioned silpdc subtypes in spf gf and af mice remained unaltered evenafter performing this assay suggesting that this enzyme assay didnot interfere with the phenotype of silpdcs figure s1cin order to further understand the role of dietary componentson raldh activity we next analyzed silpdc raldh activityin mice at diï¬erent stages of their lives after subjecting them tospecific dietary conditions we observed that raldh activityin preweaned gf mice weeks of age was significantlylower than in adult gf mice and comparable to af mice thiswas dramatically restored to the level equivalent to adult gfmice within a week after weaning figure 2a furthermorewhen mice raised in af condition were fed with normal chowdiet ncd raldh activity in cd103cd11b silpdcs waspromptly recovered within a week figure 2b mirroring thisan opposite phenomenon was observed when ncd in gf micewas replaced with afd figure 2c these results suggested thatdietary components in ncd absent in afd is required as theinitial trigger for raldh gene expression after mice are weanedthereby promoting enzyme activity as well as homeostasis ofcd103cd11b silpdcsthe above results also implied that supplementing af micewith ra the final product of the enzymatic reaction and a knownfeedback inducer of raldh activity would be sufficient indriving raldh activity in these mice indeed when adult affrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure dietary intake affects differentiation and raldh activity in cd103 silpdcs cell suspensions were prepared from silp harvested from agematchedadult ˆ¼12weekold spf gf and af mice and phenotypic and raldh activity analyses of silpdcs were carried out a representative fluorescenceactivatedcontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure cell sorting facs plots of silpdc subpopulations gated on linˆ’ cd3ˆ’b220ˆ’ cells b statistical quantification of percentage left and total numbersright of cd11cmhcii cells in mice from indicated experimental groups c graph displays percentage of silpdc subpopulations in cd11cmhcii cells bcdata are combined from four independent experiments d realtime analyses of mrna expression of indicated gene products normalized against hprt mrna levelsef representative facs plots left and quantification right of relative mean fluorescence intensity mfi of aldefluor in cd103cd11b e and cd103cd11bˆ’f silpdc subpopulations from indicated groups deab is a raldh inhibitor 01mfi is calculated by subtracting background deab mfi from aldefluor mfi relative 01mfi indicates ratio of 01mfi in experimental samples vs control data are combined from six independent experiments mean ± sem are indicated statisticalsignificance was determined by oneway anova bef and twoway anova c with turkey™s multiple comparison tests p ns not statisticallysignificantmice were supplemented in their diet with ra it resulted incomplete recovery of raldh activity figure 2d interestinglyin all the cases changes in raldh activity also correlatedwith the frequencies of cd103cd11b silpdcs suggestingits role in diï¬erentiation as well as function of these cells ofnote while the above results were obtained in a gf setting thebasic findings from these experiments could also be recapitulatedin mice raised in spf conditions thereby confirming thatmice with normal repertoire of gut flora are equally aï¬ectedby dietary components with respect to raldh activity insilpdcs figures s2a“craldh activities in different intestinalraproducing cells are differentiallyaffected by dietwe next sorted to understand whether the ‚uence of dieton raldh activity is an lpdc specific phenomenon orwhether other raldh expressing cells are also aï¬ected itis wellestablished that within the gut associated lymphoidtissues besides lpdcs ra converting enzymes are alsoexpressed in lp associated stroma cells lpscs small intestineepithelial cells iecs as well as mlndcs the ra producedfrom these cellsis known as a localsource of ra for inducing the raldh expression in cd103silpdcs “in particular iecsthe nonhematopoietic scs in secondary lymph nodescomprise three diï¬erent cell types based on the expressionof surface markers lymphatic stroma cells [lscs also calledfibroblast reticular cells frc]lymphatic endothelial cellslecs and blood endothelial cells becs amonglpscs cd45ˆ’epcamˆ’ in smallintestine the lscs thatexpressed podoplanin pdpn and are cd31ˆ’ were foundto be capable of activating raldh enzymes figure 3a aspreviously reported interestingly unlike lpdcstheraldh activity in lpscs remained comparable between gfand af mice figure 3b in contrast when iecs were analyzedthe expression of aldh1a1 raldh1 the major gene encodingfor raldh enzyme in these cells was found to be reducedin af mice figure 3c of note while the iecs are wellestablished to have raldh activity “ the baseline ofthis activity in these cells is known to be significantly lower thanlpdcs therefore our attempt to measure raldhactivity in iecs was unsuccessful due to lower sensitivity ofthe aldefluor assay however albeit comparatively lowerraldh activity on a per cell basis the cumulative contributionof iecs in ra production is understandably of larger significancesince numerically there are many more iecs than the other celltypes in the intestinefinally when mlndcs were analyzed the raldh activityin particular within the cd103cd11b dc population in afmice was found to be significantly albeit to a lesser extentlower than that of gf mice figure 3d taken together theseresults suggested that dietary components diï¬erentially ‚uenceraldh activity in diï¬erentregulatory dc populationswhereas mlndcs and iec are aï¬ected lpscs appeared toremain unaï¬ected from dietary contributions these results alsoimplied that the overall reduction of ra synthesis cumulativelyamong these cell types eventually contributed to the reducedraldh activity in the lpdcs in af miceproteins starches and minerals in diet donot ‚uence raldh activity incd103cd11b silpdcssince for this study we focused on raldh activity insilpdcs we next wished to define which dietary factorswere required to trigger the initial raldh activity in thesecells while in our initial findings we observed both thesubtypes cd103cd11b and cd103cd11bˆ’ silpdcs tohave reduced raldh activity in af mice figures 1ef thecell recovery of cd103cd11bˆ’ silpdcs from spf and gfmice were low and the level of enzyme activity in this celltype showed variability among individual mice figures 1cftherefore henceforth in this study we focused on the raldhactivity in cd103cd11b silpdcswe first confirmed that the reduction in raldh activityin cd103cd11b silpdcs was not a consequence of lowvitamin a contentin af diet thereby compromising theprecursor for the assayed reaction based on information offood compositions from the suppliers and from our perviousreport final consumption of vitamin a per day by gf andaf mice were comparable [table s1 and ] nonethelessthere remained a possibility that albeit equal consumptionthe absorption of vitamin a into the small intestine may becompromised in af mice however when gf mice were weanedon af diet supplemented with times more vitamin a inthe usual form of œoil mix or were administrated additionalœoil mix by oral gavage failed to recover the reduction inraldh activity in cd103cd11b silpdcs figure s3we thus concluded that mere unavailability of the precursorvitamin a was not a cause of reduced raldh activity inthese cellsto this end we modified the compositions of purifieddiet by removing individual food components table s2 withfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure dietary components in normal chow readily trigger and maintain raldh activity in silpdcs in mice after weaning cell suspensions prepared from silpwere subjected to aldefluor assays and raldh activity in cd103cd11b silpdcs and percentage of silpdc subpopulations in cd11cmhcii cells wereanalyzed by flow cytometry a gf mice 3weeks old before weaning preweaned gf mice weaned onto ncd for days and adult gf mice were analyzed braldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult af mice ˆ¼12weekold after feeding ncd for and days c raldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult gf mice ˆ¼12weekold after feeding afd for and weeks d raldh activity and frequencies of silpdc subpopulations in cd11cmhcii cells in adult gf af or af mice that were administered intraperitoneal injection of alltrans ra µg per mouse in soybean oil every other day for days data are combined from two to three independent experimentsmean ± sem are indicated bc statistical significance was determined by oneway anova with turkey™s multiple comparison test p p p ns not statistically significantthe presumption that taking out or adding back individualcomponents in otherwise welldefined diet may lead us towardidentifying the dietary component required to trigger raldhactivity to obtain relatively accurate results under in vivosettings the modified diets were designed to contain similaramount of vitamin a as in ncd tables s1 s2 and thefrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure raldh activity in intestinal raproducing cells is differentially regulated by diet a“c cell suspensions from mln and silp harvested from agematchedadult ˆ¼12weekold gf and af mice were subjected to aldefluor assays and raldh activity of lpscs and mlndcs were analyzed by flow cytometry arepresentative facs plot of lpsc distinguished by cd31 and pdpn from cd45epcam cells red dots in facs plot indicate aldefluor positive cells histogramsdepict the mfi of aldefluor in lpsc subtypes b graph displays the level of raldh activity in lscs data is combined from two independent experiments c iecswere isolated from small intestine si by stripping with edta and analyzed for expression of aldh1a1 raldh1 by realtime pcr upon normalization to hprt mrnalevels fold change indicates ratio target gene in experimentcontrol data are combined from four independent experiments d representative facs plots ofmlndc subpopulations distinguished by cd103 and cd11b from cd11cmhciilinˆ’ cells and the raldh activity in cd103cd11b mlndcs andcd103cd11bˆ’ mlndcs data are combined from four independent experiments mean ± sem are indicated statistical significance was determined bytwotailed unpaired ttest p p ns not statistically significantexperiments were performed primarily in gf condition in orderto eliminate the ‚uence of microbiota in addition to avoidany ‚uence from ncd during the preweaned period neonateaf mice were utilized and these mice were weaned onto eachmodified diet for “ weeksas a starting point we took advantage of two commerciallyavailable diets with welldefined dietary compositions in the socalled amino acid defined diet aadˆ— like the afd employed sofar proteins were replaced with amino acids however there wereseveral diï¬erences between their compositions table s2 whilefrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure depletion of macromolecules from purified diet do not alter the raldh activity in cd103cd11b silpdcs af mice ˆ¼4weekold were weaned ontospecific diets for ˆ¼ weeks following which the indicated analyses were carried on a a cartoon depicting experimental scheme left panel aad is a sterilized formcontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure of amino aciddefined diet aad that contains three times more vitamin a than aad where protein macromolecules are replaced with amino acidsaad_stf indicates aad from which cornstarch and maltodextrin are removed the level of raldh activity in cd103cd11b silpdcs in the indicated groups arepresented by relative aldefluor 01mfi right panel b representative facs plots left panel frequencies middle panel and absolute numbers right panel of totalcd4foxp3treg cell and cd4foxp3nrp1lo peripheral treg ptreg cell populations in silp of mice subjected to the indicated diet regimes c experimentalscheme left panel and relative aldefluor 01mfi of silpdcs in the indicated experimental groups right panel min_mix afd indicates afd mixing with the mineral mixpowder td94049 data are combined from four independent experiments mean ± sem are indicated statistical significance was determined by oneway anovawith turkey™s multiple comparison test p p p ns not statistically significantafd is a liquid diet where the fibers are provided as cellulosebedding aadˆ— diet are edible solid pellets with cellulose mixedwith the food components unlike afd aadˆ— diet containedstarches in the form of maltodextrin and corn starch and whilethe source of sugar in afd was glucose that in aadˆ— was sucrose furthermore in terms of mineral compositionthere are substantial diï¬erences between the groups the secondcommercially available diet is aadˆ—_stf which was largelysimilar to aadˆ— but was devoid of starches note the œˆ— inaadˆ— indicates a sterilizable form of the diet which is otherwisesimilar to its traditional form aad but with three times morevitamin a to account for presumed losses during sterilization byirradiation surprisingly the mice groups weaned in both aadˆ—and aadˆ—_stf showed a complete recovery of raldh activityin cd103cd11b silpdcs to an extent similar to the controlncd fed group figure 4a these results were independentof microbiota since the characteristic drop in raldh activityof cd103cd11b silpdcs in mice raised in spf conditionscould also be recovered by aad figure s2d taken togetherthese findings led to two important conclusions first starchesare not involved second antigens in the form of peptidesderived from proteins are also dispensable as far as raldhactivity in cd103cd11b silpdcs is concerned to this endwe also considered a possibility that an artifact arising fromunaccounted protein contamination in the amino acid defineddiets may be responsible for the observed recovery of raldhactivity we therefore quantified the generation of peripheralregulatory t ptreg also referred to as itreg when inducedin vitro cell population in the silp of these mice ptreg cells area type of treg cells that are extrathymically generated primarilyat mucosal sites and are distinguished from their thymic ttregcounterparts by the lack of expression of the membrane boundcoreceptor neuropilin1 nrp1 notably in a previousreport we have demonstrated that diet derived proteins are theprimary cause for the generation of cd4foxp3nrp1ˆ’ ptregcells in the small intestine and af mice display dramaticallyreduced ptreg population in silp indeed we found that whiletotal foxp3 treg populations comprising of ttreg and ptregcells remained comparable the frequencies and numbers offoxp3nrp1ˆ’ ptreg cells among total treg population couldonly be recovered in mice fed with ncd and not aadˆ—and aadˆ—_stf figure 4b therefore the recovery of raldhactivity in aadˆ— and aadˆ—_stf groups were not due to anyprotein contamination in the aadˆ— dietwe next wished to exclude the possibility that diï¬erencesin minor food components such as minerals between afdand purified diet table s2 was responsible for diï¬erences inraldh activity for that we prepared afd by supplementingwith mineral mix powder td94049 derived from aadˆ—min_mixafd as shown in figure 4c min_mixafd failedto induce raldh activity taken togetherthese resultsconcluded that proteins starches and minerals in diet werenot responsible for the induction of raldh activity incd103cd11b silpdcsoptimum glucose level in diet is requiredto induce raldh activity incd103cd11b silpdcsif the macromolecules and micromolecules in diet were notinvolved lack of which factors in afd compromise raldhactivity in cd103cd11b silpdcs to investigate furtherwe compared the food compositions between aadˆ—_stf andafd table s2 which were the closest among the four typesof diets tested in case of aadˆ—_stf this diet does not containproteins and starches as in afd but does contain unknowndietary factors responsible for the induction of raldh activityin silpdcs there are few diï¬erences between the compositionof these two diets the diet forms pellet vs liquid thecarbohydrate sources sucrose vs glucose the amount ofcarbohydrate sucrose ˆ¼ vs glucose ˆ¼ based on thisobservation as well as in order to minimize the diï¬erencesin diet forms we first generated a liquid form of afd with of sucrose afd_s500 or glucose afd_g500germ free diet gfd or aadˆ—_stf were used as positivecontrols afd with usual glucose designated as afd_g220here was used as negative control the neonate af micewere weaned onto each diet for “ weeks and were analyzedfor raldh activity in cd103cd11b silpdcs figure 5aleft panel indeed afd supplemented with sucroseresulted in significantly enhanced raldh activity in these cellsmore interestingly we observed a similar increase in raldhactivity in cd103cd11b silpdcs even when the source ofcarbohydrate was changed to glucose instead of sucrosefigure 5a middle panel these eï¬ects were found to be specificfor cd103cd11b silpdcs since raldh1 expression iniecs remained unaltered upon carbohydrate supplementationfigure 5a right panel these results suggested that regardlessofits optimum concentrationis important and glucose being a monosaccharide unit ofcarbohydrate is sufficient for the initial triggering of raldhactivity in silpdcsthe source of carbohydratein order to further understand whether the positive eï¬ectof dietary carbohydrate supplementation is specific for raldhactivity or if it can also aï¬ect diï¬erentiation of other immunecells we determined the frequencies of th1 th2 and ptregfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure supplementing glucose in af diet restores raldh activity in cd103cd11b silpdcs a cartoon depicting experimental scheme left panelneonate af mice were weaned onto specific diets for ˆ¼ weeks afd contains of glucose afd_g220 afd_g500 indicates of glucose in afd andafd_s500 indicates of sucrose in afd aad_stf was utilized as a positive control of raldh activity in lpdcs aldefluor assays were performed on cellsuspensions from silp and raldh activity of silpdc was analyzed by flow cytometry the level of raldh activity in cd103cd11b silpdcs is represented asrelative 01mfi of aldefluor middle panel aldh1a1 expression was also determined in sorted iecs relative to hprt control right panel data are combined from twoindependent experiments b“d graphs display the percentage of tbet in cd4 t cells th1 b gata3 in cd4 t cells th2 c and nrp1lo populations amongin cd4foxp3 treg cells ptreg d data are combined from at least two independent experiments mean ± sem are indicated statistical significance wasdetermined by oneway anova with turkey™s multiple comparison test p p p ns not statistically significantcells in these mice supplementation of afd with additionalsucrose or glucose did not aï¬ect tbet th1 or gata3 th2 cellsfigures 5bc neither recovered the characteristically reducedptreg population in silp figure 5dwe next directly investigated the eï¬ect of glucose on raldhactivity by employing in vitro culture conditions spldcsmlndcs and cd11c silpdcs were magnetically purifiedand cultured either without glucose in commercially availableglucosefree media without glucose but in the presence of rawith glucose or in the presence of glucose and ra for hand then measured for raldh activity in terms of baselineraldh activity as expected spldcs displayed the least whichwas significantly increased in the presence of ra glucose onthe other hand had minimal eï¬ect figure 6a although mlndcs had the highest baseline raldh activity among the threegroups tested neither ra nor glucose had an impact figure 6bin contrast silpdcs derived from 2weeks old neonatal spfmice which had low basal raldh activity at steady state wassignificantly increased in the presence of glucose alone while raitself induced slight increase of this enzyme activity the highestactivity was observed when both ra and glucose was presentfigure 6c moreover this raldh activity promoting eï¬ect ofglucose was also observed when silp mixed lymphocytes derivedfrom 8weeks old adult mice were cultured with glucose for hunder the culture conditions tested ra by itself was found tohave minimal eï¬ect on the already high raldh activity whereasthe addition of glucose in the media was able to further boost thisactivity figure 6dfinally in order to ascertain functional relevance of thesefindings we sorted to determine whether supplementationfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure glucose induces raldh activity specifically in silpdcs mlndcs and spleen dcs spldc from adult spf mice or silpdcs from 2weeks oldneonatal spf mice were either purified a“c or total single cell suspensions were isolated from adult spf mice si d cells were cultured for h in glucosefreecontinuedfrontiers in immunology wwwfrontiersinaugust volume 0cko glucose promotes silpdc raldh activityfigure media with or without treatments following which aldefluor assay was performed fluorescence intensities of aldefluor in cd8ˆ’cd11b spldcsa cd103 mlndcs b and cd103cd11b silpdcs cd were analyzed by flow cytometry raldh activity depicted in overlaid histograms and bar graphsare from the cells cultured with glucose mm or ra nm or both the line graphs indicate raldh activity from the cells cultured with different concentrations ofglucose and mm in the presence or absence of ra nm data shown is representative of at least three independent experiments mean ± sem areindicated statistical significance was determined by oneway anova with turkey™s multiple comparison test p p p ns notstatistically significantof glucose presumably through the generation of ra canfacilitate the generation of itreg cells for this we performedan in vitro itreg induction assay with magnetically purifiedcd11chi dcs with œhigh cd11c expression that were pretreated with glucose and incubated with naïve t cells undersuboptimal itreg inducing condition we observed significantincrease in itreg induction when silpdcs were pretreatedwith glucose compared to mock this eï¬ect of glucose pretreatment was specific for silpdcs and was not observedwhen mlndcs were used in the assay figures 7ab andfigure s4 these results in accordance to the results presentedin figure 6b suggested that the silp dcs are particularlymore susceptible to glucose treatment and thereby presumablythrough enhanced raldh activity acquire superior itreg cellinduction capacity compared to mln dcs it is to be notedhowever that while the purified cd11chimhcii dcs usedin this assay are presentthere aresome site specific diï¬erences with regard to the expressionof mhcii and cd11c in mln and silp compared to silpmln has significantly higher proportion of cd11cˆ’mhciiand cd11cintmchiiˆ’ with œintermediate cd11c expressionpopulations figure s4 left panels furthermore the expressionof mhcii in purified mln dcs is lower than that of silpdcs figure s4 right panels these observations raised theformal possibility that enhanced mhcii expression in silprather than glucose mediated enhanced raldh activity maybe responsible for increased itreg conversion however pretreatment with ra either alone or in the presence of glucoseresulted in equally efficient itreg induction irrespective of thesource of the dcs suggesting that the benefit of glucose pretreatment is indeed primarily due to enhanced ra productionfigures 7ab lastly when itreg induction was carried outin vitro in a dc independent manner supplementation ofthe media with excess glucose had little eï¬ect thereby furthersubstantiating the role of dc derived raldh in this processfigure s5in similar frequenciesdiscussionthere is accumulating evidence suggesting that vitamin a and itsmetabolites play a pivotal role in maintaining various biologicalprocesses “ dietary supplementation of ra in thecontext of cutaneous t cell lymphoma and acute promyelocyticleukemia have been shown to have beneficial outcome “ furthermore many studies highlight anti‚ammatoryactivities of ra at mucosal sites and tissues such as intestinalmucosa airways lung central nervous system and skin “ in addition to the eï¬ect of ra on cancer and ‚ammatorydiseases vitamin a or its metabolites play an important roleto suppress dietinduced obesity and insulin resistance “therefore a better understanding of the cellular and molecularparameters responsible for vitamin a metabolism is of greatbiological relevance in this study by identifying the role of dietand the importance of glucose consumption for establishingraldh activity early in life in small intestine dcs we makesubstantial contribution to our knowledge related to the role ofnutritional components in establishing immunological toleranceat mucosal sitesby feeding af diet to mice raised under germ free conditionwe found that small intestine cd103cd11b lpdcs require adietary component as an initial trigger for raldh activity sincevitamin a in diet is known to be essential to activate raldhand generate ra in lpdcs lpscs mlndcs mlnscs andiecs one explanation of this observation could bethe possibility that at steady state af mice consume less vitamina compared to gf mice raised on ncd however our furtherexperiments in conjunction with a previous report stronglyindicate that the availability of vitamin a and the way it is feddoes not account for the low raldh activity in af m
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"Proteasomes are found in both the cell nucleus and cytoplasm and play a major role in the ubiquitindependent and independent nonlysosomal pathways of intracellular protein degradation Proteasomes are alsoinvolved in the turnover of various regulatory proteins antigen processing cell differentiation and apoptosis Todetermine the diagnostic value of serum proteasome in antineutrophil cytoplasmic antibody ANCAassociatedvasculitis AAV we investigated patients with AAV at various stages of the diseaseMethods Serum 20Sproteasome was measured by ELISA in patients with MPOANCAassociated microscopicpolyangiitis MPA and renal involvement Thirty of the patients provided serum samples before the initialtreatment and provided samples during remission provided samples at both time pointsResults The mean serum 20Sproteasome level was significantly higher in the activevasculitis patients ± ngmL n compared to the inactivevasculitis patients ± ngmL n p and controls ± ngmL p There were significant positive correlations between the serum 20Sproteasome level and the Birmingham Vasculitis Activity Score BVAS r p the ANCA titer r p the white blood cell WBC count r p the platelet count r p and the serum Creactive protein CRP level r p There were significant negative correlationsbetween the serum 20Sproteasome level and both the hemoglobin concentration r ˆ’ p and theserum albumin level r ˆ’ p In a multiple regression analysis there was a significant positivecorrelation between the serum 20Sproteasome level and only the BVAS results p In a receiveroperating curve analysis the area under the curve for the serum 20Sproteasome level was which is higherthan those of the WBC count and the serum CRP level Conclusion The serum level of 20Sproteasome may be a useful marker for disease activity in AAVKeywords ANCAassociated vasculitis 20Sproteasome Disease activity Microscopic polyangiitis Proteasome Correspondence khiratokyomedacjp1Department of Nephrology Tokyo Medical University Ibaraki Medical Center Chuo Ami Ibaraki JapanFull 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 0cMaruyama BMC Rheumatology Page of BackgroundProteasomes are located in both the nucleus and cytoplasm of cells and they play a major role in theubiquitindependent and ubiquitinindependent nonlysosomal pathways of intracellular protein degradation[ ] Proteasomes are also involved in the turnover ofvarious regulatory proteins eg ratelimiting enzymes[] and proteins for cellcycle control [] or transcriptional regulation [] antigen processing [] cell differentiation [] and apoptosis [] The 26S proteasome is amulticatalytic enzyme with a highly ordered structurecomposed of at least different subunits arranged intwo subcomplexes a 20S core and a 19S regulator p [] The 20Sproteasome is composed of four ringsof nonidentical subunits two rings are composed ofseven alpha subunits and the other two rings are composed of seven beta subunits Three of the seven betasubunits have proteolytic sites the 1 2 and 5 subunits are associated with caspaselike trypsinlike andchymotrypsinlike activities respectively [] These 12 and 5 subunits cleave peptide bonds at postacidicˆ’basic and hydrophobic amino acid residues respectively [] However subunits 1 2 and 5 could be replaced with 1i 2i and 5i by interferongamma IFNÎ and this IFNÎinducible proteasome isotype is calledthe immunoproteasome []The serum proteasome levels of patients with malignanttumors are elevated because the proteasome is overexpressed in tumor cells In patients with multiple myelomaserum proteasome concentrations have been shown to beassociated with disease severity and activity [] theserum proteasome concentrations were significantly elevated in patients with multiple myeloma compared tocontrols in multiple myeloma versus monoclonal gammopathies of undetermined significance MGUS and in active versus smoldering multiple myeloma [] Similarlyelevated serum proteasome levels were also reported inautoimmune diseases characterized by Bcell abnormality[] In the present study to determine the diagnosticvalue of the serum proteasome concentration in antineutrophil cytoplasmic antibody ANCAassociated vasculitisAAV we investigated patients with myeloperoxidaseMPOAAV at various stages of the diseasePatientsPatients and controlsWe analyzed the cases of patients with MPOANCAassociated microscopic polyangiitis MPA and renal involvement The diagnosis of MPA was based on theEuropean Medicines Agency algorithm [] and patientswith other types of systemic vasculitis including eosinophlic granuromatosis with polyangiitis granulomatosis with polyangiitis and antiglomerular basementdisease were excludedOf the MPOAAV patients provided serumsamples before the initial treatment and providedsamples during remission provided samples both before the initial treatment and during remission The Birmingham Vasculitis Activity Score BVAS was used toevaluate patients™ disease activity and remission was defined as a BVAS of As controls healthy volunteersand patients with chronic kidney disease CKD wereinvestigated The causes of CKD were nephrosclerosisn chronic glomerulonephritis n diabeticnephrosclerosis n and autosomal dominant polycystic kidney disease n Sample collection and analysisThe serum samples measured by a commerciallyavailable enzymelinked immunosorbent assay ELISAkit Enzo Life Science Plymouth Meeting PA USin duplicate In brief 96well microtiter plates werecoated with a mouse anti20Sproteasome alpha6subunit monoclonal antibody and left overnight at °C followed by blocking with phosphatebuffered saline PBS containing bovine serum albumin for h atroom temperature RT A serum sample was thenadded to each well and the plates were incubated for h at RT A rabbit anti20Sproteasome polyclonalantibody was then added to each well and the plateswere incubated for h at RT followed by incubationwith a horseradishperoxidaselabeled goat antirabbitIgG antibody for h at RT The plates were finallyincubated with chromogen tetramethylbenzidine andhydrogen peroxide for min at RT and then addedwith N hydrochloride acid solutionBetween these steps the plates were washed five timeswith Trisbuffered saline The plates were immediatelyread on a microplate reader Sunrise Remote® TecanJapan Kanagawa Japan set at nm with nm as areference wavelength The inter and intraassay variationswere Statistical analysesAll statistical analyses were performed using PASW Statistics software ver IBM Japan Tokyo for Windows The data are expressed as means ± standarddeviations or as numbers with percentages of the totalThe chisquare test with Yates™ continuity correctionand Fisher™s exact test were used for differences in categorical variables and posthoc comparisons Bonferronicorrection were performed to detect differences amongthree groups The MannWhitney Utest was used fortwogroup comparisons and we conducted an analysisof variance ANOVA to assess differences among threeor more groups posthoc comparisons were made usingthe BonferroniDunn test Correlations were determinedusing Spearman™s univariate correlation test and a linear 0cMaruyama BMC Rheumatology Page of regression analysis The multiple linear regression analysis included the covariates shown to be significantly associated with the serum 20Sproteasome levelin thecorrelation analysis and the data are expressed as standardized regression coefficients We applied comparative receiveroperatingcharacteristic ROC curvesand the area under the curve AUC to assess the diseaseactivity accuracy of the the serum 20Sproteasome leveland inflammatory variables Pvalues were accepted assignificant at but in the comparisons of three ormore groups the critical pvalue was divided by thenumber of comparisons being madeResultsThe subjects™ characteristicsThe characteristics clinical symptoms and laboratorydata among the subjects of the three groups the activevasculitis patients the inactivevasculitis patients andthe controls are shown in Table At the testing therewas no patients treated with any immunosuppressant inboth active and inactive vasculitis but allinactivevasculitis patients had treated with corticosteroids dosesof prednisolone ± mgdaySerum 20Sproteasome levelsAs illustrated in Fig the mean level of serum proteasome in the activevasculitis patients ± ngmL was significantly higher than those in the inactivevasculitis patients ± ngmL p andthe controls ± ngmL p There weresignificant positive correlations between the serum 20Sproteasome levels and the BVAS results r p the MPOANCA titers r p theWBC counts r p the platelet countsTable Characteristics of subjectsAge yearsGender malefemaleBirmingham vasculitis activity scoreClinical symptomsFeverWeight lossArthralgiaEpiscleritis or uvitisSinusitisHearing lossAlveolar hemorrhageInterstitial lung diseaseArrhythmiaPericarditisHeart failureRapidly progressive glomerulonephritisPeripheral nerve damageLaboratory dataANCA titer UmLWhite blood cell mm3Hemoglobin conc gdLPlatelet count 104mm3Serum albumin gdLSerum creatinine mgdLSerum Creactive protein mgdLSerum 20Sproteasome mgdLDoses of prednisolone mg daily P vs Inactivevasculitis P vs ControlsMPOANCA associated vasculitisActivevasculitis n ± Inactivevasculitis n ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Controlsn ± ± ± ± ± ± ± ± 0cMaruyama BMC Rheumatology Page of Fig The serum levels of 20Sproteasome Closed circles means bars standard deviations circles the values for individual patientsr p and the serum CRP levels r p There were significant negative correlations between the serum 20Sproteasome levels and boththe hemoglobin concentrations r ˆ’ p and the serum albumin levels r ˆ’ p In the multiple regression analysis there was a significant positive correlation between the serum 20Sproteasome levels and only the BVAS results p Table In the activevasculitis patients there was no associationbetween the serum 20S proteasome levels and clinicalsymptoms except for pulmonary involvement Supplementary file S1 The mean serum 20S proteasome level inthe activevasculitis patients with interstitial lung diseasen ± ngmL was significantly higherthan those in the activevasculitis patients withoutpulmonary involvement n ± ngmLp and those in activevasculitis patients withalveolar hemorrhage n ± ngmL p There was no association between the serum 20Sproteasome levels and the percentages of crescent formation renal histological classification Berden™s classification [] or renal symptoms patients with chanceproteinuriahematuria and patients with rapidly progressive glomerulonephritisThe diagnostic potential for disease activityThe optimum cutoff levels for the disease activity ofvasculitis were identified from the ROC curves for theWBC count 7250mm3 serum CRP level mgdL and serum 20Sproteasome level ngmLFig The area under the curve AUC for the serumTable Correlation between the serum 20Sproteasome level and clinical parametersAgeBirmingham Vasculitis Activity ScoreANCA titerWhite blood cellHemoglobin concPlatelet countSerum albuminSerum creatinineSerum Creactive proteinUnivariate analysisrˆ’ˆ’Pvalue Multivariate analysis“ˆ’ˆ’ˆ’“ˆ’Pvalue““ 0cMaruyama BMC Rheumatology Page of polymyositis serum proteasome levels were correlatedwith serum creatinine kinase levels and serum proteasome levels were associated with disease activity [] Inthe present study elevated serum 20Sproteasome levelswere also demonstrated in patients with AAV Althoughthere was no relationship between the MPOANCA titersand the serum 20Sproteasome levels these elevationswere associated with disease activity ie the BVASTherefore the serum level of 20Sproteasome may be auseful marker for disease activity in AAVThe mechanisms that underlie the elevated serum proteasome observed in patients with AAV are not yetknown Several serum biomarkers are filtrated at theglomerulus and reabsorbed and catabolized by proximaltubular cells and the serum levels of such biomarkers inpatients with renal insufficiency are elevated due to lowurinary filtration Because we found no relationship between serum 20Sproteasome levels and serum creatinine in AAV patients we conclude that elevated serumproteasome is not associated with renal functionIn a previous investigationthe serum proteasomelevels in patients with multiple myeloma were elevateddue to overexpression in tumor cells but the mechanisms underlying these elevations in autoimmune diseases were not clarified [] On the other hand thestructure and function of serum proteasome in healthydonors and patients with autoimmune diseases SLE andRA were maintained in the same manner as the intracellular forms [] However rings of proteasomes inthe serum of patients with autoimmune diseases weredifferent from those in healthy donors and those ringscontained immunosubunits 2i and 5i [] Consideringthat proteasomes from nonimmunocompetent cells donot contain immunosubunits [] it was speculated thatserum proteasome in patients with autoimmune diseasesmay have its fraction structure added by an immunocompetent cell origin that is different from that in normal individuals Therefore the elevated serum proteasome levelsin AAV may also be associated with the activation of immunocompetent cells Further investigations are neededto clarify the mechanism by which the proteasome isreleased into the circulationBortezomib a proteasome inhibitor prevents the degradation of proteins marked by ubiquitination by inhibiting the 26S proteasome [] The main effects ofbortezomib are NFκB inhibition inhibition of cell proliferation by the stabilization of cyclindependent kinasesFig The comparative ROC curves for three measurements ofdisease activity Solid line serum levels of 20Sproteasome Dashdotted line WBC counts Dashed line serum CRP levels Dotted linereference line20Sproteasome level was which is higher thanthose of the WBC count and the serum CRP level On the ROC curve the serum 20Sproteasomehad sensitivity and specificity for the diseaseactivity Although the specificity of the serum 20Sproteasome level was less than that of the serum CRPlevelserum 20Sproteasome level was superior to that of the serumCRP level Table sensitivity ofthetheDiscussionPrevious studies have demonstrated that the serum20Sproteasome levels are elevated in individuals with autoimmune diseases In patients with various autoimmune diseases including systemic lupus erythematosusSLE polymyositis Sjögren™s syndrome antiphospholipidsyndrome rheumatoid arthritis RA systemic sclerosisautoimmune hepatitis and myasthenia gravis the serumproteasome levels were higher than in the controls[] The levels were especially and significantly high inthe patients with SLE polymyositis Sjögren™s syndromeRA and autoimmune hepatitis [] In patients withWhite blood cell countTable Comparative ROC curves for parameters of disease activity confidence interval“““Area under the curveSerum Creactive proteinSerum 20SproteasomePvalue Optimal cutoff levelsSensitivity Specificity 0cMaruyama BMC Rheumatology Page of the induction of apoptosis by the activation of cJunNH2terminal kinase the stabilization of proapoptotic proteinsand transcription factors and tumor suppressors and theinduction of cell death by activation of the terminalunfolded protein response [] Bortezomib has been approved for clinical use in patients with multiple myelomaand bortezomib treatment has implications for antibodymediated immune diseases as well []The efficacy of bortezomib was demonstrated in amouse model of MPOANCAassociated glomerulonephritis [] That is in antiMPOassociated glomerulonephritisinduced by immunizing MPOdeficientmice with murine MPO followed by irradiation and thetransplantation of wildtype bone marrow proteinuriaalbuminuria and hematuria were significantly reducedcompared to the controls by both standard steroidcyclophosphamide treatment and bortezomib treatment[] Moreover the percentage of glomeruli with crescent or necrosis formation was reduced by both treatments The clinical efficacy of bortezomib for AAV hasnot been determined because only one case of an AAVpatient treated with bortezomib was reported In thatcase complete remission could not be achieved by acombination treatment with corticosteroid cyclophosphamide and rituximab therefore bortezomib mgm2week for weeks was added [] After the additionof bortezomib the patient achieved complete remissionand the doses of corticosteroid could be withdrawn []Thusthe proteasome may be associated with thedevelopment of AAV and inhibition of the proteasomemay be effective for inducing the remission of AAVOur study has several limitations The study population was small and limited to MPOAAV patients withrenal involvement and thus further studies are neededto compare patients with PR3AAV or nonrenal vasculitis In addition this was a retrospective crosssectionalstudy a larger prospective longitudinal study includingvasculitis patients with relapse would provide more definitive results Since the present study was performed atone facility it is necessary to verify the accuracy of theELISA test Moreover allinactivevasculitis patientswere treated with corticosteroids at the testing so treatments themselves may affected to decreased levels in inactive vasculitis Therefore further studies are needed tocompare AAV patients without treatments at the testingor to investigate other diseases patients treated withwithout corticosteroids Finally although we did demonstrate that serum 20Sproteasome levels were elevated inour patients with AAV the cause of this elevation wasnot identified In patients with multiple myeloma elevation of serum 20Sproteasome may be associated withoverexpression in tumor cells or abnormal cellular turnover [] On the other hand elevation of serum 20Sproteasome was observed in septic patients and therelation between elevated serum 20Sproteasome levelsand increased lymphocyte apoptosis was demonstratedin critically ill patients [] In patinets with RA andSLE it was speculated that the expression of inflammatory cytokines may have influenced the elevation of theserum 20Sproteasome [] Althoug elevated serum20Sproteasome in active AAV may be reflected an acutephase response there was no significant correlation between the serum 20Sproteasome levels and serum CRPlevels in the multiple regression analysis To clarify themechanisms of the serum 20Sproteasome elevation invasculitis patients further in vitro and ex vivo investigations are neededConclusionThe serum levels of 20Sproteasome in our patients withactive MPOAAV were significantly elevated compared tothe levels in the patients with inactive MPOAAV and thecontrols Moreover the serum levels of 20Sproteasomewere related to the BVAS results The serum level of 20Sproteasome may therefore be a useful marker for diseaseactivity in AAVSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s41927020001374Additional file Supplementary file S1 The relationships betweenthe serum 20S proteasome levels and the patients™ clinical symptomsAbbreviationsAAV Antineutrophil cytoplasmic antibodyassociated vasculitisANCA Antineutrophil cytoplasmic antibody BVAS Birmingham VasculitisActivity Score MPA Microscopic polyangiitis MPO MyeloperoxidaseAcknowledgementsPart of this study was reported at the 18th International Vasculitis and ANCAWorkshop Tokyo and it was published in Rheumatology suppl as an abstractAuthors™ contributionsHM and KH designed the study executed the experiments and participatedin the data management statistical analyses and reporting logicalinterpretation and presentation of the results MY KO and RT participated inthe data collection MT and HS took part in the logical interpretation andpresentation of the results KH and MK coanized the course of the workAll authors read and approved the final manuscriptFundingThis study was supported in part by a research grant to KH from MSD KKTokyo Japan The funds for this study were used only to purchase ELISAkitsAvailability of data and materialsAll of the raw datasets used and analyzed in this study are available uponreasonable request from the corresponding authorEthics approval and consent to participateAll procedures performed in this study involving human participants were inaccordance with the ethical standards of the institutional committee andwith the Declaration of Helsinki and its later amendments orcomparable ethical standards The study protocol was approved by the 0cMaruyama BMC Rheumatology Page of Zoeger A Blau M Egerer K Feist E Dahlmann B Circulating proteasomesare functional and have a subtype pattern distinct from 20S proteasomes inmajor blood cells Clin Chem “Froment C UttenweilerJoseph S BousquetDubouch MP Matondo MBes JP Esmenjaud C Lacroix C Monsarrat B BurletSchiltz O Aquantitative proteomic approach using twodimensional gel electrophoresisand isotopecoded affinity tag labeling for studying human 20S proteasomeheterogeneity Proteomics “ Hideshima T Richardson P Chauhan D Palombella VJ Elliott PJ Adams JAnderson KC The proteasome inhibitor PS341 inhibits growth inducesapoptosis and overcomes drug resistance in human multiple myelomacells Cancer Res “ Boccadoro M Man G Cavenagh J Preclinical evaluation of theproteasome inhibitor bortezomib in cancer therapy Cancer Cell Int Fröhlich K Holle JU Aries PM Gross WL Moosig F Successful use ofbortezomib in a patient with systemic lupus erythematosus and multiplemyeloma Ann Rheum Dis “ Bontscho J Schreiber A Manz RA Schneider W Luft FC Kettritz RMyeloperoxidasespecific plasma cell depletion by bortezomib protectsfrom antineutrophil cytoplasmic autoantibodiesinducedglomerulonephritis J Am Soc Nephrol “ Novikov P Moiseev S Bulanov N Shchegoleva E Bortezomib in refractoryANCAassociated vasculitis a new option Ann Rheum Dis 2016751e9 Yousef AA Suliman GA Mabrouk MM The value of correlation of serum 20Sproteasome concentration and percentage of lymphocytic apoptosis incritically ill patients a prospective observational study Crit Care R215Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsEthics Committees of Tokyo Medical University Ibaraki Medical CenterWritten informed consent for inclusion in the study was obtained from allpatientsConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Nephrology Tokyo Medical University Ibaraki Medical Center Chuo Ami Ibaraki Japan 2Department of Intensive CareMedicine Tokyo Medical University Ibaraki Medical Center Ami IbarakiJapan 3Department of Nephrology Tokyo Medical University ShinjukuTokyo JapanReceived April Accepted May ReferencesArrigo AP Tanaka K Goldberg AL Welch WJ Identity of the 19S ˜prosome™p with the large multifunctional protease complex of mammaliancells the proteasome Nature “Hershko A Ciechanover A The ubiquitin system Annu Rev Biochem “ Murakami Y Matsufuji S Kameji T Hayashi S Igarashi K Tamura T Tanaka KIchihara A Ornithine decarboxylase is degraded by the 26S proteasomewithout ubiquitination Nature “Hershko A Roles of ubiquitinmediated proteolysis in cell cycle control CurrOpin Cell Biol “Kho CJ Huggins GS Endege WO Hsieh CM Lee ME Haber E Degradationof E2A proteins through a ubiquitinconjugating enzyme UbcE2A J BiolChem “Stoltze L Nussbaum AK Sijts A Emmerich NP Kloetzel PM Schild H Thefunction of the proteasome system in MHC class I antigen processingImmunol Today “Baz A Henry L Caravano R Scherrer K Bureau JP Changes in the subunitdistribution of prosomes MCPproteasomes during the differentiation ofhuman leukemic cells Int J Cancer “Pasquini LA Marta CB Adamo AM Pasquini JM Soto EF Relationshipbetween the ubiquitindependent pathway and apoptosis in different cellsof the central nervous system effect of thyroid hormones Neurochem Res“Jung T Grune T Structure of the proteasome Prog Mol Biol Transl Sci “ Groll M Ditzel L Lowe Löwe J Stock D Bochtler M Bartunik HD Huber RStructure of 20S proteasome from yeast at a resolution Nature “ Boes B Hengel H Ruppert T Multhaup G Koszinowski UH Kloetzel PMInterferon gamma stimulation modulates the proteolytic activity andcleavage site preference of 20S mouse proteasomes J Exp Med “Jakob C Egerer K Liebisch P Türkmen S Zavrski I Kuckelkorn U Heider UKaiser M Fleissner C Sterz J Kleeberg L Feist E Burmester GR Kloetzel PMSezer O Circulating proteasome levels are an independent prognosticfactor for survival in multiple myeloma Blood “Egerer K Kuckelkorn U Rudolph PE Rückert JC Dörner T Burmester GRKloetzel PM Feist E Circulating proteasomes are markers of cell damageand immunologic activity in autoimmune diseases J Rheumatol “ Watts R Lane S Hanslik T Hauser T Hellmich B Koldingsnes W Mahr ASegelmark M CohenTervaert JW Scott D Development and validation of aconsensus methodology for the classification of the ANCAassociatedvasculitides and polyarteritis nodosa for epidemiological studies AnnRheum Dis “ Berden AE Ferrario F Hagen EC Jayne DR Jennette JC Joh K Neumann INoël LH Pusey CD Waldherr R Bruijn JA Bajema IM Histopathologicclassification of ANCAassociated glomerulonephritis J Am Soc Nephrol“ 0c"
2
] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0“ in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global moran™s index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of pm25 and pm10 in “ respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range “ “ “ pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0“ was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0“ was associated with a increase of daily confirmed new cases wuhan rr ci1032“ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p χ2 p a μgm3 increase in pm25 was associated with a “ increase in fatality rate χ2 p a μgm3 increase in pm10 was associated with a “ increase in fatality rate no2 a 10μgm3 no2 increase lag0“ was associated with a increase in daily confirmed new cases wuhan rr ci “ xiaogan rr ci “ huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci “ estimate on deaths value cid0 ci “ kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearman™s one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci “ and deaths cid0 ci “ low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years “ and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0“ in no2 is associated with a ci “ increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053“ xiaogan rr ci “ but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations “ pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol “ httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res “ httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health “ httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med “ httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health “ https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res “ https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 “ conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop
0
lipases are very versatile enzymes and produced the attention of the several industrial processes lipase can be achieved from several sources animal vegetable and microbiological the uses of microbial lipase market is estimated to be usd million in and it is projected to reach usd million by growing at a cagr of from microbial lipases ec catalyze the hydrolysis of long chain triglycerides the microbial origins of lipase enzymes are logically dynamic and proficient also have an extensive range of industrial uses with the manufacturing of altered molecules the unique lipase triacylglycerol acyl hydrolase enzymes catalyzed the hydrolysis esterification and alcoholysis reactions immobilization has made the use of microbial lipases accomplish its best performance and hence suitable for several reactions and need to enhance aroma to the immobilization processes immobilized enzymes depend on the immobilization technique and the carrier type the choice of the carrier concerns usually the biocompatibility chemical and thermal stability and insolubility under reaction conditions capability of easy rejuvenation and reusability as well as cost proficiency bacillus spp achromobacter spp alcaligenes spp arthrobacter spp pseudomonos spp of bacteria and penicillium spp fusarium spp aspergillus spp of fungi are screened large scale for lipase production lipases as multipurpose biological catalyst has given a favorable vision in meeting the needs for several industries such as biodiesel foods and drinks leather textile detergents pharmaceuticals and medicals this review represents a discussion on microbial sources of lipases immobilization methods increased productivity at market profitability and reduce logistical liability on the environment and userkeywords microbial lipase fatty acids triglycerides protein engineering biosensor food industry candida antarctica lipase b calbintroductionthe serine hydrolases are present in abundantly and known as lipase enzyme which belong to triacylglycerol ester hydrolase family ec they can catalyze the hydrolysis and synthesis of longchain triglycerides to fatty acids diacylglycerol monoacylglycerol and glycerol known as carboxylesterases [ ] besides hydrolysis activity they display interesterification esterification aminolysis and alcoholysis activity which are contributed correspondence pchandrabbaugmailcom food microbiology toxicology department of microbiology school for biomedical and pharmaceutical sciences babasaheb bhimrao ambedkar university a central university lucknow uttar pradesh indiafull list of author information is available at the end of the in wide range industries [ ] lipase synthesizes esters from glycerol and longchain fatty acids in nonaqueous medium the microbial lipases are more valuable comparison to derive from plants or animals due to their variety of catalytic activities available high yield production and simplicity of genetic manipulation absence of seasonal fluctuations regular supply more stability safer and more convenient and the growth rate of microanisms very high in economically media [ ] the bacterial isolates offer higher activities such as neutral or alkaline ph optima and the thermostability associated to yeasts bacterial strains such as pseudomonas alcaligenes p aeruginosa p fragi p fluorescens bj10 bacillus subtilis b nealsonii s2mt and some species of fungi are penicillium expansum trichoderma penicillium chrysogenum the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreat iveco mmons licen sesby40 the creative commons public domain dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cchandra a0et a0al microb cell fact page of aspergillus niger produces lipases in higher quantities [“] the increasing awareness about animal health and quality of animal produce and increasing consumption of enzymemodified cheese emc and enzymemodified dairy ingredients emdi the lipase market has been extensively increased [ ] due to the more benefits of microbial lipases over animal and plant lipases are also motivating the market growth the request for microbial sources is projected to witness significant growth in the near future due to their wide range of food processing applications [ ] the microbial lipase market is projected to dominate due to cleaning agent segment through the forecast period the growth of industrial microbial lipases in the detergents industry is the innovative key factor to replacing harsh chlorine bleach with lipase and reduced the industrial as well as sewage pollution from fresh water [ ] the microbial lipases in the form of powder is projected to dominate the microbial lipase markets due to its stability easy to handle and easier for packaging and its transportation preferred by the consumers [ ] a0these are extensively applicable in several another industries such as dairy food and beverage animal feed cleaning biofuel pharmaceuticals textile cosmetic perfumery flavour industry biocatalytic resolution esters and amino acid derivatives fine chemicals production agrochemicals biosensor and bioremediation [“] additionally altering in the dietary patterns have led to augmented the consumption of dairy products in the region increasing in trepidations about superior hygiene in consciousness of personal hygiene contagious diseases and bleaching household industrial surfaces the manufacturers who operate on a global level and the rising in implementation of lipase enzymes drive the demand for microbial lipases in the region [ ]between the and the market scope of lipase is expected to reach million by globally at a cagr of the asia“pacific was the largest market for lipase consumption in [ ] and during the forecast period the asia“pacific market is estimated to grow at the highest cagr moreover the rising prospects in the developing markets such as india china and brazil are expected to enhance the market scope of lipases over the forecast period novozymes as denmark e i du pont de nemours and company genencor us koninklijke dsm nv netherlands and chr hansen holdings as denmark are the key industries reported for the consumption of lipases at worldwide httpwwwmarke tsand marke tscom due to the specific properties such as enantioselectivity regioselectivity and broad substrate specificity properties the lipase showing more interest between all the enzymes [ ] this present review focused on discussing the sources of microanisms immobilization methods and their potential applications of lipases including commercially availablehistorical inside or outside the cells enzymes are proteins and have ability of catalyzing the various chemical and biochemical reactions they are highly specific natural catalysts to the various types of substrates and operate under insignificant conditions of environmental factor such as temperature pressure ph with high conversion rates [ ] lipase was first discovered in pancreatic juice as an enzyme by claude bernard in which hydrolysed unsolvable oil droplets and transformed them to soluble products after that the productions of lipase have been observed in the bacteria bacillus prodigiosus b pyocyaneus and b fluorescens in and in the current scenario serratia marcescens pseudomonas aeruginosa and pseudomonas fluorescens species of bacteria have been detected for the production of lipases on large scale lipolase was the first commercial recombinant lipase industrialized from the fungus thermomycesl anugiwnosus and expressed in aspergillus oryzae in traditionally lipase has been achieved from the animal pancreas and was made applicable as digestive supplements in the form of crude or in purified grade it has been extensively used as biocatalytic procedures for the synthesis of several novel chemical compounds [“]definition of a0lipaseslipases ec are known as triacylglycerol acylhydrolase which acts on carboxylic ester bonds is the part of hydrolases family [ ] they do not require any cofactor and belongs to the class of serine hydrolases triglycerides hydrolyzed into diglycerides monoglycerides fatty acids and glycerol by using the lipases naturally fig a01a the carboxylic esters bonds can be hydrolyzed by esterases in addition to lipases [ ]the hydrolysis of ester bonds at the interface catalyzes by lipases between an unsolvable phase of substrate and aqueous phase where the enzymes keep on liquefied under natural conditions fig a0 1b however pseudomonas aeruginosa candida anatarctica b and burkholderia glumae possessed a lid but did not show interfacial activation [ ] esterification transesterification interesterification acidolysis alcoholysis and aminolysis conversion reaction takes place by lipases [ ]the presence of a lid and the interfacial activation are not the suitable criteria for to categorize a true lipase carboxylesterase simply defined that catalyzes the hydrolysis and synthesis of longchain acylglycerols 0cchandra a0et a0al microb cell fact page of fig a hydrolysis of triglyceride converts into glycerol and fatty acid b representation of a molecule of lipase with its featuresproperties and a0characteristics of a0lipasesthe molecular weight of lipases is in the range of “ a0kda and reported to be monomeric protein the position of the fatty acid in the glycerol backbone chain length of the fatty acid and its degree of unsaturation are the factors and the physical properties of lipases depend on it [ ] the sensory and nutritive values of given triglyceride also affected by these features several lipases catalyze a number of useful reactions such as esterification due to their activeness in anic solvents [ ] lipases displayed ph dependent activities generally at neutral ph or up to ph and lipases are stable chromobacterium viscosum a niger and rhizophus sp produced extracellular lipases are active at acidic ph and p nitroaeducens produced alkaline lipase and active at ph under certain experimental conditions lipases have capability to reversing the reactions which leads to esterification and interesterification in the absence of water [ ] for the expression of lipase activities the cofactors are not necessary but calcium is the divalent cation stimulates the activity [ ] co ni2 hg2 and sn2 inhibited the lipase activities drastically and zn2 mg2 edta and sds inhibited slightly the halflife values determined temperature stability profiles of lipases and lower temperature shows more stability [ ] according to the regionspecificity lipases divided into two groups and revealed with acyl glycerol substrate without display of regiospecificity only fatty acids are discharged from all three positions of glycerols in the first group of lipases [“] the fatty acids regiospecifically discharged from the positions of acylglycerols in the second group of lipase triacylglycerol hydrolysed by lipases and constructed 2monoacylglycerol and free fatty acids 3diacylglycerols in a arrhizus r delemar c cylindracea and p aeruginosa the partial stereospecificity have been detected in the hydrolysis of triacylglycerols [“] these enzymes may be used to extract optically pure esters and alcohols due to these properties at low water activity using the anic media offers an exceptional prospect over variation of the solvent so varying the properties of the solvents an enzyme™s specificity may be transformed any solvent may utilize a substantial influence on the catalytic properties of an enzyme due to the possession of soft structures and delicate [ ]kinetic model of a0lipolysisat the substratewater interface lipolysis arises so the michaelis“menten model cannot be described it in a homogeneous phase which is effective only for biocatalysis in which enzyme and substrate are soluble [ ] at an interface to describe the kinetics of lipolysis simple models has been proposed and be made up of two consecutive equilibrium [ ] the alterable adsorption of enzyme to the interface e†”e happens in the first equilibrium phase a single substrate molecule s 0cchandra a0et a0al microb cell fact page of binds by the adsorbed enzyme e in the formation of es complex as a result in the second phase of equilibrium [ ] for the enzyme“substrate complex to the michaelis menten equilibrium this latter equilibrium is equivalent ending with the discharge of the products and renovation of the enzyme in the e form the subsequent catalytic steps take place once the es complex is formed [ ] the adsorbed lipase in the vicinity of substrate concentration at the interface is at the surface concentration instead of volumetric concentration conventional in the atmosphere [ ] the rejuvenated lipase remnant adsorbed to the interface and is only unrestricted after a number of catalytic cycles in this model fig a0the activity of lipase is a utility of interfacial conformation the enzyme can be denatured as well as triggered or neutralized and the interface is a suitable spot for restraining lipolysis the directly interaction of lipase inhibitor with the enzyme and obstructs the activity of lipase on the other hand via the adsorption to the interphase or to the substrate molecules few compounds can postpone the lipolytic reaction [“]lipase inhibitors are grouped into two categoriesa synthetic lipase inhibitors including phosphonates boronic acids and fats analogues andb natural compounds βlactones and several botanical foodstuffs”plant extracts and metabolites chiefly polyphenols saponins as well as peptides and particular nutritive fibers lipases are essential enzymes for lipid absorption so the absorption of fat or obesity controlled by the lipase inhibition β lactones including orlistat are the natural compounds have the ability to inhibit the lipase activity [ ] over of total dietary fats the pancreatic lipase is responsible for the hydrolysis in several countries for the treatment of obesity orlistat is the registered drug fig lipase catalyzed different reactionslipase inhibitors from a0microbial sourcesfrom microanisms several metabolic products have potent pancreatic lipase pl inhibitory activity the several bacterial fungal and other marine species continued search of effective antiobesity agent screened to find new compounds with pl inhibitory activity [ ]lipstatinthe digestive activity of pancreatic lipases controls by the lipstatin is a βlactone molecule which also controls the absorption of fat in the small intestine lipstatin was first isolated from streptomyces toxytricini is a precursor for tetrahydrolipstatin also known as orlistat xenical and alli the only fdaapproved antiobesity medication for longterm use is a very potent inhibitor of pl [ ] lipase inhibitory activity was lost on opening of βlactone ring the catalytic hydrogenation product of lipstatin is crystalline tetrahydrolipstatin and generally known as orlistat is currently on the market as an antiobesity agent [ ]panclicinsstreptomyces sp nr produced panclicins is another class of potent pl inhibitors nformylalanyloxy or nformylglycyloxy substituent are two alkyl chains are found in panclicins too contains blactone structures panclicins a and b are alanine type while panclicins c d and e are glycine type of compounds the inhibitory activity was recognized to the amino acid moiety alaninecontaining compounds being two to three folds weaker than glycinecontaining compounds valilactonevalilactone first isolated from streptomyces albolongus mg147cf2 strain from shaken culture and jar fermentation valilactone potently inhibited hog pl with an ic50 of a0ngml it also influenced inhibitory activity of esterase from hog liver with an ic50 value of a0mgml ebelactonesebelactone a and b are two ebelactones were isolated from the fermentation broth of actinomycetes strain g7gl closely related to streptomyces aburaviensis both a and b revealed pl inhibitory activity with ic50 values of against hog pl are a0 ngml and a0 ngml respectively esterastinesterastin was isolated from actinomycetes streptomyces lavendulae md4c1 strain from the fermentation 0cchandra a0et a0al microb cell fact page of broth competitively esterastin introverted the hog pancreas lipase with ic50 value of a0ngml caulerpenynecaulerpenyne extracted and purified from an extract of caulerpa taxifolia competitively introverted the activity of lipase with ic50 values of a0mm and a0mm using creamed triolein and disseminated 4methylumbelliferyl oleate as substrates individually [ ] the inhibitory activity of caulerpenyne was independent of substrate concentration suggesting direct interaction but dependent on the lipase concentration with the lipase protein slightly than interacting with the substrate oral supervision of corn oil with caulerpenyne to rats demonstrated a reduced and hindered peak plasma triacylglycerol concentration signifying its potential as a lipid absorption inhibitor [ ]vibralactonevibralactone secreted from boreostereum virens microfungi is a scarce fused βlactonetype metabolite covalently but reversibly transforms the active site serine of the enzyme via acylation by the blactone the ic50 of the vibralactone was resolute to be a0mgml [ ]percyquininpercyquinin obtained from the cultures of basidiomycetes stereum complicatum st inhibited pl with an ic50 of a0mm is another βlactone metabolite in one study on βlactone class of compounds the stereochemistry 2s 3s of the βlactone ring was found to impart specificity for the pl while 2r 3r stereochemistry was accountable for inhibition of hmgcoa synthase sources for a0microbial lipasesmicrobial lipases found universal in nature and are commercially substantial due to the low manufacturing cost superior stability and more availability than animal and plant lipases naturally or recombinant microbial lipases are generally used in diverse bioengineering applications a wide diversity of microbial resources provides by nature microbes have more adaptation abilities and inhospitable atmospheres like dead sea antarctica alkaline lakes hot springs volcanic vents and contaminated soils which provides extraordinary potential for the lipases production with specific features [ ] an enormous spinoff with esteem to the enantioselectivity hydrolysis and the formation of carboxyl esters has produced ready availability the marine microfloras have more capabilities for the formation of enzymes and proteins active compounds mostly lipase fashioned extracellularly secretion from fungi and bacteria [ ]in numerous biocatalytic procedures candida antarctica lipase b calb is the most habitually used enzyme and have a more amount of patents candida rugosa lipase crl is another scientifically significant lipase from the yeast which is a mixture of different isoforms and is commercially accessible and this grounding is known as œgenerally recognized as safe gras and used in the food industry pla1s and pla2s from fusarium oxysporum t lanuginosus a niger and trichoderma reesei between the yeast and fungal phospholipases are used in the degumming of vegetable oils and commercialized while mostly used in the food industry are pla1s pla2s and plbs extracted from a oryzae and a niger [ ] due to their high transphosphatidylation and hydrolytic activities plds isolated from actinomycete strains are commercially available and used in several industrialized procedures mostly the bacterial genera for the production of lipases and phospholipases have been reconnoitered are pseudomonas bacillus and streptomyces followed by burkholderia chromobacterium achromobacter alcaligenes and arthrobacter some lipases producing microanisms reveal new sources and applications of industrial enzymes as shown in table a0bacterial lipaseslipase has been detected initially in b prodigiosus and b fluorescens presently serratia marcescens and p fluorescens observed today™s best lipase producing bacteria subsequently [“] the glycoproteins and lipoproteins are bacterial lipases in most of the bacteria the enzyme production is affected by the certain polysaccharides have been observed [“] some bacterial lipases are thermostable and most of the bacterial lipases are reported as constitutive and nonspecific in their substrate specificity [ ] achromobacter sp alcaligenes sp arthrobacter sp pseudomonas sp staphylococcus sp and chromobacterium sp have been exploited for the manufacturing of lipases between the bacteria fungal lipasessince ²s fungal lipases have been studied due to their affluence in thermal and ph stability substrate specificity and activity in anic solvents and downstream processing these lipases have been exploited the contemporary period machinery favors the procedure of batch fermentation and low cost extraction methods so the fungal lipases have assistances over bacteria major filamentous genera of fungi included are rhizopus aspergillus penicillium mucor ashbya geotrichum beauveria humicola rhizomucor fusarium acremonium alternaria eurotrium and ophiostoma for the production of 0cchandra a0et a0al microb cell fact page of table microbial source of a0lipase and a0their industrial applicationmicrobial sourcesfungal species fusarium solani nfccl yarrowia lipolytica aspergillus oryzae rhizomucor javanicus meih rhizomucor miehei geotrichum candidum and c antarctica candida antarctica candida rugosa candida lipolytica penicillium camembertii trichoderma lanuginosus penicillium roquefortii aspergillus niger meyerozyma guilliermondii a niger gzuf36 aspergillus flavus candida antarctica rhizomucor meihei rhizomucor meihei rhizomucor miehei candida tropicalis aspergillus oryzae penicillium abeanum rhizopus nodosus candida rugosa p chrysogenum rhizomucor meihei p chrysogenum thermomyces lanuginose m miehei c parapsilosis m miehei c antarctica m miehei rhizopus arrhizusbacterial species achromobacter sp hegn virgibacillus pantothenticus hegn pseudomonas mendocina acinetobacter radioresistens bacillus sp fh5 staphylococcus pasteuri p fluorescensapplicationsreferenceshalophilic lipase for biodiesel productiondegrades very efficiently hydrophobic and unusual substrates such as nalkanes oils fats and fatty acids as lowcost carbon sourcessaturated fatty acids synthesized faster cheese ripening flavour customized cheesenonhydrogenated solid fatscocoabutter equivalentsthrough biocatalytic processes preparation of chiral intermediates which synthesized the pharmaceutical compounds related to the elimination of bad cholesterol for the treatment of the alzheimer™s disease oils and fats enriched removal of size lubricants denim finishinghuman milk fat substitutecheese ripening fatty acid productionproduction of glycerolglycolipidssynthesis of saturated triacyl glyceridesproduced a lipase containing detergent ˜lipoprime®™production of characteristic flavor of blue cheese in dairy productsfaster cheese ripening flavor customized cheese dough stability and conditioningpromising feed lipase using cheese wheypotential of the enzyme in the synthesis of functional oilsfat stain elimination synthesis of pharmaceuticals polymers biodiesels biosurfactantspitch control in paper and pulp industry polycondensation ring opening polymerization of lactones carbonates in polymeras a biocatalyst in personal care products such as skin and suntan creams bath oils etcsurfactants for baking industry dairy products noodlesoils and fats enriched cocoa butter substitutes synthesis of bioactive moleculesdegradation of crude oil hydrocarbonsuse for docosahexaenoic acid enrichment of tuna oilleather processing and dehairing and fat removalactivated sludge treatment aerobic waste treatmentfood industry waste treatmentsurfactants for baking industry dairy products noodlesfood industry waste treatmentnonhydrogenated solid fatsused as aroma and fragrance in the food beverage and pharmaceutical industrieshydroxamic acids food additivesynthesis of short chain flavour thioester in solvent free mediumproduction of flavour esterstreatment of oily wastewaterdishwashinglaundry removal of fat strainused in detergent industryusing in oil degradationenantioselective transesterification of a racemate rs4methyl1heptyn4en3ol a component of the insecticide s2852 [ ] 0cchandra a0et a0al microb cell fact page of applicationsreferencesthe production of flavour estersused in leather processinginvolved in enantioselective degradation of aopp herbicidescommonly used detergents enhance the removal of oily stains from various types of fabricswaste water treatmentanic solventtolerant lipolytic enzymeanic solventtolerant lipase for biodiesel productionbaking industry for bread makingenhanced stability in methanolbiodegradation of oil and anics determination as chemical oxygen demand cod biodegradation of food wastewater from restaurantsfood processing and oil manufactureapplication in biocatalysisalkaline lipases able to removing fatty stains when used in a washing machinesolvay enzyme products applicable for is a nonionic andor anionic detergent formulationdetergent formulations containing alkaline lipase used in laundry detergent œtopdegrading “ of the fatty material in the waste water management table continuedmicrobial sources staphylococcus warneri and s xylosus bacillus sp brevundimonas sp qpt2 micrococcus sp bacillus cereus hss marinobacter lipolyticus haloarcula sp g41 bacillus subtilis geobacillus stearothermophilus pseudomonas aeruginosa hfe733 pseudomonas sp natronococcus sp p alcaligenes m1 pseudomonas plantarii chromobacterium viscosum acinetobacter sp bacillus thermocatenulatus lactobacillus casei lactobacillus paracasei lactobacillus rhamnosus and lactobacillus plantarumused in medical industrycheese industry for improvement of flavor penicillium roquefortii staphylococcus warneri s xylosus pseudomonas cepacia pseudomonas spcheese industry for cheese ripeningproduction of flavour estersbiodiesel fuel productionformation of ˆ’15deoxyspergualin in drug industry as antitumor antibiotic and immunosuppressive agentlipases [ ] other species such as candida rugosa candida antarctica t lanuginosus rhizomucor miehei pseudomonas mucor and geotrichum colletotrichum gloesporioides produced ul of lipase are the most productive strain identified from the brazilian savanna soil by using enrichment culture techniques [ ] a niger c rugosa h lanuginosa m miehei r arrhizus r delemar r japonicus r niveus and r oryzae are the principal manufacturers of these commercial lipases [“]purification of a0lipasesto get consistency of lipase from a large number of bacteria and fungi various novel purification technologies are available generally several steps are contains for the purification of lipases contingent upon the purity estimated for food application the extracellular microbial lipases from the culture broth eliminated by the centrifugation or filtration in the fermentation process and cells are became freed [ ] the ammonium sulphate precipitation ultrafiltration or extraction with anic solvents is concentrated the cellfree culture broth the gel filtration and affinity chromatography like several combination of numerous chromatographic approaches purified about of the using precipitation steps and then ammonium sulphate and ethanol a homogenous product produces is the final step of gel filtration the novel purification machineries such as the i membrane separation procedures ii immuno purification iii hydrophobic interaction chromatography using epoxyactivated spacer arm as a ligand and polyethylene glycol restrained on sepharose iv polyvinyl alcohol polymers as column chromatography stationary phases and v aqueous two phase systems are frequently engaged after these prepurification steps [ ] the enzyme recovery and fold purification outcomes are found acceptable using of hydrophobic interaction chromatography [ ] an 0cchandra a0et a0al microb cell fact page of acid resilient lipase has been filtered from crude profitable arrangements by size exclusion on biogelp100 and ion exchange on monoq from a niger fungi using the chromatography on hydroxyapatite octylsepharose and sephacryl s200 the lipase was purified to homogeneity from r japonicus nr400 substrates for a0lipasea chiral alcohol moiety possesses by the glycerides which is the natural substrate for lipases the lipases were mostly valuable for the resolution or asymmetrization of esters bearing a chiral alcohol moiety was assumed [“]methods for a0lipase assaydue to the wide substrate specificity of lipases a number of assay protocols are engaged for lipase assay at the lipid water interface the determination of lipase activity is the analytical of free lipase using various physiochemical approaches the determination activities can be carried as with all reactions catalyzed by enzymes and observing the vanishing of the substrate or by the product release for the determining of the hydrolytic activity several methods are presented such as titrimetry spectroscopy photometry fluorimetry and infrared chromatography radio activity interfacial tensiometry turbidimetry conductimetry immunochemistry and microscopy [ ] the triacylglycerol hydrolysis reaction catalyzed by lipases generally can be written asmultifold benefits such as increase in thermal and ionic stability are applicable using immobilized lipases which upturns its proficiency when the enzyme is immobilized it is easier to control reaction parameters like flow rate and substrates convenience [ ] for immobilization include large surface area low cost reusability good chemical mechanical and thermal stability and insolubility the desirable characteristics of solid supports used according to the interface among the enzyme and support the enzyme immobilization approaches can be classified like physical and chemical procedures the interactions among the enzymes and support are by weaker bonds like hydrogen bonds van derwalls exchanges which create these interactions adjustable in the physical method for the interface among the enzyme and support are stronger by covalent bonds the procedure created irrecoverable in chemical methods [ ]physical methodsadsorptionin the physical approaches of immobilization adsorption procedure the enzymes immobilized by van der waals bonds hydrophobic interactions hydrogen bonds and ionic bonds on the surface of the support the enzyme becomes adsorbed bound and the substrates used mostly for this procedure are cation and anion exchange resins activated carbon silica gel alumina triacylglycerols †’ diacylglycerols free fatty acids †’ monoacylglycerols free fatty acids†’ glycerols free fatty acidsthe activity of lipases can be
0
objective cannabinoids are able to reduce tumor growth in xenograft models but their therapeutic potential as anti‘cancer drugs in humans is unclear yet in vitro studies of the effect of cannabinoids on cancer cells are often car‘ried 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 prolif‘eration 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 context‘dependent 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 0csainz‘cort a0et a0al bmc res notes page of main textmaterials and a0methodsmaterialscbd was supplied by schibano pharma ag waldsch¶nengrund switzerland mccoy™s 5a medium alamarblue® ab invitrogen were bought leibovitz™s l15 medium l15 and rpmi and from thermofisher scientific barcelona spain paclitaxel ²6diamidino2phenylindole dapi dimethyl sulfoxide lglutamine penicillin“streptomycin 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 ˆ’ 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 mccoy™s 5a and l15 media respectively human gastric cancer ags cells kindly provided by francesca mateo catalan institute of oncology bellvitge institute for biomedical research l™hospitalet del llobregat spain were maintained in rpmi medium all of the media was supplemented with penicillin“streptomycin 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 shapiro“wilk test to assess data normality and nonparametrical independent samples kruskal“wallis 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 0csainz‘cort 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 œmaterials 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 dapi‘positive cells dead cells showing a higher percentage in cbd‘treated 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 0csainz‘cort 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 0csainz‘cort 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 ‘bromo‘²‘deoxyuridine cbd cannabidiol dapi ²‘diamidino‘‘phenylindole 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 wald‘sch¶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 celltec‘ub 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 implica‘tions 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 fichtner‘feigl 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 mueller‘klieser w renner k kreutz m ldha‘associated lactic acid production blunts tumor immunosur‘veillance by t and nk cells cell metab “ https doi101016jcmet201608011eastman a improving anticancer drug development begins with cell culture misinformation perpetrated by the misuse of cytotoxicity assays oncotarget “ https doi1018632 oncot arget estrella v chen t lloyd m wojtkowiak j cornnell hh ibrahim‘hashim a bailey k balagurunathan y rothberg jm sloane bf johnson j gatenby ra gillies rj acidity generated by the tumor microenvironment drives local invasion can res “ https doi1011580008‘5472can‘‘fantin vr st‘pierre j leder p attenuation of ldh‘a expression uncov‘ers a link between glycolysis mitochondrial physiology and tumor maintenance cancer cell “ https doi101016jccr200604023fisher t golan h schiby g prichen s smoum r moshe i peshes‘yaloz n castiel a waldman d gallily r mechoulam r toren a in vitro and in vivo 0csainz‘cort a0et a0al bmc res notes page of efficacy of non‘psychoactive cannabidiol in neuroblastoma curr oncol “ https doi103747co232893jeong s jo mj yun hk kim dy kim br kim jl park sh na yj jeong ya kim bg ashktorab h smoot dt heo jy han j il lee s do kim h kim dh oh sc lee d‘h cannabidiol promotes apoptosis via regulation of xiapsmac in gastric cancer cell death dis https doi101038s4141 ‘‘‘jeong s yun hk jeong ya jo mj kang sh kim jl kim dy park sh kim br na yj lee si kim hd kim dh oh sc lee d‘h cannabidiol‘induced apoptosis is mediated by activation of noxa in human colorectal cancer cells cancer lett “ https doi101016jcanle t201901011 olivas‘aguirre m torres‘l³pez l valle‘reyes js hern¡ndez‘cruz a pottosin i dobrovinskaya o cannabidiol directly targets mitochondria and disturbs calcium homeostasis in acute lymphoblastic leukemia cell death dis https doi101038s4141 ‘‘‘ pirkmajer s chibalin av serum starvation caveat emptor am j physiol cell physiol 20113012c272“c279279 https doi101152ajpce ll00091 rampersad sn multiple applications of alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays sensors “ https doi103390s1209 scott ka dalgleish ag liu wm anticancer effects of phytocannabinoids used with chemotherapy in leukaemia cells can be improved by altering the sequence of their administration int j oncol “ śledziński p zeyland j słomski r nowak a the current state and future perspectives of cannabinoids in cancer biology cancer med “ https doi101002cam41312 solinas m massi p cinquina v valenti m bolognini d gariboldi m monti e rubino t parolaro d cannabidiol a non‘psychoactive cannabinoid compound inhibits proliferation and invasion in u87‘mg and t98g glioma cells through a multitarget effect plos one 2013810e76918 https doi101371journ alpone00769 sreevalsan s joseph s jutooru i chadalapaka g safe sh induction of apoptosis by cannabinoids in prostate and colon cancer cells is phos‘phatase dependent anticancer res “ tomko a o™leary l trask h achenbach jc hall sr goralski kb ellis ld dupr© dj antitumor activity of abnormal cannabidiol and its analog o‘ in taxol‘resistant preclinical models of breast cancer front phar‘macol https doi103389fphar van der valk j bieback k buta c cochrane b dirks wg fu j hickman jj hohensee c kolar r liebsch m pistollato f schulz m thieme d weber t wiest j winkler s gstraunthaler g fetal bovine serum fbs past”pre‘sent”future altex “ https doi1014573 altex wu h‘y huang c‘h lin y‘h wang c‘c jan t‘r cannabidiol induced apoptosis in human monocytes through mitochondrial permeabil‘ity transition pore‘mediated ros production free radical biol med “ https doi101016jfreer adbio med201806023publisher™s notespringer nature remains neutral with regard to jurisdictional claims in pub‘lished maps and institutional affiliations¢ fast convenient online submission ¢ thorough peer review by experienced researchers in your field¢ rapid publication on acceptance¢ support for research data including large and complex data types¢ gold open access which fosters wider collaboration and increased citations maximum visibility for your research over 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ulcerative colitis is a type of ‚ammatory bowel disease thatcan potentially lead to cancer e age of onset of colitis istypically “ years old and it can seriously threaten thequality of life of patients e immunopathogenesis andimmunosuppressive treatment of colitis are currently theresearch topics of significant interest e research goals areto diagnose and treat colitis in order to prevent exacerbationof the disease e drugs used to treat colitis in the clinicoften have adverse eï¬ects after their longterm applicationanother crucial area of colitis research is focused on thediscovery of functional foods that can prevent colitis withoutside eï¬ects natural plants including aegle marmeloslinn also have the intervention eï¬ect on colitis a recentstudy has shown that the intestinal flora is closely related tocolitis and that the intestinal flora participates in the mucosalimmune response bacteria are an important promoter of‚ammatory bowel disease e symptoms of colitis can bealleviated by regulating the intestinal flora preventing floralimbalance and increasing the number of probiotics yak yoghurt is a natural fermented food that is rich innutrients and is common in the minority areas of theqinghai tibet plateau previous research has suggested thatyak yoghurt exerts various physiological activities such asantioxidationimmunitycholesterolreductionand 0cevidencebased complementary and alternative medicineenhancement e qinghai tibet plateau has a specificclimate and unique environment for the fermentation of yakyoghurt additionally the availability of yak milk and specialtibetan fermentation utensils eg certain fermentationmicroanisms can make the flavor and quality of yakyoghurt diï¬er greatly from ordinary fermented milk aprior study on the intestinal physiological activity of lacticacid bacterial species in yak yoghurt showed that the lacticacidproducing bacteria isolated from yak yoghurt hadantioxidant and constipation preventing eï¬ects in this studythe potential eï¬ects of lactobacillusplantarum ys4 lpys4 on oxazolidoneinduced colitiswere investigated for the first time e findings provide apossible foundation for further development of lpys4especially its application in functional food or medicineratio � solution massnormal group were daub treated with ml of ethanolwith those in the remaining four groups being daub treated with ml of oxazolidonesolvent � after treatment for days the mice wereanesthetized en the blunt head of a silicone tube wasinserted into the intestinal tract from the anus of the mouse at adepth of cm e mice in normal group were administeredwith ml of ethanol solution while those in theremaining four groups were administered with ml of oxazolidone solution mass ratio � solvent � ethanoltwenty seconds later the catheters were removed and the micewere lifted up by their tails for half a minute on the last dayof treatment day all the mice were sacrificed by decapitation and their plasma samples and colon tissues were collected e length and weight of the colon were documentedexperiment animal materials materials and methodsand reagentsexperimental strain the strain was isolated from yak yoghurt in the yushu area of qinghai province china by ourteam it was named lpys4 and stored in china center fortype culture collection cctcc wuhan china nom2016750 e negative control strain lb was purchasedfrom the cctcc no ab fifty male balbc mice weeks old were purchasedfrom the experimental animal center of chongqingmedical university certificate no syxk yu “oxazolone was purchased from sigmaaldrich co llcusa il2 il10 et1 sp ss and vip serum cytokine kitswere purchased from biolegend inc usa gsh sodmpo and mda kits were purchased from nanjing jiancheng bioengineering institute nanjing china trizol reagent oligodt18 rnase dntp mlv primer bca proteinquantitative kit aps temed sdspage pvdf membrane first antibody and second antibody were purchasedfrom ermo fisher scientific inc usa instruments and equipment imark microplate readerwas purchased from biorad usa steponeplus pcrinstrument was purchased from ermo fisher scientificinc usa tanon chemiluminescence imager waspurchased from tanon science and technology co ltdchina sas v91 statistical software package was purchasedfrom sas institute inc usalb animal grouping and intervention a total of balbcmale mice were assigned to five groups model groupnormal group lactobacillus bulgaricustreatmentgroup and highdose lpys4h and lowdoses lpys4l treatment groups and there were mice in each groupe mice in lb lpys4h and lpys4l groups were fedwith — — and — cfukg ml livingbacteria physiological saline of each corresponding straindaily by oral gavage for consecutive days and normal andmodel groups were fed with ml physiological salineafter days of treatment the abdomens of all mice wereshaved with an area of cm — cm e mouse abdomens in detection of endothelin1 et1 substance p spsomatostatin ss and vasoactive intestinal peptide vipconcentrations in serum samples e whole blood samplesof mice were allowed to clot at room temperature for h andthen centrifuged at rpmmin for min after collecting the serum samples the concentrations of et1 sssp and vip were detected using commercial kits determination of interleukin2 il2 and interleukin10il10 levels in serum samples e mouse serum sampleswere prepared according to section en the serumlevels of il2 and il10 cytokines were assessed usingcommercial kits determination of glutathione gsh malondialdehydemda myeloperoxidase mpo and superoxide dismutasesod activities in colon tissues a mixture of g colontissue and ml normal saline was prepared at weightratio after homogenizing the mixture the activities of gshmda mpo and sod in colon tissues were evaluated usingcommercial kits pathological observation of he staining e lesionsite — — cm of colon was cut with a scalpel etrimmed tissue and corresponding label were placed in neutral formalin solution for h e colon tissue wasdehydrated embedded sliced dewaxed stained and thendehydrated transparent and sealed finally the pathologicalstate of colon tissue was observed under a microscopebx43 olympus tokyo japan qpcr assay total rna was isolated using rnazol andthen diluted to the final concentration of μgμl for cnasynthesis μl of the diluted rna extract was taken andcdna was prepared using a reverse transcriptase kit enthe cdna template μl was added into μl of sybrgreen pcr master mix and μl of forward primer andreverse primer each table qpcr amplification wascarried out for cycles under the reaction conditions of95oc s °c s °c s and °c s followed by 0cevidencebased complementary and alternative medicineckitinosenosnnossequencegene nametable sequences of primers used in this studyforward ²agagagatcgggttcaca3²reverse ²cacagaactgagggtaca3²forward ²tcgtccaacttctgggctctt3²reverse ²ccttctcttcctcccctctcttc3²forward ²tcagccatcacagtgttccc3²reverse ²atagcccgcatagcgtatcag3²forward ²catagcccaggtaaagcacaat3²reverse ²gaacactccagaatcgtcaactcforward ²tcagggactacgctgcgaaag3²reverse ²aagagctggcagaccgactca3²forward ²tgcaccaccaactgcttag3²reverse ²gatgcagggatgatgttc3²Î´ctdetection gene δctgapdh measured according to the following equation ˆ’δct �cycle under the reaction conditions of °c s and°c s gapdh was used as internal control for thisdetermination and the relative mrna expression was²gapdhscf western blotting mg of tissue samples was mixedwith μl of pmsf and ml of ripa and then homogenized at rmin 4oc for min protein quantificationwas conducted using the bca protein quantitative kit andthe protein samples were diluted to μgml en thediluted protein and sample buï¬er were mixed at heatedat 100oc for min and icebathed for min subsequentlyacrylamide starting buï¬er resolving buï¬er temed aps and diï¬erentiated water were mixed in specific proportions in order to prepare sdspage separation glue andconcentration glue e prestained samples and proteinladder were placed into the sample hole of the rubber sheetrespectively and then the proteincontaining sdspageglue was subjected to vertical gel electrophoresis for minafter activation with methanol for min the pvdf wereblocked with skimmed milk in — tbst solution for hen the blocked pvdf membranes were rinsed with — tbst followed by incubation with the primary antibodyat °c for h after washing with — tbst for times thesecondary antibody was incubated at °c for h lastly theprotein bands were visualized by supersignal west picoplus chemiluminescent substrate and the images werecaptured using a chemiluminescence imager multiple comparisons were conducted using onewayanova followed by tukey™s test statistical analysis e average value of three experimental results was determined and the statistical softwaresas was used to analyze whether there was a significantdiï¬erence between each group at the level of p longest in normal group ± cm while being the results eï¬ect of lpys4 on colon parameters e experimentalresults demonstrated that the length of mouse colon was thepared to those in the remaining four groups in contrast thethe ratio of colon weightlength was the highest in normalpgml and the lowest concentrations were found for sslpys4h group and these eï¬ects were better than those of eï¬ect of lpys4 on the serum contents of et1 sp ss andvip in mice it can be seen from figure that the serumml was decreased in normal mice compared to that of theremaining four groups e colitis model mice exhibited theopposite results in which the highest concentrations wereserum concentrations of ss and vip in colitis mice andmarkedly decrease those of et1 and sp more importantlythe eï¬ects were better after treatment with lpys4hshortest in the colitis model group ± cm similarlygroup ± while being the lowest in the colitis modelgroup ± figure it was found that lpys4 couldsignificantly p attenuate the decline in colon lengthand weightlength ratio induced by colitis ± cm and± for lpys4l group ± cm and ± forlb ± cm and ± concentrations of ss ± pgml and vip± pgml in normal mice were increased comlevel of et1 ± pgml and sp ± pgobserved for et1 ± pgml and sp ± ± pgml and vip ± pgml interestingly lpys4 could significantly p improve the± ± ± and ± pglb ± ± ± and ±± pgml in colitis model mice was signifigroups p following the treatment with lpys4 anhigher in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml and the serum levels of il10 werelower in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml p gsh ± μmolmg and sod ± μmolhighest while those of mpo ± mumg and mda± nmolmg were the lowest among the five± μmolmg and sod ± μmolgprotwere significantly reduced p while those of mpo± mumg and mda ± nmolmg wereremarkably increased p in the model group mice eï¬ect of lpys4 on the serum concentrations of il2 andil10 in mice it can be seen from figure that the serumcontentand il10± pgml eï¬ect of lpys4 on the activities of mpo sod gsh andmda in mouse colon as shown in figure the activities ofincrease in il2 and a decrease in il10 cytokine level wereobserved notably the serum levels of il2 were markedlyml and the eï¬ects of lpys4 were better than those ofgroups after induced by oxazolidone the levels of gshcantly reduced and raised compared to the remaining fourgprot in the colon tissue of the normal group were the pgmlofil2 0cevidencebased complementary and alternative medicinebacmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgfigure ac colon length and colon weightcolon length of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4land mda levels p such eï¬ect was stronger comparedto lbtreated mice ± μmolmg ± μmolgprot ± mumg and ± nmolmg more± μmolgprot in colon tissue and markedly decreased those of mpo ± mumg and mda± nmolmg when compared to colitis model groupand the goblet cells were increased compared with the modelgroup among them lpys4h had the most obvious eï¬ecton improving colon tissue which indicated that lpys4 couldreduce the colon injury caused by dss and the high efficiencyeï¬ect was also enhanced with the increase of concentrationlpys4 could markedly attenuate the decline in gsh andsod levels and prevented colitisinduced increase in mpo± μmolmgspecifically treatment with lpys4h significantly increasedthesodlevelsandof pathological observation as shown in figure in thenormal group the epithelial cells of colon mucosa were intactthe ‚ammatory cells were normal without ltration andthe goblet cells were arranged orderly without congestionand edema in the model group the epithelial cells of colontissue were obviously damagedthe intestinal wall wasthickened and edema ‚ammatory cell ltration andgoblet cells were reduced after treatment with lb and lpys4 congestion edema and cell ltration were alleviated eï¬ect of lpys4 on mrna and protein expression inmouse colon as shown in figures and colitis inductioncould lead to the upregulated mrna and protein expressionof inos in mouse colon but downregulated the relativeexpression of ckit enos nnos and scf treatment withlpys4h could increase the relative expression of nnosenos ckit and scf and decrease that of inos in the colontissues of colitis mice such eï¬ects were stronger than thoseof lpys4l or lb treatment group discussione ratio of colon weightlength is employed as a vitalstandard for assessing colitis in vivo e colon length ofnormalmodellblpys4llpys4hadccb0020406080100normalmodellblpys4llpys4hcolon length cmcolon lengthadccb00100200300400500normalmodellblpys4llpys4hcolon weightcolon lengthcolon weightcolon length 0cevidencebased complementary and alternative medicineabfigure a et1 b ss c sp and d vip serum levels of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4lmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgdcfigure a il2 and b il10 serum levels of each group of mice data are presented as the mean± standard deviation a“e diï¬erentletters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4l mice treatedwith a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentration oflactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgbadabbc0050100150200250normalmodellblpys4llpys4het1 level pgmlet1aedcb00100200300400500600700normalmodellblpys4llpys4hss level pgmlssvipdabbc00100200300400500600700normalmodellblpys4llpys4hsp level pgmlspaedcb00100200300400500600700normalmodellblpys4llpys4hvip level pgmlvipadccb050100150200250300normalmodellblpys4llpys4hil2 level pgmlil2eabcd02004006008001000normalmodellblpys4llpys4hil10 level pgmlil10 0cevidencebased complementary and alternative medicineabfigure a mpo b no c gsh and d mda colon tissue levels of each group of mice data are presented as the mean± standarddeviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent testlpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a highconcentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgcdcolitis mice was shorter on average than that of control miceand the ratio of colon weightlength was lower in colitis micethan in control mice howeverit appeared thattreatment with lpys4 could attenuate the decline in colonlength and weightlength ratio induced by colitisa prior study has shown that vasoconstriction of theendothelin can lead to colonic mucosa erosion and ulceration which in turn can exacerbate the progression of colitis ss however can reduce gastrointestinal ‚ammationby suppressing the production of gastric acid and othergastrointestinal fluids us a decrease in ss level can inducethe secretion of gastrointestinal fluids thus aggravatingcolitis excessive accumulation of sp can induce colitisbut after antagonizing it has been shown to relieve colitis invivo vip inhibits no production by regulating thetranscriptional activity of inos in the colon tissue thusprotecting the intestinal mucosa besides vip can also ‚uence certain immune aspects of colitis in this studylpys4 inhibited colitis by downregulating the levels of etand sp and upregulating those of ss and vipil2 is an eï¬ector cytokine produced by cells whichhas been closely associated with colitis cells regulate the‚ammatory response that causes colitis and il2 is involved in the suppression of ‚ammatory process andseverity reduction of colitis by ‚uencing cells il is another cytokine produced by treg cells with immunoinhibitory eï¬ects which plays a significant role in thedevelopment of colitis ‚ammatory bowel diseaseibd is a chronic refractory intestinal ‚ammatory diseasemainly including ulcerative colitis uc and crohn™s diseasecd although the etiology of ibd is still unclear theimbalance between and has been recognized as themain cause of mucosal damage in ibd under the action ofdiï¬erentiation factor il10 regulatory t cells derived fromintestinal mucosa associated lymphoid tissue can correct deviation by secreting high level of il10 andmedium level of tgfβ so as to achieve the purpose oftreating ‚ammatory bowel disease to a certain extent it was observed that lpys4 could increase the level of il2thus regulating immunity and alleviating colitis and lpeabcd00100200300400normalmodellblpys4llpys4hmpo level mumgmpoadccb00100200300400normalmodellblpys4llpys4hsod level μmolgprotsodaedcb0020406080100120normalmodellblpys4llpys4hsgh level μmolmggshdabbc000510152025normalmodellblpys4llpys4hmda level nmolmgmda 0cevidencebased complementary and alternative medicinefigure observation of colon pathology in mice by he stainingys4 could inhibit the colitis and reduce the secretion of ile aggregation of neutrophils began to decline afterintestinal ‚ammation and a large amount of them enteredinto the circulation and migrated to tissues at the sametime free radicals such as reactive oxygen species and reactive nitrogen species gathered in large quantities which inturn led to damage and toxicity of colon tissue and furtheraggravated colitis after colitis the levels of sod andgsh were reduced in colon tissue while those of mda andmpo were elevated our findings also indicated that colitiscould lead to decrease in gsh and sod levels as well asincrease in mda and mpo levels [ ] in addition lpys4 attenuated colitis by inhibiting the transcriptional responses to oxidative stressnos can be divided into nnos enos and inos it hasbeen reported that no produced by enos plays a key rolein response to colonic tissue damage and excessive nogenerated by inos promotes colitis damage enoscontrols the production of no to keep the colonic tissue ina normal state which plays an important role in reducingcolitisinduced colonic injury e presence of excessiveno aggravates colon damage nnos can also controlthe level of no in tissue and protect the tissue from beingdamaged by excessive no in this study lpys4upregulated the expression of enos and nnos in the colonand downregulated that ofthereby attenuatingcolitisinosulcerative colitis not only shows hematochezia anddiarrhea but also presents colonic motility disorders it hasbeen proved that interstitial cells of cajal icc are related tocolonic motility dysfunction and directly participate in theprogression of colitis as a specific marker of gastrointestinalicc ckit is a transmembrane glycoprotein specificallyexpressed on icc cell membrane ckit gene located onchromosome 4q1213 belongs to protooncogene and itsproduct is tyrosine kinase type iii as a receptor of scfckit can regulate the proliferation and diï¬erentiation ofhematopoietic stem cells through a series of signalingpathways [ ] scf exerts a direct eï¬ect on ‚ammatorybowel disease by regulating the function and number oficc scf can interact with its ligand ckit and the dysregulation of scfkit signaling pathway may decrease theproliferation and diï¬erentiation of icc thus exacerbatingcolitis [ ] e abnormal expression of scfckitsignaling pathway can also change the physiologicalfunction of icc weaken gastrointestinal motility andaggravate intestinal dysfunction in the present studylpys4 could inhibit colitis by regulating the expressionlevels of scf and ckiticc autophagy regulation has become a new target forthe treatment of intestinal motility disorder in ulcerativecolitis because the drug treatment of colitis often hasside eï¬ects and once stopped it is easy to relapse ereforethe use of natural harmless substances through the regulation of icc prevention and treatment of colitis canmaintain longterm health a study has shown thataurantii fructusimmaturus and atractylodis macrocephalae rhizoma can inhibit the autophagy of cajal stromalcells induced by glutamate which may play an inhibitoryrole in colitis meanwhile there is also a study showingthat lactic acid bacteria can regulate icc thus regulatingnormallblpys4llpys4hmodel 0cevidencebased complementary and alternative medicineacbdthe mean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestlyfigure eï¬ect of lpys4 on a nnos b enos c inos d ckit and e scf mrna expression in mouse colon data are presented asesignificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgintestinal function and protecting the intestine isstudy also confirmed that the lpys4 can regulate the scfckitsignaling pathway and the scfckit signalingpathway is an important icc regulatory pathway erefore the lpys4 may also inhibit the colon by regulating iccaedcb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupnnosadccb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupscfaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineacbdmean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly sigfigure af eï¬ect of lpys4 on nnos enos inos ckit and scf protein expression in mouse colon data are presented as thenificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgef conclusionin this study oxazolone was used to induce colitis in balbcmice and the inhibitory eï¬ects of lpys4 on colitis weredetected rough the observation of colon tissues and serum samples of mice it was found that lpys4 treatmentcould alleviate colitis by restoring the levels of ‚ammatoryindicators closer to those measured in healthy control miceis work suggests that lpys4 is superiorquality lactic acidbacteria with a potential role in colitis treatment and provides a foundation for further research and developmentabbreviationslpys4 lactobacillus plantarum ys4lbqpcr quantitative polymerase chain reactionhelactobacillus bulgaricushematoxylineosinnormalmodellblpys4llpys4hckitenosinosnnosscfβactinadcbcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupnnosaedcb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosadccb0010203040506070normalmodellblpys4llpys4hrelative to multiple of model groupscfadccb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineendothelin1substance psomatostatinvasoactive intestinal peptideinterleukin2interleukin10et1spssvipil2il10mpo myeloperoxidasesodgshmda malondialdehydeenosnnos neuronal nitric oxide synthaseinducible nitric oxide synthaseinosscfstem cell factorsuperoxide dismutaseglutathioneendothelial nitric oxide synthasedata availabilityno data were used to support this studyconflicts of intereste authors of this manuscript state that they do not haveconflicts of interest to declareauthors™ contributionsruokun yi and fang tan contributed equally to this workruokun yi and fang tan performed the majority of theexperiments and wrote the manuscript huayi suo wenfengli xianrong zhou and jianfei mu contributed to the dataanalysis xin zhao and peng xie designed and supervised thestudy and read the final manuscriptacknowledgmentsis research was funded by national key rd program ofchina 2018yfd0502300 children™s research institute ofnational center for schooling development programmeand chongqing university of education csdp19fs01103theand technology research program ofchongqing municipal education commission kjzdk201901601 and research project of chongqing universityof education ky2015tbzc chinasciencereferences r k yi f tan w liao et al œisolation and identification oflactobacillus plantarum hfy05 from natural fermented yakyogurt and its eï¬ect on alcoholic liver injury in mice microanisms vol no p j liu f tan x h liu et al œexploring the antioxidanteï¬ects and periodic regulation of cancer cells by polyphenolsproduced by the fermentation of grape skin by lactobacillusplantarum kfy02 biomolecules vol no p t s olson b k reuter k g e scott et al œe primarydefectfrom a nonhematopoietic source journal of experimental medicinevol no pp “ ileitis originatesin experimental b manandhar k r paudel b sharma and r karkiœphytochemical profile and pharmacological activity of aeglemarmelos linn journal of integrative medicine vol no pp “ x zhou h liu j zhang et al œprotective eï¬ect of lactobacillus fermentum cqpc04 on dextran sulfate sodiuminduced colitis in mice is associated with modulation of thenuclear factorκb signaling pathway journal of dairy science vol no pp “ m c arrieta k madsen j doyle and j meddings œreducing small intestinal permeability attenuates colitis in theil10 genedeficient mouse gut vol no pp “ h suo x zhao y qian et al œerapeutic eï¬ect of activatedcarboninduced constipation mice with lactobacillus fermentum suo on treatment international journal of molecular sciences vol no pp “ y qian h suo m du et al œpreventive eï¬ect of lactobacillus fermentum lee on activated carboninduced constipation in mice experimental and eerapeutic medicinevol no pp “ x zhao y qian h suo et al œpreventive eï¬ect of lactobacillus fermentum zhao on activated carboninduced constipation in mice journal of nutritional science andvitaminology vol no pp “ x long y pan and x zhao œprophylactic eï¬ect ofkudingcha polyphenols on oxazolone induced colitis throughits antioxidant capacities food science and human wellnessvol no pp “ k zhu g huang j xie x zhou j mu and x zhaoœpreventive eï¬ect of flavonoids from wushan shencha malus doumeri leaves on ccl induced liver injury foodscience nutrition vol no pp “ x zhao j zhang s yi et al œlactobacillus plantarumcqpc02 prevents obesity in mice through the pparα signaling pathway biomolecules vol p w strober i j fuss and r s blumberg œe immunologyofmucosalmodels ofinflammation annual review of immunology vol no pp “ jl song y qian gj li and x zhao œanti‚ammatoryeï¬ects of kudingcha methanol extract ilex kudingcha cjtseng in dextran sulfate sodiuminduced ulcerative colitismolecular medicine reports vol no pp “ y qian x zhao jl song et al œinhibitory eï¬ects of resistant starch rs3 as a carrier for stachyose on dextransulfate sodiuminduced ulcerative colitis in c57bl6 miceexperimental and eerapeutic medicine vol no pp “ x y chen x zhao h w wang et al œprevent eï¬ects oflactobacillus fermentum hy01 on dextran sulfate sodiuminduced colitis in mice nutrients vol no p y qian a l lei x j liu et al œinhibitory eï¬ects oflactobacillus plantarum ys2 in dextran sulfate sodiuminduced c57bl6j mice colitis science and technology of foodindustry vol no pp “ s chen x zhao p sun j qian y shi and r wangœpreventive eï¬ect of gardenia jasminoides on hclethanolinduced gastric injury in mice journal of pharmacologicalsciences vol no pp “ l xie z h xing r x jiang et al œeï¬ect of sanpi decotionon the expression of il2 and il10 of mice with ulcerativecolitis chinese journal of experimental traditional medicalformulae vol no pp “ l hang s kumar a m blum j f urban m c fantini andj v weinstock œheligmosomoides polygyrus bakeri infection decreases smad7 expression in intestinal cd4 t cells 0cevidencebased complementary and alternative medicinewhich allows tgfβ to induce il10producing regulatorytcells that block colitis ee journal of immunology vol no pp “ j zhang q li y wei et al œprocess design of the antioxidant shuidouchi and its eï¬ect on preventing dextransulfate sodium dssinduced colitis in mice via antioxidantactivity applied sciences vol no p c fiocchi œ‚ammatory bowel disease new insights intomechanisms of ‚ammation and increasingly customizedapproaches to diagnosis and therapy current opinion ingastroenterology vol no pp j zhang r yi y qian p sun x zhao and z yangœlactobacillus plantarum cqpc06 activity prevents dextransulfate sodiuminduced colitis by regulating the il8 pathway journal of food science vol no pp “ j zhang x chen jl song et al œpreventive eï¬ects oflactobacillus plantarum cqpc07 on colitis induced bydextran sodium sulfate in mice food science and technologyresearch vol no pp “ lh chen jl song y qian x zhao hy suo and j liœincreased preventive eï¬ect on colon carcinogenesis by use ofresistant starch rs3 as the carrier for polysaccharide ofinternationallarimichthysjournal of molecular sciences vol no pp “ x feng j zhang y qian et al œpreventative eï¬ects oflactobacillus plantarum ys3 on oxazoloneinduced balbccolitis in mice applied biological chemistry vol no pp “ swimming bladdercrocea j m wang m li r tang et al œeï¬ect of yiqi jianpitongbian recipe on icc and scfckit signaling pathway incolon tissue of slow transport type constipation rats chinesearchives of traditional chinese medicine vol no pp “ j feng j gao s zhou et al œrole of stem cell factor in 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a
0
] we have filtered only research s published in english language and selected the following keywords air pollution and covid19 or sarscov2 particulate matter or pm and covid19 or sarscov2 nitrogen dioxide or no2 and covid19 or sarscov2 we choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported covid19 cases andor deaths and air pollution data related to pm25 pm10 and no2 thus excluding any letter opinion commentary review or nonrelevant s we obtained a total of eligible published research s in their final version and paper in its preprint version for some of them we chose to include only principal findings that clearly fit the aim this review particulate matter and covid19 atmospheric particulate matter pm is originated by a wide range of anthropogenic and natural sources kim it consists of a heterogeneous mixture of solid and liquid ps suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals who it has been associated with increased respiratory morbidity and mortality liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis li rhee in vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections becker and soukup recently the research group of setti gave first preliminary evidence that sarscov2 rna can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of pm it could represent a potential early indicator of covid19 although it does not give information regarding covid19 progression or severity several observations report a significant association between ambient concentrations of pm25 adhikari and yin bashir fattorini and regoli frontera jiang li vasquezapestegui wu yao zhu zoran 2020a and pm10 bashir coccia 2020b fattorini and regoli jiang li yao zhu zoran 2020a with covid19 pandemic across the most affected countries china italy and usa see table first evidences on the temporal association between air pollution and covid19 were reported in china where the outbreak was first identified zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in china the authors included over of dailyconfirmed new cases in the whole of china between january 23rd and february 29th they applied a generalized additive model gam to examine the effects of meteorological factors and air pollution on covid19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders they observed that the effect of pm25 on daily confirmed cases was greater than pm10 in particular they found that a 10μgm3 increase lag0“ in pm25 and pm10 was associated with a ci to and ci to increase in the daily counts of covid19 confirmed cases respectively jiang focused their attention on three most affected cities of china wuhan xiaogan and huanggang collecting data of daily cases and ambient air pollutant from jan 25th to feb 29th the authors by applying a multivariate poisson regression revealed a significant temporal association between pm25 increased and covid19 incidence in all the considered cities especially in huanggang wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ conversely an increase in pm10 concentrations was associated with a decrease of covid19 incidence these results were partially confirmed by findings of li who conducted a simple linear regression to compare covid19 incidence with pm concentrations in wuhan and xiaogan from jan 26th to feb 29th in they found that an increase in pm25 was correlated with an increase of covid19 incidence in both cities wuhan r2 p xiaogan r2 p while for pm10 only in xiaogan r2 p the spatial distribution of particulate matter and case fatality rate cfr of covid19 was studied by yao in cities of china including wuhan collecting data up to march 22nd first they found a significantly positive global spatial autocorrelation of covid19 cfr global moran™s index i p highlighting a high cfr clustering located in hubei province with a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product gdp per capita hospital beds per capita local indicators of spatial association lisa map values city size and population or proportion of people older than years it was found that for every μgm3 increase in pm25 and pm10 the cfr increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of pm25 and pm10 in “ respectively some studies describe the association between air pollution and covid19 across italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other european countries the 28th of july italy recorded more than total confirmed cases and deaths who most of which were distributed in the regions of northern italy especially the lombardy it is recognized as one the most air polluted areas of europe eea where the frequent pm10 annual exceedances of the who threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year baccini bontempi 2020bfocused the attention on two of the most affected regions of northern italy lombardy and piedmont the authors based on pm10 daily exceedances and covid19 confirmed cases on march 12th thus before the italian sanitary crisis observed that pm10 concentration was exceeded only few times among the lombard cities that at the beginning of the epidemic were most affected on the contrary among some piedmont cities suffering of severe pm10 pollution events covid19 incidence was lower based on their results the authors concluded that covid19 diffusion by airborne pm10 is hard to demonstrate nevertheless several research revealed how pm in particular pm25 could had a role in accelerate and vast diffusion of covid19 in northern italy for example coccia 2020b by analyzed data on italian province capitals and data of infected individuals up to april 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for pm10 in previous years and covid19 diffusion in particular cities with more than days of pm10 exceedances showed a very high average number of infected individual about infected individuals on 7th april whereas cities having less than days of pm10 exceedances showed a lower average number of infected about infected individuals frontera gave also evidences on the role of pm25 as a contributing factor of covid19 outbreak in northern italy where environmentalresearch19120201101293 0cc copat table summary table reporting reviewed results on the association between covid19 casesdeaths and air pollution pm25 pm10 and no2 references zhu data analysis generalized additive model gam aim temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 temporal association between daily confirmed cases and air pollution pm25 pm10 and no2 spatial association between fatality rate and air pollution pm25 and pm10 spatial association between deaths counts and air pollution no2 temporal association between total cases daily confirmed cases and total deaths and air pollution pm25 and pm10 temporal association between total cases daily confirmed cases and total deaths and air pollution no2 spatial description of pm10 exceedances versus covid19 cases multivariate poisson regression simple linear regression multiple linear regression descriptive analysis percentage of deaths in three no2 μmol m2concentration range “ “ “ pearson coefficient correlation pearson coefficient correlation descriptive analysis number of days of pm10 exceeding μgm3 and covid19 incidence area of study cities of china period from jan 23rd to feb 29th jiang li yao ogen zoran 2020a zoran 2020b bontempi 2020b from jan 25th to feb 29th from jan 26th to feb 29th in data up to march 22nd data up to the end of feb from jan 1st to apr 30th from jan 1st to apr 30th from feb 10th to march 12th wuhan xiaogan and huanggang china wuhan and xiaogan cities of china administrative regions in italy spain france and germany milan italy milan italy provinces of lombardy italy provinces of piedmont italy coccia 2020b data up to april 7th italian provinces fattorini and regoli data up to april 27th italian provinces pm25 a 10μgm3 pm25 increase lag0“ was associated with a increase of daily confirmed new cases pm10 a 10μgm3 pm10 increase lag0“ was associated with a increase of daily confirmed new cases wuhan rr ci1032“ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p wuhan rr ci “ xiaogan rr ci “ huanggang rr ci “ wuhan r2 p xiaogan r2 p χ2 p a μgm3 increase in pm25 was associated with a “ increase in fatality rate χ2 p a μgm3 increase in pm10 was associated with a “ increase in fatality rate no2 a 10μgm3 no2 increase lag0“ was associated with a increase in daily confirmed new cases wuhan rr ci “ xiaogan rr ci “ huanggang no association found wuhan r2 p xiaogan r2 p of fatality cases are associated with no2 μmolm2 r cid0 r r cid0 r cid0 r r cid0 r cid0 r cid0 r cid0 lombardy pm10 exceeding between and covid19 incidence between and piedmont pm10 exceeding between and covid19 incidence between and covid19 in north italy has a high association with air pollution of cities measured with days exceeding the limits set for pm10 r2 p r2 p continued on next page hierarchical multiple regression model pearson regression coefficient analysis r2 p spatial association between confirmed cases and air pollution pm10 spatial association between total confirmed cases and air pollution pm25 pm10 and no2 environmentalresearch19120201101294 0cc copat table continued references frontera frontera wu adhikari and yin bashir bashir vasquezapestegui vasquezapestegui vasquezapestegui period data up to 31st march data up to 31st march data up to april 04th from march 1st to apr 20th from march 4th to april 24th from march 4th to april 24th data up to june 12th data up to june 12th data up to june 12th area of study italian regions italian regions counties in the usa queens county new york usa california california districts of lima perù districts of lima perù districts of lima perù aim spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 prediction of risk of covid19 deaths in the long term average exposure to fine particulate matter pm25 temporal association between daily confirmed cases and total deaths and air pollution pm25 association between confirmed cases and air pollution pm25 pm10 and no2 association between deaths and air pollution pm25 pm10 and no2 spatial association between total confirmed cases and air pollution pm25 spatial association between deaths and air pollution pm25 spatial association between case fatality rate and air pollution pm25 data analysis pearson regression coefficient analysis pm25 r2 p pm10 pearson regression coefficient analysis r2 p longterm exposure increase of μgm3 in pm25 is associated with a increase in the covid19 death rate estimate on cases values cid0 ci “ estimate on deaths value cid0 ci “ kendall r cid0 spearman r cid0 zeroinflated negative binomia models negative binomial regression model spearman and kendall correlation tests spearman and kendall correlation tests no2 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 kendall r cid0 spearman r cid0 multivariate regression model crude coefficient p multivariate regression model crude coefficient p multivariate regression model crude coefficient cid0 p mortality was found significantly higher than less polluted italian regions by collecting data up to march 31st for all italian regions and performing a pearson correlation analysis they found a strong positive association both with the total number of confirmed cases r and deaths r other than with hospitalized cases r the italian situation was further highlighted by the study of fattorini and regoli in italian provinces they explored the spatial association between air pollution and covid19 cases with data up to april 27th by applying the pearson regression coefficient analysis they revealed a positive association both with pm25 and pm10 r2 p and r2 p respectively a focus on the most affected city of italy milan was conducted by zoran 2020a this city is located in the po valley basin known hotspot for atmospheric pollution at the continental scale eea the authors performed a temporal association between covid19 total cases daily new positive cases and total deaths and particulate matter from jan 1st and apr 30th by applying a person correlation in accordance with other studied they found a positive association between daily confirmed cases and pm25 r and pm10 r although they did not consider any delay time from infection to covid19 onset nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships to date the usa have more than million confirmed cases and thousand deaths who here ambient concentrations of pm and o3 were found responsible to cause between and premature deaths fann the association between air pollutants and covid19 cases and deaths was studied by bashir in the state of california from march 4th to april 24th corresponding to the beginning of the covid19 outbreak in usa based on their significant correlation found the authors state that a limited human exposure to these pollutants will contribute to defeating covid19 this conclusion seems unclear because they found a negative correlation with pm25 and pm10 environmentalresearch19120201101295 0cc copat by applying both the kendall rank correlation and spearman™s one and it is not clear if they normalized covid19 cases by population size and if they performed a day by day association or a spatial association across the country a focus on the queen county new york usa was provided by adhikari and yin they retrieved data of pm daily concentrations from two ground monitoring stations and collected data of confirmed covid19 cases and numbers of related deaths from usafacts in the period from march to april the authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of pm25 on disease outcomes over the past days they found a significant negative association among pm25 and new daily confirmed covid19 cases cid0 ci “ and deaths cid0 ci “ low pm concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as italy where pm25 monthly concentrations reached values higher than μgm3 fattorini and regoli frontera or in china where pm25 monthly concentrations reached values higher than μgm3 zhu jiang as said by the authors other gaseous pollutants such as no2 and so2 could have influenced transmission and pathogenesis of covid19 in the united states wu investigated whether longterm average exposure to fine particulate matter pm25 increases the risk of covid19 deaths by considering approximately counties in the united states of the population with an exposure prediction model the authors calculated the county level longterm exposure to pm25 averaged for to and collected covid19 deaths counts up to april 04th they conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors they found that a small longterm exposure increase of only μgm3 in pm25 is associated with a increase in the covid19 death rate confidence interval ci vasquezapestegui recently reported first evidences on the spatial relationship between particulate matter and covid19 outbreak from latin america the authors described the situation occurred in districts of lima located in the second most affected country of latin america peru in particular by applying a multivariate regression model they evaluated the association between the population exposure to pm25 concentrations in the previous years “ and cases deaths and casefatality rates of covid19 with data up to june 12th a significant association has been found both with cases and deaths crude coefficient with p and with p respectively but not with case fatality rate all these studies highlight the role of pm in triggers of the covid19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems nitrogen dioxide no2 and covid19 induced lung damage hence viral infection becomes more common after exposure to no2 zhu furthermore no2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children to increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation bahrami asl kowalska increase of chronic obstructive pulmonary disease copd ghanbari ghozikali pfeffer and increase of pulmonary heart disease related mortality chen a recent study explored the possible role of no2 in interference in angiotensin converting enzyme ace2 the expression of ace2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of covid19 alifano first observations report an association between ambient concentrations of no2 and covid19 pandemic across europe china and usa bashir fattorini and regoli jiang li et al ogen zhu et al zoran et al 2020b conversely to the other papers findings of zoran 2020b and bashir provides different findings reporting no association or a negative one between no2 and daily deaths counts in china zhu by applying the same method explained for pm observed that a 10μgm3 increase lag0“ in no2 is associated with a ci “ increase in the daily counts of covid19 confirmed cases in cities of china these findings are confirmed by jiang and li et a who applied the same method described for pm jiang revealed a significant positive association between no2 and covid19 both in wuhan and xiaogan wuhan rr ci1053“ xiaogan rr ci “ but did not found any significant association in huanggang li found a significant linear correlation both in wuhan r2 p and xiaogan r2 p ogen presented evidences on the relationship between exposure to no2 including the months of january and february shortly before the covid19 spread in europe and novel coronavirus fatality in the most affected european countries concluding that longterm exposure to no2 may be a potential contributor to mortality caused by sarscov2 he collected data concerning the number of fatality cases from administrative regions in italy spain france and germany and correlated mortality with tropospheric no2 concentrations measured by the sentinel5 precursor spaceborne satellite the major tropospheric no2 hotspot identified was located in the northern italy in all european regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards results showed that out of the fatality cases by march were in five regions located in north italy and central spain furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum no2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 the methodology used by ogen cannot support a longterm exposure investigation surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust nevertheless the study provide new insights for future investigation the italian situation was further studied by fattorini and regoli who collected data of covid19 incidence up to april 27th from italian provinces they revealed a strong spatial correlation with no2 mean levels concentrations “ pearson coefficient r2 p confirming the northern italy being a hotspot of no2 in addition to urbanized cities of central and southern italy such as rome and naples a focus on the temporal association between ground levels of no2 and covd19 cases total cases daily new positive cases and total deaths was performed by zoran 2020b for the city of milan italy in the period pre and postlockdown measures the authors nitrogen dioxide is a nastysmelling gas formed by reaction in the atmosphere of nitrogen oxides nox with other chemicals nox is naturally produced in atmosphere by lightning kang et al volcanoes oceans and biological decay thurston the major outdoor anthropogenic sources of nox are primarily emissions from transportation and fuel combustion in particular in urban areas they comes from vehicle exhaust gases and domestic heating grange maawa the nitrogen dioxide has mainly effect on the respiratory system because an increase of the outdoor concentration of no2 may significantly increase the risk of respiratory tract infection this phenomenon is particularly evident in children as they are more susceptible to no2 environmentalresearch19120201101296 0cacknowledgments c copat found no2 negative correlated with all the considered epidemiological data but the methodology used has some limitations as the delay time from infection to the covid19 onset or covid19 death was not considered as well the significant reduction of air pollution due to lockdown measures since midmarch in usa the association was also studied by bashir for the state of california as discussed above for pm the authors found a negative correlation also between no2 levels and covid19 cases and mortality nevertheless they stated that this pollutant contributes to the spread of the disease based on these scientific evidences in addition to confirming that exposure to no2 is harmful to human health and increases the risk of incurring respiratory diseases it can be stated that exposure to no2 may be one of the most important trigger for the spread and fatality caused by the covid19 disease declare references adhikari a yin j shortterm effects of ambient ozone pm25 and the authors declare no conflict of interest we have no funding to bontempi e 2020b first data analysis about possible covid19 virus airborne alifano m alifano p fez p iannelli a reninangiotensin system at the meteorological factors on covid19 confirmed cases and deaths in queens new york int j environ res publ health httpsdoi103390 ijerph17114047 heart of covid19 pandemic biochimie httpsdoi101016j biochi202004008 baccini m biggeri a grillo p consonni d bertazzi pa health impact assessment of fine p pollution at the regional level am j epidemiol “ httpsdoi101093ajekwr256 bahrami asl f leili m vaziri y salahshour arian s cristaldi a oliveri conti g ferrante m health impacts quantification of ambient air pollutants using airq model approach in hamadan iran environ res “ httpsdoi 101016jenvres201710050 bashir mf ma bj bilal komal b bashir ma farooq th iqbal n bashir m correlation between environmental pollution indicators and covid19 pandemic a brief study in californian context environ res https doi101016jenvres2020109652 becker s soukup jm exposure to urban air particulates alters the macrophage mediated inflammatory response to respiratory viral infection j toxicol environ health “ httpsdoi101080009841099157539 bontempi e 2020a commercial exchanges instead of air pollution as possible origin of covid19 initial diffusion phase in italy more efforts are necessary to address interdisciplinary research environ res httpsdoi101016j envres2020109775 diffusion due to air particulate matter pm the case of lombardy italy environ res httpsdoi101016jenvres2020109639 bontempi e vergalli s squazzoni f understanding covid19 diffusion requires an interdisciplinary multidimensional approach environ res httpsdoi101016jenvres2020109814 bremner sa anderson hr atkinson rw mcmichael aj strachan dp bland j m bower js shortterm associations between outdoor air pollution and mortality in london occup environ med “ httpsdoi 101136oem564237 cai qc lu j xu qf guo q xu dz sun qw yang h zhao gm jiang qw influence of meteorological factors and air pollution on the outbreak of severe acute respiratory syndrome publ health “ https doi101016jpuhe200609023 carugno m dentali f mathieu g fontanella a mariani j bordini l milani g p consonni d bonzini m bollati v pesatori ac pm10 exposure is associated with increased hospitalizations for respiratory syncytial virus bronchiolitis among infants in lombardy italy environ res “ https doi101016jenvres201806016 chen h chen y lian z wen l sun b wang p li x liu q yu x lu y qi y zhao s zhang l yi x liu f pan g 2020a correlation between the migration scale index and the number of new confirmed coronavirus disease cases in china epidemiol infect e99 httpsdoi101017 s0950268820001119 chen j zeng j shi c liu r lu r mao s zhang l associations between shortterm exposure to gaseous pollutants and pulmonary heart diseaserelated mortality among elderly people in chengdu china environ health httpsdoi 101186s1294001905008 chen s prettner k kuhn m geldsetzer p wang c b¨arnighausen t bloom de 2020b covid19 and climate global evidence from countries medrxiv prepr serv health sci httpsdoi1011012020060420121863 coccia m 2020a how high wind speed can reduce negative effects of confirmed cases and total deaths of covid19 infection in society ssrn scholarly paper no id social science research network rochester ny httpsdoi 102139ssrn3603380 coccia m 2020b factors determining the diffusion of covid19 and suggested strategy to prevent future accelerated viral infectivity similar to covid sci total environ httpsdoi101016jscitotenv2020138474 balakrishnan k brunekreef b dandona l dandona r feigin v freedman g hubbell b jobling a kan h knibbs l liu y martin r morawska l pope ca shin h straif k shaddick g thomas m van dingenen r van donkelaar a vos t murray cjl forouzanfar mh estimates and year trends of the global burden of disease attributable to ambient air pollution an analysis of data from the global burden of diseases study lancet lond engl httpsdoi101016s0140673617305056 “ conticini e frediani b caro d can atmospheric pollution be considered a co factor in extremely high level of sarscov2 lethality in northern italy environ pollut barking essex httpsdoi101016jenvpol2020114465 croft dp zhang w lin s thurston sw hopke pk van wijngaarden e squizzato s masiol m utell mj rich dq associations between source cohen aj brauer m burnett r anderson hr frostad j estep k conclusion the scientific evidences collected in the literature highlight the important contribution of chronic exposure to air pollution on the covid19 spread and lethality although the potential effect of airborne virus exposure it has not been still demonstrated in particular it seems that pm25 and no2 are more closely correlated to covid19 than pm10 the lower correlation of pm10 with covid19 incidence and mortality can be due to the impossibility of particulate matter greater than μm to reach type ii alveolar cells where is located the cell entry receptor ace2 for sarscov2 nevertheless differences between countries such as the implementation of different lockdown restrictions stage of infection topographic sociodemographic and socioeconomic characteristics level of air pollution and meteorological factors may have contributed to obtain some contrasting finding although most of the revised studies support the relationship between air pollution and covid19 the manifold limitations of this review are the small number of papers collected and the great diversity of methodologies used sometimes lacking in some parts which makes the results difficult to compare the authors who first investigated this association although with great effort and rapidity of analysis dictated by a global emergency sometimes do not include all confounding factors whenever possible such as control policy urbanization rate availability of medical resources population size weather lifestyles sociodemographic and socioeconomic variables in addition to date incidence data are underestimated in all countries and to a lesser extent mortality data for this reason the cases included in the considered studies cannot be considered conclusive more studies are needed to better clarify the role of air pollution during the covid19 pandemic particularly studies that consider the multiplepollutants to strengthen scientific evidences and support firm conclusions useful to implement pandemic application plans to adequately prevent new health emergencies for a long time we have known that reducing outdoor and indoor air pollution in cities or countries can have a significant effect on health almost immediately and the benefits can far outweigh the costs surely the health emergency that the world is experiencing right now highlights how environmental research is a fundamental reference point to improve the knowledge concerning diseases of infectious origin and how all the intellectual and economic resources are to be spent to accelerate actions aimed to implement environmental policies act to reduce air pollution and develop new urban planning interventions influences or multidisciplinary studies declaration of competing interest the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper environmentalresearch19120201101297 0cc cop
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Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly citedAccumulating evidence has supported an increased risk of osteoporotic fracture in postmenopausal women and elderly mendiagnosed with diabetes mellitus However it is not uncommon for young and middleaged male patients diagnosed with type diabetes mellitus T2DM to suï¬er from oste ia or osteoporosis Few studies focused on this population group are availableThe aim of this study is to evaluate bone metabolic status and investigate the ‚uence of T2DM on bone metabolism in yearold men Anthropometric assessment and blood samples were obtained from patients with T2DM and nondiabeticvolunteers Serum parathyroid hormone PTH and bone turnover markers BTMs including serum procollagen type I Nterminal peptide PINP osteocalcin OC and crosslinked Ctelopeptide of type I collagen CTX were analysed Nosignificant diï¬erences were observed based on age body mass index systolic blood pressure serum calcium phosphoruscreatinine total protein and albumin levels when comparing T2DM and control groups Fasting blood glucose HbA1ctriglyceride TG total cholesterol and lowdensity lipoprotein cholesterol were significantly increased while highdensitylipoprotein cholesterol was significantly decreased in the T2DM group Compared with controls diabetic patients showed lowerserum PINP OC and PTH levels whereas serum CTX levels were similar between the two groups Moreover HbA1c levelswere positively correlated with PINP and inversely associated with PTH levels TG levels were negatively correlated with OC orCTX levels Furthermore multiple linear regression revealed a positive correlation between HbA1c and PINP levels Theseresults also revealed a negative association between HbA1c and PTH and between TG and OC levels even after adjusting forexpected confounder factors Collectively these findings indicated that young and middleaged male patients with T2DMshowed a lower turnover state resulting from bone formation inhibition Glucose and lipid metabolic disorders may aï¬ect boneformation through diï¬erent pathways IntroductionType diabetes mellitus T2DM is a common chronic metabolic disease caused by insufficient insulin secretion andoractivity leading to chronic hyperglycaemia Its high prevalence has resulted in a heavy burden on social financialand health care systems [] There is a large amount of evidence revealing an increased risk of fracture in diabeticpatients particularly hip fracture [ ] Recent metaanalyses indicated that hip fracture risk increases times in patients with T2DM [ ] In addition studies haddemonstrated that severe vertebral fracture in patients withT2DM was associated with increased allcause mortality [] Osteoporotic fracture has been increasingly recognizedas another complication of T2DM High morbidity and mortality make the two diseases be more serious global healthproblem The association between osteoporosis and T2DMshould be paid close attentionOsteoporosis is a skeletal chronic metabolic disease characterized by low bone mass and destroyed bone microarchitecture resulting in the high risk of fragility fracture []Therefore bone metabolism should be further studied inpatients with T2DM Bone metabolism is a dynamic cyclicalprocess where osteoblasts are involved in bone formationand osteoclasts are involved in bone resorption [] Metabolites known as bone turnovers markers BTMs are generated 0cJournal of Diabetes Researchfrom bone tissue and cells during the dynamic process andreflect bone metabolism during a relatively short period oftime [] and are thus better at predicting more recentchanges Specifically procollagen type I Nterminal peptidePINP is the degradation product during the formation oftype I collagen secreted by osteoblasts serum osteocalcinOC is released by osteoblasts during bone formation crosslinked Ctelopeptide of type I collagen CTX is abreakdown product during the degradation of mature typeI collagen secreted by osteoclasts [] Consequently PINPand OC are key markers of bone formation and CTX is akey marker for bone resorption The International Osteoporosis Foundation IOF recommends PINP and CTX as thereference markers for bone formation and bone resorptionrespectively due to their high sensitivity and specificity[] Recently these BTMs have been used to assess bonemetabolism evaluate the clinical efficacy of osteoporosistherapies and predict fracture risk [] Additionally BTMsare shown to be associated with energy metabolism []which is closely related to glucose metabolism Studying theeï¬ect of glucose metabolism disorders on BTMs is importantto evaluate bone metabolic status in T2DMMost research has focused on studying postmenopausalwomen and elderly men since these two groups of individualsare at a high risk for fractures especially those diagnosedwith T2DM Bone formation and bone mass are highest inthe third decade and then decrease with age [ ] However oste ia or osteoporosis in young and middleagedmale patients with T2DM is not uncommon in clinical practice Yet only a few studies focused on these populationgroups are available It is important to study how bonemetabolism disorders aï¬ect younger patients with T2DMTherefore young and middleaged male patients withT2DM were recruited as the subjects in the study presentedhere We aim to assess bone metabolism by determiningserum PINP OC CTX and parathyroid hormone PTHlevels and investigate the association among these markersand glucose metabolism The goal is to explore the ‚uenceof T2DM on bone metabolism which may allow for an accurate assessment of fracture risk and an earlier management ofbone metabolism disorders Materials and Methods Participants The study presented here is a crosssectional study conducted in men aged years oldPatients with T2DM who were admitted to the TianjinMetabolic Diseases Hospital from December to February were included in the T2DM group Nondiabeticmale volunteers from the physical examination centre wererecruited and included in the control group during thesame periodbloodfastingThe diagnosis of T2DM was based on the guidelinesprovided by the World Health anization [] includ°FBGž level ‰¥ mmolling mgdl or h blood glucose ‰¥ mmoll mgdlduring an oral glucose tolerance test OGTT Diabeticpatients were treated with oral antidiabetic agents or incombination with insulin Exclusion criteria included theglucosepresence of kidney disease eGFR mLmin173 m2hepatic disease ALT or AST ‰¥ times than the upperreference cancer rheumatic diseases rheumatic arthritisand rheumatoid arthritis other bone metabolic diseasesosteitis and osteomalacia hypercalcemia or other endocrine diseases Cushing™s syndrome primary hyperparathyroidism and thyroid dysfunction Participants takingmedications that may ‚uence bone metabolism were alsoexcluded These medications included glucocorticoids calcium vitamin D antiosteoporosis drugs steroids and thyroid hormonesThis study was conducted following the Declaration ofHelsinki and was approved by the Ethics Committee of the Tianjin Medical University Chu HsienI Memorial Hospital Each participant signed a written informedconsent form Clinical Measurements Anthropometric and biochemical assessments were performed in all participants Diabeticduration height weight body mass index BMI and bloodpressure data were collected BMI was calculated by the formula as weight in kg divided by height squared in m2All overnight fasting blood samples were obtained in themorning Serum samples were separated by centrifugationand stored at °C Blood calcium phosphorus total protein albumin alanine aminotransferase aspartate aminotransferase alkaline phosphatase ALP serum creatinineuric acid urea nitrogen haemoglobin A1c HbA1c FBGinsulin CpeptidetriglycerideTG highdensity lipoprotein cholesterol HDLc andlowdensity lipoprotein cholesterol LDLc were measuredusing standard methods Serum parathyroid hormonePTH and BTM levels including PINP OC and CTXwere measured using an IDSiSYS automated analyserRoche Germany The intraassay and interassay coefficients of variation CVs of BTMs were below and respectivelycholesterolTCtotal Statistical Analyses The statistical analyses were performed with SPSS SPSS Inc Chicago IL USA Normality testing was conducted in all continuous variablesVariables with normal distributions were described as mean± standard deviation and the diï¬erences were determinedusing Student™s ttest between the two groups Those withskewed distributions were expressed as median interquartilerange and diï¬erences between groups were assessed usingthe Mann“Whitney U test The Pearson or Spearman correlation analysis was used to determine the correlation betweenblood glucose or lipid and bone metabolism markers Multiple linear regression analyses were conducted to evaluate theassociation between HbA1c TG and BTMs P value was considered statistically significant ResultsA total of diabetic patients were included in the T2DMgroup The mean age of these patients was ± yearsand the mean diabetic duration was years ranging from to years The mean FBG was ± mmoll 0cJournal of Diabetes ResearchTable Comparison of characteristics between diabetic patientsand controlsVariablesPatients with T2DMn ± Nondiabeticcontrols n P ± ””””””” ± ± ± ± ” ± ± ± ± ± ± ± ± ± ± ± ± ± Age yDiabeticduration yHeight cmWeight kgBMI kgm2SBP mmHgDBP mmHgFBG mmollHbA1c INS mIUlCP ngmlTG mmollTC mmollHDLcmmollLDLcmmollCa mmollP mmollTP glALB glALT IUlAST IUlALP IUlCr μmollSUA μmollBUNmmolly years T2DM type diabetes mellitus BMI body mass index SBP systolicblood pressure DBP diastolic blood pressure FBG fasting blood glucoseHbA1c haemoglobin A1c INS fasting insulin CP fasting Cpeptide TGtotaltriglyceride TCcholesterol HDLc highdensity lipoproteinlowdensity lipoprotein cholesterol Ca calcium Pcholesterol LDLcalaninephosphorus TPtotalaminotransferase ASTalkalinephosphatase Cr serum creatinine SUA serum uric acid BUN blood ureanitrogen P value was considered significant ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± protein ALBaspartatealbumin ALTaminotransferase ALPand the mean HbA1c value was ± A total of nondiabetic volunteers were recruited in the control groupthat had a mean age of ± years and a mean FBG of ± mmollBaseline clinical characteristics of the two groups areshown in Table No significant diï¬erences were observedbetween the control or T2DM groups for age P height P weight P BMI P orsystolic blood pressure P There were also no significant diï¬erences between the two groups for serum calciumP phosphorus P creatinine P total protein P albumin P or ALPP As expected patients in the T2DM groupshowed significantly higher FBG levels P comparedwith the control group In addition significantly higher TGP TC P and LDLc P levelsand significantly lower HDLc P levels wereobserved in diabetic patients compared with controlsComparison of BTMs and PTH between diabetic patientsand controls is shown in Table There were significantdecreases in serum PINP P OC P andPTH P levels in patients with T2DM comparedwith controls In contrast serum CTX levels were similarbetween the two groups P Moreover univariate correlation analyses were performed to investigate the association between blood glucoseor lipid and bone metabolism markers The results revealedthat HbA1c was positively correlated with PINP rs P and inversely associated with PTH r ˆ’P There was a significant negative correlationbetween OC or CTX and TG rs ˆ’ P rs ˆ’ P levels Figure There was no significantassociation observed between PINP and TG or between OCand HbA1c levels Age was negatively correlated with PINPrs ˆ’ P OC rs ˆ’ P andPTH r P but not with CTX levels TheBTMs and PTH levels did not correlate with BMI bloodpressure calcium or phosphorous levelstheFurthermore multiple linear regression analyses wereperformed to examinecrosssectional associationbetween blood glucose or lipid and BTMs after adjustingfor expected confounder factors Serum PINP OC orPTH levels were used as dependent variables while HbA1cor TG levels were used as independent variables Thesefindings indicated that HbA1c was positively correlatedwith PINP P and inversely associatedwith PTH ˆ’ P after adjusting for ageBMI systolic blood pressure TG HDLc LDLc calciumand phosphorus Our results also showed a significantnegative correlation between TG and OC ˆ’ P after adjusting for age BMI systolic blood pressure HbA1c HDLc LDLc calcium and phosphorusTable All independent variables used in multiple linearanalyses are shown in Table S1 DiscussionMost previous studies investigating postmenopausal womenand elderly men have shown that the markers for bone formation andor resorption were reduced in patients withT2DM compared with nondiabetic individuals [] indicating a lower bone turnover state It is unclear whetheryoung and middleaged diabetic patients shared similarresults In this study we focused on young and middleaged male patients with T2DM Results demonstrated thatdiabetic patients had significantly lower serum PINP andOC levels compared with the control individuals In contrast serum CTX levels were not significantly diï¬erentbetween the two groups Results indicated that inhibition 0cJournal of Diabetes ResearchTable Comparison of BTMs and PTH between diabetic patients and controlsPatients with T2DM VariablesPINP ngmlOC ngmlCTX ngmlPTH pgmlT2DM type diabetes mellitus PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone P value was considered significant ± Nondiabetic controls ± Plmgn PNIPlmgn COrs P HbA1c ars “P lmgp HTPlmgn XTC𝛽r “P HbA1c brs “P TG mmolldTG mmollcFigure Correlation between serum glucose or lipid levels and BTMs or PTH a Correlation between PINP and HbA1c b Correlationbetween PTH and HbA1c c Correlation between OC and TG d Correlation between CTX and TG HbA1c haemoglobin A1c TGtriglyceride PINP procollagen type I Nterminal peptide OC osteocalcin CTX crosslinked Ctelopeptide of type I collagen PTHparathyroid hormone r Pearson™s correlation coefficient rs Spearman™s correlation coefficient P value was considered significantTable Multiple linear regression analyses between serum glucose or lipid and bone metabolism markersIndependent variablesDependent variablePINPOCPTHHbA1c haemoglobin A1c TG triglyceride PINP procollagen type I Nterminal peptide OC osteocalcin PTH parathyroid hormone P value wasconsidered significantUnstandardized coefficients Standardized coefficients HbA1cTGHbA1cPof bone formation phase rather than resorption led to alower bone turnover state in young and middleaged malepatients with T2DM Moreover this study demonstratedthat HbA1c was an independent factor for PINP suggesting the ‚uence of glycaemic control on PINP in youngand middleaged male patients with T2DM Early glycaemic control may reduce the risk of fracture by delayingbone formation reduction 0cJournal of Diabetes ResearchReduced serum OC levels were previously reported inmale patients with T2DM [“] Bezerra dos SantosMagalhaes further demonstrated a weak negative correlation between FBG and OC levels [] Whereas serumPINP was not available in these studies A recent studyrevealed a decrease in serum PINP levels in patients withimpaired fasting glucose and diabetes [] which was in linewith our research Further analyses revealed that serum PINPlevels were significantly reduced in younger diabetic patients years old compared with the older patients ‰¥ yearsold but serum CTX was also significantly decreased []The controversial conclusions may be related to diï¬erencesin age race diabetic duration and glycaemic control Astudy by Kulkarni [] shared a similar relationshipbetween HbA1c and PINP levels Additionally a largescale crosssectional study performed in Germany indirectly supported this conjecture The authors revealed thatchances for metabolic syndrome or T2DM significantlydecreased with the higher serum PINP and CTX levelsin men aged years old [] However two largescale studies performed in China one involving men andwomen aged years old [] and the other includingmen aged years old [] indicated that serum OCwas negatively correlated with chances for T2DM evenafter adjusting age BMI waist circumference blood pressure FBG and TG As described by these studies theclose relationship between glucose and BTMs has beeninvestigated but needs further understandingIn addition compared with controls diabetic patientsshowed higher TG TC and LDLc and lower HDLc levelswhich may represent a high probability of lipid metabolismdisorders in patients with T2DM Further analyses investigating the correlation between lipid and BTMs revealed a significant negative correlation between serum TG and OClevels High TG levels may reduce serum OC levels andinhibit bone formation in young and middleaged malepatients with T2DM These observations were similar towhat was found in a recent male populationbased studywhere serum TG levels were also inversely correlated withOC levels [] Some research investigating male diabeticpatients showed no relationship between serum OC levelsand blood lipid metabolism [ ] These findings are contradictory to one another Diï¬erences in age race and metabolic status may account for these controversial resultsThe impact of blood glucose and lipid metabolism disorders on BTMs needs further studies to elucidate mechanismsIt is known that hyperglycaemia can lead to osmotic diuresiswhich causes renal calcium leakage and a negative calciumbalance Improved blood glucose control contributes to thereduction of urinary calcium levels [] The calciumsensing defect and secondary chronic hypomagnesaemiainduced by osmotic diuresis may be responsible for impairedPTH secretion [] The pathological regulation of PTH onBTMs in patients with T2DM is not clear In this studyserum PTH levels were decreased and were negatively associated with HbA1c levels in T2DM implying that diabeticpatients especially those with poor glycaemic control hadlower PTH levels These observations were in line with previous studies [ ] Relative hypoparathyroidism may disrupt bone metabolism in patients with T2DM Previousstudies demonstrated that low PTH levels directly inhibitedosteoblast activity and contribute to bone demineralizationIn the nondiabetic population PTH inhibited transcriptionalsuppression of sclerostin produced by osteocytes As a Wntantagonist sclerostin inhibited Wntcatenin signallingand osteoblast activity However the regulation of PTH onsclerostin may be impaired in diabetes [] As mentionedabove the negative relationship between blood glucose andbone metabolism is probably explainedOtherwise chronic ‚ammatory conditions and turbulence of adipokines increased the risk of osteoporosis inpatients with T2DM [] Advanced glycation endproductsAGEs were accumulated in diabetes and played a key rolein chronic ‚ammatory complications [] Previous studieshave shown that BTMs were suppressed by hyperinsulinemiaand the accumulation of AGEs [] AGEs promoted theproduction of both ‚ammatory cytokines and reactive oxygen species ROS in the body by activating ligands furthertriggering chronic ‚ammation and bone resorption []In vitro studies reported that AGE2 and AGE3 inhibitedthe maturation of human marrow mesenchymal stem cellsMSCs and their diï¬erentiation into cartilage and bone tissues resulting in decreased osteoblasts [] Moreover theformation and accumulation of AGEs inhibited synthesis ofosteocalcin and osteoblastic ossein matrix [] increasednonenzymatic crosslinked folding of the collagen fibres[] and disturbed osteoblast development A recent studyindicated that hyperglycaemia directly inhibited the diï¬erentiation of osteoblasts and then decreased bone formationenhanced osteoclast activity and increased bone absorptioneventually leading to a reduction of bone mass [ ] Glucose and insulin signalling involved receptor activation of thenuclear factor κB ligandosteoprotegerin RANKLOPGpathway [ ] Analyses revealed thatlower serumRANKL levels were associated with higher TG levels []This inverse relationship may explain the results generatedin this study Furthermore adiponectin a recently uncoveredadipocytokineis produced exclusivity in adipose tissueResearch shows that adiponectin stimulated osteoblast proliferation diï¬erentiation and mineralization [] Howeverserum adiponectin concentrations decreased in patients withT2DM [] The turbulence of adipocytokines may lead to animbalance of bone metabolismAntidiabetic agents may have diï¬erent eï¬ects on bonemetabolism Agents that may have an eï¬ect include thiazolidinediones TZDs sodiumglucoselinked transporter2SGLT2 inhibitors insulin and glucagonlike peptide1receptor agonists GLP1 RA A previous work shows thatrosiglitazone a type of TZDs promoted osteoblastosteocyteapoptosis and led to a negative balance in bone metabolism[] Analyses demonstrated a gender diï¬erence when itcame to the eï¬ects of TZDs on fracture in patients withT2DM and confirmed that TZDs only increased fracture riskin female patients and not male patients [] SGLT2 inhibitors improved blood glucose levels by promoting urinaryglucose excretion which may aï¬ect urinary calcium excretionand bone metabolism Canagliflozin treatment was associatedwith a higher fracture rate in patients with T2DM [] A 0cJournal of Diabetes Researchmetaanalysis indicated no relationship between three SGLT2inhibitors canagliflozin dapagliflozin and empagliflozin andfracture risk Clinical studies on adverse skeletal events ofSGLT2 inhibitors are still lacking Few studies have assessedthe association between insulin injection and BTMs Severalstudies reported an increased fracture risk in insulintreatedpatients with T2DM [] A high incidence of hypoglycaemicevents and falling [] may be the main reasons in olderadults Longterm disease and the presence of multiple diabetic complications may also disrupt bone metabolism Nosignificant diï¬erences were observed between diabetic patientsunder treatment with n or without n TZDswith n or without n SGLT2 inhibitors andwith n or without n insulin in this study Liraglutide and exenatide two GLP1 RAs may improve skeletalblood supply increase bone mineral density BMD andreduce the risk of osteoporosis and fracture in animal andhuman studies [ ] However the bone protective eï¬ectsbehind this require clinical studies There were no significantdiï¬erences observed between diabetic patients under treatment with n or without n GLP1 RAs in ourstudy In addition there were also no significant diï¬erencesbetween patients under treatment with n or withoutn dipeptidyl peptidase4 DPP4 inhibitors withn or without n insulin secretagogues withn or without n metformin and with n or without n alphaglucosidase inhibitors AGI in thepresent study Table S2 As a multiple metabolic diseasethe treatment of T2DM is complex and requires additionalclinical studies to evaluate the ‚uence of these therapies onbone metabolismOne advantage of this study is that it focused on male diabetic patients aged years old where BTMs varied withsmall changes and there was a restriction on gender and agebeing an ‚uence on the results With this it was easier toinvestigate the relationship between blood glucose lipidsand bone metabolism However this study still faces somelimitations First the crosssectional design prevents onefrom drawing a causal relationship and failed to explorechanges in BTMs after improving blood glucose and lipidmetabolism disorders Further prospective research mayoï¬er additional information about this Second the samplesize number between the two groups was unequal and thenumber of controls used was inadequate Besides this studywas a singlecentre study that only analysed patients with relatively severe diabetes Therefore the results presented heremay not be generalizable to all young and middleaged malepopulations diagnosed with T2DM Largescale and multicentre studies remained to verify these issues Third the‚uence of antidiabetic agents on bone metabolism remainscontradictory Consequently potential confounder factorsmay exist Fourth serum levels of bonespecific alkalinephosphatase BAP vitamin D or steroids all of which ‚uence bone metabolism were not determined in this studySerum ALP is mainly derived from liver isoform LAPand its specificity for bone metabolism is lacking [] BAPis a more bonespecific marker of bone formation while thecurrent immunoassays available for BAP still show up to crossreactivity toward LAP [] As recommended bythe IOF [] serum PINP was preferred for bone formationbecause of high specificity in our study Vitamin D promotesthe absorption of calcium and may aï¬ect bone metabolismHowever relatively limited data about the eï¬ect of vitaminD on BTMs are available A prospective partial interventionstudy in postmenopausal women with T2DM shows that25OHD was positively correlated with PINP especially inpatients taking alfacalcidol [] The MINOS study a prospective study of men aged years revealed thatserum 25OHD was not associated with BTMs in men under years of age n [] The relationship between vitamin D and BTMs still needs further research Fifth we didnot take BMD into consideration BMD altogether withBTMs may be helpful to evaluate bone metabolism BMDreflects mineral density of bone and is the cumulative resultof longterm bone metabolic activities This study mainlyfocused on the impact of T2DM on BTMs and evaluatedthe recent changes of bone metabolism Further studiesshould be conducted to investigate the longterm eï¬ect ofT2DM on BMD ConclusionsThis study demonstrated that young and middleaged malepatients with T2DM showed a lower turnover state resultingfrom bone formation inhibition HbA1c levels were positively correlated with PINP levels and inversely associatedwith PTH levels These findings also revealed a negative correlation between TG and OC levels even after adjusting forexpected confounder factors Glucose and lipid metabolismdisorders may aï¬ect bone formation through diï¬erent pathways The study presented here provides evidence of T2DM‚uencing bone metabolism in young and middleagedmen The improvement of blood glucose and lipids may bebeneficial to bone metabolism and reduce fracture risk inpatients with T2DMData AvailabilityThe data used to support the findings of this study are available from the corresponding author upon requestConflicts of InterestThe authors declare that there is no conflict of interestregarding the publication of this paperAcknowledgmentsThis work was supported by Scientific Research Funding ofTianjin Medical University Chu HsienI Memorial Hospitalgrant numbers 2018ZDKF07 We gratefully acknowledgethe participants in this studySupplementary MaterialsTable S1 multiple linear regression between serum glucoseor lipid levels and BTMs or PTH Table S2 the informationaboutthe patients with T2DMSupplementary Materialsthe medications of 0cJournal of Diabetes ResearchReferences[] K Ogurtsova J D da Rocha Fernandes Y Huang œIDFDiabetes Atlas Global estimates for the prevalence of diabetesfor and  Diabetes Research and Clinical Practicevol pp “ [] A V Schwartz D E Sellmeyer K E Ensrud œOlderwomen with diabetes have an increased risk of fracture a prospective study Journal of Clinical Endocrinology and Metabolism vol no pp “ [] L Forsen H E Meyer K Midthjell and T H Edna œDiabetesmellitus and the incidence of hip fracture results from theNordTr¸ndelag Health Survey Diabetologia vol no pp “ [] Y Fan F Wei Y Lang and Y Liu œDiabetes mellitus and riskof hip fractures a metaanalysis Osteoporosis Internationalvol no pp “ [] M Janghorbani R M Van Dam W C Willett and F B HuœSystematic review of type and type diabetes mellitus andrisk of fracture American Journal of Epidemiology vol no pp “ [] I KostoglouAthanassiou P Athanassiou A Gkountouvasand P Kaldrymides œVitamin D and glycemic control in diabetes mellitus type  Therapeutic Advances in Endocrinologyand Metabolism vol no pp “ [] H Miyake I Kanazawa and T Sugimoto œAssociation ofbone mineral density bone turnover markers and vertebralfractures with allcause mortality in type diabetes mellitusCalcified Tissue International vol no pp “ [] E S Siris R Adler J Bilezikian œThe clinical diagnosis ofosteoporosis a position statement from the National BoneHealth Alliance Working Group Osteoporosis Internationalvol no pp “ [] M B Greenblatt J N Tsai and M N Wein œBone turnovermarkers in the diagnosis and monitoring of metabolic bonedisease Clinical Chemistry vol no pp “ [] S Vasikaran for the IOFIFCC Bone Marker Standards Working Group R Eastell œMarkers of bone turnover for theprediction of fracture risk and monitoring of osteoporosistreatment a need for international reference standards Osteoporosis International vol no pp “ [] H W S Cabral B F G Andolphi B V C Ferreira œTheuse of biomarkers in clinical osteoporosis Revista da Associa§£o M©dica Brasileira vol no pp “ [] P Iglesias F Arrieta M Pi±era œSerum concentrationsof osteocalcin procollagen type Nterminal propeptide andbetaCrossLaps in obese subjects with varying degrees of glucose tolerance Clinical Endocrinology vol no pp “ [] J M Wishart A O Need M Horowitz H A Morris  andB E C Nordin œEï¬ect of age on bone density and bone turnover in men Clinical Endocrinology vol no pp “ [] P Szulc P Garnero F Munoz F Marchand and P D Delmas œCrosssectional evaluation of bone metabolism inmen Journal of Bone and Mineral Research vol no pp “ [] K G M M Alberti P Z Zimmet and WHO ConsultationœDefinition diagnosis and classification of diabetes mellitusand its complications Part diagnosis and classification ofdiabetes mellitus provisional report of a WHO consultationDiabetic Medicine vol no pp “ [] J StarupLinde and P Vestergaard œBiochemical bone turnover markers in diabetes mellitus a systematic review Bonevol pp “ [] L Achemlal S Tellal F Rkiouak œBone metabolism inmale patients with type diabetes Clinical Rheumatologyvol no pp “ [] S V Kulkarni S Meenatchi R Reeta R Ramesh A R Srinivasan and C Lenin œAssociation of glycemic status with boneturnover markers in type diabetes mellitus InternationalJournal of Applied Basic Medical Research vol no pp “ [] K B dos Santos Magalh£es M M Magalh£es E T DinizC S Lucena L Griz and F Bandeira œMetabolic syndromeand central fat distribution are related to lower serum osteocalcin concentrations Annals of Nutrition and Metabolismvol no pp “ [] K L HollowayKew L L F De Abreu M A Kotowicz M ASajjad and J A Pasco œBone turnover markers in men andwomen with impaired fasting glucose and diabetes CalcifiedTissue International vol no pp “ [] E Lerchbaum VSchwetz M Nauck H V¶lzkeH Wallaschofski and A Hannemann œLower bone turnovermarkersthepopulationbased study of health in Pomerania NutritionMetabolism and Cardiovascular Diseases vol no pp “ in metabolicsyndromediabetesand[] H Shu Y Pei K Chen and J Lu œSignificant inverse association between serum osteocalcin and incident type diabetesin a middleaged cohort DiabetesMetabolism Research andReviews vol no pp “ [] A Tan Y Gao X Yang œLow serum osteocalcin level is apotential marker for metabolic syndrome results from a Chinese male population survey Metabolism vol no pp “ [] X Y Ma F Q Chen H Hong X J Lv M Dong and Q YWang œThe relationship between serum osteocalcin concentration and glucose and lipid metabolism in patients with type diabetes mellitus the role of osteocalcin in energy metabolism Annals of Nutrition and Metabolism vol no pp “ [] Y Chen Q Zhao G Du and Y Xu œAssociation betweenserum osteocalcin and glucoselipid metabolism in ChineseHan and Uygur populations with type diabetes mellitus inXinjiang two crosssectional studies Lipids in Health andDisease vol no p [] N C Thalassinos P Hadjiyanni M Tzanela C Alevizaki a
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"25 Thus our results of a 2-year DFS rate of 80% and OS rate of 96% appear favorable by comparison. However it is prudent to be cautious because we lost 20 of 81 patients from the survival analysis because of consent withdrawal and a direct comparison of outcomes data among trials cannot account for differences in study populations eligibility and staging criteria and provisions for data collection and analysis. The spectrum of protein levels for ERCC1 and RRM1 significant correlation of levels between both molecules and distribution of patients into the 4 gene expression categories in the current study is consistent with previous experience.91213161826 However the current analysis method for biomarker evaluation (ie antibody-based assessment of in situ protein levels) is not suitable for general clinical implementation for several reasons. First ERCC1 has multiple isoforms that cannot be specifically distinguished by the available reagents and only 1 isoform appears to be involved in platinum-induced DNA damage repair.27 Second the monoclonal antibody 8F1 which is consistently used for ERCC1 protein expression analysis detects a second and unrelated protein that shares a common epitope with ERCC1.28“30 This observation may account for the highly batch-dependent performance of this antibody1827 which may explain the significantly lower ERCC1 values in the current study compared with prior results.16 Third protein levels for RRM1 in particular and to a lesser degree for ERCC1 appear to be influenced by the specimen processing and handling procedures used at collection sites.26 Finally although the method for immunofluorescence-based quantitative detection of both molecules performs well if all specimens to be analyzed are processed simultaneously there is considerable interassay variability if specimens need to be processed individually over an extended period of time as required for real-time patient decision-making.18 However it is important to note that the biochemical biophysical and cell biological evidence for ERCC1 and RRM1 as predictive molecules for platinum and gemcitabine efficacy remains undisputed.510“12273132 A small number of recent clinical trials have used ERCC1 prospectively for therapeutic decision-making. These include 2 randomized phase 3 trials in patients with advanced-stage NSCLC (1 published [NCT00499109]18 and the other terminated and unpublished [NCT00801736]) and 2 adjuvant trials 1 of which was a terminated and not yet published phase 2 trial [TAilored Post-Surgical Therapy in Early Stage NSCLC (TASTE) NCT00775385] and the other an ongoing phase 3 trial [International TAilored Chemotherapy Adjuvant trial (ITACA); EudraCT 2008-001764-36]. Results from the first trial (NCT00499109) demonstrated no improvement in patient survival; however the authors raised the possibility of a false-negative result because of an inexplicably divergent survival in an internal control group.18 The second trial (NCT00801736) and third trial (NCT00775385) were terminated early after the discovery of ERCC1 isoforms27 and specificity problems with the 8F1 antibody.28“30 The fourth trial is using ERCC1 and tumor thymidylate synthase mRNA expression levels for treatment assignment compared with a cisplatin-based control treatment with OS as the primary endpoint and a planned accrual of 700 patients. Results from these trials will help to further delineate the feasibility and technical issues mentioned above. The results of the current study demonstrated the feasibility of our biomarker-based decision algorithm in a multiinstitutional cooperative group environment for patients with surgically resected NSCLC. We identified that the current practice of evaluation and treatment for these patients may present an obstacle to rapid molecular-based decision-making. Although encouraging efficacy data emerged from this trial bioassays that specifically measure platinum-induced DNA damage repair must be developed before further clinical trials are launched that seek to tailor the use of these agents. "
1
"evaluate the clinicopathologic characteristics of Lymph Node metastasisbetween investing layer of Cervical fascia and deep fascia of infrahyoid strap Muscles LNCM in papillary thyroidcarcinoma PTCMethods Retrospective review of patients with PTC who underwent thyroidectomy and central compartment neckdissection CND from January to January was performed in two tertiary referral academic medicalcenters A total of consecutive patients with PTC who underwent thyroidectomy and CND were included inthe retrospective review The LNCM was resected as a separate specimen by the surgeon and the clinicopathologiccharacteristics of the patients were recorded Multivariate logistic regression analysis was performed to identify riskfactors for LNCM metastasisResults Of PTC patients patients had lymph nodes in the LNCM Among them caseswere confirmed to be positive in the LNCM In total the metastasis rate of LNCM in PTC patients was Univariate analysis revealed that the metastasis of LNCM were more likely to have a primary site in theinferior pole extrathyroidal extension ETE central cervical metastasis level III and level IV metastasis Multivariateanalysis further showed tumor location in the inferior pole ETE level III and level IV metastasis conferred asignificantly increased odds ratio for LNCM metastasisConclusion Attention should be paid to the lymph tissue in the LNCM for PTC patients especially in presence of aprimary site in the inferior pole ETE level III and level IV metastasisKeywords Thyroid carcinoma Surgery Central compartment neck dissection Recurrence Suprasternal spaceIntroductionPatients with papillary thyroid carcinoma PTC have afavorable prognosis with central neck locoregional recurrence varying from to [] The goal of a prophylacticor therapeutic central compartment neck dissection pCNDor tCND is to decrease the incidence of local recurrenceby removing all lymphatic tissue within the levels VI and Correspondence wugaosongwhueducn1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei People™s Republic of ChinaFull list of author information is available at the end of the VII compartments which are generally the first and themost commonly involved with metastasis [] For patientswithout evidence of lymph node metastasis on preoperativeevaluation the additive value of a pCND at the time ofthyroidectomy is controversial Some authors advocatepCND considering high rate “ of occult metastaticnodal disease in cN0 PTC [] while other authors considerthat there is no highlevel evidence in favor of pCND []The performance of pCND is dependent on the weightgiven to the risks and benefits of pCND [] Consideringthe oncologic benefits of CND and the risks of a repeat 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 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of neck operation performing pCND is recommended toevery patient in China [ ]Although American Thyroid Association ATA guideline has defined the boundary of central neck compartment there is also significant variability in terms of theextent of CND In routine clinical practice CND canrange from sampling a few nodes in the paratrachealregion to a complete clearance from left carotid artery toright carotid artery and down to and including the uppermediastinum [] Owing to the variant extent of CNDsome central compartments are easily to be neglectedFor thyroid carcinoma patients with specific clinicopathologic characteristics incomplete lymph node dissectionmay result in increased recurrence reoperation andreoperationassociated complications [] Lymph Nodebetween investing layer of Cervical fascia and deep fasciainfrahyoid strap Muscles LNCM has not beenofreported The LNCM compartment is defined as followssuperiorly by the hyoid bonelaterally by the carotidarteries anteriorly by the investing layer of cervicalfascia and posteriorly by the deep fascia of infrahyoidstrap muscles LNCM space includes suprasternal spaceand intrainfrahyoid strap muscle spaceAnatomically LNCM is located anterior to the strapmuscles We consider that what is special about the concept of the LNCM is that it is belong to level VI but is aneasily overlooked anatomical area by a strap musculatureinvolving the sternohyoid and sternothyroid musclesduring selective or modified neck dissection Although themetastasis in LNCM was seldom it did occur in somePTC patients with regional recurrence As part of LCNMsuprasternal space metastasis for thyroid cancer wereinvestigated in three studies [“] Thus we routinelydetected the suprasternal space and intrainfrahyoid strapmuscle space Fig This study was performed to identify the clinicopathologic characteristics and indication forlymph node metastasis dissection in the LNCMMaterials and methodsPatientsA retrospective review from the clinical and histopathologydatabase of two tertiary referral academic medical centersTongji Hospital of Huazhong University of Science andTechnology and Zhongnan Hospital of Wuhan Universityfrom January to January were conducted In theinstitutions preoperative examinations consisted of athorough physical examination neck ultrasound a clinicalevaluation of thyroid nodules and neck lymph nodes Fineneedle aspiration cytology FNAC were performed in patients who were suspected thyroid nodules or lymph nodeWith a pathological confirmation of PTC all the patientsreceived a thyroidectomy with CND Accordingly a pCNDwas performed for cN0 patients and a therapeutic CNDwas performed for cN1 patients The inclusion criteria forthe patients were as follows the clinical history completely recorded the LNCM was resected as a separatespecimen by the surgeon PTC patients who underwentthyroidectomy plus CND with or without lateral neckdissection A total of consecutive PTC patients wereenrolled The medical ethic committee of ZhongnanHospital of Wuhan University approved the procedure andinformed written consent was obtained from all patientsSurgical approachAll the operations were performed by the same seniorsurgeon Gaosong Wu with the patients under generalFig Four subdivisions Level VI 1st Level VI 2nd Level VI 3rd and Level VI 4th of central neck compartment are divided by deep fascia ofinfrahyoid muscles pretracheal visceral fascial and right recurrent laryngeal nerve 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of anesthesia Thyroidectomy was performed with a standard technique of fine capsular en bloc dissection andresection from inferior pole to superior pole [“]Intraoperative neuromonitoring was employed for all ofthe thyroidectomies [] Superior parathyroid glandswere identified and preserved in situ inferior parathyroidglands were protected in situ or autotransplanted in thesternocleidomastoid muscle according to three certaintypes based on their blood supply and location [ ]After the incision of the investing layer of cervicalfascia the interval between sternohyoid and sternothyroid muscles and the space anterior to the sternohyoidmuscle above the clavicle and the sternum weredetected If there was fibrofatty tissue Instead of the enbloc removal of the entire centrallymph nodes theLNCM was resected as a separate specimen if occurredThe presence or absence of lymph node metastasis wasdefined according to postoperative pathological reportsWhile dissecting paratracheal lymph nodes intraoperative neuromonitoring was employed to detect RLN fromdistally to proximally minimizing morbidity from injuryto RLN during compartment nodal dissection LNCMand other compartment lymphatic tissue were processedfor routine hematoxylin and eosine HE separatelyThe pathologic results were independently determinedby two qualified pathologists without any prior knowledge of the patients™ clinical dataData collection and statistics analysisTo determine the relation between LNCM metastasis andclinicopathologic factors such as age sex primary tumorsite lateral cervical lymph node metastasis level VI metastasis the chisquare test and Fisher™s exact test were usedas appropriate Multivariate logistic regression analysiswas performed to identify risk factors for LNCM metastasis of PTC P was considered statistically significantAll calculations were performed using SPSS for windows Postthyroidectomy hypocalcemia lasting for morethan months was considered as permanent VCP All patients were followed up every “ months postoperativelyResultsPatients detected with LNCMAfter reviewing patients who underwent thyroidectomy plus CND with or without lateral neck dissectionfrom January to January patients were detected with LNCM and patients wereabsent of LNCM The average tumor size of LNCM was cm and the mean number of lymph nodes sampledfrom LNCM was ranging from to Table showsthe comparison of clinicopathologic characteristics between the present LNCM group and the absent groupIn univariate analysis Hashimoto™s disease p multifocality leisions p the tumor located ininferior portion p extrathyroidal extensionETE p central cervical metastasis p level III and level IV metastasis p were significantly associated with high prevalence of LNCMPatients with metastatic LNCMAmong a total of patients with LNCM metastaticLNCM was found in patients Table compares the clinicopathologic characteristics between themetastatic LNCM group and the nonmetastatic LNCMgroup Three hundred eightythree patients were confirmed free of LNCM metastasis of themwith clinically negative node performed pCND and of them with clinically positive performed tCND All thepatients in the metastatic LNCM group performedtCND Lateral neck dissection was performed in cases in the metastatic LNCM group and cases in the nonmetastatic group all lateral neckdissection wastherapeutically performed Univariateanalysis was performed for the patients with and patients without metastatic LNCM Age at diagnosisgender and tumor size coexistent thyroid disease tumorfocality and level II metastasis were not correlated withLNCM metastasis Univariate analysis identified tumorlocated in the inferior pole central cervical metastasisETE level III and level IV metastasis as significant predictors of LNCM metastasis in our study population Multivariate analysis further showed that tumor location ETElevel III and level IV metastasis conferred a significantlyincreased odds ratio for LNCM metastasis Table ComplicationsThe median followup time was months range “ of patients had voice changes all of whomrecovered within “ months Temporary vocal cord paralysis was confirmed in patients by laryngoscope andthirteen permanent hypocalcemia was observed aftersurgeryDiscussionIn order to achieve the best chance of cure and effectivedisease control thoroughness of dissection has to betaken into account We prospectively performed comprehensive CND for PTC patients who underwent thyroidectomy and CND In addition data were analyzed for PTC patients to investigate the clinicopathologic characteristics for LNCM metastasis The occurrence rate ofLNCM was and of the patients harbored metastatic LNCM In total the positiverate of the LNCM was In this studymultivariate analysis revealed that a primary site in the inferior pole ETE level III and level IV metastasis were ofhigher LNCM metastasis rate which was consistent withthe findings by the previous report oflymph node 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for patients who had lymph nodes in LNCM compared tothose who did notVariablesAge mean ± SDAbsent n Present n P value‰¤ GenderFemale MaleTumor size cm ‰¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimoto™s diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes No LNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid Musclesmetastasis between sternocleidomastoid and sternohyoid muscle []Several studies have emphasized the importance of similar compartment in neck dissection for thyroid carcinomaSun pioneered the confirmation of the significantinvolvement of lymph node metastasis between sternocleidomastoid and sternohyoid muscle LNSS in lateralneck dissection [] which anatomically classified as part ofthe space of Burns They concluded that the positive rate ofLNSS was in clinically nodepositive cN PTCwhich was correlated with a primary site in the inferiorpole the lateral nodal metastasis level III and level IV nodalmetastasis [] Then Homma [] reported two casesof PTC patients with level III and IV lymph node metastases as well as metastasis in the suprasternal space Yu et al[] investigated the clinical significance of the suprasternalspace lymph node SSLN in pathological nodepositivepN PTC patients They concluded that metastasis rate ofSSLN was and the high SSLN metastasis of PTC wascorrelated with primary cancer site in the inferior thyroidpole strap muscle invasion level IV metastasis and LNSSmetastasis In our experience LNCM was rarely occurredin the central neck compartment and the positiveLNCM in PTC patients was infrequent as well Notably among the patients with pN PTC themetastasis rate of LNCM was which was muchlower than the metastasis incidence of SSLN described by Yu [] According to their results onefifth patients with pN PTC were performed incompleteCND and remained metastatic lymph nodes 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Table Univariate analysis of demographic and clinicopathologic factors for positive LNCMVariablesAge mean ± SDMetastasis n Nonmetastasis n ‰¤ GenderFemale MaleTumor size cm ‰¥ cmCoexistent thyroidNodular goiterYes NoToxic goiterYes NoHashimoto™s diseaseYes NoTumor focalityMultifocality UnifocalityTumor locationInferior portionUpperMiddle portionExtrathyroidal extensionYes NoCentral cervical metastasisYes NoLateral cervical metastasisLevel IIYes NoLevel IIIYes NoLevel IVYes NoLNCM Lymph Node between superficial layer of deep Cervical fascia and deep fascia of infrahyoid MusclesP value The total number of lymph nodes in the central neckcan range from to [] There is no consensus on thenumber of nodes removed or examined that would constitute an adequate dissection Aimed to allow surgeons tomore accurately report the extent of lymphadenectomyTable Multivariate analysis of predictors for LNCM positivityVariablesTumor locationp valueCentral cervical metastasisExtrathyroidal extensionLevel III metastasisLevel IV metastasisOR CI “ “ “ “ “LNCM Lymph Node between superficial layer of deep Cervical fascia and deepfascia of infrahyoid Musclesvisceralwe divide the central neck compartment into four subdivisions by deep fascia of infrahyoid strap muscles pretrachealfascial and right RLN Fig Theproposed LNCM compartment is bounded superiorly bythe hyoid bone laterally by the carotid arteries anteriorlyby the investing layer of the cervical fascia and posteriorlyby the deep fascia of infrahyoid strap muscles which is defined as Level VI 1st In the current study suprasternalspace composed part of the LNCM Fig Compared toSSLN reported by Yu [] LNCM encompasseslymph nodes in the suprasternal space and lymph nodesbetween sternohyoid and sternothyroid muscles Figs and LNCM can fall under the normal subdivisions ofthe central compartment Subdivisions can actually recordthe extent of the CND which is able to provide detailedinformation for the possible second operation 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Fig LNCM coverage area in vivo Lymph nodes between sternohyoid and sternothyroid muscles a and lymph nodes in the suprasternal space bIncluding LNCM as an anatomical part of the centralneck allows for removal of previously unrecognized micrometastatic disease in of PTC patients with the inferiorportion lesions ETElevel III and level IV metastasisDissection of the LNCM space is less invasive and easy toachieve and is not timeconsuming It is at the entrance ofcentral neck compartment which is easy to expose and haslow risk of damaging RLN or parathyroid With the application ofintraoperative neuromonitoring and in situpreservation or autotransplantation of parathyroid theoccurrence of vocal cord paralysis and permanenthypoparathyroidism in the current study were lowerin this study [ ] Therefore in cases where LNCMspace metastasis is suspected or preoperative ultrasoundand CT suggests LNCM metastasis greater attentionshould be paid to the nodal tissue in the LNCM space inthyroid carcinoma patients These patients might benefitfrom a reduced risk of regional recurrence central neckreoperative morbidity and improved decision making inrelation to the use of radioiodine ablationThere are severallimitations in the present studyThe retrospective design is a limitation of the studyAnd this was two tertiary referral centers retrospective review and routine prophylactic nodal surgerywas offered in China however it is not standard elsewhere in the world which is a major limitation Aprospective randomized trial with a long time followup period may help to further evaluate the clinicalsignificance of LNCM in PTC patientsConclusionsIn summary additional dissection of nodes in theLNCM were accessible and might not increase morbidity Therefore attention should be paid to the lymphtissue between investing layer of cervical fascia and deepfascia ofinfrahyoid strap muscles for PTC patientsespecially in presence of inferior portion lesions ETElevel III and level IV metastasisAcknowledgementsThe authors thank the studied patients for their willingness to cooperatewith our studyAuthors™ contributionsGaosong Wu Study concepts and design Qianqian Yuan Study designmanuscript preparation and editing Jinxuan Hou Data analysis andmanuscript editing Yiqin Liao Data acquisition Lewei Zheng Manuscriptpreparation Fang Lu Data acquisition Kun Wang Quality control of dataand algorithms The authors read and approved the final manuscriptFundingThe authors have no support or funding to reportAvailability of data and materialsNot applicableEthics approval and consent to participateThis research was comprised of human participants and was approved byMedical Ethics Committee of Wuhan University Zhongnan Hospital Informedconsent was obtained from all individual participants included in the studyConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Thyroid and Breast Surgery Zhongnan Hospital of WuhanUniversity Donghu Road Wuhan Hubei People™s Republic of China 2Department of Thyroid and Breast Surgery Tongji Hospital of TongjiMedical College of Huazhong University of Science and Technology Jiefang Avenue Wuhan Hubei People™s Republic of China Received April Accepted August ReferencesLang BH Ng SH Lau LL Cowling BJ Wong KP Wan KY A systematic reviewand metaanalysis of prophylactic central neck dissection on shorttermlocoregional recurrence in papillary thyroid carcinoma after totalthyroidectomy Thyroid “So YK Seo MY Son Y Prophylactic central lymph node dissection forclinically nodenegative papillary thyroid microcarcinoma influence onserum thyroglobulin level recurrence rate and postoperative complicationsSurgery “Hughes DT Rosen JE Evans DB Grubbs E Wang TS Solórzano CCProphylactic central compartment neck dissection in papillary thyroid 0cYuan Journal of Otolaryngology Head and Neck Surgery Page of Cancer and effect on Locoregional recurrence Ann Surg Oncol “ McHenry CR Is prophylactic central compartment neck dissection indicatedfor clinically nodenegative papillary thyroid Cancer the answer isdependent on how the data are interpreted and the weight given to therisks and benefits Ann Surg Oncol “Selberherr A Riss P Scheuba C Niederle B Prophylactic œfirststep centralneck dissection level does not increase morbidity after Totalthyroidectomy Ann Surg Oncol “Hartl DM Leboulleux S Al Ghuzlan A Baudin E Chami L Schlumberger M Optimization of staging of the neck with prophylactic central andlateral neck dissection for papillary thyroid carcinoma Ann Surg “ McAlister ED Goldstein DP Rotstein LE Redefining classification ofcentral neck dissection in differentiated thyroid cancer Head Neck“ Miller JE AlAttar NC Brown OH Shaughness GG Rosculet NP Avram AM Location and causation of residual lymph node metastasis aftersurgical treatment of regionally advanced differentiated thyroid CancerThyroid “Sun G Wang Y Zhu Y Wang Y Xu K Wei W Lymph node metastasisbetween sternocleidomastoid and sternohyoid muscle in clinically nodepositive papillary thyroid carcinoma Head Neck “ Homma A Hatakeyama H Mizumachi T Furusawa J Kano S Sakashita T Lymph node metastasis in the suprasternal space from thyroidpapillary cancer Int Cancer Conf J “ Yu S Ge J Sun B Wei Z Lei S Lymph node metastasis in suprasternal spacein pathological node“positive papillary thyroid carcinoma Eur J Surg Oncol“ Wu G Kong D Thyroidectomy with Wu Gaosong's ProcedureVideoEndocrinologr httpsdoi101089ve20150050 Wu G Wang K Intraoperative Neuromonitoring and Protection of theSuperior Laryngeal Nerve with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160070 Wu G Cui Q Wang K Carbon Nanops for Identifying Lymph Nodesand Protecting Parathyroid Glands in Thyroid Lobectomy with IpsilateralCentral Compartment Lymph Nodes Dissection VideoEndocrinology httpsdoi101089ve20160064Kong D Cui Q Gaosong W A Novel Classification of Parathyroid Glands andTheir Preservation in Thyroidectomy VideoEndocrinology httpsdoi101089ve20170093 Wang K Wu G Intraoperative Neuromonitoring in Selective Neck Dissectionfor Thyroid Cancer SND IIa Vb with Wu Gaosong's ProcedureVideoEndocrinology httpsdoi101089ve20160082 Yuan Q Wu G Hou J Liao X Liao Y Chiang F Correlation betweenelectrophysiological changes and outcomes of vocal cord function in recurrent laryngeal nerves with visual integrity during thyroidectomyThyroid “ Cui Q Li Z Kong D Wang K Wu G A prospective cohort study of novelfunctional types of parathyroid glands in thyroidectomy Medicine 9552e5810Tavares MR Cruz JA Waisberg DR Toledo SP Takeda FR Cernea CR et alLymph node distribution in the central compartment of the neck ananatomic study Head Neck “ Wang K Cai H Kong D Cui Q Zhang D Wu G The identificationpreservation and classification of the external branch of the superiorlaryngeal nerve in thyroidectomy World J Surg “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"dysregulation of bcl2 is a pathophysiology observed in haematological malignancies forimplementation of available treatmentoptions it is preferred to know the relative quantificationof bcl2 mrna with appropriate reference genes for the choice of reference genes”i reference genes were selected by assessing variation of genes from rnaseq datasets of haematological malignancies followed by filtering based on their go biological processannotations and proximity of their chromosomal locations to known disease translocationsselected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using genorm normfinder bestkeeper and reffinderii commonly used reference genes were obtained from literature through extensive systematic review levels of bcl2 mrna was assessed by qpcr normalized either by novel reference genes from this study or gapdh the most cited reference gene in literature andcompared the analysis showed ptcd2 ppp1r3b and fbxw9 to be the most unregulatedgenes across lymphnodes bone marrow and pbmc samples unlike the reference genesused in literature bcl2 mrna level shows a consistent higher expression in haematologicalmalignancy patients when normalized by these novel reference genes as opposed togapdh the most cited reference gene these reference genes should also be applicable inqpcr platforms using taqman probes and other model systems including cell lines and rodentmodels absence of sample from healthynormal individual in diagnostic cases call for carefulselection of reference genes for relative quantification of a biomarker by qpcrbcl2 can beused as molecular diagnostics only if normalized with a set of reference genes with stable yetlow levels of expression across different types of haematological malignanciesintroductionoverexpression of bcl2 bcell lymphoma a mitochondrial membrane protein has beenobserved in several haematological malignancies due to genetic and epigenetic mechanismsa1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation dwivedi n mondal s p k s t ssachdeva k bathula c relativequantification of bcl2 mrna for diagnostic usageneeds stable uncontrolled genes as reference one e0236338 101371 pone0236338editor pedro v baptista universidade nova delisboa portugalreceived may accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0236338copyright dwivedi this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0236338 august one 0cfunding the study is funded by glue grantscheme number btpr23078med2912532017by department of biotechnology httpdbtindiagovin govt of india awarded to sd and md thefunders had no role in study design data collectionand analysis decision to publish or preparation ofthe manuscriptcompeting interests the authors have declaredthat no competing interests existbcl2 molecular diagnostics with novel reference genesresulting in evasion of apoptosis giving the malignant cells a longer life span and survival benefits at times of nutrient deficiency hypoxia and growth factor deprivation [“] estimationof level of bcl2 along with other antiapoptotic genes are essential to avail efficient treatmentoptions by rchop regimen of cyclophosphamide doxorubicin vincristine and prednisone and rituximab or venetoclax in different haematological malignancies [ ] byvisualization of chromosomal aberrations using karyotyping or fish fluorescence insituhybridization bcl2 levels can be inferred indirectly detection of expression of bcl2protein by immunohistochemistry a standard pathological testing procedure for dlbcl hasnot been adopted in the clinics for bone marrow tissues of liquid cancers due to sample inconsistency and challenging procedure of capturing low concentrations of biomarkers western blotting for the very nature of the method cannot be adopted for high throughputpathological testing elisa for detection of bcl2 in human plasma remains limited sinceonly one splice isoform of the mitochondrial membrane protein is available in soluble formthus bringing down the effectiveness of the assay bcl2 at the mrna level can be determined without ambiguity by next generation sequencing nanostring and microarray though increasing time and expense of pathological testing in clinical trials relative quantification by qpcr quantitative polymerase chain reactioncan be successfully used due tothe availability of appropriate controls in untreated or normal groups [ ] although beingtime and costeffective it suffers misinterpretation in pathological setting since the relativequantification depends only on the rg reference gene used due to the absence of normalsamplesnormalization with a rg which shows varying expression across samples can often lead towrong s as seen with the use of glyceraldehyde3phosphate dehydrogenasegapdh as rg in gene expression studies of pulmonary tuberculosis and cd8 tcellsunder inactivated or activated condition similarly abl protooncogene abl1 therecommended rg for gene expression studies with leukemic patients was found to haveextremely low expression in neutrophils making it unsuitable as rg for the specific casesuch discrepancies have prompted researchers to analyze gene expression across multiple tissues or pancancer database like tcga to propose normalization factors using multiple rg candidatesthis study through a systematic review of literature in haematological malignancies concluded that mostly conventionally used œhousekeeping genes are still being deployed s1table and s1 fig despite their varied expression based on cell type developmental stage andexperimental conditions with rare exceptions [ ] none of the genes thus identified could be used to relatively quantify bcl2 as molecular diagnostics since compared to thefpkm fragments per kilobase of transcript per million mapped reads value of the antiapoptotic genes across databases s2 fig most of the rgs from the literature are not onlyhigher but also varied significantly s3 and s4 figs with few exceptions inspired by genomewide search for rgs from publicly available rnaseq or microarray data in human and otheranisms [“] we report here a set of novel candidate rgs obtained from an unbiasedsearch of genes in haematological malignancies to be used to normalize bcl2 andother antiapoptotic genes in qpcr as molecular diagnosticsmaterials and methodsethics statementthe study was performed in compliance with ethical practices and was approved by narayanahealth academics ethics committee narayana health hospitals ethics approval numbernhhaeccl2017152a one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genessystematic review of commonly used rgsliterature search was carried out in pubmed databasepubmed as detailed in s5 figaccording to prisma preferred reporting items for systematic reviews and metaanalysesguidelines selection of stable genes proteincoding genes identified from publicly available datasets table using ensembldb annotation package within r statistical software were categorised into four quartiles based on their median expression values across all samples geneswith median expression in middle two quartiles q2 and q3 in all datasets were consideredas q1 and q4 representing extreme ends of the expression spectrum are not preferred as rgcandidates for normalization of molecular diagnostic markersto determine the stability of a gene following statistical measures were employed“i cv �xsx where �x and σx are mean and standard deviation of a variable x respectively and ii normality pvalue as measured by shapirowilks test where a pvalue less than signifies thatthe distribution is away from normal cv although used most frequently isn™t a robustmeasure as it is affected by outliers to solve this a third parameter was used mad medianabsolute deviation medianjx 00 xj where x is the median of x after normalization withmedian mad is a better measure for understanding the spread of the distribution as itdepends on medians a parameter less prone to deviations by outlierslow or comparable statistical variation across samples represented by low values of cvand mad and a normal distribution high value of normality pvalue or low values of “pvalue are characteristics of an ideal rg therefore genes with median expression values inmiddle quartiles q2 and q3 were shortlisted and clustered based on their cv mad and “pvalue normalized to their respective zscores using pam partitioning around medsalgorithm required optimal number of clusters was calculated using silhouette graphicalmethod for each tissue sample the gene cluster with the lowest med value of parameters was selected and the genes at the intersection of the four clusters were shortlisted the list was further filtered by analysing and eliminating genes based on stop words in theirgo gene ontology annotation such as transcription factors nuclear receptor or other nuclearlocalization dna binding activity response to external stimuli translational and transcriptionalactivation since genes with such characteristics regulated by environmental conditions areunsuitable as rg candidates next genes were ranked in ascending order of their mean euclidqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffifficv þ mad2 þ ð1 00 pþ2ean distance d ¼all parameters replaced by their zscores in thisthreeparameter hyperspace for each dataset average of d across four datasets was taken to calculate the mean euclidean distance �d genes with �d median were selected for furthertable list of rnaseq databasesdatasetdiseasetcgalamlamltargetaml paediatric amlgdcdlbcdlbclmmrfmmmultiplemyeloma� both primary and recurrent tumor only 1st visit recordstissuebloodbonemarrowlymphnodesbonemarrowsamples n sourcedownload location� tcga research networkwwwcancergovtcgaschmitz multiple myeloma researchfoundationgdccancergovaboutdatapublicationsdlbclresearchthemmrf fpkm data for gdcdlbc dataset was available as log2 transformed normalized value which was converted to fpkm101371 pone0236338t001 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesanalysis locus of genes associated with pathogenic translocations were identified [ ] andcandidate rgs in close proximity of such loci within bands in the same arm of chromosomewere eliminated by an automated method further only genes with nonzero fpkm value in allsamples from four datasets were retained then each gene was given a composite quartile ranking cqr the sum of quartile indices from each dataset and genes with cqr value median expression in 2nd quartile in at least two datasets were shortlisted s6 figdesign of primersbcl2 primers bcl2 has two known splice isoforms membranebound bcl2α and aless studied soluble bcl2β lacking the transmembrane domain at the ™ cterminal most reported primers amplified only bcl2α or larger amplicon s2 table hence new primers were designed table rg primers primers for shortlisted genes were designed table s3 table using primerbank and idt sample detailsrna was isolated from peripheral blood or bone marrow samples from patient or normalindividuals s7 fig with their informed consent ethics approval number nhhaeccltable primers details of rgs and bcl2primeracy1accession nonm_000666ankrd26nm_014915jmjd4nm_001161465ptcd2nm_0247545ppp1r3bnm_024607fbxw9nm_032301nanpnm_1526673plekhm3nm_0010804753tsga10nm_025244nat1nm_001160174ric8bnm_018157gapdhnm_0012897453bcl2nm_0006572sequence ™ ™fw 'cactgacaaccgctatatccgrv 'ctcatgcagccgttcatcgtfw 'tctcggcaagatccacaaagcrv 'aatgtagagccgtcctgttcafw 'gtctgtcaatgtctgtgggagrv 'caggtgtgtgtcgcagagt3'fw 'tatgggacactgcacatcac3'rv 'ggctgaccatcctcttgttta3'fw 'agaacctcgcatttgagaagac3'rv 'tctgaaccggcataagtgtcc3'fw 'tagggcggtgcgatgattc3'rv 'cggattttggcggactgaga3'fw 'ggtccgcctacttctattaacg3'rv 'tctctgctctccacctacaa3'fw 'gatgatatcagcccagccttag3'rv 'ggacttcctggatcccataaac3'fw 'tactcagcgacaccttgctaa3'rv 'ccagatcattgagggttccac3'fw 'gggagggtatgtttacagcac3'rv 'acatctggtatgagcgtccaa3'fw 'atagtgttcaacagtcagatggc3'rv 'gcaagcgcaagtcaaagca3'fw 'tcgacagtcagccgcatcttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw 'ggaggattgtggccttcttt3'rv 'gcccaatacgaccaaatccgttga3'fw forward primer rv reverse primer101371 pone0236338t002amplicon length bptm ˚camplification factor one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genes2017152a subjects with hepatitis bc or hiv and pregnant or lactating women wereexcluded from the studypbmcbmmc peripheral blood mononuclear cells bone marrow mononuclear cellswere separated by layering of blood or bone marrow diluted to with 1x pbs gibco„¢germany above ficollpaque plus histopaque himedia india followed by centrifugation at rcf for mins with brakes off resultant buffy coat was washed twice with 1x pbs andonce with 1x penstrep himedia india before culturing at cell density of to millioncellsml of rpmi himedia india with fbs gibco„¢ germany brazil origin and1x penstrep for subculturing the lymphocyte populationrna cdna and qpcrfrom ffpe formalinfixed paraffinembedded blocks “ curls were deparaffinized inxylene at ˚c followed by proteinase k himedia india treatment prior to rna isolationeither from lymphocytes or from deparaffinized retrospective samples rna was isolatedby trizol„¢ ambion us method and quantified with qubit rna br assay kit thermofisher scientific us before converting to cdna using superscript iv ssiv thermo fisherscientific us as per manufacturers™ instructions with notemplate control ntc qpcrwas performed in triplicates for each sample using kapa sybr green universal reagentssigma aldrich us cdna dilution and primers in a 5μl reaction mix qpcr condition preincubation at ˚c for minutes followed by amplification for cycles“denaturation at ˚c for sec amplification at ˚c for sec and extension at ˚c for sec inroche lightcycler ii machineoptimization of primersprimers were optimized for qpcr as required by the miqe guidelines all primers wereused at four different final concentrations forwardreverse 200nm200nm 200nm100nm100nm200nm and 100nm100nm with pooled cdna template obtained from six normalhealthy volunteers to yield single amplification product primer efficiency was checked using atwofold fivepoint dilution of the template primer efficiency was obtained from standardcurve using the formula amplication factor ¼ 00� table ��slope 00 stability analysis of candidate rgsmean of cq quantification cycle of ntc were subtracted from cq values of each gene inqpcr experiments to obtain δcq cq sampleˆ’mean cq ntc and relative expression aseˆ’δcq for each replicate where e is the amplification factor of corresponding genestability of expression of the candidate rgs was analysed using three independent algorithms“genorm normfinder and bestkeeper and the webbased reffindertool that integrates all three algorithms plus the delta ct method algorithm genorm wasrun using the slqpcr r package whereas authorsupplied r package and excel worksheet were used for normfinder and bestkeeper analysis respectively mean cq values foreach gene for all samples were used as input for bestkeeper and reffinder whereas fenorm and normfinder relative expression values were used since normfinder uses amodelbased approach to quantify inter and intragroup variations the malignant and nonneoplastic or healthynormal samples were used as two groups for normfinder analysiscomprehensive stability rank of each gene was calculated as the geometric mean of stabilityrank given by each method one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesexpression analysis of bcl2rq relative quantification of bcl2 expression was calculated either as ratio of relativeexpression of bcl2 with relative expression of gapdh or the normalization factor which isgeometric mean of relative expression of three candidate rgsrq ðgapdhþ ¼ e 00 dcqðbcl2þe 00 dcqðgapdhþrq ðproposedþ ¼ e 00 dcqðbcl2þgeo mean e 00 dcqðptcd2 ppp1r3b fbxw9þresults and discussionquantification by qpcr could be the choice of pathology laboratories for a quick and costeffective platform for singlegene expression level with appropriate rg towards this effort macrae performed a genome wide search and statistical analysis using rnaseq datafrom leukemia patients in a more recent pancancer study publicly available geneexpression data from microarray studies were analysed to identify a few rg candidates thatshowed minimal variation between malignant and normal samples and were validated in droplet digital pcr on bone marrow samples of all patients we have used types of haematological malignancy samples encompassing bone marrow pbmc and ffpe blocks along with nonneoplastic bone marrow and healthy pbmc samples subsequent to using much wider publiclyavailable data from samples in aml dlbcl and multiple myeloma databases furtherwe have employed an improved statistical analysis including clustering technique described inmethods section instead of an ad hoc approach of selection of top few genes from the clusterswe used important biological considerations to further prune the list of candidate rgssystematic review of commonly used rgs from literaturesystematic review of s yielded rgs used in haematological malignancies througha selection of genes by different analysis methods s4 table and b usage of known rgs inqpcr s1 table fpkm values of all these rgs when examined in public databases showedvaried expression among different types of haematological malignancies s3 and s4 figs withmaybe the exception of pggt1b however since other genes selected in the literatureshowed higher expression and correlated extreme variation we could not depend on the assayand proceeded to select novel rgs with an unbiased approachselection of candidate rgsstatistical analysis stepwise filtration of the number of genes from each dataset is summarized in s6 fig and also in graphical abstract fig shows gene clusters plotted in cv normalized mad and 1pvalue hyperspace for four datasets cluster marked in green in eachfigure represents the cluster with least med value s5 table for the three parametersselected clusters in the four datasets had an overlap of genes indicating large number ofgenes involved in housekeeping processes and hence showing lesser intersample variationacross diverse datasets common genes were pruned further to by go biological processterm filtration disease association and cqr to lead to a final of genes s6 table that weretaken through experimental validation melt curve analysis and efficiency check with pooledcdna from six healthy volunteers narrowed it down to genes with stable median expression and single amplification product of expected size for each table primers for geneswhich did not qualify the efficiency check were eliminated as they failed to show single amplification peak after repeated trials with new experimental conditions and even new primersequences s3 table one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig statistical analysis of candidate genes genes plotted in the cv normalized mad and “pvalue hyperspace for the fourdatasets a tcgalaml b targetaml c gdcdlbc and d mmrfmm cluster shown in green represents the chosencluster with least value of meds101371 pone0236338g001expression of genes with efficient primers were analysed on samples by qpcr usingobserved cq values preliminary stability analysis of the genes were done with online reffinder tool to select top stable genes ptcd2 ppp1r3b fbxw9 nanp ric8b jmjd4plekhm3 nat1 ankrd26 tsga10 as rg candidatessssstability analysis of candidate rgs results of bestkeeper algorithm used independentlyor as part of reffinder were comparable whereas results of genorm or normfinder analysisdiffered as they used different inputs geometric mean of stability ranks assigned in each algorithm was used to create comprehensive stability ranking of all the candidate rgs s7 tableand fig the analysis shows ptcd2 ppp1r3b and fbxw9 to be most stable across all analysed patient samplesptcd2 pentatricopeptide repeatcontaining protein codes for a mitochondrial proteininvolved in rna binding maturation and respiratory chain function though its exact one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig stability rank of candidate reference genes101371 pone0236338g002molecular function is not well understood [ ] ppp1r3b protein phosphatase1 regulatorysubunit3b encodes for a catalytic subunit phosphatase regulatory subunit 3b which isinvolved in hepatic glycogen dysregulation in type diabetes [“] fbxw9 fboxwdrepeatcontaining protein is a cytosolic protein involved in ubiquitination and proteasomedegradation expression analysis of bcl2accurate determination of bcl2 expression among few antiapoptotic markers in patients withhaematological malignancies is emerging as a critical diagnostic test for clinicians to suggest efficacious therapy options fpkm values of rgs common and novel from the publicly availabledatabases when compared fig with bcl2 indicated the novel rgs to be better normalizationcandidate for bcl2 in qpcr assays in pathology labs due to less and stable expressioncomparison of relative expression of gapdh versus the proposed normalization facteometric mean of relative expression of the three rg candidates clearly show a large variation in gapdh expression “ across malignant samples fig 4a s8 table granted itspopularity the expression stability of gapdh has been proven to differ in different conditionsdue to its involvement in apoptotic cell death through ubiquitin ligase membrane trafficking upregulation in aml involvement in nonhodgkin™s bcell lymphomas and inconsistency in several other cancers on the other hand proposed rgs havelesser variation “ and their expressions are consorted with each other making them better candidate as rg compared to gapdh this behaviour is translated to bcl2 expressionrq in malignant samples when normalized with gapdh fig 4b evidently normalizationwith gapdh underestimates relative quantification of bcl2 compared to normalization withproposed rgs with a statistically significant difference in median values p wilcoxonrank sum test between the two schemes bcl2 quantification in haematological malignanciesby qpcr is overtly reliant on rg since availability of œadjacent normal sample is ruled outabove results clearly demonstrate how the quantification may go off limit due to a wrongchoice of rg one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig candidate reference genes in hematological malignancy datasets expression values of candidate genes in four datasets a tcgalamlb targetaml c gdcdlbc and d mmrfmm101371 pone0236338g003broader applicability of proposed reference genesthough primary objective of this study is to discover rg candidates for bcl2 diagnostics in aclinical setting the rgs may have broader utility in other experimental platforms or modelsystems in the systematic review we found a number of research s [“] that haveused taqman probes instead of sybr green whereas our validation experiment was carriedout using sybr green probes however studies in different contexts such as a tropical oilseedplant or measurement of expression of various adenosine receptors in breast cancer tissue and in experiments using human reference rna sybr green pcr assays wereobserved having fair concordance with taqman pcr from these evidences we believe thatstability of proposed rgs is not likely to differ between sybr green and taqman qpcr assaysto assess variation of these stable rgs in cell lines we analyzed rpkm values of proteincoding genes across cell lines of haematopoietic and lymphoid tissue origin frombroad institute cancer cell encyclopedia and found the proposed rgs presenting muchlesser variations in expression compared to the common rgs gapdh abl1 b2m gusband actb in cell lines as well s8 figboth transgenic and wild type and occasional rat models are widely used in leukemia andlymphoma research [ ] usability of rgs common between clinical and animal studies one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesfig relative expression of chosen reference genes and relative quantification of bcl2 a relative expression of chosen reference genes solidlines and gapdh dashed line across patient samples b relative quantitation of bcl2 expression with respect to the candidate reference genesand gapdh in malignant patient samples101371 pone0236338g004will thus be of immense advantage we find that the proposed rgs“ptcd2 ppp1r3b andfbxw9 have “ sequence similarity and identity with corresponding genes in mice andother commonly used rodent models s9 table suggesting the genes playing similar role incellular function thereby displaying stability similar to that in humans hence normalizationfactor derived from the expression of these rgs may be applicable in murine and other rodentmodels as well with suitable design of primers encompassing conserved regionsbeyond detection of gene expression at mrna level it may be worthwhile to explore theapplicability of protein counterpart of the stable rgs in western blot as control for proteindetection by design we have chosen rgs that are of moderate expression level in middlequartiles of expression among other genes and they may not be detectable by western blotunless a larger amount of sample is loaded which is often not feasible with clinical sampleshowever it may be an interesting proposition to predict stable reference proteins for use inwestern blot by statistical analysis of proteomics data and associated systematic review ofliterature one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesour results indicate that genes ptcd2 ppp1r3b and fbxw9 render more reliability toqpcrbased diagnostic test of bcl2 in haematological malignancies the can beextended to other biomarkers in liquid cancer as well as for research with other model systemssuch as cell lines and rodentssupporting informations1 table list of reference genes in literaturedocxs2 table list of bcl2 primers from literaturedocxs3 table list of unqualified primersdocxs4 table literature explaining analysis and selection of reference genedocxs5 table zscore med valuesdocxs6 table list of selected genesdocxs7 table individual and combined stability rank and scores of candidate reference genesdocxs8 table relative expression of gapdh and the proposed normalization factordocxs9 table sequence similarity and identity with corresponding genes in mice rat andguinea pigdocxs1 fig rgs found in literature with more than one citationtiffs2 fig fpkm values of bcl2 family of antiapoptotic genes in the four datasetstiffs3 fig fpkm values of rgs found in relevant literature with more than one citationtiffs4 fig fpkm values of rgs found in relevant literature with a single citationtiffs5 fig workflow according to prisma guidelines for systematic review for commonlyused reference genestiffs6 fig statistical analysis workflowtiffs7 fig patient samples used in the studytiff one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference geness8 fig variation in stable rgs in cell lines and animal modeltiffs1 graphical abstracttiffacknowledgmentsauthors acknowledge prof joy kuri chair department of electronic science and engineering indian institute of science bangalore for providing the computational resourcesauthor contributionsconceptualization sujan k dhar manjula dasdata curation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdeva christopher bathula vishnupriyan kformal analysis sujan k dharfunding acquisition sharat damodar manjula dasinvestigation nehanjali dwivedi sreejeta mondal smitha p k sowmya tmethodology nehanjali dwivedi sreejeta mondal smitha p k sowmya t vishnupriyank manjula dasproject administration manjula dasresources nataraj k s sharat damodarsoftware sujan k dharsupervision manjula dasvalidation nehanjali dwivedi sreejeta mondal smitha p k sowmya t kartik sachdevachristopher bathula vishnupriyan kvisualization manjula daswriting “ original draft sreejeta mondal sujan k dharwriting “ review editing nehanjali dwivedi sreejeta mondal smitha p k sujan kdhar manjula dasreferences perini gf ribeiro gn pinto neto jv campos lt hamerschlak n bcl2 as therapeutic target forhematological malignancies vol of hematology and oncology biomed central ltd gratiotdeans j merino r nuñez g turka la bcl2 expression during tcell development early lossand late return occur at specific stages of commitment to differentiation and survival proc natl acad sciu s a oct “ 101073pnas912210685 pmid merino r ding l veis dj korsmeyer sj nuñez g developmental regulation of the bcl2 protein andsusceptibility to cell death in b lymphocytes embo j feb “ pmid li l li y que x gao x gao q yu m prognostic significances of overexpression myc andorbcl2 in rchoptreated diffuse large bcell lymphoma a systematic review and metaanalysis scirep “ 101038s41598017177655 uchida a isobe y asano j uemura y hoshikawa m takagi m targeting bcl2 with venetoclaxis a promising therapeutic strategy for œdoubleproteinexpression lymphoma with myc and bcl2 one 101371 pone0236338 august one 0cbcl2 molecular diagnostics with novel reference genesrearrangements haematologica jun “ 103324haematol2018 pmid baro´ c espinet b salido m garcı´a m sa´nchez b florensa l cryptic ighbcl2 rearrangementswith variant fish patterns in follicular lymphoma leuk res feb “ 101016jleukres201009011 pmid hofman p heeke s alixpanabières c pantel k liquid biopsy in the era of immunooncology is itready for primetime use for cancer patients suppressed immune microenviron repert brain metastases from patients with resected nsclc “fatani s h mukhtar m h ali a s correlation between serum antiapoptotic bcl2 level and its immunohistochemical expression in relation to apoptosis in gastric cancer j mol biomark diagn albitar m zijun xy wang y manman d tzankov a visco c myc and bcl2 mrna expressionas determined by ngs predicts survival in dlbcl in gcb but not in abc subgroup blood nov 134supplement_15092“ derenzini e rossi a agostinelli c rossi m melle f motta g integration of nanostring profilingand functional characterization of oxidative and replicative stress biomarkers identifies poor prognosis mycbcl2 positive diffuse large bcell lymphoma subsets providing opportunities for precisiontherapies blood nov 132supplement “zhang f yang b zhang k hou ml lu xc li yx ccnd1bcl2 gene network a direct target of amifostine in human acute megakaryocytic leukemia cells chem biol drug des may “101111cbdd12889 pmid patel vm balakrishnan k douglas m tibbitts t xu ey kutok jl duvelisib treatment is associated with altered expression of apoptotic regulators that helps in sensitization of chronic lymphocyticleukemia cells to venetoclax abt199 leukemia sep “ 101038leu2016382 pmid bomben r ferrero s d™agaro t dal bo m re a evangelista a a bcell receptorrelated genesignature predicts survival in mantle cell lymphoma results from the fondazione italiana linfomi mcl trial haematologica apr “ 103324haematol2017184325pmid dheda k huggett jf chang js kim lu
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complex disease caused by coordinated alterations of multiple signaling pathways TheRasRAFMEKERK MAPK signaling is one of the bestdefined pathways in cancer biology and its hyperactivation isresponsible for over human cancer cases To drive carcinogenesis this signaling promotes cellular overgrowthby turning on proliferative genes and simultaneously enables cells to overcome metabolic stress by inhibitingAMPK signaling a key singular node of cellular metabolism Recent studies have shown that AMPK signaling canalso reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components RAFKSRfamily kinases which affects not only carcinogenesis but also the outcomes of targeted cancer therapies againstthe MAPK signaling In this review we will summarize the current proceedings of how MAPKAMPK signalingsinterplay with each other in cancer biology as well as its implications in clinic cancer treatment with MAPKinhibition and AMPK modulators and discuss the exploitation of combinatory therapies targeting both MAPK andAMPK as a novel therapeutic interventionKeywords RasRAFMEKERK signaling AMPK signaling Interplay Tumorigenesis Cellular metabolism RAFMEKERKinhibitors AMPK inhibitors AMPK activators Autophagy Targeted therapyIntroductionThe RasRAFMEKERK MAPK signaling is a fundamental pathway in cell biology and its alteration causeshuman cancers or developmental disorders Given its crucial roles in physiology and pathology this pathway hasbeen extensively studied for over two decades Unfortunately the regulation of MAPK signaling remains ambiguous till now by virtue of its intrinsic complexity anddiverse crosstalks with other signalings Here we focus onthe complicated interplays between the MAPK and theAMPK signalings in cellular carcinogenesis and their implications in current targeted cancer therapies We hopethis review would provide a conceptual framework for Correspondence yuanjiminszhospitalcom hujianchengnccscomsg1Department of Urology Shenzhen People™s Hospital The Second ClinicalMedical College Jinan University The First Affiliated Hospital SouthernUniversity of Science and Technology Shenzhen Guangdong China4Cancer and Stem Cell Program DukeNUS Medical School College RoadSingapore SingaporeFull list of author information is available at the end of the developing more effective therapeutic approaches againsthyperactive MAPK signalingdriven cancersThe RasRAFMEKERK MAPK signaling and itsaberrant activation in cancersThe RasRAFMEKERK MAPK signalingThe RasRAFMEKERK MAPK mitogenactivated protein kinase signaling is a central pathway that regulatescellular proliferation differentiation and survival Thissignaling pathway was discovered in the 1970s“1980swhen Ras small GTPases were identified as first oncogenes from sarcoma viruses [“] Later studies on viraloncogenes had also led to the discovery of a Nterminaltruncated version of RAF SerThr kinase RAF1 or CRAF[“] In contrast the other two components of this signaling pathway MEK mitogenactivated protein kinasekinase and ERK mitogenactivated protein kinase wereidentified as cytoplasmic protein kinases activated by mitogens in the 1990s [“] Following these discoveries 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 0cYuan Journal of Hematology Oncology Page of RAF was identified as the upstream kinase of MEK in and the first direct effector of Ras in [ ]resulting in the delineation of the whole MAPK signalingpathway which is considered as a milestone in our understanding of how cell senses external stimuliThe first component of MAPK signaling Ras smallGTPases have three gene isoforms Hras Kras and Nras that encode four proteins with splicing isoforms ofKras giving rise to Kras4A and Kras4B Although allRas proteins possess highly homologous sequences theyhave quite different activities tissue expression patternsand effector preferences which lead to their differentialphysiological and pathological functions [“]The downstream of Ras small GTPases is the RAFMEKERK kinase cascade [] The first kinases in thiscascade RAFKSR kinase suppressor of Ras family kinases include three RAF isoforms ie CRAF BRAF andARAF and two close pseudokinases ie KSR1 and KSR2All RAF isoforms have highly homologous sequences andsimilar structures with three conserved regions conservedregion CR1 contains RASbinding domain RBD anda Cysrich domain [ ] conserved region CR2 ischaracterized by a SerThrrich sequence conserved region CR3 comprises of a putative kinase domain with aNterminal acidic motif NTA [“] and a Cterminalregulatory tail [“] Nevertheless RAF isoforms havevariable kinase activities with an order as BRAFCRAFARAF likely by virtue of their distinct NTA motifs andAPE motifs that contribute to the dimerizationdriventransactivation of RAFs [“] In contrast to RAF isoforms KSR proteins replace the RBD at the Nterminusfused sterile αmotif and Prorichwith a coiledcoilstretch that are responsible for recruiting proteins to theplasma membrane upon stimulation and lack the catalyticlysine in VAIK motif of kinase domain which impairs theircatalytic activity [ ] Given their associations withMEK and ERK as well as low kinase activity KSR proteinshave been thought as scaffold proteins in a long termHowever recent studies have indicated that KSR proteinscan also function as allosteric activators to stimulate thecatalytic activity of RAF proteins through dimerization[ “] The sidetoside dimerization of RAFKSRfamily kinases is critical not only for their activation butalso for their catalytic activity towards downstream kinases [ “] MEKs MEK1 and MEK2 are the second kinases of the RAFMEKERK kinase cascade whichhave both redundant and nonredundant functions [] These two dualspecific kinases comprise a shortregulatory Nterminus and a canonic kinase domain TheNterminal regulatory region of MEK12 contains a docking site for substrate ERKs a nuclear export sequence thatcontrols the cytoplasmicnuclear shuttling of proteins anda negative regulatory sequence that forms a helix andlocks kinase in an inactive conformation [ ]Further through its kinase domain MEK12 forms a facetoface heterodimer with RAFKSR or a homodimerheterodimer with itself which is indispensable for its activation stimulated by RAF and for its activity towards ERKs[ ] Like MEKs the terminal kinases of MAPKsignaling ERKs also include two highly homologousmembers ERK1 and ERK2 which have a central kinasedomain flanked by short N and Cterminal tails Thesetwo isoforms also have redundant functions albeit different expression patterns [“] However unlike RAFs andMEKs that have very limited substrates ERKs recognizeand phosphorylate numerous substrates that include transcription factors protein kinases and phosphatases andother functional proteins [“]It should be noted that active Ras also turns on othersignaling pathways such as PI3KAKTmTORC whichregulate different cellular functions [] In this reviewwe focus only on the MAPK signaling given its dominant role in cancer biologyHyperactive RasRAFMEKERK MAPK signaling incancersThe MAPK signaling plays a crucial role in cell biologyand is tightly regulated in normal cells Upon engagement of receptor tyrosine kinases RTKs or other stimulations Ras small GTPases are activated by GTPGDPexchange factors GEFs which in turn recruit RAFMEK complexes to the plasma membrane and triggerthe RAFMEKERK kinase cascade through facilitatingRAFRAF or KSR RAFMEK and MEKMEK interactions as well as subsequent phosphorylations [] ActiveERKs are further translocated into the nuclei or stay inthe cytoplasm where they phosphorylate a number ofsubstrates that regulate cell functions [“ ]On the other hand active MAPK signaling also turns onsome negative feedback loops which help cells return toquiescent status [“] An aberrant activation ofMAPK signaling frequently induces human cancers ordevelopmental disordersthough an extremely highMAPK signaling may induce cell death or senescenceunder some conditions [“]its upstream activators or componentsHyperactive MAPK signaling exists in over ofcancers which is caused directly by genetic alterationsofincludingRTKs Ras and BRAF or indirectly by those independent of Ras or RAF [“] and significantly promotesdisease progression [] Since genetic alterations ofRTKs in cancers have been extensively reviewed in recent years [“] here we focus on oncogenic mutations of Ras and BRAF As a small GTPase Ras cyclesbetween active GTPbound status and inactive GDPbound status which is regulated by GEFs and GTPaseactivating proteins GAPs Oncogenic Ras mutationscan be mainly classified into two groups mutations 0cYuan Journal of Hematology Oncology Page of on glycine or G1213 that impair GAP associations and mutations on glutamine Q61 that diminish the intrinsic GTPase activity of Ras [] both ofwhich lead to an extended halflife of GTPloaded RasOncogenic Ras mutations have both isoform andcancertype preferences Kras is mostly mutated in allcancers followed by Nras and Hras and its mutations prevailin pancreatic cancers whilethose of Nras in myeloma and melanomas and Hrasin adrenal gland cancers [ ] This phenomenon mayreflect underlying fundamental signaling landscapes andRAS mutants interplay with these landscapes As thedownstream effector of Ras RAF is another dominanttarget of oncogenic mutations in the MAPK signalingpathway Similarly RAF mutations have isoform preference in cancers as Ras mutations with BRAF CRAF ARAF which may arise from their different basal activities Overall a single point mutation that converts Val into Glu in the activation loop of BRAF accounts for cases [] Although BRAF V600E exists only in of all cancers it is highly prevalent in some tissuespecific cancers such as melanoma thyroid cancer and histiocytosis [“] albeit theunderlying molecular mechanisms remains unknown Incontrast to Ras and RAF MEK and ERK have rare mutations in cancers though their mutations have been shownto be responsible for some RAF inhibitor RAFiresistantcases in current cancer therapies [“]Targeting the RasRAFMEKERK MAPK signalingpathway for cancer therapy promising but challengingGiven their high prevalence in cancers great efforts havebeen made to develop specific inhibitors against oncogenicRas and RAF mutants in the last decades These inhibitorsthat have been approved for clinic treatment of RasRAFmutated cancers or under clinical trials are listed in Table However none of these inhibitors can effectively target thelarge portion of Ras mutants in cancers Since having no attractive docking sites suitable for designing highaffinity andselective small molecule inhibitors Ras mutants have beenthought as œundruggable cancer drivers in a long term Untilrecently a group of covalent small inhibitors that are dockedinto a previously unknown pocket of GDPbound Ras andare linked to the adventive cysteine of RasG12C have beendeveloped and achieved encouraging outcomes for treatingRasG12Cdriven cancers as a single agent in clinical trials[“] Fig To further enhance their efficacy theseRasG12C inhibitors are also undergoing clinical evaluationwhen combined with SHP2 Src homology region domaincontaining phosphatase2 inhibitors that block the pathwayreactivation caused by the relief of negative feedback loops[ ] Clinical Trial NCT04330664 In addition these inhibitors have also been further developed into RasG12C degraders by conjugating with ligands of ubiquitin E3 ligaseswhich effectively deplete Ras mutant proteins in cancer cells[ ] though their efficacy in vivo remains unknown Unlike RasG12C the majority of Ras mutants remain œundruggable at present []It has been shown that Ras activates downstream effectors through direct interactions Therefore disruptingRaseffector interactions might be an alternative approach that can effectively block cancer growth drivenby Ras mutations Such a type of small moleculeblockers include rigosertib sulindac and MCP110 andamong which the therapeutic efficacy of rigosertib combined with nivolumab for Rasmutated cancers is beingdetermined by phase III clinical trials currently []Clinical Trial NCT04263090 Howeverit has to benoted that these inhibitors impair the MAPK signalingin both Rasmutated cancers and normal tissues andthereby their therapeutic index may not be highGenetic studies have revealed that the ablation of theRAFMEKERK kinase cascade but not other effectorpathways is a most efficient approach to inhibit thegrowth of Rasmutated cancers [] which leads to extensive developments of specific inhibitors against thiskinase cascade for treating Rasmutated cancers Moreover these inhibitors should be also effective for treatingRAFmutated cancers Indeed a number of RAFMEKERK inhibitors have been developed and applied to clinical trials for treating RasRAFmutated cancers [ “] At present three RAF inhibitors and three MEKinhibitors have been approved to treatlatestageBRAFV600Eharboring cancers as a single agent or incombination with other chemotherapeutics and exhibited excellent efficacies [ “] Fig HoweverRasmutated cancers possess intrinsic resistance to bothRAF and MEK inhibitors [] and even BRAF V600Eharboring cancers develop acquired resistance after “months treatment [ ] Mechanistic studies haveshown that active Ras facilitates the RAF dimerizationon plasma membrane which leads to both intrinsic andacquired resistance to RAF inhibitors [“] Toovercome the drug resistance arising from enhancedRAF dimerization the secondgeneration RAF inhibitorssuch as PLX8394 BGB283 TAK580 and CCT3833have been developed and are undergoing clinical evaluations Clinical Trials NCT02428712 NCT02610361NCT03905148 NCT02327169 NCT02437227 Thesenovel RAF inhibitors reduce the RAF dimerizationdriven resistance through distinct mechanismsPLX8394 and BGB283 impair RAF dimerization uponloading on RAF proteins [“] TAK580 bindsto and inhibits both protomers in RAF dimers [] CCT3833 inhibits both RAF and upstream kinases ofRas and thereby prevents the activation of Ras by the relief of negative feedback loops [ ] Besides thesesecondgeneration RAF inhibitors a unique RAFMEK 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancersTargetKRasG12CDescriptionPhase I results showed ORR of nonsmall cell lung cancer NSCLC harboring KRas G12CCompoundAMG510Development stagesPhase IIINCT04303780MRTX849JNJRigosertibRasPhase IIINCT03785249Phase IIINCT04330664Evaluation of clinical activity of MRTX849 alone and combined with TNO155SHP2 inhibitor in KRas G12C mutated cancersPhase I NCT04006301 Safety and PK of JNJ74699157Phase IIINCT04263090Evaluation of safety and clinical efficacy of Rigosertib plus Nivolumab PD1 Abin KRas mutated NSCLCBRAFVemurafenib ApprovedLatestage or unresectable melanoma expressing BRAF V600E in ErdheimChester disease ECD with BRAF V600E mutation in DabrafenibApprovedEncorafenibApprovedLatestage or unresectable melanoma expressing BRAF V600E in Combination with trametinib for the treatment of unresectable or metastatic melanoma withBRAF V600EK in Combination with trametinib for the treatment of metastatic NSCLC with BRAF V600E in Combination with trametinib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with trametinib for the treatment of anaplastic thyroid cancer ATC that cannotbe removed by surgery or has spread to other parts of the body with BRAF V600E in Combination with binimetinib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Combination with cetuximab EGFR Ab for the treatment of metastatic colorectal cancerwith BRAF V600E in PLX8394BGB283TAK580Phase IIINCT02428712PLX8394 with cobicistat CYP3A inhibitor was well tolerated and showed promising activityin BRAFmutated refractory cancersPhase I NCT02610361Phase IIINCT03905148Evaluation of safety and PK of BGB283 alone and combination with mirdametinibPhase I NCT02327169Phase I NCT03429803TAK580 is the inhibitor of BRAF V600E and dimersTreatment in pediatric lowgrade gliomaCCT3833Phase I NCT02437227 CCT3833 is a panRAF inhibitor of mutant BRAF CRAF and SRC kinasesRAFMEKRO5126766Phase I NCT00773526Phase I NCT03681483Phase I NCT03875820Phase I NCT02407509RO5126766 is a dual inhibitor for both RAF and MEKTreatment of advanced KRasmutant lung adenocarcinomasEvaluation of safety and PK of RO5126766 with VS6063 FAK inhibitor or everolimusmTOR inhibitorRO5126766 showed activity across Ras and RAFmutated malignancies with significantresponse in lung and gynecological cancersMEK12TrametinibApprovedCobimetinib ApprovedPhase IIINCT03989115BinimetinibApprovedSelumetinibApprovedMirdametinib Phase IINCT03962543Phase IINCT02022982Phase IIINCT03905148A singleagent oral treatment for unresectable or metastatic melanoma with BRAFV600EK in Combination with dabrafenib for the treatment of unresectable or metastatic melanomawith BRAF V600EK in Combination with dabrafenib for the treatment of metastatic NSCLC with BRAF V600E in Combination with dabrafenib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with dabrafenib for the treatment of ATC that cannot be removed by surgeryor has spread to other parts of the body with BRAF V600E in In combination with vemurafenib to treat advanced melanoma with BRAF V600EK in Doseescalation of combination of RMC4630 SHP2 inhibitor and cobimetinibCombination with encorafenib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Selumetinib was approved for neurofibromatosis type with symptomatic inoperable plexiformneurofibromas according to NCT01362803Evaluation of mirdametinib in the treatment of symptomatic inoperableneurofibromatosis type1 NF1associated plexiform neurofibromas PNsCombination of mirdametinib with palbociclib in the treatment of KRasmutant nonsmall cell lung cancer NSCLCEvaluation of safety and PK of BGB283 alone and combination with mirdametinibSHR7390Phase I NCT02968485 Evaluation of safety and PK of SHR7390 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancers ContinuedTargetDescriptionEvaluation of safety and PK of CS3006CompoundCS3006Development stagesPhase I NCT03516123Phase I NCT03736850ERK12UlixertinibMK8353LY3214996ASTX029ATG017KO947Phase IIINCT01781429Phase I NCT04145297Phase IINCT03698994Phase I NCT03454035Phase I NCT01358331Phase I NCT03745989Phase I NCT02972034Phase I NCT04081259Phase I NCT04391595Phase I NCT02857270Phase IINCT04386057Phase IIINCT03520075Responses to ulixertinib in NRas BRAF V600 and nonV600 BRAF mutant cancersEvaluation of ulixertinib alone or combined with hydroxychloroquine palbociclibCDK46 inhibitor in MAPK mutated cancersMK8353 was optimized from SCH772984 for better pharmacokinetics and exhibitedinhibition of BRAF V600 mutant cancersEvaluation of combination of MK8353 with selumetinib or pembrolizumab PD1 Abin advanced malignanciesEvaluation of treatment of MK8353 alone or combined with abemaciclibCDK46 inhibitorHydroxychloroquine in advanced malignanciesEvaluation of safety and PK of ASTX029Phase I NCT04305249 Evaluation of safety and PK of ATG017Phase I NCT03051035 Evaluation of safety and PK of KO947dual inhibitor RO5126766 has been developed and exhibited a strong potential against both Ras and RAFmutated cancers in phase I clinical trials [“] Thisallosteric inhibitor docks on MEK and prevents the release of MEK from RAF as well as the subsequent phosphorylation of MEK by RAF [] which gives it muchmore advantages than all other known RAF inhibitorsaccording to the regulatory mechanism of the RAFMEKERK kinase cascade [] As to small molecule inhibitors that target the terminal kinase ERK although anumber of them have been developed and are undergoing clinical trials [ ] their therapeutic values fortreating RasRAFmutated cancers remain unknownLike MEK inhibitorsthese ERK inhibitors may notachieve a good therapeutic index as single agents byvirtue of their inhibitory role in both malignant and normal tissues However they may contribute to antiRasRAF cancer therapy as synergetic agents combined withRasRAF inhibitorsOveralltargeting hyperactive MAPK signaling hasachieved exciting outcomes for treating RasRAFmutated cancers However although some effective smallmolecule inhibitors have been developed and applied toclinical treatment drug resistance and side effects remain remarkable challenges and there is still a long wayto develop a longeffective approach with manageableside effects for treating RasRAFmutated cancersAlthough hyperactive MAPK signaling has a dominantrole in cancer biology it is finetuned by other signalingssuch as PI3KAKTmTORC and AMPK during diseaseprogression [] These signaling interplays have important impacts on both cancer progression and clinicaltreatment based on MAPK inhibition In this review wewill focus on the crosstalk between MAPK and AMPKsignalingsAMPK signaling and its roles in cancer biologyAMPK signaling and cellular metabolismAMPK AMPactivated protein kinase is an energy sensorthat monitors the AMPADPATP ratio in eukaryotic cellsThis atypical protein kinase was firstly discovered as acontaminant during the purification of acetylCoA carboxylase ACC a wellstudied substrate of AMPK for fattyacid FA synthesis nowadays [“] Fig Howeverthe phosphorylation of ACC by AMPK in response to thehigh AMPATP ratio had not been revealed until a decadelater [] and the enzyme was thus named as AMPKthereafter [] Fig Biochemical studies have shownthat AMPK consists of three subunits including the catalytic α subunit and the regulatory β and γ subunits [“] Fig In mammals AMPK subunits are encoded asseveral isoforms α1 α2 β1 β2 γ1 γ2 γ3 which arepreferentially expressed in specific tissues or anisms[ ] For instance the α2 subunit associatesonly with β1 in type I muscle fibers while it binds to bothβ1 and β2 in type II muscle fibers [ ] Also theliver formulation of AMPK subunits differs among speciesas that α1β2γ1 is dominant in human whereas α1β1γ1and α2β1γ1 in dog and rat respectively [] Althoughan isoform replacement of AMPK subunits may not extensively affect the basal activity of AMPK as adaptive reit alters AMPK™ssponses such as exercise do []subcellular locations and sensitivity as well as interactionswith other signaling pathways [] The anismtissue 0cYuan Journal of Hematology Oncology Page of Fig Target hyperactive RasRAFMEKERK MAPK signaling for cancer therapy The RasRAFMEKERK MAPK signaling functions downstream ofreceptor tyrosine kinases RTKs Upon engagement by their ligands RTKs activates guanine exchange factors Sos proteins which load GTP to RasGTPases Then GTPbound Ras GTPases recruit RAFMEK heterodimers in cytosol to plasma membrane where they form transient tetramers throughthe sidetoside dimerization of RAFs The RAF dimerization not only turns on RAFs but also loosens RAFMEK heterodimerization and facilitates MEKhomodimerization on RAF dimer surface which leads to the activation of MEKs by RAFs Once MEKs are activated they phosphorylate ERKs and thenactive ERKs phosphorylate a number of downstream effectors In cancer cells hyperactive RasRAFMEKERK MAPK signaling arising from geneticmutations of Ras GTPases and BRAF can be targeted by small molecular inhibitors of Ras G12C BRAFV600E MEK and ERKstagespecific selectivity of subunit isoforms complicatesAMPK™s regulationAs a key sensor of cellular energy stress the activity ofAMPK is predominantly regulated by cellular AMPADPATP that competitively binds to the γ subunit of AMPKand thus promotes or inhibits the phosphorylation ofThr172 on α subunit by the tumor suppressor liver kinaseB1 LKB1 or the dephosphorylation of this site by phosphatases [ ] Fig Besides adenine nucleotidesintracellular calcium ions activate AMPK through calciumcalmodulindependent protein kinase kinase CAMKK2 also called CAMKKβFig which acts downstream of the hormoneactivated receptors such as muscarinic receptors and ghrelin receptor onendothelial cells or neuron cells [“] On the otherhand AMPK can be inhibited by a metabolite of glucosefructose 16bisphosphate FBP which binds to the aldolase and prevents the interaction of AMPK with LKB1 inglucoserich environments [] Fig Active AMPK[“]has more than downstream substrates that regulatethe metabolism of lipids cholesterol carbohydrates andamino acidsActive AMPK promotes the oxidation of fatty acidsand inhibits the synthesis of fatty acids and cholesterolwhich involves largely in acetylCoA AMPK phosphorylates and inhibits HMGCoA reductase HMGR that requires acetylCoA in its reduction reaction [ ] Fig Also AMPK phosphorylates ACC that converts acetylCoA to malonylCoA and therefore slowsdown the de novo fatty acid FA synthesis and increasesthe FA oxidation [] Fig Alternatively AMPK regulates the lipid metabolism through altering the mitochondria structure and function In the mitochondriaAMPK phosphorylates Akinase anchoring protein AKAP1 a key scaffold protein for protein kinase APKA and facilitates the phosphorylation of a mitochondriafusion factor dynaminrelated protein DRP1 by PKA which promotes mitochondrial fusion 0cYuan Journal of Hematology Oncology Page of Fig AMPK signaling and its downstream effectors AMPK is activated by liver kinase B1 LKB1 or calciumcalmodulindependent protein kinasekinase CAMKK2β through phosphorylation on Thr172 of α subunit and is inactivated through dephosphorylation of this site by proteinphosphatases in response to changes of cellular AMPADPATP ratio Downstream effectors activated by AMPK are indicated as arrows and thoseinhibited by AMPK are shown as barheaded linesand oxidative phosphorylation [] Moreover AMPKaccelerates the mitochondria biogenesis likely throughphosphorylating and activating the transcriptional activator proliferatoractivated receptor gamma coactivator1alpha PGC1α [ ] Fig However upon energy stress AMPK plays an opposite role in mitochondria biology Under this condition AMPK is essential forthe fragmentation of mitochondria AMPK phosphorylates mitochondrial fission factor MFF on Ser129 andthereby facilitates the translocation of DRP1 from cytosol to mitochondria membrane in energy stressdrivenmitochondria fission [ ] Then AMPK promotesthe clearance of damaged mitochondria through autophagy In this process AMPK binds directly to and phosphorylates the unc51like autophagy activating kinase ULK1 Autophagyrelated gene ATG9 and Beclin which triggers the autophagosome formation [“] Fig Active AMPK directly regulates the carbohydrate metabolism or indirectly through altering the fatty acid metabolism as described above Activation of AMPKstimulates the expression and plasma membrane translocation of solute carrier family member GLUT proteins and thereby facilitates glucose import [ “] Fig Intracellularly AMPK phosphorylates andactivatesis6phosphofructo2kinasePFK2thatfructose 26bisphoresponsible for the synthesis ofsphate a potent stimulator of glycolysis and thus accelerates glycolysis []Fig Furthermore AMPKappears to phosphorylate and inhibit glycogen synthasein the liver which dampens glycogen synthesis and thusindirectly enhances glycolysis []Active AMPK maintains cellular amino acid homeostasis mainly by controlling the activity of mammaliantarget ofrapamycin complex mTORC1 ThemTORC1 is a central sensor of cellular amino acids thatsamples amino acids in both cytosol and lysosome [] Upon activation by amino acids mTORC1 stimulates protein synthesis by phosphorylating ribosomalprotein S6 kinase B1 S6K and eukaryotic translationinitiation factor 4E binding protein 4EBP1 whichenhances the consumption of cellular amino acidsMoreover active mTORC1 blocks cellular autophagy byphosphorylating ULK1 and impairs the recycling ofamino acids [] Both effects of mTORC1 lead to a remarkable drop of cellular amino acid reservoir ActiveAMPK has been shown to inhibitthe activity ofmTORC1 direct and indirectly upon energy stresswhich limits the expenditure of amino acids Alternatively active AMPK can restrict protein synthesis byphosphorylating and thereby inhibiting eukaryotic translation elongation factor eEF2 kinase a key regulator 0cYuan Journal of Hematology Oncology Page of of protein synthesis [] To restore cellular amino acidreservoir active AMPK stimulates cellular autophagy asdiscussed above which degrades surplus or dysfunctional proteins into amino acids [] In addition it isworth noted that cellular amino acids can affect theactivity of AMPK reversely Dependent on conditionscontexts either amino acids may inhibit or stimulate theactivity of AMPK though underlying molecular mechanisms remain ambiguous [“]YAPactivity which impairsAMPK signaling in cancer biologyIt is well known that AMPK is a putative substrate oftumor suppressor LKB1 [ ] Fig Therefore AMPK has been generally considered as a key effector that mediates the tumorsuppressive function ofLKB1 Indeed a genetic ablation of the AMPK α subunitin mice accelerates Mycdriven lymphomagenesis throughfacilitating a metabolic shift to aerobic glycolysis []Simultaneously AMPK inhibitorsAMPKi promoteepithelialtomesenchymal transition EMT in breast andprostate cancers [] These studies validate AMPK as atumor suppressor under certain circumstances Furthermechanistic studies have demonstrated that AMPK prevents cancers through phosphorylating multiple targetsthat play indispensable roles on different layers of diseaseprogression AMPK phosphorylates angiomotin like AMOTL1 an adaptor protein in the HippoYap pathway and thus blocks Yes1 associated transcriptional regucells™latorproliferation and survival [] AMPK also phosphorylates TSC complex subunit TSC2 and regulatory associated protein of MTOR complex Raptor and therebyinactivates mTORC1 [ ] which in turn elevatescellular autophagy activity and inhibits cancer initiationTo bypass this inhibitory effect cancer cells can activatethe MAGE family member A MAGEA36tripartitemotif containing TRIM28 ubiquitin ligase complexthat targets the AMPK α subunit for degradation and thusreactivates mTORC1 to restrict cellular autophagy []Moreover AMPK is able to phosphorylate enhancer ofzeste polycomb repressive complex subunit EZH2and thereby disrupts the polycomb repressive complex PRC2 which relieves the epigenetic silence of tumorsuppressors in cancers [] Alternatively AMPK phosphorylates and stabilizes another epigenetic master regulator Tet methylcytosine dioxygenase TET2 whichfunctions as a putative tumor suppressor to preventtumorigenesis [] Altogether these findings indicatethat AMPK has a pronounced antitumor activity as itsupstream kinase LKB1 doescancerkillsandLICsstressleukemia TALL oncogenic Notch signaling induces ahigh level of aerobic glycolysis which needs to be restrained by AMPK and loss of AMPK results in energystressdriven apoptosis of leukemic cells and slows downdisease progression [] Similarlyin acute myeloidleukemia AML metabolic stress elevates the ROS leveland induces DNA damage in leukemiainitiating cellsLICs and AMPK confers metabolic stress resistance toLICs [] AMPK knockout or pharmaceutical inhibitionunder metabolicinhibitsleukemogenesis Moreover AMPK plays a determinantrole in maintaining the NADPH homeostasis in cancercells upon energy stress which is critic
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" new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease In addition to the personto person transmission dynamic of the novel respiratory virus it has been recently studied the role of environmental factors in accelerate SARSCoV2 spread and its lethality The time being air pollution has been identified as the largest environmental cause of disease and premature death in the world It affects body™s immunity making people more vulnerable to pathogens The hypothesis that air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography can acts both as a carrier of the infection and as a worsening factor of the health impact of COVID19 disease has been raised recently With this review we want to provide an update state of art relating the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality The Authors who first investigated this association often used different research methods or not all include confounding factors whenever possible In addition to date incidence data are underestimated in all countries and to a lesser extent also mortality data For this reason the cases included in the reviewed studies cannot be considered conclusive Although it determines important limitations for direct comparison of results and more studies are needed to strengthen scientific evidences and support firm s major findings are consistent highlighting the important contribution of PM25 and NO2 as triggering of the COVID19 spread and lethality and with a less extent also PM10 although the potential effect of airborne virus exposure it has not been still demonstrated Introduction A new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease The scientific community has come together to implement pharmaceutical and nonpharmaceutical intervention measures designed to contain SARSCoV2 global spread Nevertheless on March 11th WHO™s Directeneral announced that COVID19 can be characterized as a pandemic SARSCoV2 is primarily transmitted from persontoperson through close contact approximately m by aerosol respiratory droplets smaller than μm in diameter wwwwhoint Indoor environments might be especially hazardous because of their reduced ventilation Morawska lack ultraviolet light which rapidly inactivates the virus and because it can become less diluted than it would in outdoor environments Schuit It is also known how the virus can survive and being infectious in aerosols for hours and on surface up to days van Doremalen et al similarly with transmission dynamics known for SARSCoV1 associated with nosocomial spread and superspreading events Chen et al 2020ab Beyond the causality it is uncertain even if certain demographics of the population are more vulnerable to SARSCoV2 infection Based on recent reports male gender advancing age and comorbidities seem to be correlated with death and severe illness Harris et al Furthermore COVID19 seems to be associated with an increasing rate of thromboembolic events in hospitalized patients Llitjos Mechanisms of social and economic interactions are additionally supposed to be involved in the diffusion dynamic of COVID19 in the diverse parts of the world or of the same country such as the living conditions the healthrelated behaviour KhalatbariSoltani et al Corresponding author Email address ccopatunictit C Copat 101016jenvres2020110129 Received July Received in revised form August Accepted August EnvironmentalResearch1912020110129Availableonline24August2020001393512020ElsevierIncAllrightsreserved 0cC Copat the commercial exchanges Bontempi 2020a or the migration scale index H Chen It seems that these diffusion dynamics have particularly affected the COVID19 spread at the early stage Among the environmental parameters some climate condition such as temperature humidity sunlight and wind revealed a reduction of the COVID19 spread S Chen Coccia 2020a and air pollution seems to have a role in airborne transmission of SARSCoV2 and severity of COVID19 Domingo Nevertheless to better understand COVID19™s diffusion patterns an interdisciplinary multidimensional approach should be encourage in order to develop firm s Bontempi Air pollution has been identified as the largest environmental cause of disease and premature death in the world GBD Ambient particulate matter PM induces its proinflammatory and thrombogenic effects through the generation of oxidative stress by its chemical compounds and metals Li Signorelli The recent identification of environmentally persistent free radicals EPFRs in the PM resulting from a mixture of combustion sources theorize its role in the increase of disease severity of lower respiratory tract infections LRTI Jaligama Scientific evidences support that short and longterm exposures to ambient air pollutants are associated with a broad of adverse health outcomes Ferrante and Conti Fiore such as higher mortality rates greater hospital admissions and increased outpatient visits Bremner Cohen Dehghani Dockery It has markedly detrimental consequences on asthma bronchitis pneumonia and COPD Dick Perng and Chen Raji Vignal Yarahmadi et al where bacteria and viruses are the most accepted causative factors that harm airway stability driving to infection exacerbation Furthermore air pollution represents an aggravating factor for infection diseases caused by some viral infections Domingo and Rovira such as respiratory syncytial virus RSV influenza A and B para influenza virus type pneumonia and influenzalike illness Carugno Croft Fukuda Huang Huh Liang Lin Silva Somayaji It determines an increase in the rate of hospitalizations and access to emergency department visits Studies related to the epidemic of SARSCoV coronavirus identified in November from the Guangdong province of southern China reported similar associations Cai Cui Kan Several Authors suggest that outdoor air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography could operate both as a carrier of the infection and as a worsening factor of the COVID19 severity Conticini Frontera Isaifan Martelletti and Martelletti This association is getting stronger thanks to the results of the numerous studies that have been launched all over the world and summarized with this review Most of the reviewed studies support that chronic exposure to air pollution might led people more susceptible to COVID19 disease leading to widespread COVID19 spread and lethality Nevertheless as suggested by Bontempi 2020b the potential effect of airborne virus exposure due to PM10 remain unclear With this review we want to provide an updated state of art of the recently epidemiological studies dealing with understanding the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality Fig PRISMA Flow Diagram of identification screening and inclusion of studies EnvironmentalResearch19120201101292 0cC Copat Method We have conducted a systematic review of the literature concerning the relationship between some air pollutants PM25 PM10 and NO2 and COVID19 outbreak The research was performed in compliance with the PRISMA criteria Preferred Reporting Items for Systematic Reviews and MetaAnalyses and the Flow Diagram is showed in Fig The research was conducted between April and July 6th in PubMed database It was used the Advanced Search Builder and the keywords were searched in [Title OR Abstract] We have filtered only research articles published in English language and selected the following keywords Air pollution and COVID19 or SARSCOV2 Particulate matter or PM and COVID19 or SARSCOV2 Nitrogen dioxide or NO2 and COVID19 or SARSCOV2 We choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported COVID19 cases andor deaths and air pollution data related to PM25 PM10 and NO2 thus excluding any Letter Opinion Commentary Review or nonrelevant articles We obtained a total of eligible published research articles in their final version and paper in its preprint version For some of them we chose to include only principal findings that clearly fit the aim this review Particulate Matter and COVID19 Atmospheric particulate matter PM is originated by a wide range of anthropogenic and natural sources Kim It consists of a heterogeneous mixture of solid and liquid particles suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals WHO It has been associated with increased respiratory morbidity and mortality Liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis Li Rhee In vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections Becker and Soukup Recently the research group of Setti gave first preliminary evidence that SARSCoV2 RNA can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of PM it could represent a potential early indicator of COVID19 although it does not give information regarding COVID19 progression or severity Several observations report a significant association between ambient concentrations of PM25 Adhikari and Yin Bashir Fattorini and Regoli Frontera Jiang Li VasquezApestegui Wu Yao Zhu Zoran 2020a and PM10 Bashir Coccia 2020b Fattorini and Regoli Jiang Li Yao Zhu Zoran 2020a with COVID19 pandemic across the most affected countries China Italy and USA see Table First evidences on the temporal association between air pollution and COVID19 were reported in China where the outbreak was first identified Zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in China The Authors included over of dailyconfirmed new cases in the whole of China between January 23rd and February 29th They applied a Generalized Additive Model GAM to examine the effects of meteorological factors and air pollution on COVID19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders They observed that the effect of PM25 on daily confirmed cases was greater than PM10 In particular they found that a 10μgm3 increase lag0“ in PM25 and PM10 was associated with a CI to and CI to increase in the daily counts of COVID19 confirmed cases respectively Jiang focused their attention on three most affected cities of China Wuhan XiaoGan and HuangGang collecting data of daily cases and ambient air pollutant from Jan 25th to Feb 29th The Authors by applying a multivariate Poisson regression revealed a significant temporal association between PM25 increased and COVID19 incidence in all the considered cities especially in HuangGang Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Conversely an increase in PM10 concentrations was associated with a decrease of COVID19 incidence These results were partially confirmed by findings of Li who conducted a simple linear regression to compare COVID19 incidence with PM concentrations in Wuhan and XiaoGan from Jan 26th to Feb 29th in They found that an increase in PM25 was correlated with an increase of COVID19 incidence in both cities Wuhan R2 p XiaoGan R2 p while for PM10 only in XiaoGan R2 p The spatial distribution of particulate matter and case fatality rate CFR of COVID19 was studied by Yao in cities of China including Wuhan collecting data up to March 22nd First they found a significantly positive global spatial autocorrelation of COVID19 CFR Global Moran™s index I p highlighting a high CFR clustering located in Hubei Province With a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product GDP per capita hospital beds per capita local indicators of spatial association LISA map values city size and population or proportion of people older than years It was found that for every μgm3 increase in PM25 and PM10 the CFR increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of PM25 and PM10 in “ respectively Some studies describe the association between air pollution and COVID19 across Italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other European countries The 28th of July Italy recorded more than total confirmed cases and deaths WHO most of which were distributed in the regions of Northern Italy especially the Lombardy It is recognized as one the most air polluted areas of Europe EEA where the frequent PM10 annual exceedances of the WHO threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year Baccini Bontempi 2020bfocused the attention on two of the most affected regions of Northern Italy Lombardy and Piedmont The Authors based on PM10 daily exceedances and COVID19 confirmed cases on March 12th thus before the Italian sanitary crisis observed that PM10 concentration was exceeded only few times among the Lombard cities that at the beginning of the epidemic were most affected On the contrary among some Piedmont cities suffering of severe PM10 pollution events COVID19 incidence was lower Based on their results the Authors concluded that COVID19 diffusion by airborne PM10 is hard to demonstrate nevertheless several research article revealed how PM in particular PM25 could had a role in accelerate and vast diffusion of COVID19 in Northern Italy For example Coccia 2020b by analyzed data on Italian province capitals and data of infected individuals up to April 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for PM10 in previous years and COVID19 diffusion In particular cities with more than days of PM10 exceedances showed a very high average number of infected individual about infected individuals on 7th April whereas cities having less than days of PM10 exceedances showed a lower average number of infected about infected individuals Frontera gave also evidences on the role of PM25 as a contributing factor of COVID19 outbreak in Northern Italy where EnvironmentalResearch19120201101293 0cC Copat Table Summary table reporting reviewed results on the association between COVID19 casesdeaths and air pollution PM25 PM10 and NO2 References Zhu Data analysis Generalized Additive Model GAM Aim Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Spatial association between fatality rate and air pollution PM25 and PM10 Spatial association between deaths counts and air pollution NO2 Temporal association between total cases daily confirmed cases and total deaths and air pollution PM25 and PM10 Temporal association between total cases daily confirmed cases and total deaths and air pollution NO2 Spatial description of PM10 exceedances versus COVID19 cases Multivariate Poisson regression Simple linear regression Multiple linear regression Descriptive analysis percentage of deaths in three NO2 μmol m2concentration range “ “ “ Pearson coefficient correlation Pearson coefficient correlation Descriptive analysis Number of days of PM10 exceeding μgm3 and COVID19 incidence Area of Study cities of China Period From Jan 23rd to Feb 29th Jiang Li Yao Ogen Zoran 2020a Zoran 2020b Bontempi 2020b From Jan 25th to Feb 29th From Jan 26th to Feb 29th in Data up to March 22nd Data up to the end of Feb From Jan 1st to Apr 30th From Jan 1st to Apr 30th From Feb 10th to March 12th Wuhan XiaoGan and HuangGang China Wuhan and XiaoGan cities of China administrative regions in Italy Spain France and Germany Milan Italy Milan Italy provinces of Lombardy Italy provinces of Piedmont Italy Coccia 2020b Data up to April 7th Italian Provinces Fattorini and Regoli Data up to April 27th Italian provinces PM25 A 10μgm3 PM25 increase lag0“ was associated with a increase of daily confirmed new cases PM10 A 10μgm3 PM10 increase lag0“ was associated with a increase of daily confirmed new cases Wuhan RR CI1032“ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p χ2 p A μgm3 increase in PM25 was associated with a “ increase in fatality rate χ2 p A μgm3 increase in PM10 was associated with a “ increase in fatality rate NO2 A 10μgm3 NO2 increase lag0“ was associated with a increase in daily confirmed new cases Wuhan RR CI “ XiaoGan RR CI “ HuangGang no association found Wuhan R2 p XiaoGan R2 p of fatality cases are associated with NO2 μmolm2 R cid0 R R cid0 R cid0 R R cid0 R cid0 R cid0 R cid0 Lombardy PM10 exceeding between and COVID19 incidence between and Piedmont PM10 exceeding between and COVID19 incidence between and COVID19 in North Italy has a high association with air pollution of cities measured with days exceeding the limits set for PM10 R2 p R2 p continued on next page Hierarchical multiple regression model Pearson regression coefficient analysis R2 p Spatial association between confirmed cases and air pollution PM10 Spatial association between total confirmed cases and air pollution PM25 PM10 and NO2 EnvironmentalResearch19120201101294 0cC Copat Table continued References Frontera Frontera Wu Adhikari and Yin Bashir Bashir VasquezApestegui VasquezApestegui VasquezApestegui Period Data up to 31st March Data up to 31st March Data up to April 04th From March 1st to Apr 20th From March 4th to April 24th From March 4th to April 24th Data up to June 12th Data up to June 12th Data up to June 12th Area of Study Italian regions Italian regions counties in the USA Queens county New York USA California California districts of Lima Perù districts of Lima Perù districts of Lima Perù Aim Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Prediction of risk of COVID19 deaths in the long term average exposure to fine particulate matter PM25 Temporal association between daily confirmed cases and total deaths and air pollution PM25 Association between confirmed cases and air pollution PM25 PM10 and NO2 Association between deaths and air pollution PM25 PM10 and NO2 Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Spatial association between case fatality rate and air pollution PM25 Data analysis Pearson regression coefficient analysis PM25 R2 p PM10 Pearson regression coefficient analysis R2 p Longterm exposure increase of μgm3 in PM25 is associated with a increase in the COVID19 death rate Estimate on cases values cid0 CI “ Estimate on deaths value cid0 CI “ Kendall r cid0 Spearman r cid0 Zeroinflated negative binomia models Negative binomial regression model Spearman and Kendall correlation tests Spearman and Kendall correlation tests NO2 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient cid0 p mortality was found significantly higher than less polluted Italian regions By collecting data up to March 31st for all Italian regions and performing a Pearson correlation analysis they found a strong positive association both with the total number of confirmed cases R and deaths R other than with hospitalized cases R The Italian situation was further highlighted by the study of Fattorini and Regoli in Italian provinces They explored the spatial association between air pollution and COVID19 cases with data up to April 27th By applying the Pearson regression coefficient analysis they revealed a positive association both with PM25 and PM10 R2 p and R2 p respectively A focus on the most affected city of Italy Milan was conducted by Zoran 2020a This city is located in the Po valley basin known hotspot for atmospheric pollution at the continental scale EEA The Authors performed a temporal association between COVID19 Total cases Daily New positive cases and Total Deaths and particulate matter from Jan 1st and Apr 30th by applying a Person correlation In accordance with other studied they found a positive association between daily confirmed cases and PM25 R and PM10 R although they did not consider any delay time from infection to COVID19 onset Nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships To date the USA have more than million confirmed cases and thousand deaths WHO Here ambient concentrations of PM and O3 were found responsible to cause between and premature deaths Fann The association between air pollutants and COVID19 cases and deaths was studied by Bashir in the state of California from March 4th to April 24th corresponding to the beginning of the COVID19 outbreak in USA Based on their significant correlation found the Authors state that a limited human exposure to these pollutants will contribute to defeating COVID19 This seems unclear because they found a negative correlation with PM25 and PM10 EnvironmentalResearch19120201101295 0cC Copat by applying both the Kendall rank correlation and Spearman™s one and it is not clear if they normalized COVID19 cases by population size and if they performed a day by day association or a spatial association across the country A focus on the Queen county New York USA was provided by Adhikari and Yin They retrieved data of PM daily concentrations from two ground monitoring stations and collected data of confirmed COVID19 cases and numbers of related deaths from USAFacts in the period from March to April The Authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of PM25 on disease outcomes over the past days They found a significant negative association among PM25 and new daily confirmed COVID19 cases cid0 CI “ and deaths cid0 CI “ Low PM concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as Italy where PM25 monthly concentrations reached values higher than μgm3 Fattorini and Regoli Frontera or in China where PM25 monthly concentrations reached values higher than μgm3 Zhu Jiang As said by the Authors other gaseous pollutants such as NO2 and SO2 could have influenced transmission and pathogenesis of COVID19 In the United States Wu investigated whether longterm average exposure to fine particulate matter PM25 increases the risk of COVID19 deaths by considering approximately counties in the United States of the population With an exposure prediction model the Authors calculated the county level longterm exposure to PM25 averaged for to and collected COVID19 deaths counts up to April 04th They conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors They found that a small longterm exposure increase of only μgm3 in PM25 is associated with a increase in the COVID19 death rate confidence interval CI VasquezApestegui recently reported first evidences on the spatial relationship between particulate matter and COVID19 outbreak from Latin America The Authors described the situation occurred in districts of Lima located in the second most affected country of Latin America Peru In particular by applying a multivariate regression model they evaluated the association between the population exposure to PM25 concentrations in the previous years “ and cases deaths and casefatality rates of COVID19 with data up to June 12th A significant association has been found both with cases and deaths Crude coefficient with p and with p respectively but not with case fatality rate All these studies highlight the role of PM in triggers of the COVID19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems Nitrogen dioxide NO2 and COVID19 induced lung damage Hence viral infection becomes more common after exposure to NO2 Zhu Furthermore NO2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children To increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation Bahrami Asl Kowalska increase of chronic obstructive pulmonary disease COPD Ghanbari Ghozikali Pfeffer and increase of pulmonary heart disease related mortality Chen A recent study explored the possible role of NO2 in interference in Angiotensin converting enzyme ACE2 The expression of ACE2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of COVID19 Alifano First observations report an association between ambient concentrations of NO2 and COVID19 pandemic across Europe China and USA Bashir Fattorini and Regoli Jiang Li et al Ogen Zhu et al Zoran et al 2020b Conversely to the other papers findings of Zoran 2020b and Bashir provides different findings reporting no association or a negative one between NO2 and daily deaths counts In China Zhu by applying the same method explained for PM observed that a 10μgm3 increase lag0“ in NO2 is associated with a CI “ increase in the daily counts of COVID19 confirmed cases in cities of China These findings are confirmed by Jiang and Li et a who applied the same method described for PM Jiang revealed a significant positive association between NO2 and COVID19 both in Wuhan and XiaoGan Wuhan RR CI1053“ XiaoGan RR CI “ but did not found any significant association in HuangGang Li found a significant linear correlation both in Wuhan R2 p and XiaoGan R2 p Ogen presented evidences on the relationship between exposure to NO2 including the months of January and February shortly before the COVID19 spread in Europe and novel coronavirus fatality in the most affected European countries concluding that longterm exposure to NO2 may be a potential contributor to mortality caused by SARSCoV2 He collected data concerning the number of fatality cases from administrative regions in Italy Spain France and Germany and correlated mortality with tropospheric NO2 concentrations measured by the Sentinel5 Precursor spaceborne satellite The major tropospheric NO2 hotspot identified was located in the Northern Italy In all European regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards Results showed that out of the fatality cases by March were in five regions located in north Italy and central Spain Furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum NO2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 The methodology used by Ogen cannot support a longterm exposure investigation Surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust Nevertheless the study provide new insights for future investigation The Italian situation was further studied by Fattorini and Regoli who collected data of COVID19 incidence up to April 27th from Italian provinces They revealed a strong spatial corr
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"Severe iodine deficiency impacts fertility and reproductive outcomes The potential effects of mildtomoderate iodine deficiency are not well known The aim of this study was to examine whether iodine intake wasassociated with subfecundity ie months trying to get pregnant foetal growth and adverse pregnancyoutcomes in a mildtomoderately iodinedeficient populationMethods We used the Norwegian Mother Father and Child Cohort Study MoBa and included pregnancieswith data on iodine intake and pregnancy outcomes Iodine intake was calculated using an extensive foodfrequency questionnaire in midpregnancy In addition urinary iodine concentration was available in a subsampleof pregnancies Associations were modelled continuously by multivariable regression controlling for a range ofconfounding factorsResults The median iodine intake from food was μgday and the median urinary iodine was μgLconfirming mildtomoderate iodine deficiency In nonusers of iodine supplements n low iodine intake “ μgday was associated with increased risk of preeclampsia aOR CI at vs μgday p overall preterm delivery before gestational week aOR at vs μgday p overall and reduced foetal growth ˆ’ SD ˆ’ ˆ’ difference in birth weight zscore at vs μgday p overall but not with early preterm delivery or intrauterine death In planned pregnanciesn having an iodine intake lower than μgday was associated with increased prevalence ofsubfecundity aOR at μgday vs μgday p overall Longterm iodine supplementuse initiated before pregnancy was associated with increased foetal growth SD on birth weightzscore p and reduced risk of preeclampsia aOR p but not with the other adverseContinued on next page Correspondence annelisebrantsaeterfhino2Department of Environmental Health Division of Infection ControlEnvironment and Health Norwegian Institute of Public Health PO Box Sk¸yen NO0213 Oslo NorwayFull 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 0cAbel BMC Medicine Page of Continued from previous pagepregnancy outcomes Urinary iodine concentration was not associated with any of the dichotomous outcomes butpositively associated with foetal growth n p overall Conclusions This study shows that a low iodine intake was associated with restricted foetal growth and a higherprevalence of preeclampsia in these mildtomoderately iodinedeficient women Results also indicated increasedrisk of subfecundity and preterm delivery Initiating iodine supplement use in pregnancy may be too lateKeywords Mildtomoderate iodine deficiency Iodine intake Iodine supplement Pregnancy cohort Foetal growthPreeclampsia Preterm delivery Subfecundity The Norwegian Mother Father and Child Cohort Study MoBaBackgroundIodine is an essential micronutrient and an integral partof the thyroid hormones triiodothyronine T3 and thyroxine T4 The thyroid hormones regulate multiplemetabolic processes that are important in growth metabolism and reproduction Thyroid dysfunction hasbeen linked to menstrual disturbances reduced fecundityto become pregnant miscarriagegestationinduced hypertension preterm delivery andreduced foetal growth []abilityieIodine deficiency is highly prevalent in both low andhighincome countries even though deficiency is easilypreventable through salt iodization strategies as recommended by the World Health anization WHO []Worldwide iodine nutrition is recognized as one of thekey determinants of thyroid dysfunction [] Recent findings in two populationbased cohort studies have indicated that even mildtomoderate iodine deficiency mayaffect thyroid function in pregnant women [ ] Inaddition an abrupt increase in iodine intake caused byintroduction of salt iodization programmes or iodinesupplement use might temporarily affect thyroid function in populations that are mildtomoderately iodinedeficient [ ] While it is well documented that severeiodine deficiency poses reproductive risksincludingabortions stillbirths and impaired neurodevelopmentthe potentialimpact of mildtomoderate iodine deficiency on fertility and pregnancy outcomes remainslargely unknown []It has been well known for many decades that iodinedeficiency reduces fecundity in livestock [] but we haveidentified only one study that has investigated this association in humans [] The study sample included women trying to get pregnant and a low urinary iodineconcentration UIC μgg creatinine wassignificantly associated with delayed conception compared to aUIC within the normal range ‰¥ μgg creatinine []A few studies have investigated associations betweeniodine status and risk of adverse pregnancy outcomesie preeclampsia preterm delivery pregnancy loss andor birth anthropometrics in mildtomoderate iodine deficiency [“] but the studies were underpowered toidentifyforpotential minorchangesrisksinthe effect ofdichotomous outcomes and most have reported nullfindings For birth weight some studies have reportedreduced birth weight in mildtomoderate iodine deficiency [ ] but a recent systematic review found noevidence ofiodine supplement or saltiodization on prenatal growth in mildtomoderate iodine deficiency [] However the quality of the evidencewas assessed as very low [] Therefore there is still amajor knowledge gap as to whether mildtomoderateiodinepregnancyoutcomesdeficiencyfertilityaffectsandIn the present study we used data from the Norwegian Mother Father and Child Cohort Study MoBaa large pregnancy cohort with detailed informationabout food intake supplement use and a number ofobstetric outcomes [] We have previously documented that the MoBa pregnant women were mildtomoderately iodine deficient at a group level definedby WHO criteria [] and that there was a large variation in iodine intake between participants due to fewfood sources mainly milk and fish and supplementuse [] We also found that iodine intake was associated with thyroid function in pregnancy and that alow maternaliodine intake in pregnancy was associated with poorer child neurocognitive development atages and years [ ] In MoBa three different exposures are available as measures of iodine intake calculated iodine intake from food reported useof iodinecontaining supplements and UIC in a subsample of women Consequently this large prospective study offered a unique opportunity to add newknowledge about the role of mildtomoderate iodinedeficiency on subfecundity and adverse pregnancyoutcomesMethodsThe aim of the current study was to examine if iodine intake was associated with subfecundity ie months trying to get pregnant stillbirth preeclampsia preterm delivery and birth anthropometrics in alarge cohort of mildtomoderately iodinedeficientwomen 0cAbel BMC Medicine Page of Subjects and designThis study is based on MoBa a prospective populationbased pregnancy cohort initiated and maintained by theNorwegian Institute of Public Health [] Women pregnant in their first trimester were recruited from all overNorway during years to and were asked toanswer questionnaires available in Norwegian only atregular intervals during pregnancy and after birth Pregnancy and birth records from the Medical Birth Registryof Norway MBRN are linked to the MoBa database[] The women consented to participation in ofthe pregnancies The cohort now includes children mothers and fathers The currentstudy is based on version of the qualityassured datafiles released for research in and restricted to participants recruited from to because the MoBafood frequency questionnaire FFQ was included in thedata collection from March To be included in thecurrent study participants had to have responded to ia baseline questionnaire Q1 around gestational weekGW covering general health and sociodemographicinformation and ii the FFQ Q2 around GW andiii to be registered in MBRN with a singleton deliveryWe excluded women who reported use of thyroid medication at any time during pregnancy Given the largesample size and low rates of missing values ‰¤ onlypregnancies with information on all covariates were included FFQs with more than three blank pages or withcalculated energy intakes outside the range “ MJday were excluded [] Some women participate inMoBa with more than one pregnancy The final studypopulation comprisedpregnancieswomen for the analyses of pregnancy outcomes and planned pregnancies for the analysis of subfecundity UIC was available in a subsample of pregnanciesand was measured in GW A flow chart of inclusion isillustrated in Fig Exposure variable iodine intakeThe MoBa FFQ is a comprehensive semiquantitativequestionnaire specifically designed and validated forMoBa [ ] Participants responded to the FFQaround GW and were asked to report their averageintake since becoming pregnant GW “ Food frequencies were converted to food amounts using standard Norwegian portion sizes and daily intakes of energyand nutrients were calculated using FoodCalc [] andthe Norwegian food composition table [] Data on thecontent of more than food supplements was collected from suppliers [] Participants with unrealisticenergy intakes in the FFQs ie or MJday ormore than three blank pages were excluded from thestudy sample Iodine intake measured by the FFQ haspreviously been validated and it shows good agreementwith days weighed food diary [] and urinary iodineconcentration UIC [ ] Use of supplements was reported in the FFQ and in the general questionnaires fordifferent time periods Timing of the first reported usewas coded in four categories never weeks “ beforepregnancy GW “ and GW Although we assessed iodine intake in pregnancy withan FFQ covering the average food intake in the first “months of pregnancy we propose that this iodine intakeFig Flow chart of inclusion Only complete cases were included had missing values on one or more covariates Asterisk indicates that thefood frequency questionnaire FFQ was in use from 0cAbel BMC Medicine Page of also can serve as an indicator of habitual iodine intakeprior to pregnancyUrine samples were collected at the routine ultrasoundexamination offered free of charge to all Norwegianwomen in GW UIC was determined at the NationalInstitute for Health and Welfare in Helsinki Finland byinductively coupled plasma“mass spectrometry using anAgilent ICPMS system Agilent Technologies IncSanta Clara CA USA The limit of quantification was μgL and the linearity was excellent up to μgLr The coefficient of variation was “Pregnancy and birth outcomesAll outcomes except subfecundity were based on information in the MBRN The subfecundity outcome whichapplied only to planned pregnancies was based on reported time months to conception reported in the general questionnaire in GW Subfecundity was defined as months trying to getpregnant for planned pregnancies of the womenhad reported that pregnancy was planned and also reported time to pregnancy The wording of the questionwas œHow many months did you have regular intercourse without contraception before you became pregnant Women with in vitro fertilization were notexcludedIntrauterine death was defined as death before birth or death during birth It also includedregistered intrauterine deaths where the time of deathwas not specified Abortions oflive foetuseswere not includedPreeclampsia was defined if any of the following conditions were checked off in the pregnancy record iHELLP syndrome ie haemolysis elevated liver enzymes and low platelet count ii eclampsia iii earlyivonset preeclampsia diagnosed before weeksmild preeclampsia orsevere preeclampsiaInNorway all pregnant women receive free antenatal careBlood pressure measurement and proteinuria analysisare carried out at each antenatal visit According toguidelines issued by the Norwegian Society of Obstetricsand Gynaecology the diagnostic criteria for preeclampsia are blood pressure after weeks gestationcombined with proteinuria greater than dipstick onat least two occasionsvPreterm delivery was defined as delivery before GW and as early preterm when delivered before GW Gestational age in days was determined based onthe routine ultrasound examination given free of chargeto all women in GW or it was calculated based ontime from the first day of the last menstruation periodin the few women where ultrasound data was missing Preterm delivery was also categorized by deliveryinitiationie spontaneous preterm delivery pretermlabour or preterm prelabour rupture of the membranesor iatrogenic preterm delivery induced or primary caesarean delivery on maternal or foetal indicationsBirth weight was examined as four outcomes crudebirth weight gram standardized birth weight zscoreie birth weight adjusted for child sex and gestationalage based on all deliveries in MBRN small for gestational age SGA gestational age and sexadjusted zscore percentile and large for gestational ageLGA gestational age and sexadjusted zscore percentile Outcomes on birth weight and gestational ageat birth were recoded to missing if birth weight for gestational age and sex was ± standard deviations fromthe mean n since these data suggested misreporting of either birth weight or gestational lengthHead circumference was examined as a crude measurecm Recorded head circumference cm wassuspected as misreporting and recoded to missingPlacenta weight was examined as a crude measuregram Recorded placenta weight g wassuspected as misreporting and recoded to missingOther variablesCovariates were included in the statistical modelsbased on previous knowledge and directed acyclicgraphs DAGs see Additional file Figure S1 Maternal age at the time of birth was obtained from thebirth registry Maternal prepregnancy body massindex BMI education ‰¤ “ ‰¥ years marital status marriedcohabitant yesno parity previous pregnancies ‰¥ weeks ‰¥ history ofchronic illness asthma diabetesinflammatory boweldisease rheumatic disease epilepsy multiple sclerosisor cancer before or during pregnancy yesno smoking before pregnancy no occasional daily use ofin vitro fertilization in current pregnancy yesnoand use of a folic acid supplement within the intervalfrom weeks before to weeks after conception yesno were obtained from questionnaire GW Maternal energy intake fibre intake as marker of ahealthy diet use of probiotic milk products yesnoand total intake of the omega3 fatty acids EPA andDHA were calculated based on the FFQ GW Also use of dietary supplements other than the onescommonly recommended for pregnant women ieother than vitamin D folic acid and iron was obtained from the FFQ yesno Information on smoking in pregnancy was obtained from questionnaire andif available questionnaires GW and child™s age months three categories no reportedsmoking in pregnancy reported occasional smokingor stopped smoking before GW and daily smokingat any time in pregnancy and had not stopped smoking before GW 0cAbel BMC Medicine Page of Statistical methodsStatistical analyses were performed in STATA version Stata Corp College Station TX Associations were estimated by linear regression analysesfor continuous outcomes and logistic regression fordichotomous outcomesIn sensitivity analyses forthe outcome subfecundity Cox regression was usedto modelcontinuousvariableto pregnancytimeasaAssociations between iodine from food and outcomes and UIC and outcomes were modelled flexiblywith restricted cubic splines Since some motherswere included with more than one pregnancy we specified person clusters by using the option vcecluster person_ID in models in STATA which relaxes the assumption of independence of the observations and produces robust estimates of variance pvalues are reported for overall associations betweencontinuous exposures and outcomes testing H0 noassociation by testing the coefficients of all splinetransformations equal to zero In addition tests fornonlinearity were performed by testing the coefficients of the second and third spline transformationsequal to zero Covariates were included in the modelsbased on DAGs Continuous covariates eg maternalage BMI and energy intake were modelled flexiblyby restricted cubic splines if there was evidence ofnonlinear associations determined by inspecting theestimated associations while controlling for other covariates in the model otherwise they were modelledlinearly We reportthe specific covariates for eachoutcome in the respective tables and figures Tabularresults of the graphs included in this paper are provided in Additional file Tables S1S3firstneverreported useIodine supplement use was modelled as any reported iodine supplement use in GW “ and timing ofstarted beforeconception started in GW “ started in GW “Potential effect modification by iodine intake from foodwas explored including an interaction term between iodine from food modelled by restricted cubic splines andthe supplement use variable Potential interactions wereexplored by testing all interaction coefficients equal tozero If the interaction terms were not statistically significant iodine from food was not included in the finalmodels Women in the control group were all nonusersof iodinecontaining supplements In the sensitivity analysis we restricted the control group to women who hadreported use of dietary supplements other than thestandard recommended ones The use of this restrictedcontrol group could control for the behaviour of takingan extra vitaminmineral supplement and could to someextentin themultisupplementsother nutrientscontrolalsoforWe did not include power calculations as no relevantcomparableestimates wereavailableineffectpopulationsA p value was considered statistically significantand results are reported including robust confidence intervals CI Only participants with completedata on all covariates were included in the analyses dueto the low rate of missing values in total of eligibleparticipants had missing on one or more covariatesResultsThe median calculated iodine intake from food was μgday IQR μgday Table Seventyfourper cent had an iodine intake from food lower than theestimated average requirement for pregnant women defined by the Institute of Medicine ie μgday[] and only reached the recommended intake inpregnancy by the WHO ie ‰¥ μgday [] withoutincluding supplements The median UIC measured inn was μgL and had UIC μgLThis is well below the WHO recommendation iemedian UIC ‰¥ μgL for pregnant women and median ‰¥ μgL for nonpregnant []Some groups of women could be identified as having aparticularly low UICfor example all nonusers ofiodinecontaining supplements of all participantsmedian UIC μgL Furthermore noniodine supplement users who consumed less than dL milkyoghurtper day of all participants had a median UIC of μgL and those who excluded dairy products entirelyfrom their diet of all participants had a medianUIC of μgLIodine intake from food correlatedstrongly with the reported intake of milkyoghurtSpearman r and moderately with the intake oflean fish Spearman r The women with availableUIC measurements of the total study populationhad a similar calculated iodine intake from food by theFFQ and equal frequency distribution of reported iodinesupplementthe women without UICmeasurementsuseasThere were marginal variations in iodine intake fromfood use of iodinecontaining supplements and UIC bybackground characteristics Table Table S4 in Additionalfile shows background characteristics bycategories of iodine intake from food and use of iodinecontaining supplements Iodine from food was weaklycorrelated with fibre intake Spearman r ˆ’ afteradjusting for energy intake indicating a weak negativeassociation with this indicator of a healthy dietParticipants in MoBa that were excluded from thestudy sample due to missing values on one or more ofthe covariates n did not differ in iodine intake from food UIC or any of the outcomes prevalenceofpreeclampsiasubfecundityintrauterinedeath 0cAbel BMC Medicine Page of Table Iodine exposures by characteristics of the study population n Study populationStudy sample n Maternal age at delivery mean SD years “‰¥ Prepregnancy BMI mean SD kgm2 ““ Parity or moreMaternal education ‰¤ years“ years yearsMarriedcohabitant YesNoSmoking in pregnancy NoOccasionallyDailyChronic illness NoYesCouples incomeLowMediumHighMissingIodine supplement in pregnancy NoYesReported use in GW “Vitamin D supplement Multivitaminmultimineral Folic acid beforeearly pregnancy Maternal energy intake median IQR MJ Iodine from foodmedian IQR μgday Iodine supplementGW “ UICa n median IQR μgL 0cAbel BMC Medicine Page of Table Iodine exposures by characteristics of the study population n ContinuedStudy populationIodine from foodmedian IQR μgdayIodine supplementGW “ UICa n median IQR μgLIodine from food median IQR μgday “‰¥ 96b 114b 129baUrinary iodine concentration UIC was measured in a subsample of n pregnant women in mean gestational week SD Iodine intake from foodand use of iodinecontaining supplements were comparable in this subgroup versus the whole study samplebRestricted to nonusers of iodinecontaining supplementspreterm delivery or birth weight zscore The prevalence of iodine supplement use was lower in excludedparticipants vs p Descriptive statistics of the pregnancy and birth outcome variables is provided in Table There were someoverlaps between the outcomes Of the preeclampticpregnancies were preterm deliveries and resulted in SGA infants while of preeclampticpregnancies were both preterm and SGASubfecundityThe association between iodine intake from food and subfecundity was Ushaped and iodine intake in the intervalbetween approximately and μgday was associatedwith the lowest likelihood of subfecundity Fig Compared with an intake of μgday reference OR the aOR at μgday was CI and at μgday aOR was CI p overall There was no data in MoBa on supplement useTable Pregnancy and birth outcomes n before months prepregnancy thus supplement use wasnot included as a variable in the model for subfecundityand women were included in the analysis regardless oftheir reported iodine supplement use later However insensitivity analyses women who reported use of iodinecontaining supplements in the time period “ weeks before conception were excluded and this did notchange the results results not shown Time to pregnancyin months was also modelled as a continuous variable byCox regression and the findings were consistent with theresults for subfecundity In the subsample of women withUIC measurements GW who did not report currentsupplement use at the time of UIC sampling there was noassociation between UIC in pregnancy and prevalence ofsubfecundity n p Intrauterine death preeclampsia and preterm deliveryIn nonusers of iodinecontaining supplementsiodineintake from food lower than μgday was associatedStudy sampleaMedian IQRTime to pregnancy monthsbGestational length weeksBirth weight gBirth weight zscore by gestational age and sexPlacenta weight gHead circumference cmSubfecundity months bIntrauterine deathPreeclampsiaPreterm delivery GW Spontaneous preterm delivery weeksEarly preterm delivery weeksSmall for gestational age percentileStudy sampleaLarge for gestational age percentileaSmall differences in numbers are explained by missing databOnly for planned pregnancies with available data on time to pregnancy cPrevalence in the subsample with UIC measurements n ˆ’ Number with outcome range“““ˆ’ “““Percent with outcome 100c 0c 27c 29c 22c 004c 82c 106c 0cAbel BMC Medicine Page of Table For preeclampsia longterm supplement useie use initiated before pregnancy was associated witha decreased prevalence aOR CI p and the effect estimate was not attenuatedwhen restricting the reference group Regarding pretermdelivery the results were not consistent Longterm supplement use was associated with an increased risk ofpreterm delivery also when restricting to spontaneouspreterm delivery whereas more shortterm supplementuse initiated in pregnancy was associated with a decreased risk of early preterm deliveryThere was no evidence of effect modification by habitual iodine intake from food for any of the associationsstudied between iodine supplement use and outcomesAlso supplement use was not associated with iodine intake from food Thus models presented were not adjusted for iodine intake from foodIn light of results in previous studies in MoBa [ ]we additionally tested a potential confounding effect ofuse of milk products containing probiotic bacteria yesno in the models with the outcomes preeclampsia andpreterm delivery and for preeclampsia a potential confounding effect of vitamin D supplement use The resultsdid not change and therefore these variables were notincluded in the final modelsChild anthropometrics at birthA low iodine intake from food less than μgdayas well as a low UIC below μgL was associatedwith lower birth weight and lower birth weight zscoreadjusted for gestational length child sex and standardized Fig Compared with an intake of μgdayreference mean zscore was SD lower at μgday CI ˆ’ ˆ’ and SD lower at μgday CI ˆ’ ˆ’ p overall In fullterm babies born in GW a SD difference in zscore corresponds to g A low iodine intake from foodwas also associated with a reduced risk of being LGAie having a birth weight in the top percentile forchild sex and gestational age at birth and an increasedrisk of being SGA percentile Fig Results didnot change when restricting the definition of SGA to thebelow percentiles or on birth weight zscore resultsnot shown The curve shapes indicated similar associations for UIC but they did not reach statistical significance Fig Iodine intake from food was also associated with placenta weight and head circumference but when adjusting for child birth weight the associations were nolonger present Additional file Figure S6 Again thecurve shapes indicated similar associations for UIC butthey did not reach statistical significance results notshownFig Habitual iodine intake from food GW “ and estimatedprevalence of subfecundity months trying to get pregnant inplanned pregnancies n subfecundity Theassociation was modelled by logistic regression adjusting formaternal age BMI parity education smoking before pregnancyenergy intake and fibre intake The curve represents the estimatedprevalence when all covariates are set to their means and theshaded area illustrates the robust confidence interval Thehistogram shows the distribution of the exposure For the crudeassociation see Additional file Figure S2with increased prevalence of preeclampsia and pretermdelivery but not with early preterm delivery or intrauterine death Fig Compared to an intake of μgdayreference an intake of μgday was associated withan increased risk of preeclampsia aOR CI and an increased risk of preterm deliveryaOR CI At an intake of μgday the adjusted odds ratio of preeclampsia was CI and for preterm delivery it was CI Fig For preterm delivery less than week excludingparticipants with preeclampsia did not change the resultsresults not shown When subdividing into spontaneousand iatrogenic preterm delivery the shape of the associations looked similar but only remained significant foriatrogenic PTD p Additional file Figure S4We did not examine associations between UIC andintrauterine death prevalence or early preterm delivery prevalence due to lack of power to detectpotential differences in the subsample of women withavailable UIC data We found no associations betweenUIC and preterm delivery or preeclampsia Additionalfile Figure S5Use of an iodinecontaining supplement in GW “was borderline associated with a reduced risk of intrauterine death however the association was attenuatedwhen restricting the reference group ie nonuse ofiodinecontaining supplements to only include womenwho reported use of dietary supplements other than theones commonly recommended for pregnant women 0cAbel BMC Medicine Page of Fig Iodine from food and adverse pregnancy outcomes in nonusers of iodinecontaining supplements Sample size intrauterine death n intrauterine deaths preeclampsia n preeclampsia and preterm delivery n preterm and earlypreterm The associations were modelled by logistic regression adjusting for maternal age BMI parity education smoking in pregnancy energyintake and fibre intake For the crude associations see Additional file Figure S3Use of an iodinecontaining supplement was associated with increased birth weight but the estimates wereattenuated when restricting the reference group to participants using nutrient supplements other than thestandard recommended ones Table Then only longterm use initiated before pregnancy remained statistically significant However use ofiodinecontainingsupplements was associated with a reduced risk of beingSGA aOR p and longtermuse was associated with an increased risk of being LGAaOR p Overall the results indicated that use of iodine supplements increased birthweight and especially longterm use SD or gin fullterm babiesDiscussionThe main finding in this uniquely large pregnancy cohortis that a low iodine intake lower than μgday wasconsistently associated with reduced foetal growth acrossall three measures of exposure ie iodine from food UICand iodine supplement use This strengthens the evidenceof a causal relationship Also both a low iodine intakefrom food lower than μgday and no iodine supplement use were associated with an increased risk of preeclampsia This association was not detected for UIC butUIC was only measured in a subsample of women anda single spot UIC provides a very poor measure of iodine status at the individuallevel Thus the analyseswith UIC as the measure of exposure were underpowered to detect small differences in risk for dichotomousoutcomes A low iodine intake from food was associated with an increased risk of preterm delivery but theresults for supplement use on this outcome were notconsistent We also found indications that low iodineintake from food was associated with an increased riskof subfecundity but the design of this study includingonly women who had already succeeded in becomingpregnant and measuring the exposure after the outcome was far from optimal to study the associationwith this outcome 0cAbel BMC Medicine Page of Table Use of iodinecontaining supplements and pregnancy and birth outcomesNumberCrude modelsOdds ratio CIAdjusted modelsaRestricted controlsbp value Odds ratio CI p value Odds ratio CIIntrauterine death Any iodine supplement use GW “First report of iodine supplementNever nonsupplement userBefore pregnancycGW “GW 13451b ref ref ref Preeclampsia Any iodine supplement use GW “First report of iodine supplementNever nonsupplement userBefore pregnancycGW “GW 13451b ref ref ref Preterm delivery GW Any iodine supplement use GW “First report of iodine supplementNever nonsupplement userBefore pregnancycGW “GW 13405b ref ref ref Early preterm delivery GW Any iod
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" 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 patient™s 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 d™hebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall d™hebron 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
"Number of female samples with mutations (Mutation frequency in 36 female samples) Number of male samples with mutations (Mutation frequency in 40 male samples) ABL1 0(0.0%) 0(0.0%) 0(0.0%) AKT1 0(0.0%) 0(0.0%) 0(0.0%) ALK 0(0.0%) 0(0.0%) 0(0.0%) APC 0(0.0%) 0(0.0%) 0(0.0%) ATM 0(0.0%) 0(0.0%) 0(0.0%) BRAF 2(2.6%) 1(2.8%) 1(2.5%) CDH1 0(0.0%) 0(0.0%) 0(0.0%) CDKN2A 0(0.0%) 0(0.0%) 0(0.0%) CSF1R 0(0.0%) 0(0.0%) 0(0.0%) CTNNB1 3(3.9%) 3(8.3%) 0(0.0%) EGFR 32(42.1%) 22(61.1%) 10(25.0%) ERBB2 1(1.3%) 0(0.0%) 1(2.5%) ERBB4 0(0.0%) 0(0.0%) 0(0.0%) FBXW7 0(0.0%) 0(0.0%) 0(0.0%) FGFR1 0(0.0%) 0(0.0%) 0(0.0%) FGFR2 0(0.0%) 0(0.0%) 0(0.0%) FGFR3 0(0.0%) 0(0.0%) 0(0.0%) FLT3 0(0.0%) 0(0.0%) 0(0.0%) GNAS 0(0.0%) 0(0.0%) 0(0.0%) HNF1A 0(0.0%) 0(0.0%) 0(0.0%) HRAS 0(0.0%) 0(0.0%) 0(0.0%) IDH1 0(0.0%) 0(0.0%) 0(0.0%) JAK3 0(0.0%) 0(0.0%) 0(0.0%) KDR 0(0.0%) 0(0.0%) 0(0.0%) KIT 0(0.0%) 0(0.0%) 0(0.0%) KRAS 4(5.3%) 1(2.8%) 3(7.5%) MET 0(0.0%) 0(0.0%) 0(0.0%) MLH1 0(0.0%) 0(0.0%) 0(0.0%) MPL 0(0.0%) 0(0.0%) 0(0.0%) NOTCH1 0(0.0%) 0(0.0%) 0(0.0%) NPM1 0(0.0%) 0(0.0%) 0(0.0%) NRAS 0(0.0%) 0(0.0%) 0(0.0%) PDGFRA 0(0.0%) 0(0.0%) 0(0.0%) PIK3CA 2(2.6%) 0(0.0%) 2(5.0%) PTEN 1(1.3%) 1(2.8%) 0(0.0%) PTPN11 0(0.0%) 0(0.0%) 0(0.0%) RB1 0(0.0%) 0(0.0%) 0(0.0%) RET 0(0.0%) 0(0.0%) 0(0.0%) SMAD4 1(1.3%) 1(2.8%) 0(0.0%) SMARCB1 0(0.0%) 0(0.0%) 0(0.0%) SMO 0(0.0%) 0(0.0%) 0(0.0%) SRC 0(0.0%) 0(0.0%) 0(0.0%) STK11 0(0.0%) 0(0.0%) 0(0.0%) TP53 17(22.4%) 6(16.7%) 11(27.5%) VHL 0(0.0%) 0(0.0%) 0(0.0%) The sequenced data were processed and mutations identified using Ion Torrent Suite Software v3.0 with a plug-in œvariant caller. In order to eliminate error base calling three filtering steps were used to generate reliable variant calling as described in the Materials and Methods. The Sequence read distribution across 189 amplicons generated from 76 FFPE specimens were normalized to 300000 reads per sample (Fig. 1). Using a strict standard variant calling we identified mutations in the following genes as listed in : BRAF EGFR ERBB2 KRAS PIK3CA PTEN SMAD4 and TP53. .0095228.g001 Sequence read distribution across 189 amplicons generated from 76 FFPE specimens normalized to 300000 reads per sample. A. Distribution of average coverage of each amplicon. Data are showed as mean ±SD. B. Number of amplicons with a given read depth sorted in bins of 100 reads. (blue bars present number of target amplicons within read depth red line presents % of target amplicons >?=? read depth). The samples were classified based on their origin as lung adenocarcinoma lung large cell carcinoma lung squamous cell carcinoma and lung neuroendocrine carcinoma. The different stages the cancers have progressed to were scored based on ˜American Joint Committee on Cancer/Tumor size Lymph Nodes affected Metastases (AJCC/TNM) ' system (Ia Ib IIa IIb IIIa IIIb) and as metastasizing and non-metastasizing lung cancers. Also cancers were sorted out as from heavy smokers light-smokers and non-smokers to check the correlation of smoking with the accumulation of these mutations. The detailed list of missense point mutations insertions and deletions profiled on 737 loci of 76 lung cancer samples is provided in Table S1. Out of the mutations identified in our sample set BRAF (2.6%) EGFR (42.1%) ERBB2 (1.3%) KRAS (5.3%) PIK3CA (2.6%) PTEN (1.3%) SMAD4 (1.3%) and TP53 (22.4%) incurred the highest rates of mutations (). The mutation frequencies at their different differentiation levels () at different AJCC staging () of the metastatic and non-metastatic lung cancers () and from patients with different smoking habits () are outlined in the Tables. Detailed sequencing analysis in the exons and functional domains of these genes was hence performed. .0095228.t002 Mutations (including Missense point mutations/deletion/insertion) frequencies in 45 genes (737 loci) in lung adenocarcinoma (AC) lung large cell carcinoma lung squamous cell carcinoma and other two lung cancer samples (lung neuroendocrine carcinoma and unknown lung cancer). Genes Number of samples with mutations in 76 samples (Mutation frequency) Number of AC samples with mutations (Mutation frequency in 52 AC samples) Number of SC samples with mutations (Mutation frequency in 20 SC samples) Number of LCC samples with mutations (Mutation frequency in 2 LCC samples) Number of other samples with mutations (Mutation frequency in 2 other samples) ABL1 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) AKT1 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) ALK 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) APC 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) ATM 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) BRAF 2(2.6%) 1(1.9%) 1(5.0%) 0(0.0%) 0(0.0%) CDH1 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) CDKN2A 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) CSF1R 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) CTNNB1 3(3.9%) 3(5.8%) 0(0.0%) 0(0.0%) 0(0.0%) EGFR 32(42.1%) 30(57.7%) 1(5.0%) 0(0.0%) 1(50.0%) ERBB2 1(1.3%) 1(1.9%) 0(0.0%) 0(0.0%) 0(0.0%) ERBB4 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) FBXW7 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) FGFR1 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) FGFR2 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) FGFR3 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) FLT3 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) GNAS 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) HNF1A 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) HRAS 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) IDH1 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) JAK3 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) KDR 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) KIT 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) KRAS 4(5.3%) 4(7.7%) 0(0.0%) 0(0.0%) 0(0.0%) MET 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) MLH1 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) "
1
Pathway‘specific model estimation for improved pathway annotation by network crosstalkMiguel Castresana‘Aguirre Erik L L SonnhammerPathway enrichment analysis is the most common approach for understanding which biological processes are affected by altered gene activities under specific conditions However it has been challenging to find a method that efficiently avoids false positives while keeping a high sensitivity We here present a new network‘based method ANUBIX based on sampling random gene sets against intact pathway Benchmarking shows that ANUBIX is considerably more accurate than previous network crosstalk based methods which have the drawback of modelling pathways as random gene sets We demonstrate that ANUBIX does not have a bias for finding certain pathways which previous methods do and show that ANUBIX finds biologically relevant pathways that are missed by other methodsImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisImprovements in molecular biology have led to an increase in highthroughput data which typically produces lists of differentially expressed genes or proteins These lists are useful for identifying genes with important roles in certain conditions However more insight about the biological mechanisms is often needed eg which functional gene sets are related to genes in the result list The study of the relation between a query gene set differentially expressed gene list and functional gene sets pathways is called pathway enrichment analysisThere are four generations of pathway enrichment analysis approaches Overrepresentation analysis ORA calculates how many genes from a list of genes extracted based on a threshold or criteria eg differentially expressed genes are in a certain pathway1 Statistical significance is assessed repeating this process with a background list of genes eg all the genes in the microarray This is known as Gene Enrichment Analysis GEA and famous tools like DAVID2 use it Similar but taking into account all the genes in the experiment and the gene expression values is the Functional Class Scoring algorithms FCS3 for which known algorithms include Gene Set AnalysisGSA4 and Gene Set Enrichment Analysis GSEA5 However both FCS and ORA have limitations They both consider genes as independent which is often not true only taking into account their overlap and not their associations or interactions6 Another issue with overlapbased methods is their low coverage since they are heavily dependent on pathway knowledge which is still incomplete leading to a high rate of false negatives7 Pathway topologybased methods use the same steps as FCS with additional pathway topology information However the reliance on gene overlap leads to similar limitations as ORA and FCSWe could consider the network crosstalk enrichment tools as the fourth generation They rely on a network such as a functional association network like Funcoup8 or STRING9 These networks integrate different experiments from different data types into a single network providing information about gene to gene functional associations which is translated into links in the network With this limitations such as gene independency and low coverage of overlapbased methods are overcome Association between two sets is measured in terms of links between them in the network known as crosstalk In the past few years different ways to assess enrichment between two gene sets have been published like NEA10 EnrichNet11 CrosstalkZ12 NEAT13 NEArender14 BinoX7 and GeneSetDPGeneSetMC15 EnrichNet defines a network enrichment score based on network distances between two gene sets using random walks with restart but is not able to calculate statistical significance Department of Biochemistry and Biophysics Science for Life Laboratory Stockholm University Box Solna Sweden email eriksonnhammerdbbsuseScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cof the enrichment The tools NEA and CrosstalkZ assess significance using statistical tests assuming that crosstalk between nonenriched gene sets is normally distributed but this is often not the case Moreover they rely on network randomizations to obtain null model parameters which makes them computationally very slow Computational time is reduced in BinoX which also applies network randomization but uses the binomial distribution to calculate statistical significanceThe methods NEAT NEArender and GeneSetDPGeneSetMC do not use network randomization NEAT calculates the expected number of links between two gene sets based on their degrees and then uses the hypergeometric distribution to assess statistical significance NEArender computes the expected number of links in the same way as NEAT but uses a chisquare test to assess statistical significance GeneSetDP uses dynamic programming to calculate an exact distribution of the expected number of links to a pathway for a certain gene set size GeneSetMC does this approximately using MonteCarlo sampling which is faster These two algorithms are however not implemented to allow large scale pathway enrichment analysisThe null model assumption of NEAT NEArender and BinoX is that compared gene sets are expected to behave like random gene sets For real pathways that are very nonrandom eg highly intraconnected this can lead to underestimating the expected level of crosstalk and produce a high false positive rate FPR To avoid this it is important that the method can cope with the nonrandomness of pathways To this end we have developed a novel networkbased pathway enrichment analysis algorithm called ANUBIX Adaptive NUll distriButIon of Xtalk which is based on scoring random gene sets against real pathways to build its null model We show that ANUBIX clearly outperforms recent network crosstalk methods like BinoX NEArender and NEAT in terms of avoiding False Positives FP showing that it can model expected network crosstalk to pathways more preciselyMaterial and methodsOur networkbased pathway enrichment analysis tool ANUBIX depends on a global functional association network We used the network Funcoup version with a link confidence cutoff of containing genes and links With those genes cid31g1 g2 gnˆ’ gncid30 ˆˆ S and all the pairwise links between them form a symmetric matrix A with dimensions SxS such thataij 1if gi is connected to gj and i � j otherwise aij A gene set Q and a pathway P are a subset of the total number of genes for a certain proteome such that Q P Š† S Notice that S Š† Q we can have some genes from the proteome that are not in the network The crosstalk between Q and P is measured with the degree k cid31iˆˆQcid31jˆˆPaijThe null model is built based on the expected crosstalk between a random gene set of the same size as the original gene set Q and pathway P Since the network connections are binary each link is considered as a Bernoulli trial Y ˆ¼ Bcid31pcid30 where p is the probability of observing a link We also calculate n QP ˆ’ Q ˆ P all the possible links between Q and P We count the links each gene from Q has to the pathway P meaning that if two linked genes are in Q and also in the P we count that link twice boosting the cases where we find overlap Each of these Bernoulli trials are assumed to be independent and the sum of them follows a binomial distributionIn the binomial distribution the mean and variance are defined as µ np and Var npcid311 ˆ’ pcid30 respectively This means that µ ‰¥ Var which may not be true when the random variable is overdispersed leading to an underestimation of its variance16The betabinomial distribution has been extensively used as an alternative to handle overdispersed binomiallike random variables1718 Here the probability of success p is not fixed as it is in the binomial distribution but follows a beta distribution Betaα β with parameters α and β The marginal distribution of the betabinomial is described in Eq a0fcid31kn α βcid30 cid29 nk cid28 Bk α n ˆ’ k βBα βTo estimate the optimal parameters of the betabinomial we use maximum likelihood estimation MLE19 where the loglikelihood is Eq a0lcid31kn α βcid30 logLcid31kn α βcid30 logcid29 n logcid29 nk cid28 logBα k β n ˆ’ k ˆ’ logBα βk cid28 logŴα k logŴβ n ˆ’ kˆ’logŴα β n ˆ’ logŴα ˆ’ logŴβ logŴα βThe negative loglikelihood is optimized with the Nelder and Mead method20 The factorial term in the loglikelihood is removed since it does not depend on the parameters to be optimized Once we have the betabinomial parameters α β of our null distribution we calculate if the crosstalk between Q and P is enriched The null and alternative hypotheses areH0 No more links between Q and P than expected by chanceH1 More links between Q and P than expected by chanceBecause of the discrete nature of the null distributions ordinary pvalues are conservative and therefore mid pvalues were used2122 Mid pvalue is defined as half the probability of the observed statistic plus the probability of observing more extreme values22 The workflow of the ANUBIX algorithm is depicted in Fig a0Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Workflow of ANUBIX The algorithm assesses the significance of the network crosstalk between a query gene set and a pathway A null distribution is generated for each pathway to model the expected crosstalk of random gene sets of the same size as the original gene set This distribution is then fit to a betabinomial distribution to calculate the probability of reaching at least the number of observed links or more between the query gene set and the pathway Software Inkscape version inksc apeIt is important to point out that the networkbased approaches ANUBIX NEAT NEArender and BinoX test three different types of null hypothesis ANUBIX which takes only enrichment into account computes a one tailed test NEAT computes two onetailed tests for enrichment and depletion and takes the minimum pvalue of them multiplied by to emulate a twotailed test BinoX and NEArender compute both enrichment and depletion but only perform one onetailed test since the hypothesis test changes depending on whether the observed number of links is above or below the expected crosstalkPathways To generate the false positive and true positive benchmarks we used KEGG v70123 pathways and REACTOME v6224 pathways for Homo sapiens REACTOME pathways have a deep hierarchical structure including many small pathways on the lower levels that are very specific To reduce Reactome™s specificity we resolved its hierarchy by collapsing lower level pathways below a certain pathway size to their parents until obtaining an average pathway size similar to KEGG pathways genes per pathwayPerformance measures In the FP benchmark we generated random gene sets and tested them against KEGG and REACTOME pathways To make these gene sets representative of real experiments we took the average size of MSigDB25 gene sets which is genesIn the True Positive TP benchmark we bisected the KEGG pathways and REACTOME pathways into two parts Each part gets a similar number of genes and links7 To be able to benchmark GEA we emulated some overlap between the two bisected parts This overlap corresponded to the average overlap between the MSigDB gene sets and the pathway measured individually for each of the pathways in KEGG and REACTOMECorrection for multiple hypothesis testing was done using the Benjamini“Hochberg procedure26Stability Our null distributions are based on random sampling We take random samples of genes from the genome This stochastic procedure makes the null distributions different every time they are generated Since the pvalues are computed from the null distribution their values may change To analyze stability we generated the null distribution times for the crosstalk between the same gene set to the same pathways for increasing numbers of random samples For each sample size we computed the coefficient of variation CV which is the ratio between the standard deviation SD and the mean We required a CV lower than to limit the dispersion of the mean of the null distribution and this was reached at random samples Once the number of random samples were chosen we measured how much the pvalues were varying in each run For that we ran a randomly selected MSigDB gene set times To compute the confidence interval of the pvalues we used the central limit theorem and applied normal distribution statistics to compute themUsed programs ANUBIX bitbu cketsonnh ammer group anubi xBinoX bitbu cketsonnh ammer group binox NEAT cranrproje ctwebpacka gesneatneatpdfNEArender cranrproje ctwebpacka gesNEAre nderNEAre nderpdfGeneSetDP githu bcomstati stica lbiot echno logygenes etdpScientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Overdispersion of KEGG and REACTOME pathways null distributions when sampling random gene sets of size from the proteome The dispersion for each pathway is calculated as the ratio between the variance and the mean of the crosstalk null distribution For each pathway database we illustrate the dispersion values through a boxplot and also by showing the dispersion distribution Software R version wwwrproje ctFigure a0 Observed crosstalk distribution fit with binomial and betabinomial distributions random gene sets of size were used to generate a null distribution of crosstalk to the A œBetaalanine metabolism B œProstate cancer and C œAlzheimer™s disease pathways Betabinomial shows a much better fit to the observed link distribution than the binomial Software R version wwwrproje ctResultsTo correctly assess the statistical significance of an observed network crosstalk between two gene sets eg one experimental gene set and one known pathway it is paramount that the null distribution appropriately models the crosstalk of random query gene sets Note that it is not necessarily appropriate to assume that the pathway gene set behaves like a random gene set ie the null distributions need to model crosstalk between random query gene sets versus real pathway gene sets It is also paramount to model the expected crosstalk distribution with an appropriate distribution Previous methods such as BinoX or NEAT use binomial and hypergeometric distributions respectively which are not appropriate for overdispersed distributions since they do not allow the variance of the distribution to be greater than the mean To showcase this we generated null distributions for KEGG and REACTOME pathways by sampling gene sets of size from the proteome In Fig a0 we show the dispersion for each pathway as the ratio between the variance and the mean of the crosstalk null distribution We observe that almost all of these distributions suffer from overdispersion meaning that the variance of the distribution is greater than the mean Therefore statistical models that cannot cope with overdispersion are not appropriate to model the null distribution of most pathwaysTo visualize the overdispersion in detail we chose pathways that are in different quartiles of the dispersion distribution We show their null distributions in Fig a0 Figure a03A shows the œBetaalanine metabolism Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Pvalue uniformity test of ANUBIX Binox GEA GeneSetDP NEArender and NEAT random gene sets of genes were tested for crosstalk enrichment against the KEGG pathway œProstate cancer A Reported pvalues are plotted against theoretical quantile rank A perfect method should adhere to the diagonal B Distributions of the FPR for all KEGG and REACTOME pathways tested with ANUBIX BinoX NEArender NEAT and GEA Green distribution for enriched tests and red distribution for depleted The dashed line at FPR denotes the expected FPR level The black triangle and circle represent the mean FPR for enrichment or depletion respectively Software R version wwwrproje ctpathway whose dispersion value is in the first quartile Figure a03B shows the œProstate cancer pathway with a dispersion in the second quartile and Fig a03C shows the œAlzheimer™s disease pathway with a dispersion in the fourth quartile The high variance relative to the mean gives a very poor fit with the binomial distribution yet the betabinomial distribution gives a very good fit This underestimation of variance by the binomial distribution would lead to many false positives With a few pathways there is no overdispersion in the data but these can fit a betabinomial equally well as a binomialBenchmark for false positives Since the null model in ANUBIX is based on random gene sets we expect the pvalue distributions when tested with random query gene sets to behave uniformly for any pathway For almost all pathways we observed a virtually perfectly uniform distribution when plotting ANUBIX pvalues of random gene sets against each KEGG pathway full results at Supplementary Fig a0 A few pathways deviated somewhat from uniform which is the result of the betabinomial fit not being able to model the null distribution with enough precision A second type of deviation from perfect uniform distribution is caused by staggering of observed pvalues This is relatively frequent and arises because the support of the test statistics is limited to a few values and therefore unavoidable We also generated the pvalue distributions for gene sets of size and size against each KEGG pathway Supplementary Fig a0 and respectively which gave similar results However some pathways seem to be affected by the size of the gene set ANUBIX was compared to the top networkbased methods BinoX NEAT and NEArender and a recently published method GeneSetDP For comparison we also tested a popular overlapbased pathway enrichment method GEA Because GeneSetDP and GenesetMC are too computationally heavy for large scale analysis we first tested all the gene sets against one individual pathway We only used GeneSetDP because GeneSetMC produces similar pvalues Pvalues were plotted versus quantiles of a uniform distribution For an unbiased method the pvalues would lie on the diagonal y x Figure a04A shows that for the œProstate cancer pathway Pvalues of ANUBIX adhere to the diagonal much better than for BinoX NEAT NEArender and GEA while performing equally well as GenesetDPFor crosstalk to random gene sets we expect of the pvalues to be lower than However for the œProstate cancer pathway BinoX had of its pvalues lower than NEAT and NEArender GEA whose coverage is small7 had of its pvalues below and highly discrete taking on only four possible values for œProstate cancer due to few overlapping genes ANUBIX and GeneSetDP find a correct fraction of the pvalues with and GeneSetDP under respectivelyWe also ran ANUBIX BinoX NEAT NEArender and GEA for the random gene sets against all pathways in the KEGG database and REACTOME database Full results in Supplementary Data and Data respectively GeneSetDP was not included as it is not implemented to run at a large scale NEAT NEArender and BinoX can also give statistical significance when gene sets have fewer links to a pathway than expected by chance known as depletion To make a more consistent benchmark where all methods can be compared equally we only considered enrichment and depleted pathways were treated as nonsignificant The average FPR for all KEGG pathways was with ANUBIX with BinoX with NEAT with NEArender and with GEA For REACTOME almost the same FPR values were obtained ANUBIX BinoX NEAT NEArender and GEA Roughly the same FPR levels came from significant depletions for BinoX NEAT and NEArender However the averaging of the FPR levels for all pathways does not show the real problem of these methods Some pathways could give very nonconservative pvalues while other pathways could give very conservative pvalues To show how each method performs for each of the pathways we plot the distribution of the FPR fraction of pvalues below for each pathway as violin plots in Fig a04B Since GEA Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cand ANUBIX cannot test for depletion they only have the enriched case A perfect method would have all points close to the dashed line at FPR ANUBIX produces FPR values close to this line meaning that the model is robust GEA greatly underestimates FDR and produces almost no false positives but this leads to very poor sensitivity as shown below NEArender NEAT and BinoX produce similar FPR distributions that are very spread out ie the FPR tends to be very different for different pathways For the tests performed per pathway some pathways reach an FPR of for enriched cases and similar for depleted Summing these two can lead to a total FPR above if we take both enriched and depleted cases into account which is very nonconservative The plot also shows that for some pathways these methods are overly conservative giving considerably lower FPR than they should In other words methods like BinoX NEAT and NEArender have a huge variation in the quality of their pvalues depending on the pathway under studyBinoX is implemented in a web server called PathwAX27 where users can submit a query gene set to test for network crosstalk enrichment By analogy we studied false positive rates assuming independence between gene sets where each user submits a single gene set ie multiple testing correction is only performed for number of pathways each query is compared to random gene sets were used against the KEGG database A FDR threshold of was used and enrichment and depletion were grouped separately as shown in Fig a05A The top pathways with highest FPR for BinoX were plotted full results in Supplementary Data all having a highly nonconservative behaviour for BinoX NEAT and NEArender Every time a user submits a random gene set the chance of getting one of these pathways is very high on average if we take both enriched and depleted cases into account In contrast ANUBIX and GEA have less than FPR We observed a very high correlation between perpathway FPR values for BinoX NEAT and NEArender above for each pairwise comparison This indicates that the pathway enrichment analysis results obtained with these methods are highly similar They all had low Pearson correlation to ANUBIX with for BinoX for NEAT and for NEArender The corresponding Spearman correlations were and As for the pathways we noticed that there is a high overlap between some of them For instance the œAlzheimer™s disease and œParkinson™s disease pathways share of their genes The œAlzheimer™s disease the œParkinson™s disease and the œHuntington™s disease pathways have of the genes in common from the union between them Further the œOxidative phosphorylation the œNonalcoholic fatty liver disease the œAlzheimer™s disease the œParkinson™s disease and the œHuntington™s disease have of the genes in common from the union between them Therefore if there is significant crosstalk to one of them crosstalk to the other pathways is very likely The high dependency between some pathways points to opportunities for further improvement of pathway definitions Further exploration was performed in these pathways™ topologies to understand their tendency to generate many FPsWe computed the fraction of intralinks for each pathway as the ratio between the number of internal links and the total number of links We plotted this ratio against the FPR Fig a05B A higher fraction of intralinks means that more links are within the pathway than to the outside suggesting a more isolated pathway The Spearman correlation coefficient between the fraction of intralinks and FPR for BinoX was indicating that the fraction of internal links plays a major role in causing false positives This dependence is also observed with NEAT with a correlation of and with NEArender at However ANUBIX had a correlation of only and GEA This indicates that methods like NEAT NEArender and BinoX cannot deal properly with pathways that are clearly not random and behave more as isolated communitiesAdditionally we calculated the number of maximal cliques each of the KEGG pathways has and we observed a correlation with the FPR for BinoX with a spearman correlation of These maximal cliques were computed using the igraph package in R We considered cliques as all complete subgraphs and a clique is considered maximal if we cannot add more nodes to it This indicates that the higher the number of maximal cliques in a pathway meaning a less random pathway in terms of topology the higher the FPR isBenchmark of true positives Besides a correct FPR it is also important to verify that the power of the method is sufficient for a high true positive rate TPR To this end we devised a benchmark by splitting each KEGG and REACTOME pathway into two parts and then measured each method™s ability to reconnect these parts The splitting into parts included giving an amount of gene overlap between the two parts emulated based on the average overlap between MSigDB gene sets and KEGG and REACTOME pathways We compared the methods by their Receiver Operating Characteristic ROC curves Figure a06A shows only the tests that are statistically significant FDR and only considering enrichment ANUBIX has a TPR of of the enrichment tests as significant without having any FP BinoX has a TPR of with FPR NEArender a TPR of with FPR and NEAT a TPR of with FPR GEA whose coverage is low gives only TPR and no FPs Figure a06B shows the ROC curve for all the enriched tests performed also including insignificant results This shows the coverage of each method ANUBIX recovers of the TP tests without suffering any FPs BinoX NEArender and NEAT have similar curves recovering and of the enriched TP tests respectively GEA can here maximally find of the TP tests since only those tests have some gene overlap This benchmark shows that GEA has very low coverage of what it can potentially find We note that the maximal TPR obtained by GEA corresponds to the amount of significantly enriched crosstalks obtained when running all of MSigDB against KEGG pathways see Pathway annotation of MSigDB gene setsStability and robustness Considering that the null distributions are based on random sampling we studied the number of iterations required to reach a coefficient of variation CV of Figure a07A shows how many pathways pass that threshold depending on different amounts of random samples of the pathways had a CV lower than when using random samples to model the null distribution To verify that this number of random samples is sufficient for every pathway we computed the enrichment of one randomly selected MSigDB Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Analysis of why certain pathways are very prone to produce false positives random gene sets of genes were tested independently for crosstalk enrichment against the KEGG pathways A The top ten pathways that produce the highest false positive rate FPR with BinoX and the FPR obtained with other methods B Fraction of intralinks for each of the KEGG pathways against FPR The size of the point reflects the total number of links in each pathway Software R version wwwrproje ctgene set to all KEGG pathways times The null distributions are thus generated times for each pathway and we would expect some changes in the pvalues between runs Figure a07B shows the standard deviation of the pvalues We observe that the pvalues almost did not vary showing that random samples are enough Moreover because of sampling the pvalue is not an exact pvalue but a point estimate of it we also provide with the confidence interval of each of the pvalues Supplementary Data Compute time Our method relies on random sampling to model the null distribution which makes ANUBIX computationally intensive To benchmark its speed we did runs each time with a randomly chosen biological gene set extracted from MSigDB against KEGG REACTOME and KEGG plus REACTOME We measured the compute time for each of the networkbased methods see Fig a0 With this benchmark we can show that ANUBIX is fast when running single gene sets One should take into account that ANUBIX and BinoX need a precomputation step before running the actual analysis However the ANUBIX precomputation step takes around a0s whereas in BinoX it takes around a0min To compute the randomized network for BinoX Scientific RepoRtS 101038s4159802070239zVol0123456789wwwnaturecomscientificreports 0cFigure a0 Receiver Operating Characteristic ROC curve For the TP tests each KEGG and REACTOME pathway is divided into two and a TP is interpreted as the crosstalk between two parts from the same pathway For the FP tests random gene sets of size are tested for enrichment against KEGG and REACTOME pathways A ROC curve for only the significantly enriched tests FDR B ROC curve for all enriched tests Software R version wwwrproje ctFigure a0 Stability analysis of ANUBIX A Fraction of KEGG pathways with Coefficient of variation CV below for different number of iterations B ANUBIX pvalues are stable”their variance is low and proportional to the magnitude of the pvalue A randomly chosen MSigDB gene set DAIRKEE_CANCER_PRONE_RESPONSE_BPA was run times against KEGG pathways Standard deviation of the logpvalue is plotted against the meanlogpvalues for each pathway Software R version wwwrproje ctwe used iterations A drawback for ANUBIX compared to methods like BinoX or NEAT is that the computation time for large scale analyses take more time For instance the time required to compute the large scale pathway annotation study for the MSigDB gene sets against KEGG pathways took a0min for ANUBIX using cores a0min for NEArender a0min for BinoX and a0min for NEAT Compute times were measured on an i77700 CPU a0GHz with a0Gb RAMPathway annotation of MSigDB gene sets We carried out a largescale pathway analysis study by running MSigDB gene sets against KEGG pathways using ANUBIX BinoX NEAT NEArender and GEA Full results are in Supplementary Data In total crosstalk tests were done per method and to get a more fair comparison between different methods we only considered enriched crosstalk considering that ANUBIX and GEA can only test for enrichmentNEArender BinoX and NEAT found the highest number of significantly FDR enriched crosstalks with and of all pairs respectively followed by ANUBIX with and GEA with Many MSigDB gene sets thus appear to have a high occurrence of pathway enrichments Even if we do not know whether those enrichments are TPs or FPs we show above Figs a0 and 5A that BinoX NEArender and NEAT are prone to produce FPsThe Venn diagram in Fig a0 shows that the overlap between BinoX NEAT and NEArender is very high having of their significant pathway annotations in common This was expected since all these methods consider pathways as random The overlap is even higher between NEAT and NEArender because they compute Scientific RepoRtS 101038s4159802070239zVol1234567890wwwnaturecomscientificreports 0cFigure a0 Compute time when running a random experimental gene set from MSigDB different gene sets were tested against KEGG REACTOME and KEGG plus REACTOME pathways for each of the networkbased methods Since ANUBIX allows parallelization we also added another run with cores The error bars show the variability in compute time for each of the methods in each of the databases The BinoX precomputation step is not included since it takes a0min Software R version wwwrproje ctFigure a0 KEGG pathway annotation for MSigDB gene sets with five methods The Venn diagram shows the number of shared pathway annotations at FDR Note that ANU
2
distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedVon Hippel“Lindau disease is an autosomal dominant inherited syndrome predisposing to a variety of highly vascularised tumorsin diï¬erent ans Although bilateral pheochromocytoma was reported in patients with von Hippel“Lindau disease the coexistence of primary hyperparathyroidism is not a common condition We report an observation of a primary hyperparathyroidism secondary to an ectopic secretion of intact parathyroid hormone in a 17yearold girl with von Hippel“Lindau diseaseand bilateral pheochromocytoma She presented with a newly diagnosed diabetes mellitus and a severe arterial hypertensionBlood tests disclosed hypercalcemia with increased intact PTH level Cervical ultrasound and sestamibi scintigraphy were normalTwentyfourhour urinary normetanephrine level was highly elevated pointing to a catecholaminesecreting tumor e abdominal computed tomography showed bilateral adrenal masses MIBG scintigraphy exhibited a high accumulation of the tracerin both adrenal tumors Genetic testing revealed a mutation of the VHL gene e patient underwent a bilateral adrenalectomye postoperative outcome was marked by normalization of blood pressure blood glucose calcium and PTH levels In our casethe elevation of intact PTH and its spontaneous normalization after surgical treatment of pheochromocytomas confirms itsectopic secretion IntroductionPheochromocytomas are uncommon neuroendocrinetumors that arise from chromaffin cells of the adrenalmedulla and produce excessive amounts of catecholamines epinephrine norepinephrine and dopamine []Although most pheochromocytomas are sporadic morethan are associated with an inherited mutation andthis frequency can be as high as if diagnosed before years of age [] In childhood pheochromocytomasare mostly due to genetic causes including von Hippel“Lindau VHL disease multiple endocrine neoplasiatype MEN2 hereditary pheochromocytoma paraganglioma syndrome and rarely neurofibromatosis type [] Compared to adults VHL disease is reported to bethe most frequent genetic disorder causing pheochromocytomasbilateral[] Whileinchildrenpheochromocytomas were reported in patients with VHLdisease the coexistence of primary hyperparathyroidismis not a common condition []Herein we report a case of a VHL disease with bilateralintactpheochromocytoma and an ectopic secretion ofparathyroid hormone in an adolescent girl Presentation of CaseA 17yearold girl was referred to our department for theinvestigation of a newly diagnosed diabetes mellitus and asevere arterial hypertension She was the first child ofconsanguineous parents Her past medical history wasunremarkable Her family history was notable for type diabetes and dyslipidemia but no history of pheochromocytoma paraganglioma unexplained sudden death or 0cCase Reports in Endocrinologycondition that may lead to thinking about VHL disease wasreportedAs symptoms the patient complained of headachespalpitations diaphoresis and hot flashes since one monthShe reported also asthenia and body weight loss of fivekilograms in two weeksOn examination she had a body weight of kg abody height of cm a body mass index of kgm2 ablood pressure of mmHg without orthostatichypotension a regular pulse of bpm and a largeabdominal caf´eaulait spot Figure yroid abdominal and neurological examinations were normalTwentyfourhour blood pressure monitoring confirmed the diagnosis of hypertension and the presence ofpeaks of mmHg Electrocardiogram showed asinus tachycardia Capillary glucose level was glwithout ketosisFundoscopy showed grade hypertensive retinopathywithout any other abnormalitiese results of biological investigations are shown inTable e diagnosis of clinically suspected pheochromocytomawas confirmed by the dosage of urinary methoxylated derivatives at times the upper limit of normal In addition thediagnosis of diabetes mellitus dyslipidemia and primaryhyperparathyroidism were madethe first on thee abdominal computed tomography CT showed tworight measuringadrenal masses — — mm with a spontaneous density of HUheterogeneously enhanced in the arterial time showing areasof necrosis with an absolute washout of the second onthe left measuring — mm with the same characteristicsas the first one Figure ere were neither other localizations norlymph nodes MetaiodobenzylguanidineMIBG scintigraphy exhibited a high accumulation of thetracer in both adrenal tumors with no other localizationCervical ultrasound and 99mTcsestamibi scintigraphy werenormal Cardiac ultrasound was normale diagnosis of multiple endocrine neoplasia type 2awas highly suspected and the patient underwent a molecularinvestigation DNA analysis did not find a RET protooncogene mutation However it showed a missense mutationc191G C pArg64Pro in exon of the VHL gene onchromosome e diagnosis of bilateral pheochromocytoma in the setting of VHL disease was established Abdominal CT scan and craniospinal magnetic resonanceimaging with contrast did not show any cysts or othertumorse patient was treated with an alpha blocker prasozinea calcium channel blocker a beta blocker and insulin at adaily dose of unitskg She underwent a bilateral adrenalectomy in two steps e pathological examinationconfirmed bilateral adrenal pheochromocytomas Replacementtherapy with hydrocortisone was initiated aftersurgerye postoperative outcome was determined by thespontaneous normalization of blood pressure blood glucose calcium and PTH levels e patient remainedasymptomatic with no evidence oflocal recurrence ordistant metastasis during the months of followup efamily screening for VHL has not been performed yet DiscussionVHL disease is a dominantly inherited familial cancersyndrome caused by a germline mutation in the VHL tumorsuppressor gene and predisposing to a variety of benign andmalignant neoplasms most frequently retinal central nervous system and spinal hemangioblastomas renal cell carcinoma RCC pheochromocytoma and pancreatic tumors[ ] While central nervous system and retinal hemangioblastomas are the earliest expressions of the VHL syndrome pheochromocytoma may be the first manifestationof the disease especially in children and adolescents as it wasthe case of our patient [“] e frequency of pheochromocytoma in VHL syndrome is about to []Families with VHL disease have been divided into twosubtypes VHL type and VHL type based on the likelihood of developing a pheochromocytoma e presence ofpheochromocytoma defines types VHL disease is latteris subdivided based on the risk of developing RCC Type 2Aand 2B families have a low and high incidence of RCCrespectively whereas VHL type 2C kinds are characterizedby the development of pheochromocytoma without anyother manifestations of the disease [] However late onsetof other attacks is possible and a followup even spaced isrequired []Genotypephenotype correlations have been documented for this disorder and specific mutations are associated with the appearance of tumors in certain ansWhile most type families were reported to be more likelycarrying a missense mutation in the VHL gene most type families are aï¬ected by truncating or deletion mutations[] In our case the presence of pheochromocytomas andthe missense mutation in VHL gene suggested type VHLdisease Moreover the mutation found in our patientpArg64Pro has been described in patients with isolatedpheochromocytoma associated with RCC or with pancreaticneuroendocrine tumor [“] However our patient didnot present any sign of RCC or pancreatic neuroendocrinetumor during the months of followup is evaluationmay be early in our case as RCC and pancreatic neuroendocrine tumors in patients with VHL disease generallyappear at more advanced ages around “ years []Pheochromocytoma in VHL disease tends to be seen at ayounger age and is more frequently multifocal as in ourpatient and may be extraadrenal [ ] In most publishedcases the mean age at presentation was about years butvery young cases have been described the youngest before years [ ] In addition VHLassociated pheochromocytomas are less likely to be associated with symptoms orbiochemical evidence of catecholamines production compared with those occurring in patients without VHL [ ]In a report of the National Institute of Health about patients with VHL disease and pheochromocytomas a totalof tumors were identified Of these originatedoutside the adrenal gland and of the patients wereasymptomatic without hypertension orevidence of 0cCase Reports in EndocrinologyFasting glucose level mmollGlycated hemoglobin Total cholesterol mmollTriglycerides mmollNatremia mmollKalemia mmollCreatinine mgLCalcium mgLPhosphate mgLAlbumin glIntact PTHˆ— μgLPTH parathormone TSH thyroid stimulating hormone FT4 freeT4 NMN normetanephrines MN metanephrinesˆ—e intact PTH dosage was madeTSH mUILFT4 ngdlCalcitonin ngL hurine NMN μg24 hurine MN μg24 hurine sodium mmol24 h hurine potassium mmol24 h hurine calcium mg24 h hproteinuria g24““using 3rd generation chemiluminescence immunoassayReference ranges‰¤““““““““““““Figure A large abdominal caf´eaulait spotTable Biological parameters before and after surgeryBefore surgeryAfter surgery””””””””Figure Abdominal computed tomography showing the two adrenal masses two white arrowsincreased catecholamines production [] is was not thecase of our patient who had sustained severe hypertensionassociated with the classic symptoms of pheochromocytomapalpitation sweating and hot flashes hypokalemia and asecondary diabetes mellitus In our case as reported in theliterature [ ] a remarkable remission of diabetes 0cCase Reports in EndocrinologySoahighmellitus and an improvement of lipid profile were noticedafter tumor removal confirming the secondary character ofthese two metabolic disorders In fact catecholamine excessaï¬ects insulin secretion decreased glucose uptake in theperipheral tissues and increased insulin resistance leading toimpaired fasting glucose or overt diabetes mellitus []Moreover the increase in catecholamine production may beresponsible for decreased inhibition of lipolysis by insulinand decreased activity of lecithincholesterol acyltransferasean enzyme which breaks down free cholesterol []e risk of malignancy is low Less than of allpheochromocytomas in VHL disease are malignant [] Inour case neither distant metastasis nor lymph nodes werefound but a long term followup should be carried outMeasurement of plasma or urinary metanephrines andnormetanephrines is the gold standard in diagnosingpheochromocytoma and can also provide important diagnostic information [] In fact in a study carried out byEisenhofer [] comparing the clinical and biochemical characteristics of pheochromocytomas in multipleendocrine neoplasia type versus the VHL syndrome andincluding and patients with these disorders respectively VHL patients almost exclusively produced normetanephrinesnormetanephrinestometanephrines ratio is expected in patients with VHL disease as was found in our patient Furthermore in comparison with MEN2 tumors VHL tumors had lower totaltissue contents of catecholamines and expression of tyrosinehydroxylase TH the ratelimiting enzyme in catecholamine synthesis ey also had much lower expression ofphenylethanolamine Nmethyltransferase PNMT the enzyme that converts norepinephrine to epinephrine andtissue stores of epinephrine [] Regarding the histopathological features VHLassociated pheochromocytomas arecharacterized by a thick vascular tumor capsule and are incontrast to MEN not associated with adrenal medullarhyperplasia []While bilateral pheochromocytomas were reported inpatients with VHL disease the coexistence of primary hyperparathyroidism as in our case is not a common condition[] Hypercalcemia associated with pheochromocytoma hasbeen documented and is thought to be caused by severalmechanisms First elevated catecholamines can activate thePTH receptor resulting in catecholamineinduced osteoclastic bone resorption but in contrast to our case the PTHlevel is not elevated [] Secondly hypercalcemia can be dueto the production of PTHrelated peptide PTHrp by thetumor which was doubtless not the case in our patient as thelevel of intact PTH was elevated [] Furthermore intactPTH in our case was quantified using a 3rd generationchemiluminescence immunoassay which does not recognizePTHrpird parathyroid adenoma can be a part of multipleendocrine neoplasia that was however rarely reported inVHL disease [] e negativity of the topographic investigations and the spontaneous normalization of intact PTHafter surgical treatment are against this hypothesis Finallyalthough the exact physiopathological mechanism is notclear the fact that both serum calcium and PTH levels wereelevated before surgery and became normal after the removal of the pheochromocytomas strongly suggests that thetumor itself was secreting PTH or a substance that stimulatesexcessive PTH secretion by the parathyroid glands Only fewcases of ectopic hormonal secretion by pheochromocytomasuch as adrenocorticotropic hormone ACTH calcitoninparathyroid hormone PTH vasoactive intestinal peptideVIP and growth hormonereleasing hormone GHRHwere reported [ ] Unfortunately immunohistochemistry assay for PTH in the tumor tissue was not available toclarify this question in our case Conclusionis report highlights the rare case of ectopic intact PTHsecretion by a bilateral pheochromocytoma in an adolescentgirl with VHL disease We consider that controlling calciumand PTH after adrenalectomy is useful if the topographicassessment of primary hyperparathyroidism is negativeTo the best of our knowledge our patient is the secondyoungest reported childhood VHL case in the literaturepresenting with a bilateral pheochromocytoma secretingectopic intact PTH Genetic testing and a meticulous followup are necessary for the diagnosis ofthe associatedcomorbidities in VHL diseaseData Availabilitye data used to support the findings of this study areavailable from the corresponding author upon requestConflicts of Intereste authors declare that they have no conflicts of interestAcknowledgmentse authors thank Professor Anne Barlier Laboratory ofMolecular Biology Hospital La Conception MarseilleFrance for her help in molecular studyReferences[] J W M Lenders QY Duh G Eisenhofer œPheochromocytoma and paraganglioma an endocrine societyclinical practice guideline e Journal of Clinical Endocrinology Metabolism vol no pp “ [] E Sbardella T Cranston A M Isidori œRoutine geneticscreening with a multigene panel in patients with pheochromocytomas Endocrine vol no pp “ [] S G Waguespack T Rich E Grubbs œA current reviewof the etiology diagnosis and treatment of pediatric pheochromocytoma and paraganglioma e Journal of ClinicalEndocrinology Metabolism vol no pp “[] M Barontini G Levin and G Sanso œCharacteristics ofpheochromocytoma in a to 20yearold population Annals of the New York Academy of Sciences vol no pp “ [] T Arao Y Okada T Tanikawa œA case of von HippelLindau disease with bilateral pheochromocytoma renal cell 0cCase Reports in Endocrinology[] O Mete A S Tischler R D Krijger œProtocol for theexamination of specimens from patients with pheochromocytomas and extraadrenal paragangliomas Archives ofPathology Laboratory Medicine vol no pp “[] N O Atuk T McDonald T Wood œFamilial Pheochromocytoma Hypercalcemia and von HippelLindauDisease a ten year study of a large family Medicine vol no pp “ [] K Takeda N Hara M Kawaguchi T Nishiyama andK Takahashi œParathyroid hormonerelated peptideproducing nonfamilial pheochromocytoma in a child International Journal of Urology vol no pp “ [] J KirkbyBott L Brunaud M Mathonet œEctopichormonesecreting pheochromocytoma a francophone observational study World Journal of Surgery vol no pp “ [] L V Neto G F Taboada L L Corrˆea œAcromegalysecondary to growth hormonereleasing hormone secreted byan incidentally discovered pheochromocytoma EndocrinePathology vol no pp “ carcinoma pelvic tumor spinal hemangioblastoma and primary hyperparathyroidism Endocrine Journal vol no pp “ [] A N A V D HorstSchrivers W J Sluiter R C Kruizinga œe incidence of consecutive manifestations in VonHippelLindau disease Familial Cancer vol no pp “ [] R R Lonser G M Glenn M Walther œvon HippelLindau disease e Lancet vol no pp “[] A D ‡akır H Turan A Aykut A Durmaz O Ercan andO Evliyao˘glu œTwo childhood pheochromocytoma cases dueto von HippelLindau disease one associated with pancreaticneuroendocrine tumor a very rare manifestation Journal ofClinical Research in Pediatric Endocrinology vol no pp “ [] S P Rednam A Erez H Druker œVon hippellindauand hereditary pheochromocytomaparagangliomasyndromes clinical features genetics and surveillance recommendations in childhood Clinical Cancer Research vol no pp e68“e75 [] G F C Fagundes J Petenuci D M Lourenco œNewinsights into pheochromocytoma surveillance of young patients with VHL missense mutations Journal of the Endocrine Society vol no pp “ [] F Hes R V D Luijt A Janssen œFrequency of VonHippelLindau germline mutations in classic and nonclassicVon HippelLindau disease identified by DNA sequencingSouthern blot analysis and multiplex ligationdependentprobe amplification Clinical Genetics vol no pp “ [] T Krauss A M Ferrara T P Links œPreventivemedicine of von HippelLindau diseaseassociated pancreaticneuroendocrine tumors EndocrineRelated Cancer vol no pp “ [] E Wittstr¨om M Nordling and S Andr´easson œGenotypephenotype correlations and retinal function and structure invon HippelLindau disease Ophthalmic Genetics vol no pp “ [] G Eisenhofer M M Walther TT Huynh œPheochromocytomas in von HippelLindau syndrome and multiple endocrine neoplasia type display distinct biochemicaland clinical phenotypes e Journal of Clinical Endocrinology Metabolism vol no pp “ [] M M Walther R Reiter H R Keiser œClinical andgenetic characterization of pheochromocytoma in von HippelLindau families comparison with sporadic pheochromocytomaofpheochromocytoma Journal of Urology vol no pp “ naturalhistoryinsight[] J Cha M Khurram L Gellert P Epstein N Baregamian andC Hendrickson œCase of reversible diabetes mellitus in thesetting of benign Pheochromocytoma Journal of Clinical andTranslational Endocrinology Case Reports vol pp “[] M L Good P Malekzadeh S M Ruï¬ œSurgical resection of pheochromocytomas and paragangliomes is associated with lower cholesterol levels World Journal of Surgeryvol no pp “ [] B Mesmar S PoolaKella and R Malek œe physiologybehind diabetes mellitus in patients with pheochromocytomaa review of the literature Endocrine Practice vol no pp “ givesinto 0c'
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neutrophils account for “ of circulating leukocytes and are the first immune cells recruitedto an ‚ammatory site they play an important role in the innate immune response topathogens as patients with neutropenia are highly susceptible to bacterial and fungal infections neutrophils perform numerous functions that target microbes including phagocytosis the releaseof antimicrobial peptidesproteases and netosis interestingly neutrophils have garneredconsiderable interest for their emerging and prominent roles in modulating cancer growth andmetastatic progression the roles played by neutrophils in the cancer setting are diverse andcomplex leading to the concept of neutrophil heterogeneityplasticity and the notion that distinctneutrophil subsets might existgranulopoiesisdiï¬erentiationand mobilization of maturefrom the bone marrow intocirculation this process begins with the commitment of granulocytemonocyte myeloidsegmented neutrophilsregulatedprocessthatinvolvestheisatightlyedited bybrahm segaluniversity at buffalo united statesreviewed byye liuniversity of texas md andersoncancer center united statesconnie jackamancurtin university australiacorrespondencepeter m siegelpetersiegelmcgillcaspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in immunologyreceived may accepted july published august citationhsu be shen y and siegel pm neutrophils orchestrators of themalignant phenotypefront immunol 103389fimmu202001778frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypegmps which progressthrough a series ofprogenitorsneutrophil progenitors myeloblast promyelocyte myelocytemetamyelocyte band cell untilthey become a matureneutrophil in cancer dysregulated granulopoiesis hasled to theidentification of diï¬erent neutrophil subsets that play a role intumor progression preneus comprise a neutrophil precursorpopulation that retain their proliferative capacity and expandin the bone marrow and spleen of tumor bearing mice preneus diï¬erentiate into immature and mature neutrophilswith the former found to accumulate in growing tumors anearly stage committed unipotent neutrophil precursor nep hasalso been identified and their adoptive transfer into humanizedmice promoted solid tumor growth by inhibiting t cellactivation two neutrophil subsets highdensity neutrophilshdns and lowdensity neutrophils ldns were identified invarious tumor models by diï¬erential density centrifugation hdns represent mature segmented neutrophils whereas ldnscomprise a heterogeneous mixture of mature and immatureneutrophils increasing mobilization of ldns into theperipheral blood was associated with enhanced tumor growthand metastasis “functionsand antitumorigenicin addition to the identification of distinct neutrophil subsetsneutrophils exhibit plasticity in response to tumorderivedfactors in a manner similar to macrophages neutrophils havebeen classified into two categories n1 and n2 to describetheir prorespectively in vivo evidence has shown that tumorassociatedneutrophils tans can change their function from a protumor phenotype n2 to an antitumor n1 phenotypewith the addition of a tgf inhibitor arguing that tgfis an important factor driving the n2 phenotype incontrast signals associated with an antitumor n1 phenotypeinclude type iinterferons and those propagated by themet receptor however this categorization is likelyto represent an oversimplification of neutrophil diversityneutrophil polarization similar to macrophages could alsorepresent a continuum of diï¬erent neutrophil phenotypespresent in the tumor microenvironment these advancesregarding the degree of neutrophil heterogeneityplasticityobserved in the cancer setting have sparked an intense andrenewed interestin this cell population while there areongoing discussions in the field regarding the relationshipsubsets we directbetween pmnmdscs and neutrophilto excellentthe readerthatfully discussthese we will briefly discuss antitumorrelationshipsneutrophilreview will primarilyroles of neutrophils and neutrophildiscussassociated functionsgrowth andmetastatic progressionfunctions howeverin promotingthe recentreviewstumorthisantitumor neutrophil functionscan participateantitumorneutrophilsmechanisms thattumor growth or eliminate cancercells a wellstudied neutrophilassociated function isin a variety oflimittheir ability to generate reactive oxygen species ros tolimit tumor progression upon tumor cell contact mousederived neutrophils can release hydrogen peroxide to eliminatemetastatic cancer cells in vitro subsequentlyit wasdemonstrated that expression of trpm2 transient receptorpotential cation channel subfamily m2 on tumor cells increasedtheirsensitivity to neutrophilmediated h2o2dependentcytotoxicity this occurred through a mechanism that involveda transient increase in ca2 mobilization within cancer cells trpm2 upregulation in tumor cells occurred followingan epithelialtomesenchymaltransition emt and cancercells that have undergone an emt were more susceptible toneutrophilmediated killing more recently an interactionbetween the receptor for advanced glycation end productsrage which is expressed on tumor cells and cathepsin gpresent on murine neutrophils was shown to mediate in vitrotumor cell cytotoxicity in a h2o2dependent manner the release of neutrophil ros is also dependent on the tumormicroenvironment in hypoxic tumor microenvironments theability of murine neutrophils to kill tumor cells in vivo throughthe release of ros is greatly diminished thus neutrophilshave the capacity to mediate rosdependent direct tumorcell killingcausingthe interplay of neutrophils with otherimmune celltypes can also indirectly limittumor progression tumorassociated neutrophils suppress the protumorigenic role ofil17 secreting Îδ t cells by inhibiting their proliferationlow glutathione levels in Îδ17 t cells rendered them sensitiveto neutrophilderived rosenhanced oxidativestress and reduced proliferation in earlystage humanlung cancer a subset ofimmature neutrophils have beenidentified as having antigenpresenting functions and act topromote antitumor immunity by stimulating the secretionofinaddition to neutrophilt cellinteractions communicationbetween neutrophils and monocytes can also elicit antitumoreï¬ects nonmetastaticifnÎproducing monocytes to the lungs ifnÎ release activatestmem173sting within neutrophils whichstimulatesneutrophilmediated killing of disseminated cancer cells in thelungs ‚ammatory cytokinesfrom t lymphocytescan mobilizecancercellsneutrophils have been shown to ltrate deposits of prostatecancer cells within bone metastases importantly neutrophilsimpaired bone metastasis progression by inhibiting stat5signal transducer and activator of transcription functionwithin prostate cancer cells resulting in their apoptotic cell death recently neutrophils have been reported to be involvedin antibodymediated trogocytosis a process that mechanicallydisrupts the plasma membrane of antibodyopsinized cancercells leading to a lyticnecrotictype cell death iga antibodiesagainst receptors expressed by cancer cells her2 egfr couldenhance neutrophilmediated trogocytosis of cancer cells if thecd47sirpα innate immune cell checkpoint was simultaneouslyblocked taken together these results demonstrate thatneutrophils can impair tumor growth and metastasis using acombination of direct and indirect cancer cell killing mechanismssupplementary table frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure neutrophil functions that promote tumor growth and metastasis to support primary tumor growth neutrophils can mediate t cell suppression and altermacrophage differentiation neutrophil release of timp1 enhances tumor cell invasion by inducing epithelialtomesenchymal transition once in circulation circulatingtumor cells interact with neutrophils which enables tumor cell proliferation secretion of various pro‚ammatory markers such as il8 il1 or mmps can mediateincreased tumor cell extravasation in addition neutrophils can inhibit intraluminal nkmediated killing of circulating cancer cells leading to increased extravasation atthe metastatic site various systemic and microenvironmental factors can promote neutrophil ltration neutrophils can awaken dormant cancer cells by promotingecm remodeling and angiogenesis lastly continued growth of the metastatic lesion is facilitated by key neutrophildependent mechanisms which includeangiogenesis proliferation immune suppression and immune exclusion csf1 colony stimulating factor timp1 tissue inhibitor of matrix metalloprotease pdl1programmed death ligand tgf transforming growth factor ros reactive oxygen species mmp matrix metalloproteinases gmcsf granulocyte macrophagecolony stimulating factor angptl2 angiopoetin like2 fgf2 fibroblast growth factor ltb4 leukotriene b4 inos inducible nitric oxide synthase net neutrophilextracellular trap caf cancerassociated fibroblastneutrophil functions thatpromote primary tumor growthneutrophils promote primary tumor growth by variousmechanisms figure netosis is a process that involvesthe extrusion of neutrophilderived chromatin structures thatare decorated with neutrophil granule constituents whichform extracellular structures called neutrophil extracellulartraps nets normally netosis and net productionhave been described in the context of a neutrophil™s ability tocapture and kill bacteria extracellularly however netshave been shown to play an important role in the growth of aprimary tumor tumor microenvironmental changes includingtumorassociated coagulation and enhanced thrombosishave been linked to enhanced tumor growth several recentstudies suggest that netosis may play an important role inthese processes lps stimulation was shown to increase c3arexpression within neutrophils enhance netosis and increasecoagulation these events were correlated with n2 neutrophilpolarization and increased tumor growth interestingly ithas recently been shown that immature neutrophils preferentiallyrespond to cancer cell derived c3a to promote their migration subsequently it was shown that breast cancer cells thatexpressed high levels of gcsf and il1 exhibited highneutrophil counts and tumorassociated thrombosis which wasdependent on net formation pharmacological blockadefrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeof il1 receptor signaling reduced net formation attenuatedtumorassociated thrombosis and impaired tumor growth nets can also directly ‚uence cancer cell proliferationneutrophil elastase ne present within nets activates tumorcells to increase mitochondria biogenesis and atp productionthereby further enhancing the growth of cancer cells in addition to the impact of nets neutrophils canalso interact with other immune cells through additionalmechanisms to promote tumor growth neutrophilderived roscan inhibit t cell proliferation creating an immunosuppressiveenvironment that is supportive of tumor growth phenotypiccharacterization and singlecell rna sequencing identified aneutrophil subset that is cd84hi which exhibited potent t cellsuppressive activity and increased ros production in amodel of gastric cancer neutrophils were activated by tumorderived gmcsf that resulted in elevated programmed deathligand pdl1 expression these pdl1 neutrophils wereable to suppress t cell function and promote tumor growth secretion of mmp9 matrix metalloproteinase fromltrating neutrophils activates latent tgf and induces tcell suppression and tumor growth in a colorectal cancer model siglecfhigh neutrophils in lung adenocarcinoma createdan immunosuppressive environment by promoting macrophagediï¬erentiation causing the release of high levels of rosand enabling tumor progression together these findingsindicate that neutrophils that ltrate diverse primary tumorscan modify the local environment in diï¬erent ways to favortumor growthneutrophil functions thatpromote metastasisthe ability of cancer cells to leave the primary tumor anddisseminate to distant ans represents the deadliest aspectof cancer progression indeed the emergence of metastaticcancer accounts for ˆ¼ of cancer related deaths themetastatic cascade represents a series of barriers to cancercells and neutrophils have been found to assist cancer cells insuccessfully navigating several of these distinct steps figure supplementary table local invasionintravasationltrating neutrophils within primary tumors are associatedwith an increase in emt enhanced metastasis and pooroutcomes mechanisticallyof matrixmetalloprotease timp1 secreted by neutrophils induced anemt and consequently increased the migration and invasion oftumor cells cancer cells that had undergone an emt expressedcd90 which enhanced timp1 secretion by neutrophils in acontactdependent manner inhibitortissuesurvival in circulationextravasationthe ability of circulating tumor cells ctcsto surviveis criticalfor metastasis formation the formation ofheterotypic cancer cell”neutrophil clusters was found to greatlyincrease metastatic fitness using a 4t1 breast cancer modelit was demonstrated that ctcneutrophil interactions reliedon vcam1 dependent adhesion which enhanced cancercell proliferation and increased metastasis indirectlyneutrophils can also inhibit nk cellmediated tumor clearancein circulation thereby increasing the intraluminal survival ofdisseminated tumor cells in this study 4t1 breast cancer cellswere injected subcutaneously to mobilize murine neutrophilsly6gfollowing which d2a1 breast cancer cells wereinjected intravenously mice bearing 4t1 cells exhibited reducedclearance of d2a1 cells from the lungs when compared to micethat were not injected with 4t1 cells depletion of nk cellsresulted in enhanced d2a1 cancer cell accumulation in the lungswhile neutrophil depletion had the opposite eï¬ect cancer cells that have survived in circulation must exitthe bloodstream and extravasate into tissue parenchyma neutrophils have been shown to regulate the extravasationprocessthrough several mechanisms neutrophilderivedfactors can diminish the integrity of the endothelial barrierpermitting cancer cellsil8il1 and matrix metalloproteasesmmp8 and mmp9released from neutrophils activated endothelial cells reducedendothelialtransendothelialmigration and accelerated the rate of cancer cell extravasation to extravasate more easilyincreasedfunctionbarriersites“netosis and net constituents can support cancer cellextravasation through enhanced trapping of ctcs withinmetastaticimportantly blocking netosisdecreases cancer cell adhesion and inhibits metastatic spread tothe lung and liver furthermore changes within specificmetastatic microenvironments such as exposure to ozoneor redox imbalance triggered netosis and led to increasedentrapment of cancer cells in the lung and enhanced metastasis collectively these studies show that neutrophils play animportant role in enhancing tumor cell survival and increasedextravasation which promote cancer metastasisrecruitmentearly seedingsurvivalsystemic and tumorderived factors have been implicatedin neutrophilin the premetastatic nichetumorderived il1 induces Îδ t cells to produce il17aand granulocytecolony stimulating factor gcsf whichresults in the recruitment of immunosuppressive neutrophilsto the lung gmcsf and il5 have been shown topromote the expansion and recruitment of prometastaticneutrophils in the lungs of obese mice which promotes lungmetastasis angiopoetinlike2 angptl2 secreted byosteosarcoma cells implanted in the tibia stimulates lungepithelial cells which led to the accumulation of neutrophilsin the lung and enhanced lung metastatic burden in the lung neutrophils secrete ltb4increases theinitiating cellsproliferation of ltb4rpositive metastasis activation of notch1 in colorectalcellsdrives tgf2dependent recruitment of immunosuppressiveneutrophils within the liver which enabled the formation of livermetastases cancerthatnets also support early cancer cell seeding and colonizationof metastases induction of nets by ovarian tumorderivedfactors has been shown to be important in promoting metastasisfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeto the omentum in the liver nets have also beenshown to promote metastasis by activating cancerassociatedfibroblasts growth in the metastatic siteneutrophils have been shown to promote the growth ofmetastases after seeding minor subclones of breast cancer cellsthat secrete il11 and figf cfosinduced growth factor cansupport the formation of polyclonal metastases composed ofdriver and passenger subpopulations these il11 producingsubclones activated il11 responsive mesenchymal stromal cellswhich induced chemokine secretion and subsequent recruitmentof prometastatic neutrophils tumor cellderived gmcsfwas shown to stimulate neutrophils to synthesize and secretetransferrin an iron transport protein which has mitogenicactivity that promotes lung metastatic growth when taken up bycancer cells a recurring function of prometastatic neutrophils is theirability to create an immunosuppressive microenvironmentthat support metastasis within lung metastasesinduciblenitric oxide synthase inos producing neutrophils havebeen shown to limit cd8 t cell dependent antitumorresponses by promoting immune suppression recently p53deficient cancer cells were found to increase the expressionof wnt ligands which in turn upregulated il1 productionfrom tumorassociated macrophages high il1 levelsengaged Îδ17 t cells which subsequently enhanced neutrophilrecruitment that promoted the formation of lung metastases furthermore loss of elf5 e74like transcription factorexpression in triplenegative breast cancer led to increased ifnÎ signaling resulting in the expansion of immunosuppressiveneutrophils in addition to tumorderived factors a lackof systemic testosterone levels can lead to an impairmentof antitumor neutrophil functions a shift toward immatureneutrophils was observed in castrated male mice leading toincreased neutrophilderived ros and suppression of nk cellactivation that promoted increased lung metastatic burden intwo melanoma models recently a role for net formationhas been described for the continued growth of establishedmetastases nets released during cancer progressionwas shown to limitthe ability of nk and cytotoxic tcells to eliminate cancer cells specifically net formationimpaired direct contact between the cancer cells and cytotoxicimmune cells nk and t cells inhibition of netosis with aprotein arginine deiminase pad4 inhibitor synergized withimmune checkpoint inhibitors to control tumor growth andmetastasis proangiogenic functions have long been ascribed forneutrophils which revealed that neutrophilderived proteasessuch as mmp9 could release stored angiogenic factors vegffgfs that were stored in the local environment to enable bloodvessel formation recently a diï¬erent mechanism bywhich neutrophils enhance angiogenesis has been describedthe synthesis and secretion of fibroblast growth factor fgf2 by neutrophils in the liver microenvironment drivesangiogenesis and growth of nascent colorectal cancerderivedhepatic metastases dormantresidual disease andtherapy resistanceneutrophils have also been implicated in awakening dormantcancer cells lpsinduced tissue ‚ammation led to metastaticoutgrowth of dormant tumor cells in a neutrophildependentmanner mmp9 produced by neutrophils can trigger thegrowth of dormant cancer cells by remodeling extracellularmatrix and releasing potent angiogenic factors ne andmmp9 which are enzymes associated with nets can cleavethe extracellular matrix ecm leading to integrinmediatedsignaling which awakens dormant cancer cells and promotescancer cell growth severalstudies have shown that neutrophils promoteresistance to therapy doxorubicin and paclitaxel resistant breastcancer cells express more il17 and cxcr2 ligands whichincreases neutrophil recruitment a neutrophilenrichedsubtype characterized in triple negative breast cancer tnbcdetermined that neutrophils were largely immunosuppressiverendering these tumors resistant to immune checkpoint blockadetherapy in a genetically engineered mouse model ofsarcoma neutrophils promote resistance to radiation therapyby activating mitogenactivated protein kinase mapk pathway in addition cd177 neutrophil ltrates in colorectalcancer patients are associated with adverse outcome in patientsreceiving bevacizumab [antivascular endothelial growth factora vegf a] furthermore lysyl oxidaselike loxl4expressing neutrophils that ltrated colorectal cancer livermetastases were found to identify patients that were resistant toantiangiogenic therapy metabolic programming inneutrophilsrecentinteresthasbeenconceptin thethereofimmunometabolism and the realization that altered cellularmetabolism in ltrating immune cells can have a significantimpact on tumor growth and metastasis neutrophilsare typically viewed as a cell type that is heavily reliant onglycolysis to perform their eï¬ector functions consistentwith this notion neutrophils have very few mitochondria andinhibitors of oxidative phosphorylation oxphos do notalter their rates of oxygen consumption howeverduring tumor progression neutrophils have been shown toundergo a metabolic switch which involves the upregulationof genes associated with oxphos fatty acid metabolism andglycolysis figure neutrophils isolated from lewis lungcarcinoma exhibit increased flux through oxphos glycolysisand increased atp production compared to naïve neutrophilssuggesting that multiple metabolic strategies are engaged intumor ltrating neutrophils recently upregulation offatp2 fatty acid transport protein in neutrophils was shownto increase lipid accumulation in these cells fatp2 regulated theuptake of arachidonic acid which was subsequently convertedto prostaglandin e2 neutrophilderived prostaglandin e2 wasfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure metabolic changes in cancerassociated neutrophils neutrophils which possess few mitochondria are reliant on glycolysis to generate atp to fueleffector functions such as phagocytosis generation of reactive oxygen species and netosis in cancer neutrophils upregulate oxidative phosphorylation oxphosand fatty acid transporters to mediate many neutrophil functions including migration and t cell suppression under nutrient limiting conditions such as low glucoseneutrophils can reprogram their metabolism to break down fatty acids or utilize certain amino acids glutamate proline to fuel protumorigenicprometastaticfunctions ppp pentose phosphate pathway glut glucose transporter mct monocarboxylate transporter tca tricarboxylic acid cycle fatp2 fatty acidtransport protein aa arachidonic acid pge2 prostaglandin e2found to be important or neutrophilmediated cd8 t cellsuppression and tumor growth metabolic flexibility refers to the ability of a cell to shiftbetween one metabolic program to another in response tochanging metabolic demands or nutrient supply high metabolicflexibility increases the cell™s ability to survive various andeverchanging metabolic microenvironments neutrophilsubpopulations can also exhibit metabolic flexibility figure in breast cancer splenic neutrophils can engage mitochondrialdependent fatty acid oxidation as a predominate fuel sourceto support ros production and maintain t cell suppression under glucoselimiting conditions similar to certain tumormicroenvironments immature ldns have been shown to utilizeoxphos to generate atp that is required to support theirprotumorigenic functions indeed immature ldns can supportnetosis under nutrient limiting conditions via mitochondrialdependent amino acid catabolism which is importantforefficient breast cancer liver metastasis in addition thelongevity of neutrophils could also be altered due to the enhancedmetabolic flexibility the ex vivo halflife of mouse circulatinghdns and ldns was and h respectively suchobservations raise the intriguing possibility that under certainconditions distinct neutrophil subsets may not be as shortlived as previously thought these studies argue that increasedmetabolic flexibility in distinct neutrophil populations may beimportant for cellular functions that can ‚uence tumor growthand metastatic progressionclinical importance futureperspectives on treatmentin keeping with their protumorigenicmetastatic functions thepresence of neutrophils across diï¬erent cancers was shownto be strongly associated with adverse patient outcomes frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeamong certain subtypes of breast cancer er the presence ofa neutrophil ltrate in the primary tumor is also indicativeof worse patient outcomes furthermore in patients withadvanced cancers serum il8 levels and neutrophil ltrationare associated with worse overall survival and diminishedresponse to immune checkpoint inhibitors the mobilization of neutrophils into circulation also hasprognostic significance the neutrophiltolymphocyte rationlr is an important risk stratification and treatment selectiondiagnostic tool for cancer patients a high nlr is associated withpoor prognosis in many solid human cancers “ a highnlr is also associated with decreased overall survival in patientswith tnbc or metastatic breast cancer an important and unanswered question with respect to thenlr is the type of neutrophil that is being detected in thesepatients are they high or lowdensity neutrophils interestinglyldns have been identified in patients with breast cancer lungcancer head and neck cancers urologic cancers and lymphoma “ in patients with advanced lung cancerit wasreported that higher proportion of ldns predictedpoorer survival these observations are in keeping withthe protumorigenic and prometastatic functions associatedwith ldnn2 neutrophils while most studies reveal a negativeprognostic impact of neutrophils in cancer there was one studythat associated the presence of a cd16high cd62dim neutrophilsubset with increased survival of head and neck squamous cellcarcinoma patients these observations highlight the needfor better markers that are capable of discriminating betweenneutrophils that exert antitumor vs those that mediate protumormetastatic eï¬ectsmechanistic insights have greatly advanced our knowledgeoftumorderived factorstumor growth andmetastasis in a neutrophildependent manner additional studiesimpactthatreferences sipsas nv bodey gp kontoyiannis dp perspectives for the management offebrile neutropenic patients with cancer in the 21st century cancer “ 101002cncr20890 kolaczkowska e kubes p neutrophil recruitment and function in healthand ‚ammation nat rev immunol “ 101038nri3399inde visser ke neutrophilssb wellenstein md coï¬eltcancer neutral no more nat rev cancer 101038nrc201652 “ cowland jb borregaard n granulopoiesis and granules of humanneutrophils immunol rev “ 101111imr12440 evrard m kwok iwh chong sz teng kww becht e chenbone marrow neutrophilstraffickinganalysisspecializedpopulationsexpansioninofal developmentaljetrevealsand 101016jimmuni201802002functionseï¬ectorimmunity“79e8 zhu yp padgett l dinh hq marcovecchio p blatchley a wu r identification of an early unipotent neutrophil progenitor with pro tumoralactivity in mouse and human bone marrow cell rep “41e8 101016jcelrep201807097 sagiv jy michaeli j assi s mishalian i kisos h levy l phenotypicdiversity and plasticity in circulating neutrophil subpopulations in cancercell rep “ 101016jcelrep201412039focused on characterizing the phenotypic and functional role ofneutrophils in cancer it may be possible to develop strategies thatspecifically target those neutrophil subsets that actively promotetumor growth and metastasis while sparing those neutrophilsthat possess antitumor and antimicrobial functions finallythe emerging concept of metabolic flexibility that is exhibited bycertain neutrophil subsets may aï¬ord new ways of targeting theseprotumorigenicmetastatic neutrophilsauthor contributionsbh ys and ps wrote the review and prepared the figuresall authors contributed to the and approved thesubmitted versionfundingwork from the authors laboratory cited in this review wassupported by an operating grant to ps from the cancer researchsociety and the terry fox research institute and québecbreast cancer foundation grant bh acknowledgessupport from the charlotte and leo karassik foundation phdfellowship and the rolande and marcel gosselin graduatestudentship ys holds an entrance studentship from thegoodman cancer research centre ps is a mcgill universitywilliam dawson scholarsupplementary materialthe supplementary materialfor this can be foundonline at httpswwwfrontiersins103389fimmu202001778fullsupplementarymaterial coï¬elt sb kersten k doornebal cw weiden j vrijland k hau cs il17producing gammadelta t cells and neutrophils conspireto promote breast“ 101038nature14282cancer metastasis nature hsu be tabaries s johnson rm andrzejewski s senecal j lehuede c immature lowdensity neutrophils exhibit metabolic flexibility thatfacilitates breast cancer liver metastasis cell rep “15e6 101016jcelrep201905091 fridlender zg sun j kim s kapoor v cheng g ling l polarizationof tumorassociated neutrophil phenotype by tgfbeta œn1 versus œn2tan cancer cell “ 101016jccr200906017 ohms m möller s laskay t an attempt to polarize human neutrophilstoward n1 and n2 phenotypes in vitro front immunol 103389fimmu202000532jablonska j leschner s westphal k lienenklaus s weiss s neutrophilsresponsive to endogenous ifnbeta regulate tumor angiogenesis andgrowth in a mouse tumor model j clin invest “ 101172jci37223 finisguerra v di conza g di matteo m serneels j costa s thompsonaa met is required for the recruitment of antitumoural neutrophilsnature “ 101038nature14407 ostuni r kratochvill f murray pj natoli g macrophages and cancer frommechanisms to therapeutic implications trends immunol “ 101016jit201502004frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotype brandau s moses k lang s the kinship of neutrophils and granulocyticmyeloidderived suppressor cells in cancer cousins siblings or twins semincancer biol “ 101016jsemcancer201302007 vols s sionov rv granot z always look on the bright side antitumor functions of neutrophils curr pharmac design “ granot z henke e comen ea king ta norton l benezra r tumorentrained neutrophils inhibit seeding in the premetastatic lung cancer cell “ 101016jccr201108012 gershkovitz m caspi y fainsodlevi t katz b michaeli j khawaled s trpm2 mediates neutrophil killing of disseminated tumor cells cancer res “ 10115800085472can173614 gershkovitz m fainsodlevi t khawaled s shaul me sionov rvcohendaniel l microenvironmental cues determine tumor cellsusceptibility to neutrophil cytotoxicity cancer res “ 10115800085472can180540 sionov rv fainsodlevi t zelter t polyansky l pham ct granotz neutrophil cathepsin g and tumor cell rage facilitate neutrophilanti8e1624129 1010802162402x20191624129cytotoxicity oncoimmunologytumor mahiddine k blaisdell a ma s crequergrandhomme a lowell caerlebacher a relief of tumor hypoxia unleashes the tumoricidal potentialof neutrophils j clin invest “ 101172jci130952 mensurado s rei m lanca t ioannou m goncalvessousa n kubo h etal tumorassociated neutrophils suppress protumoral il17 gammadeltat cells through induction of oxidative stress plos biol 16e2004990 101371 pbio2004990 singhal s bhojnagarwala ps o™brien s moon ek garfall al rao as etal origin and role of a subset of tumorassociated neutrophils with antigenpresenting cell features in earlystage human lung cancer cancer cell “ 101016jccell201606001et hagerling c gonzalez h salari k wang cy lin c roblescooperationtumor prevents metastatic progressionaliinduced byof breast cancer proc natl acad sci usa 101073pnas1907660116eï¬ector monocyteneutrophilimmunethe primary “ costanzogarvey dl keeley t case aj watson gf alsamraae myu y neutrophils are mediators of metastatic p
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"Marek™s disease MD is a chicken neoplastic disease which brings huge economic losses to theglobal poultry industry The wild type p53 a tumor suppressor gene plays a key role in blocking cell cyclepromoting apoptosis and maintaining the stability of the genome However the mutant p53 losses its tumorinhibitory role and become an oncogene when a mutation has happenedResults The mutation rate of p53 was in the experimentally and naturally infected chickens The mutationsincluded pointmutations and deletions and mostly located in the DNAbinding domain The mutated p53 wasexpressed in various tumor tissues in an infected chicken The mutant P53 proteins were notably accumulated inthe cytoplasm due to the loss in the function of nuclear localization Unlike the study on human cancer theconcentrations of P53 in the serums of MD infected chicken were significantly lower than the control groupConclusions The p53 mutations were apparent in the development of MD P53 and P53 antibody level in serumcould be a useful marker in the diagnosis and surveillance of MDKeywords Marek™s disease p53 P53 antibodyBackgroundMarek™s disease MD is a lymphoproliferative neoplasticdisease caused by the chicken Marek™s disease virusMDV or named Gallid alphaherpesvirus The infection caused by this virus may lead to lymphocyte proliferation tumor formation immunosuppression paralysisand mononuclear cell infiltration in peripheral nervesgonads and immune ans [ ] As one of the mosthighly contagious tumor diseases in chickens the data ofOIE [] reported by about half of the world has shownthat this disease accounts for the loss of up to “ billionUS dollars annually in the global poultry industry [] Correspondence liusidsdaueducn1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong ChinaFull list of author information is available at the end of the Described often as the œguardian of the genome [] andthe œcellular gatekeeper [] p53 is the most relevantand important tumor suppressor gene with the highestmutation frequency in human and animal tumor diseases [] The chicken p53 gene has a fulllength reading frame ™ and ™ untranslated regions and a polyadenylation signal which encodes amino acidsThese amino acids share a homology to the aminoacids of human P53 []p53 is divided into the wild type and the mutant typeThe wild type is a normal tumor suppressor gene whilethe mutant p53 is an oncogene transformed from atumor suppressor gene due to spatial conformationalchange As a result the mutant P53 protein losses itsability in regulating cell growth apoptosis and DNA repair [] which often a prerequisite for tumorigenesis and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cZhang BMC Veterinary Research Page of disease progression [] The proportion of p53 mutations in tumor tissue varies between “ []The wild type P53 has a very short halflife while themutant P53 protein is prolonged This abnormality ofP53 can lead to the accumulation of P53 antibody inserum as well as the P53 protein in tumor tissue []Several studies have demonstrated that the P53 antibodyplays a predictive role in tumorigenesis and its manifestation in serum is an early event in the development ofmalignant tumors in humans [“] The level of P53antibody in serum correlates significantly with commonclinical neoplastic diseases but it has been barely detectable in the serum of healthy subjects [] This correlation may also exist in chicken and currently there is aknowledge gap concerning the role of mutant p53 inMarek™s disease in chicken Therefore this study aimedto investigate the role of p53 as a tumor marker inassisting the clinical diagnosis and prognosis of MDResultsHistopathological and immunohistochemical analysis ofp53 expressionHistopathological examination revealed the evidence ofliver cells undergoing necrosis in the infected SPFchicken Such liver tissues demonstrated focal infiltration and hyperplasia of lymphoid tumor cells Fig 1aIn addition the lymphoid follicle in the bursa of Fabricius was atrophied diffuse infiltration and proliferationof lymphoid tumor cells between the follicles were observed Fig 1b In the livers of the clinically infectedchickens the cytoplasm of the lymphoid tumor cellsunderwent multifocal proliferation which was stainedbrown by the immunohistochemicalFig 2aPositive staining was also detected in the tumor cells inIHCthe spleen and the bursa of Fabricius tissues Fig 2b and cIn contrast no positive stained cells were observed in thecontrol group Fig 2dMutations in the p53The mutation rate of p53 was in the infectedpoultry There were two types of p53 mutations deletions and point mutations detected which was consistent with the results reported in a previous study []Among the mutated p53 genes the base sites withhigh mutation frequency were and There was no mutation found in the control groupMost of the mutations were located in the core domainand the Cterminal domain The mutation analyses wereshown in Table P53 antigen and P53 antibody levels for MDThe concentrations of the P53 antigen of the clinicaland experimental MDVinfected group were significantlylower than the control group However the P53 antibody levels in the experimental infection group were significantly higher than the control group These analyseswere shown in Fig DiscussionThe human P53 is widely acknowledged as an intranuclear phosphorylated protein The wide type P53 normally exists in the nucleus for an extremely short period[] On the other hand the mutated P53 has a prolonged halflife to “ h as it is not digested quicklyand therefore accumulates inside tumor cells [] Thisallows the detection of mutant P53 protein via IHC forFig Histopathological observation of diseased chickens infected with MDV a Different sizes and shapes of focal infiltration and hyperplasia oflymphoid tumor cells were observed in the liver tissues HE — b Diffuse infiltration and proliferation of lymphoid tumor cells between thefollicles were observed in the bursa of Fabricius HE — 0cZhang BMC Veterinary Research Page of Fig Immunohistochemical staining of p53 in infected chickens a Liver lymphoid tumor cells with cytoplasm expression HE — b Spleen lymphoid tumor cells with cytoplasm expression HE — c Bursa of Fabricius lymphoid tumor cells with cytoplasm expression HE —d Liver Negative controls were incubated with PBS HE —which IHC has now become an important modality forthe detection of various tumor biomarkers P53 proteinrepresents an effective substitute marker for TP53 mutation status [] but at present P53 IHC is mainly applied for tumor diseases in humans P53 IHC allows theassessment of the stage and grade of cancers with only afew studies reported in poultry tumor diseasesIn this study the immunohistochemical analysis revealed that P53 was present in the liver spleen and thebursa of Fabricius of infected chicken with MDV Interestingly P53 was notably expressed in the cytoplasmThe potential explanation of this phenomenon could bethat the mutated P53 protein lost its function in nuclearlocalizationtheaccumulatedinandeventuallycytoplasm Furthermore p53 has been regarded as theœhotspots given that p53 mutations have been frequently detected in a variety of tumors [] In thisstudy several types of p53 mutations were demonstratedin natural and experimental infections of MDV The fivemost frequent mutation sites were and The altered codons of mutant p53 comparedwith the wildtype were ACGACA GCAGCC CGCCGG GCCGCA and ACCACA but these were all synonymous mutationsIn our study a short form of p53 transcript was detected in a clinical case The deleted sequence was located at the “ bp in the reading frame of thereference sequence resulting in missense mutations fromTable Primers used toamplify chicken p53 cDNAPrimerp531Nucleotide sequence™GTGGCCGTCTATAAGAAATCAGA3™™AAAAAGGGGGCGTGGTCAGT3™p532Annealing temperature „ƒSize of fragments amplifiedlocation bp 0cZhang BMC Veterinary Research Page of Fig Levels of p53 antigen and antibody in the serum of study groups The Yaxis represented the concentrations of antigen or antibody andthe Xaxis represented the different groups of samples Each group consisted of seven samples Statistical significance was designated as p or p the position to the termination The short form ofp53 transcript was also found in the cases infected byavian leukosis virus [] A series of missense mutationsfrom the position to the termination due to a base deletion was found in an experimentally infected chicken Inaddition this study found that the p53 was mutated in of poultry oncology with the mutation region concentrated mainly in the DBD The DBD allows the specificrecognition of target sequences [] Once the p53 is deleted or point mutations in this region occursit mayaffect the formation of tetramers which in turn leads tothe conversion of wild type P53 into a mutant type resulting in the loss of normal functionThe conformation of mutant P53 extends its halflifeto several hours in humans The accumulated mutantP53 then acts as a target antigen which elicits an autoimmune response [] However in our study the levelsof serum P53 antigen in clinical and experimentalMDVinfected groups were significantly lower than thecontrol group contrary to the findings in human cancerresearch Only the serum P53 antibody concentrationsof the experimentalinfected group were significantlyhigher than the control group This might be that P53as a tumor suppressor was largely consumed in response to the occurrence of MD Moreover tumorigenesis promoted mutation of p53 and further reduced P53concentrationConclusionsOur study revealed p53 was mutated and expressed inMDV infection which suggested that these mutationswere playing an important role in the development ofMD The mutant p53 was expressed in the tumor cellsof various tissues of the infected chicken Mutant P53protein lost its nuclear localization function and transferred from the nucleus to the cytoplasm Unlike studiesin human cancer the concentration of P53 was significantly lower in the natural and experimental MDV infectionnovelinnovations for the diagnosis and monitoring of MD inpoultryresults maygroup OurprovideMethodsNatural infection of MDV in chickensSeven 160dayold egglaying hens were obtained from alocal flock These hens were confirmed to have acquiredMDV infection naturally with the absence of other common viral diseases by PCR detection Their serums werecollected for the detection of the P53 antigen and antibody Their tissue samples including liver spleen pancreas and bursa of Fabricius were collected and fixedwith formaldehyde for h before histopathologicaland immunohistochemical studiesExperimental infection of chickens with MDVThe number of experimental animal was referred to thenumber of clinical samples Twenty 1dayold SPF chickens were obtained from the Shandong Academy of Agricultural Sciences Poultry Institute SPF Chicken ResearchCenter Jinan China They were randomly divided intotwo equal groups as an infection group and a controlgroup The two groups of chickens were raised separately in isolators with filtered air under positive pressureOn the first day of the experiment each of the chickenin the infection group was inoculated intraabdominallyia with plaqueforming units PFU of vvMDV 0cZhang BMC Veterinary Research Page of GX0101 which normally cause tumors at the tenthweek The chickens in the control group were inoculatedwith PBS In the tenth week serums were collected fromseven chickens in each group The experiment endedafter ten weeks and all chickens were put into a euthanasia box Thirty percent of carbon dioxide CO2 by volume was infused into the euthanasia box per minuteuntil all chickens lost their consciousness Then theCO2 flow rate was increased to for one minuteand the euthanasia box was kept airtight for ten minutesWhen all chickens were confirmed dead they were dissected and their livers and spleens were collected forhistopathology and immunohistochemistry studyHistopathology and ImmunohistochemistryThe fixed liver spleen pancreas and bursa of Fabriciuswere dehydrated waxed and cutinto µm slicesfollowed by Haematoxylin and eosin HE staining for ahistopathology examination In IHC processing tissueswere cut into sections of µm thickness and mountedon microslides treated with polyLlysine The sections were deparaffinized in xylene and rehydrated in agraded series of ethanol solutions into PBS then werepretreated in citrate buffer molL pH °Cfor antigen retrieval by microwaving and cooled at roomtemperature for min [] Endogenous alkaline peroxidase was quenched with hydrogen peroxide solutionin methanol for min [] Nonspecific antibody binding sites were obliterated by incubating the sections with fetal bovine serum for min at room temperatureFollowing this the antiP53 polyclonal antibody BOSTER China as a primary antibody was diluted into and immersed overnight at °C in a black humidchamber The secondary antibodies were HRP horseradish peroxidaseconjugated goatIgGCWBIO China Immunoreactivity was then visualizedwith diaminobenzidine DAB staining The sectionswere counterstained with hematoxylin and mountedNegative controls were incubated with PBS instead ofthe primary antibody in the immunohistochemicalanalysisantimouseTotal RNA isolation and reverse transcriptionThe total RNA ofliver and spleen tissue samples mgtissue were isolated by using TRIzol reagentTakara Japan according to the manufacturer™s instructions Extracted total RNA 1ug was reverse transcribedto cDNA with PrimeScript„¢ RT Reagent Kit RocheSwitzerland According to the published complementaryDNA cDNA sequence of the chicken p53 GeneBankaccession number nm205264 specific primers were designed to amplify the DNAbinding domain DBD ofp53 as shown in Table Using PCR Polymerase ChainReaction techniques p53 cDNA genes sequences wereamplified The PCR reaction was performed by using athermal cycler Takara Japan under the following conditions „ƒ for min followed by cycles of „ƒfor s „ƒ for s „ƒ for s and a final „ƒTable Mutations of p53 genes in MDSample InfoNCMutation analysisBase mutation sitesNDMD CMD CMD CMD CMD CMD CMD EMD EMD EMD EMD EMD E TC AC C G AG AC gA CG CA AC CG CA CG CA delete TC TC AC CG CG CA“ delete CA CA TG AC CG CA gA CA CA AG AC CG CA CT gA AC CG CA gA gT CT CG CA gANC was SPF chicken without diseases E was experimental chicken C was clinical chickenAmino acid mutationsSite from toNDMutation area Y A E G R HNDND Stop StopNDND N S T I G DND R L V M E Y H YCore domainCterminal domainCore domainCore domainCore domainCore domainCterminal domainCore domainC terminal domain 0cZhang BMC Veterinary Research Page of for min extension cycle DNA templates that were acquired from the MDV positive chickens were amplifiedusing primer pair p5312 The SPF chickens wereregarded as negative controls DNA fragments were successfully amplified with sizes of bp The target fragment was gel extracted and connected to the pEASYT1vector Then the recombinant plasmid was transformedinto bacteria and sequencing analysis was made Finallythe sequencing results were compared with the wild typep53 cDNA sequences reported previouslyEnzymelinked immunosorbent assay ELISA for P53 andP53 antibodyThe P53 antigen and antibody levels of chicken weremonitored by ELISA Mlbio China In order to eliminate subjective interference the assessors were blinded tothe background of the samples The detection rangeswere “ pgml for P53 antigen and pgmlfor P53 antibody Samples were diluted five time with aspecial diluent and all processes were implemented inaccordance with the instructions The absorbance ODof each sample was finally measured at a wavelength of nm The sample concentration was calculated depending on the standard curveStatistical analysisStatistical analyses were conducted with GraphPadPrism Version San Diego CA USA The differences in the levels of P53 antigen and antibody were antwotailed Student™s Ttestalyzed by usingStatistical significance was designated as p or p theAbbreviationscDNA Complementary DNA DAB Diaminobenzidine DBD DNAbindingdomain ELISA Enzymelinked immunosorbent assay HE Haematoxylin andeosin IHC Immunohistochemical HRP Horse radish peroxidasePCR Polymerase chain reaction PFU Plaque forming unitsAcknowledgementsNot applicableAuthors™ contributionsHXZ carried out laboratory work wrote the manuscript and performed thedata analyses MDL designed the experiment wrote the manuscript andperformed the data analyses HZ SLC YL SNJ and YNS carried out laboratorywork SDL conceived and supervised this work revised manuscript All authorsread and approved the final manuscriptFundingThe work was supported by grants from the National natural sciencefoundation project NO The funding body was solely involved infunding and had no role in the design of the study the collection analysisand interpretation of the data or in writing the manuscriptEthics approval and consent to participateThe sample were collected and handled in accordance with the goodanimal practices required by the Animal Ethics Procedures and Guidelines ofthe People™s Republic of China Informed consent to participate wasobtained from the chicken farmer owner All animal protocols andprocedures were performed according to the Chinese Regulations ofLaboratory Animals and were approved by the Animal Ethics Committee ofShandong Agricultural UniversityConsent for publicationNot applicableAvailability of data and materialsThe data supporting the findings are included in the manuscriptCompeting interestsThe authors declare that they have no competing interestsAuthor details1College of Animal Science and Veterinary Medicine Shandong AgriculturalUniversity Daizong Street Taian Shandong China 2China AnimalHealth and Epidemiology Center Nanjing Road QingdaoShandong ChinaReceived January Accepted August ReferencesJarosinski KW Tischer BK Trapp S Marek™s disease virus lytic replicationoncogenesis and control Expert Rev Vaccines “Reddy MS Book Review Marek™s Disease An Evolving Problem Vet Pathol“Boodhoo N Gurung A Sharif S Behboudi S Marek™s disease in chickens areview with focus on immunology Vet Res Lane DP p53 guardian of the genome Nature “Bertzbach LD Kheimar A Ali FAZ Kaufer BB Viral Factors Involved inMarek™s Disease Virus MDV Pathogenesis Curr Clin Microbiol Rep “Levine AJ p53 the cellular gatekeeper for growth and division Cell “Soussi T B¨gue A Kress M Stehelin D May P Nucleotide sequence of acDNA encoding the chicken p53 nuclear oncoprotein Nucleic Acids ResZilfou JT Lowe SW Tumor suppressive functions of p53 Review ColdSpring Harb Perspect Biol 20091a001883Stiewe T Haran TE How mutations shape p53 interactions with thegenome to promote tumorigenesis and drug resistance Drug Resist Updat“K¶bel M Piskorz AM Lee S Lui S LePage C Marass F Rosenfeld N MesMasson AM Brenton JD Optimized p53 immunohistochemistry is anaccurate predictor of TP53 mutation in ovarian carcinoma J Pathol Clin Res“ Zekiye H B¼lent T Taner E p53 antibody is it an indicator ofdedifferentiated thyroid cancer Ann Nucl Med “ Balogh GA Mailo D Nardi H Corte MM Vincent E Barutta E Lizarraga GLizarraga P Montero H Gentili R Serological levels of mutated p53 proteinare highly detected at early stages in breast cancer patients Exp Ther Med“Sabapathy K Lane DP Understanding p53 functions through p53antibodies J Mol Cell Biol “Kunizaki M Fukuda A Wakata K Tominaga T Nonaka T Miyazaki TMatsumoto K Sumida Y Hidaka S Yasutake T Sawai T Hamamoto RNanashima A Nagayasu T Clinical Significance of Serum p53 Antibody inthe Early Detection and Poor Prognosis of Gastric Cancer Anticancer Res“ Yue Q Yulong G Liting Q Shuai Y Delong L Yubao L Lili J Sidang LXiaomei W Mutations in and Expression of the Tumor Suppressor Gene p53in EggType Chickens Infected With Subgroup J Avian Leukosis Virus VetPathol “ Bykov VJN Eriksson SE Bianchi J Wiman KG Targeting mutant p53 forefficient cancer therapy Nat Rev Cancer “ Yemelyanova A Vang R Kshirsagar M Lu D Marks MA Shih IeM Kurman RJImmunohistochemical staining patterns of p53 can serve as a surrogatemarker for TP53 mutations in ovarian carcinoma an immunohistochemicaland nucleotide sequencing analysis Mod Pathol “ 0cZhang BMC Veterinary Research Page of Takagi M Ohashi K Morimura T Sugimoto C Onuma M The presence ofthe p53 transcripts with truncated reading frames in Marek™s diseasetumorderived cell lines Leuk Res “ Arlt C Ihling CH Sinz A Structure of fulllength p53 tumor suppressorprobed by chemical crosslinking and mass spectrometry Proteomics “Thierry S Analysis of p53 Gene Alterations in Cancer A Critical View Years of p53 Research “Stiasny A Freier CP Kuhn C Schulze S Mayr D Alexiou C Janko C Wiest IDannecker C Jeschke U Kost BP The involvement of E6 p53 p16 MDM2and Gal3 in the clinical outcome of patients with cervical cancer OncolLett “Shen FX Ma GP Cheng AC Wang MS Li CF Sun KF Chang H Zhu DK JiaRY Chen XY Sun T Development and application of an indirectimmunohistochemical method for the detection of duck plague virusvaccine antigens in paraffin sections and localization in the vaccinatedduckling tissues Poult Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"
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"Dietary macronutrients may indirectly affect body weight through their interactions with the fat massand obesity associated FTO gene This study aimed to investigate the association between FTO gene rs9939609polymorphism with macronutrients intake in overweight adultsMethods This study was carried out on overweight adults of Shiraz Iran Dietary intake was assessed using avalidated 168item semiquantitative food frequency questionnaire FFQ The FTO gene was genotyped forrs9939609 polymorphism The association between dietary macronutrients and the FTO genotype were assessedusing linear regression after adjustments for sex age physical activity and the serum levels of triglycerides fastingblood sugar FBS and low density lipoprotein LDLResults The higher intake of carbohydrates P fat P and calorie P were significantlyassociated with rs9939609 AA genotype P Carriers of the AA genotype of rs9939609 had significantlyhigher calorie fat and carbohydrate intake than the carriers of the TT genotype after adjusting for age and sex P P and P respectively Further adjustments for physical activity TG LDL and FBS did notchange these resultsConclusion The amounts of dietary calorie carbohydrate and fat intake were associated with FTO genotypeFurther studies are warranted to confirm these associations and to identify the underlying mechanismsKeywords FTO Polymorphism Genotype Macronutrient Carbohydrate Protein Fat FiberIntroductionThe prevalence of obesity as a healthrelated problemhas been dramatically increased in both developed anddeveloping countries [ ] More than of adults™population of the United States are obese [] Obesity isassociated with other chronic diseases such as cancerhypertension dyslipidemia cardiovascular disease type diabetes and psychological disorders [] Obesity is a Correspondence sdoaeeyahoocom2Cancer Research Center Shahid Beheshti University of Medical SciencesTehran Iran3Research Center of Health and Environment Guilan University of MedicalSciences Rasht IranFull list of author information is available at the end of the multifactorial disorder caused by genetics lifestyle andenvironmental factors [ ]The role of some genes in obesity has been reported inmany studies [“] The fat mass and obesity associatedFTO gene is located on the chromosome region16q122 and was reported to be strongly associated withobesity [ ] The FTO gene is widely expressed in several tissues such as brain visceral fat liver and hypothalamus Several studies reported that FTO genotypehas a strong association with body mass index BMIand obesity [ ] FTO rs9939609 polymorphism is associated with the increased risk of obesity People withrs9939609 FTO variant alleles homozygous AA and The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMehrdad Lipids in Health and Disease Page of heterozygous AT are predisposed to greater adipositythan are those with wildtype alleles TT The minorallele frequency of rs9939609 is much different based onethnicity ie it is about and inEuropean Chinese Japanese and African populationsrespectively []FTO gene has an important role in regulation of foodintake energy balance appetite and basal metabolic rateBMR [ ] Polymorphisms in the intron regions ofFTO gene may act as a regulator of other genes such asIroquois homeobox IRX3 and obesityassociated single nucleotide polymorphisms of FTO were associatedwith expression of IRX3 but not FTO in human brains[] On the other hand FTO genotypes may influencethe association of dietary macronutrients with BodyMass Index BMI body weight food intake energy balance appetite and hormone secretion [“] Dietarymacronutrients including carbohydrate fat and proteinas the main sources of energy play key roles in regulation of body weight and BMI [ ] However the effects of polymorphisms in obesityrelated genes on theamount of macronutrients™ intake is not clear So thisstudy aimed to investigate the interactions between theamount of dietary carbohydrate protein and fat withthe FTO genotype in overweight adultsMethodologyThis study was carried out from September to October on randomly selected participants referred to the Shohadaye Valfajr health center ShirazIran Participants were overweight adults aged to years with BMI between to kgm2 The Inclusioncriteria was defined as healthy people with overweightwillingness to participation in the study not participating in a weight management programs during two pastmonths and no weight loss greater than over the last months Participants with alcohol or drugs addictionn smoking certain weightrelated diseases including specific psychological or neurological disorders insulin resistancerenalfailure and infectious diseases n and pregnant orlactating women n were excluded Thus the finalnumber of participants in this study was All participants signed a consent form before participation in thestudythyroid diseasesliver diseasesAnthropometric measuresThe height of the participants was measured with a calibrated tape line fastened to a wall and without shoeswith a precision of cm A bio impedance analysisBIA scale BC418 Tanita Cooperation Tokyo Japanwas then used to measure anthropometric indices suchas BMI skeletal muscle percentage SM body fatBF skeletal muscle SM and body fat percentageBF after entering their height age and genderGenotypingDNA was extracted from whole peripheral blood sampleusing the DNA extraction kitCinnagen CompanyTehran Iran and were stored at ˆ’ °C before genotyping The concentration of the extracted material wasassessed using spectrophotometer by the NanoDrop®ND1000 UVVis Spectrophotometer Nanodrop technologies Rockland USA FTO gene was genotyped forrs9939609 polymorphism via tetraprimer amplificationrefractory mutation systempolymerase chain reactionTetraARMS PCR The sequences of the primers arepresented in supplementary file Macronutrients™ intakeUsual Macronutrients™ intakes of the participants wereassessed using a validated 168item semiquantitativefood frequency questionnaires FFQ [] The FFQ wasconsisted of food items with standard portion sizescommonly consumed by Iranian people Facetoface interviews were conducted by a trained dietitianDietary intake was analyzed using the Nutritionist4software program which was modified for Iranian foods[] Daily intakes of calorie were measured for eachperson by using the US Department of Agriculture foodconsumption database which was modified for IranianfoodsPhysical activityA validated international physical activity questionnaireIPAQ was used to measure participants™ physical activity [] Results obtained from IPAQ were expressed asmetabolic equivalents MET per minuteLaboratory measurementThe levels of serum triglyceride TG total cholesterolTC high density lipoprotein HDL lowdensity lipoprotein cholesterol LDL and glucose were measuredafter h of an overnight fastingStatistical analysisThe ShapiroWilk normality normality test was used todetermine if the quantitative variables had a normal distribution ANOVA test was used to compare demographic anthropometric measurements macronutrients™intake and physical activity between different FTO genotypes The post hoc Tukey™s test was then used to identify significant differences of calorie and macronutrientsintake between three genotypes Linear regression wasused to adjust the effects of confounders including agesex PA TG TC LDL and FBS Statistical analyses wereperformed using SPSS version IBM SPSS IBM 0cMehrdad Lipids in Health and Disease Page of Corp Chicago USA The results were considered statistically significant at P Ethics approval and consent to participateThis study has been approved by ethics review board of ShirazUniversity of Medical Sciences Code irsumsrec1395100ResultsAll data were normally distributed according to theShapiroWilk normality test Regarding FTO rs9939609genotype about half of the participants were heterozygote n about of them were TT wild type n and about of them were AA homozygote n The genotype distribution of the study populationwas in HardyWeinberg equilibrium Significant differences were found in BMI P fat mass P calorie intake P fat intake P and carbohydrate intake P status of three FTOgenotypes Table Carriers of the AA genotype ofFTO rs9939609 polymorphism had significantly highercalorie fat and carbohydrate intake than the carriers ofthe TT genotype P P and P respectively Table Linear association of FTO rs9939609 genotype withintake carbohydrate fatthe level of macronutrients™protein and fiber was then assessed after adjustmentthe effects of confounders This association remainedsignificant for carbohydrate calorie and fat intake afteradjustment for age and sex P P and P respectively model Further adjustments forphysical activity TG LDL and FBS did not change theresults P P and P respectivelymodel Table DiscussionThe present study evaluated the associations betweenrs9939609 FTO gene polymorphism with caloriefatcarbohydrate protein and fiber intake The results identified that there was a significant association betweenFTO genotype with calorie carbohydrate and fat intakeThis association remained significant for calorie carbohydrate and fat intake after adjustments for sex agephysical activity LDL HDL and FBS In carriers of AAgenotype of rs9939609 polymorphism dietary carbohydrate fat and calorie intake were higher than TT carriers However the results of recent studies about theassociation between dietary macronutrients and FTOpolymorphism were inconsistent [“] Oyeyemi et alin a casecontrol study on people with obesity estimated as BMI ‰¥ and controls identified kcalTable characteristics of the subjects categorized by FTO rs9939609 genotypes N VariablesMale sex NAT n TT n Age years mean SDWeight kgHeight m mean SDBMI kgm2 mean SDFat Mass kg mean SDFM mean SDFFM kg mean SDFFM mean SDIPAQ METminute mean SD±±±±±±±±±Calorie intake Kcal mean SD ±Fat gday mean SDProtein gday mean SDCarbohydrate gday mean SDFiber gday mean SDFBS mg dL mean SDLDLC mg dL mean SDHDLC mg dL mean SDT Chol mg dL mean SD±±±±±±±±±±±±±±±±±±±±±±±±±±AA n ±±±±±±±±± ±±±±±±±±±TG mgdL mean SDAbbreviations BMI body mass index HDL highdensity lipoprotein FFM fat free mass IPAQ International Physical Activity Questionnaire LDL lowdensitylipoprotein T Chol total cholesterol TG triglycerides FBS fasting blood sugar FAT fat intake carbohydrate carbohydrate intake Protein protein intake Fiberfiber intake±±±P value 0cMehrdad Lipids in Health and Disease Page of TTTable Tukey test for comparison the calorie andmacronutrient intake between three genotypesAAVariableˆ’Calorieˆ’ˆ’ˆ’ˆ’ATˆ’ˆ’ˆ’ˆ’P valueCarbohydrateProteinFatFiberPvalueP valued more energy intake per risk A allele of rs9939609 []Timpson reported higher calorie and fat intakeamong rs9939609 AA genotype carriers They suggestthat FTO polymorphism may influence on appetite andfood intake [] Some other studies also reported thatcarriers of risk allele FTO received higher energy intake[] Consistent with the results of this study Daya et alreported that carriers of ATAA genotype had higher fatintake times and had higher risk of obesity compared with TT genotype [] The FTO variants were reported to be associated with intake of energydensefoods such as fatrich foods [] FTO gene variantsplayed important roles in appetite regulation food intake tendency to choose energydense food high fatand high carbohydrate diet [] The carriers of A alleleFTO rs9939609 had energydense food choices higherbody weight and overeating behaviors [] On theother hand Qi in a crosssectional study on whitepopulation n found a lower energy intake perA risk allele ß ˆ’ kcald [] Another study foundno association between a highfat diet and a high carbohydrate diet with the FTO gene in rats [] Drabsch in a systematic review reported that there is noconsistent evidence that the FTO gene SNPs are associated with total energy carbohydrate and fat intakes[] The cause of this discrepancy between the studiesremained unclear However the relationship betweenFTO genotype and dietary intake seems to be very complex and many factors may have a role in this associationTable Association of FTO genotypes with macronutrients™intakevariablesCalorieFATProteinCarbohydrateFiberR2 Model Beta PModel Beta PR2Model adjusted for age and sexModel Further adjustments for physical activity the serum levels oftriglycerides fasting blood sugar FBS and low density lipoprotein LDLPvalue such as the obesity [] level of physical activity []serum leptin [] and other dietary components [] However only overweight subjects were includedbecause of the possible effect of BMI on the associationbetween FTO genotype and dietary intakeRegarding to dietary carbohydrate the AA genotypecarriers had higher carbohydrate intake than TT genotype carriers which was in line with the results of theprevious studies [“] Sonest found that FTOgenetic variants are associated with the amounts ofcarbohydrate intake Some study reported that carbohydrate intake especially glucose intake increased FTOgene expression [ ] In homozygous people for therisk allele of FTO gene rs9930506 polymorphism higherdietary carbohydrate intake had a positive associationwith FTO gene expression []This study found no association between protein intake and FTO genotype While some studies indicatedthat protein intake was associated with FTO genotype[ ] However another study reported that leucineintake increased FTO gene expression [] Doaei et alfound that higher protein intake upregulated the FTOgene and also indicated that only in A allele carrier []The mechanism of the interactions between the FTOgenotype and dietary macronutrients is not fully understood The FTO gene polymorphisms may change theamounts of macronutrients™ intake On the other handthe association of FTO polymorphisms with obesity maybe influenced by dietary intake It was observed that theA risk allele of FTO rs9939609 polymorphism had nosignificant association with obesity in subjects whosedietary fat intake was below of total energy but increased central and total adipose tissues in subjects withfat intake higher than [] Another study reportedthat the risk allele carriers who received Mediterraneandiet for years had lower BMI compared with the others[] Dietary macronutrients may also change the levelof FTO gene expression NowackaWoszuk indicated that a highfat diet could increase FTO gene expression in white adipose cells in rats [] Ronkainen investigated the association between fat intake andthe FTO gene expression They found that a highfat dietcould suppress FTO expression []Some studies suggested that FTO play a crucial role inregulating energy homeostasis FTO gene is expressed inhypothalamus that controls feeding and energy expenditure [ ] Interestingly FTO expression level in hypothalamus is regulated by dietary intake It was reportedthat a highfat diet can downregulate FTO expression inshortterm and up regulate it in longterm [ ]On the other hand the FTO gene is related with guthormones such as orexigenic hormone acylghrelin satiety hormone peptide YY that regulate food intake andappetite [] FTO gene polymorphism AA genotype 0cMehrdad Lipids in Health and Disease Page of influence on circulating PYY3“ and acylghrelin levelsthat lead to increased food intake especially energydense foods and reduced satiety [ ] In rs9939609AA carriers suppression of acylated ghrelin led to overeating and obesity [] So it is plausible that FTO genepolymorphisms could change appetite and food intakethat may lead to weight gain and obesityAuthors™ contributionsMM and MHE designed the study involved in the data collection analysisand drafting of the manuscript MM SD and MGh were involved in theanalysis of the data and writing the manuscript All authors read andapproved the final manuscriptFundingFunding for this study was provided by Shiraz University of Medical SciencesStudy strengths and limitationsThe main strength of this study was the relatively highsample size of overweight adults and adjustments forsugar and lipid profiles as the possible factors affectingdietary intake This study also included only overweightsubjects because of the possible effect of BMI on the association between FTO genotype and dietary intake Inaddition information on a wide range of potential confoundersmodifiers and their potential effects were takeninto account The present study also has several limitations to acknowledge First the study was limited bycrosssectional design Second dietary intake was determined according to a selfreported questionnaire thisparameter was not measured objectively although similarto many prior epidemiological studiesAvailability of data and materialsNot applicableEthics approval and consent to participateThis study has been approved by Local ethics review boards at ShirazUniversity of medical sciences irsumsrec1395100Consent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interestsAuthor details1Department of Clinical Nutrition School of Nutrition and food SciencesShiraz University of Medical Sciences Shiraz Iran 2Cancer Research CenterShahid Beheshti University of Medical Sciences Tehran Iran 3ResearchCenter of Health and Environment Guilan University of Medical SciencesRasht Iran 4Student research committee Cancer Research Center ShahidBeheshti University of Medical Sciences Tehran IranReceived January Accepted August forcalorieremainedsignificantConclusionThe genotype of FTO may influence the amount of dietary intake in overweight people FTO gene rs9939609polymorphism was associated with dietary intake Theintake of calorie carbohydrate and fat intake were associated with FTO gene polymorphisms and this associationandmacronutrients after adjustments for sex age physicalactivity LDL HDL and FBS In AA carriers dietarycarbohydrate fat calorie was higher than TT carriersGenetic profile can play a key role in future nutritionalrecommendations especially for weight management andalso for prevention of dietrelated chronic diseases Diettherapy in people with risk allele of FTO rs9939609polymorphism may require to consider their desire toeat more carbohydrate fat and calorie Further studiesare needed to increase understanding of the underlyingmechanisms of the association between FTO gene anddietary intakeSupplementary informationSupplementary information accompanies this paper at httpsdoi101186s1294402001372xAdditional file AcknowledgementsThis study was conducted at the Department of Public Health Nutrition ofthe Shiraz University of Medical Sciences Shiraz IranReferencesHaslam DW James WP Obesity Lancet “Ogden CL Carroll MD Kit BK Flegal KM Prevalence of Childhood and AdultObesity in the United States “ JAMA “Alwan A Global status report on noncommunicable diseases WorldHealth anization Fall T Ingelsson E Genomewide association studies of obesity andmetabolic syndrome Mol Cell Endocrinol “Fruhbeck G GomezAmbrosi J Muruzabal FJ Burrell MA The adipocyte amodel for integration of endocrine and metabolic signaling in energymetabolism regulation Am J Physiol Endocrinol Metab “Fredriksson R Hagglund M Olszewski PK Stephansson O Jacobsson JAOlszewska AM Levine AS Lindblom J Schiöth HB The obesity gene FTO isof ancient origin upregulated during food deprivation and expressed inneurons of feedingrelated nuclei of the brain Endocrinology May “Doaei S Hajiesmaeil M Aminifard A MosaviJarrahi SA Akbari MEGholamalizadeh M Effects of gene polymorphisms of metabolic enzymeson the association between red and processed meat consumption and thedevelopment of colon cancer a literature review J Nutritional Sci Doaei S Kalantari N Mohammadi NK Izadi P Gholamalizadeh M EiniZinabH Salonurmi T Jarrahi AM Rafieifar S Janipoor R Sadeghypor M Upregulation of FTO gene expression was associated with increase in skeletalmuscle mass in overweight male adolescents Arch Med Sci Sep155Hakanen M Raitakari OT Lehtimäki T Peltonen N Pahkala K Sillanmäki LLagstrom H Viikari J Simell O Ronnemaa T FTO genotype is associatedwith body mass index after the age of seven years but not with energyintake or leisuretime physical activity J Clin Endocrinol Metab Apr “Thorisson GA Smith AV Krishnan L Stein LD The international HapMapproject web site Genome Res Nov “Smemo S Tena JJ Kim KH Gamazon ER Sakabe NJ GómezMarín C AneasI Credidio FL Sobreira DR Wasserman NF Lee JH Obesityassociatedvariants within FTO form longrange functional connections with IRX3Nature Mar5077492371“ Antonio J Knafo S Kenyon M Ali A Carson C Ellerbroek A Weaver CRoberts J Peacock CA Tartar JL Assessment of the FTO gene 0cMehrdad Lipids in Health and Disease Page of Ronkainen J Huusko TJ Soininen R Mondini E Cinti F Mäkelä KAKovalainen M Herzig KH Järvelin MR Sebert S Savolainen MJ Fat massandobesityassociated gene Fto affects the dietary response in mouse whiteadipose tissue Sci Rep Mar Poritsanos NJ Lew PS Fischer J Mobbs CV Nagy JI Wong D Rüther UMizuno TM Impaired hypothalamic Fto expression in response to fastingand glucose in obese mice Nutr Diab 2011110e19 Doaei S Kalantari N Izadi P Salonurmi T Jarrahi AM Rafieifar S Azizi Tabesh GRahimzadeh G Gholamalizadeh M Goodarzi MO Interactions between macronutrients™ intake FTO and IRX3 gene expression and FTO genotype in obeseand overweight male adolescents Adipocyte Jan “ Olszewski PK Fredriksson R Olszewska AM Stephansson O Alsiö JRadomska KJ Levine AS Schiöth HB Hypothalamic FTO is associated withthe regulation of energy intake not feeding reward BMC Neurosci Dec1011“ Razquin C Martinez JA MartinezGonzalez MA A 3year intervention with aMediterranean diet modified the association between the rs9939609 genevariant in FTO and body weight changes Int J of Obesity “ McTaggart JS Lee S Iberl M Church C Cox RD Ashcroft FM FTO isexpressed in neurones throughout the brain and its expression is unalteredby fasting PLoS One 2011611e27968 httpsdoi101371journalpone0027968Stratigopoulos G Padilla SL LeDuc C A Watson E Hattersley AT McCarthyMI Zeltser LM Chung WK Leibel RL Regulation of FtoFtm gene expressionin mice and humans Am J Physiol Integr Comp Physiol 2008294R1185“ Wang P Yang FJ Du H Guan YF Xu TY Xu XW Su DF Miao CYInvolvement of leptin receptor long isoform LepRbSTAT3 signalingpathway in brain fat massand obesityassociated FTO downregulationduring energy restriction Mol Med May ““ Batterham RL Cohen MA Ellis SM Le Roux CW Withers DJ Frost GS GhateiMA Bloom SR Inhibition of food intake in obese subjects by peptide YY3“ N Engl J Med Sep “ Wardle J Carnell S Haworth CM Farooqi IS O™Rahilly S Plomin R Obesityassociated genetic variation in FTO is associated with diminished satiety JClin Endocrinol Metab “ httpsdoi101210jc2008 Velders FP De Wit JE Jansen PW Jaddoe VW Hofman A Verhulst FCTiemeier H FTO at rs9939609 food responsiveness emotional control andsymptoms of ADHD in preschool children PLoS One Nov e49131Karra E O™Daly OG Choudhury AI Yousseif A Millership S Neary MT ScottWR Chandarana K Manning S Hess ME Iwakura H A link between FTOghrelin and impaired brain foodcue responsivity J Clin Invest Aug “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationspolymorphisms rs1421085 rs17817449 and rs9939609 in exercisetrainedmen and women the effects of a 4week hypocaloric diet J Int Soc SportsNutr Dec Blundell JE Lawton CL Cotton JR Macdiarmid JI Control of humanappetite implications for the intake of dietary fat Annu Rev Nutr “Ludwig DS The glycemic index physiological mechanisms relating toobesity diabetes and cardiovascular disease Jama “Sonestedt E Roos C Gullberg B Ericson U Wirfält E OrhoMelander M Fatand carbohydrate intake modify the association between genetic variationin the FTO genotype and obesity Am J Clin Nutr Sep “Esfahani FH Asghari G Mirmiran P Azizi F Reproducibility and relativevalidity of food group intake in a food frequency questionnaire developedfor the Tehran lipid and glucose study J Epidemiol “ Azar M Sarkisian E Food composition table of Iran National Nutrition andfood research institute Tehran Shahid Beheshti University Press [Persian] VasheghaniFarahani A Tahmasbi M Asheri H Ashraf H Nedjat S Kordi RThe Persian last 7day long form of the international physical activityquestionnaire translation and validation study Asian journal of sportsmedicine Jun22106 Oyeyemi BF Ologunde CA Olaoye AB Alamukii NA FTO gene associatesand interacts with obesity risk physical activity energy intake and timespent sitting pilot study in a Nigerian population J Obes May212017 Villagran M Petermann R Mardones L Garrido MA Natalia MM Associationbetween the polymorphism rs9939609 of the FTO gene with energy intakemacronutrients and alcohol consumption in the Chilean populationMedium Chile Dhurandhar NV Schoeller D Brown AW Heymsfield SB Thomas DSørensen TI Speakman JR Jeansonne M Allison DB Energy balancemeasurement when something is not better than nothing Int J Obes “ Daya M Pujianto DA Witjaksono F Priliani L Susanto J Lukito W Malik SGObesity risk and preference for high dietary fat intake are determined byFTO rs9939609 gene polymorphism in selected Indonesian adults Asia PacJ Clin Nutr Mar281183Livingstone MB Robson PJ Black AE Coward WA Wallace JM McKinley MCStrain JJ McKenna PG An evaluation of the sensitivity and specificity ofenergy expenditure measured by heart rate and the Goldberg cutoff forenergy intake basal metabolic rate for identifying misreporting of energyintake by adults and children a retrospective analysis Eur J Clin Nutr Mar573455“ Zheng Y Huang T Zhang X Rood J Bray GA Sacks FM Qi L Dietary fatmodifies the effects of FTO genotype on changes in insulin sensitivity JNutr May “ Hardy DS Racette SB Hoelscher DM Macronutrient intake as a mediatorwith FTO to increase body mass index J Am Coll Nutr 201433256e66 Qi L Kraft P Hunter DJ Hu FB The common obesity variant near MC4Rgene is associated with higher intakes of total energy and dietary fatweight change and diabetes risk in women Hum Mol Genet Nov “ Zhong T Duan XY Zhang H Li L Zhang HP Niu L Angelica sinensissuppresses body weight Gaiand alters expression of the FTO gene in highfatdiet induced obese mice BioMed Res Int Drabsch T Gatzemeier J Pfadenhauer L Hauner H Holzapfel C Associationsbetween single nucleotide polymorphisms and total energy carbohydrateand fat intakes a systematic review Adv Nutr Jul “ Dorling JL Clayton DJ Jones J Carter WG Thackray AE King JA Pucci ABatterham RL Stensel DJ A randomized crossover trial assessing the effectsof acute exercise on appetite circulating ghrelin concentrations andbutyrylcholinesterase activity in normalweight males with variants of theobesitylinked FTO rs9939609 polymorphism Am J Clin Nutr Nov “Katus U Villa I Ringmets I Vaht M Mäestu E Mäestu J Veidebaum T HarroJ Association of FTO rs1421085 with obesity diet physical activity andsocioeconomic status a longitudinal birth cohort study Nutr MetabCardiovasc Dis NowackaWoszuk J PruszynskaOszmalek E Szydlowski M Szczerbal INutrition modulates Fto and Irx3 gene transcript levels but does not altertheir DNA methylation profiles in rat white adipose tissues Gene “ 0c"
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"dramatic spread of Coronavirus Disease COVID19 has profound impacts on every continent and life Due to humantohuman transmission of COVID19 nuclear medicine staffs also cannot escape the risk of infection from workplaces Everystaff in the nuclear medicine department must prepare for and respond to COVID19 pandemic which tailored to the characteristics of our profession This provided the guidance prepared by the Korean Society of Nuclear Medicine KSNM incooperation with the Korean Society of Infectious Disease KSID and Korean Society for HealthcareAssociated InfectionControl and Prevention KOSHIC in managing the COVID19 pandemic for the nuclear medicine department We hope that thisguidance will support every practice in nuclear medicine during this chaotic periodKeywords Coronavirus COVID19 Nuclear medicine Prevention and control Practice guideline HoYoung Leedebobkrgmailcom Department of Nuclear Medicine CHA Bundang Medical CenterCHA University of Medicine Professor Pocheon Republic ofKorea Department of Nuclear Medicine Seoul National UniversityBundang Hospital Professor Seongnam Gyeonggido Republic ofKorea Department of Nuclear Medicine Samsung Medical CenterSeoul Republic of Korea Department of Nuclear Medicine Seoul National UniversityHospital Seoul Republic of Korea Department of Nuclear Medicine Chosun University HospitalGwangju Republic of Korea Department of Nuclear Medicine Korea University Guro HospitalSeoul Republic of Korea Department of Nuclear Medicine Hanyang University Guri HospitalSeoul Republic of Korea Department of Nuclear Medicine National Cancer CenterGoynag Republic of Korea Department of Nuclear Medicine Seoul Medical CenterSeoul Republic of Korea Division of Nuclear Medicine Department of RadiologyEunpyeong St Mary™s Hospital College of Medicine The CatholicUniversity of Korea Seoul Republic of Korea Department of Nuclear Medicine Soonchunhyang University SeoulHospital Bucheon Republic of Korea Department of Nuclear Medicine Inje University Haeundae PaikHospital Busan Republic of Korea Department of Nuclear Medicine Keimyung University DongsanMedical Center Daegu Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityCheonan Hospital Cheonan Republic of Korea Department of Nursing Soonchunhyang University BucheonHospital Bucheon Republic of Korea Division of Infectious Disease Department of Internal MedicineKangdong Sacred Heart Hospital Hallym UniversityChuncheon Republic of Korea Department of Nuclear Medicine Soonchunhyang UniversityBucheon Hospital Bucheon Republic of Korea Department of Nuclear Medicine Korea University Anam Hospital Korean Society of Nuclear Medicine Quality Control CommitteeSeoul Republic of KoreaBucheon Republic of Korea 0cIntroductionSince the first reports of Coronavirus Disease COVID in Wuhan China the infection had spread worldwiderapidly and COVID19 has reached pandemic levels InSouth Korea since its outbreak in February COVID has affected profoundly every aspect of communities Thehumantohuman transmission of COVID19 provides challenges for all healthcare facilities and healthcare providersIn the face of the COVID19 pandemic the Korean Societyof Nuclear Medicine KSNM Korean Society of InfectiousDisease KSID and Korean Society for HealthcareAssociated Infection Control and Prevention KOSHIC haveprepared the guidance for the nuclear medicine department tominimize confusion and ensure that nuclear medicine physicians and technicians continue to provide their services whileprotecting the patients and workers and preventing the transmission of the virus The Quality Control Committee ofKSNM reviewed several reports and recommendations previously published by the European Association of NuclearMedicine EANM [] Society of Nuclear Medicine andMolecular Imaging SNMMI American Society of NuclearCardiology ASNC [] International Atomic Energy AgencyIAEA and others [“] This guidance is basically in compliance with the COVID19 guidelines of the Korea Centersfor Disease Control and Prevention KCDC [“] Finallythis document was prepared in cooperation with KSID andKOSHIC KSNM emphasize that this guidance must be considered in the context of following the state and hospital infection control policies and flexibly applied according tochanges in circumstances and evidenceGeneral Principles During COVID19PandemicIn a pandemic situation such as COVID19 if necessarythe condition of the scheduled patient can be checked inadvance to adjust the examination schedule Nonurgent elective studies or therapy should be postponed in COVID19confirmed or COVID19suspectedpatients Rescheduling the studiestherapy must be donein a discussion with the referring clinicians Only urgent studies or therapy could be performed inCOVID19confirmed or COVID19suspected patientswhenever clinically appropriate The priority of studytherapy should be based on a casebycase indepth discussion between nuclear medicine physicians and referring clinicians In case of performing the urgent studiestherapy consult with the infection control offices of eachinstitution to comply with the infection control rules ofownNucl Med Mol Imaging “ COVID19suspected patients should undergo COVID testing before performing the studiestherapy Lung ventilation scan should not be performed in anyCOVID19confirmed or COVID19suspected patients Lowdose radioiodine therapy may be considered in caseof acute hyperthyroidism patients who are unable to tolerate antithyroid medications As lowdose radioiodinetherapy lower than GBq of I131 can be performedin an outpatient setting in South Korea COVID19infected patient can be administrated lowdoseradioiodine in the isolation room or negative pressureroom without any additional monitoring related toradioiodine therapyConsideration During the StudyTherapy Patient transportationScheduling COVID19confirmed or COVID19suspected patient as last study of the day to preventcrossinfection in the nuclear medicine department Ensure that other patients or caregivers should notaccess the nuclear medicine department to minimizethe exposure to COVID19 patient during the studytherapy Transfer the COVID19infected patient to the nuclear medicine department using negative pressuretransport bag to minimize exposure and contact todroplet COVID19 patients should wear masks at all timesof procedures If necessary add gowns gloves etc Devices and scanner management Mainly use disposable instruments or items Do notreuse disposable items such as oxygen masks nasalprongs suction tubes or suction lines The protocolfor reusable devices is as follows Cleaning After use the equipment contaminated with blood bodyfluids secretions and feces should be delivered to awashing room with care not to contaminate the surrounding environment The washing place should be separated from the spaceused for cleaning other items or other patients After immersing the contaminated equipment in a washing spacewash the product carefully to avoid splashing Wash enough to remove blood body fluids secretionsand feces from remaining 0cNucl Med Mol Imaging “ Staff undertaking cleaning should wear KF94 or N95masks longsleeved waterproof gowns goggles or faceshields hats shoe covers or rubber boots and doublegloves outer gloves are rubber gloves Disinfection and sterilization Depending on the risk level of the device according tothe Spaulding Classification of medical equipmentdevices noncritical devices require lowlevel disinfectionsemicritical devices require highlevel disinfectionsterilization and critical devices must be sterilized Disinfectants and sterilization methods by device classification should be followed in accordance with the notificationof the Ministry of Health and Welfare Be sure to check the disinfectant manufacturer™s recommendations The recommended disinfection process suchas dilution and application time of disinfectant and theeffective period and concentration of disinfectant arestrictly followed Laboratory and scan room management Only the minimum number of staffs should be placedin the nuclear medicine department All participatingstaffs should wear appropriate personal protectiveequipment PPE eye protection with goggles or faceshield medical protective masks N95KF94 or equivalent respirator disposable latex gloves disposablegown disposable shoe covers etc Cover the scanner couch or other equipment with aplastic cover to prevent contamination Every effort should be made to minimize theCOVID19 exposure to medical staff during injection of radiopharmaceuticalsSelect the protocol with the shortest duration of uptake time and scan time to minimize the time spentby the COVID19 patient in the departmentIn case of studies requiring an uptake phaseCOVID19 patients should be waiting in separatespace If possible COVID19 patients wait in negative pressure transport bag If negative pressuretransport bag is not available use bed or stretcherin waiting room with disposable cover Considerusing standard radiopharmaceutical dose to shortenthe procedure time After the completion of image acquisition the scanroom and patient™s space area should be disinfectedaccording to the standard protocol After image acquisition remove the plastic cover ofthe scanner and disinfect the scanner surface Remove and discard PPE adequately when leavingthe camera room or care area and immediately perform hand hygieneIn case of performing the radiolabeling of theCOVID19 patients™ blood products every processwith infectious materials openingstirringmixingdispensing COVID19 patient™s blood sampleradiolabeling etc should be done in class II biosafety cabinet according to the Biosafety Level Regulation Disinfection of laboratory with properdisinfectants ethanol hydrogen peroxide or ppm sodium hypochlorite should bedone Used PPE and disposable covers are removed withcaution not to contaminate the clean area and disposed in a container for biosafety waste Employee management All employees should be trained in the preventionand management of COVID19 infection and adhereto the rules of infection prevention Considering the skill level fatigue etc of the working staff sufficient personnel are allocated to securethemPriority from exemption is given to employees withhighrisk underlying diseases such as diabetesmellitus chronic obstructive pulmonary diseaseCOPD endstage renal disease ESRD chroniccardiac disease etc or pregnant women Cleaning and environmental management General principle Personnel responsible for cleaning or disinfectionshould complete the infection preventioneducation Employees should wear PPE KF94 or N95 respirators fullbody protective clothing or aprons goggles or face shields shoe covers or rubber bootsdouble gloves outer gloves are rubber gloveswhen cleaning or disinfectingIf there are organic substances on the surface of theenvironment it cannot be properly disinfectedTherefore wipe the surface before disinfecting theenvironmentIn order to prevent the possibility of microbialspraying cleaning should be performed using acleaning solution or a mop moistened with a disinfectant rather than a cleaning method using abroom or a vacuum cleanerInstead of spraying disinfectants thoroughly cleanthe surface of the environment using a clean towelmoistened with the disinfectant or a commerciallyavailable disinfecting tissue towel 0cNucl Med Mol Imaging “ Use cleaning tools as disposable as possible or exclusively However when the cleaning tool isreused the used cleaning tool is sterilized usingan appropriate disinfectant and then dried andstored Disinfection of a patient™s space areaIn the case of the space area used by the patientmark the place where contamination was confirmedbefore cleaning and disinfecting the surface andseal the contaminated object to prevent others frombeing exposed Ventilation before during and after cleaningdisinfection disinfection after ventilation for hbased on air cycles per hour Wear PPE Wipe with a cloth cloth etc wet withthe diluted disinfectant Wipe the touched wall surface and all frequently used areas and keep it for atleast min After then wipe the surface with acloth dampened with clean water cloth etcResumption of use Consider the characteristics ofeach type of disinfectant used and the purpose of thefacility After disinfection the virus is killed but thedecision at the time of resumption of use cannot beapplied in batch due to different characteristics ofdisinfectants so it is necessary to consider the precautions for each productFor details on disinfecting methods such as surfacedisinfection and washing refer to œDisinfectionGuidelines to Prevent the Spread of COVID19 atPublic and Multipurpose Facilities 3rd editionRefer to the method of disinfecting the patient spaceareaSelect an environmental disinfectant Select an approved or declared disinfectant by the Ministry ofEnvironment and follow the usage usage and precautions for each product Disinfectant list of the Ministry of Environmenthttpecolifemegokr Precautions when using environmentaldisinfectants Select the disinfectant after confirming informationsuch as approval from the Ministry of Environmentand Environment When using environmental sterilizers make sure tofollow the manufacturer™s recommendations suchas checking the expiration date safe usage for eachproduct and precautions and preparing the diluentaccording to the manufacturer™s instructions The disinfecting method of sprayinginjecting disinfectant is not applied to surface disinfectionbecause it causes aerosol infection increased riskof inhalation and the range of contact between thedisinfectant and the surface is insufficient so thedisinfecting effect is insufficient Disinfectant hazard information must be checkedand used carefully Do not mix different disinfectants Do not placenear flammable materials Disinfectant should beused in a wellventilated area As the disinfection effect may decrease over timedilute as much as necessary and use it immediatelyDo not store the remaining amount and discard itimmediately Laundry managementStore clean laundry in a separate space Employees handling laundry should be trained toprevent infection Employees handling contaminated laundry shouldwear PPE N95 masks or equivalent respiratory protection gowns gloves overshoes etc and performhand hygiene after removing PPE The laundry used for the patient is disposed of according to the relevant regulations see WasteManagement Act Medical Institution LaundryManagement Rules etc Thoroughly ensure that pathogens are not exposed topersonnel handling the laundry or surrounding environment during the entire process of collectingtransporting and washing laundry Waste management Waste related to COVID19 patients is managed bythe rules of hospital infectious control policySharp tools such as needles or blades are collectedin containers for impervious and nonpermanentwaste and containers should be stored in the placewhere the items are usedSimple infectious waste contaminated or possiblycontaminated with COVID19 patients™ sample isautoclaved and discarded Radioactive waste shouldbe discarded in compliance with national regulationwith caution not to contaminate the staff or areaConclusionConsidering that outbreaks of novel viruses have been periodically appearing these days nuclear medicine staffs should getused to guidance and policies for infectious disease in working 0cNucl Med Mol Imaging “place to protect patients worker themselves and furthermorevaluable medical resources Basically this guidance can beapplied in case of any other humantohuman transmissiondisease for operating the nuclear medicine department Alsoalways bear in mind the rapid change in the situation thisguidance should be used in conjunction with the currentgovernment and local hospital policiesCompliance with Ethical StandardsConflict of InterestJiIn Bang HoYoung Lee Young Seok ChoHongyoon Choi Ari Chong Jae Sun Eo Ji Young Kim Tae SungKim HyunWoo Kwon Eun Jeong Lee Eun Seong Lee Hye LimPark Soo Bin Park Hyekyung Shim BongIl Song Ik Dong YooKyung Jae Lee Hong Jae Lee Su Ha Han Jin Seo Lee Jung Mi Parkand Sung Hoon Kim declare that they have no conflict of interestEthical Approval This work does not contain any studies with humanparticipants or animals performed by any of the authorsInformed Consent Not applicableReferences Paez D Gnanasegaran G Fanti S Bomanji J Hacker M SathekgeM et al COVID19 pandemic guidance for nuclear medicine departments Eur J Nucl Med Mol “ Skali H Murthy VL AlMallah MH Bateman TM Beanlands RBetter N et al Guidance and best practices for nuclear cardiologylaboratories during the coronavirus disease COVID19 pandemic an information statement from ASNC and SNMMI J NuclCardiol “ httpsdoiorg101007s12350020021232 Huang HL Allie R Gnanasegaran G Bomanji J COVID19“nuclear medicine departments be prepared NuclMedCommun MossaBasha M Medverd J Linnau K Lynch JB Wener MHKicska G et al Policies and guidelines for COVID19 preparedness experiences from the University of Washington Radiology httpsdoiorg101148radiol2020201326 Zhang X Shao F Lan X Suggestions for safety and protectioncontrol in Department of Nuclear Medicine during the outbreak ofCOVID19 Eur J Nucl Med Mol “ Buscombe JR Notghi A Croasdale J Pandit M O'Brien J GrahamR et al COVID19 guidance for infection prevention and controlin nuclear medicine Nucl Med Commun “ Standard guideline for healthcareassociated infection control andprevention Korean Center for Disease Control and Prevention andKorean Society for HealthcareAssociated Infection Control andPrevention httpcdcgokrCDCcmscontentmobile2675626_viewhtml Accessed 2nd Jun Korean Society for HealthcareAssociated Infection Control andPrevention Korean Center for Disease Control and Preventionhttpwwwcdcgokrboardesmida20507020000bid0019actviewlist_no366579 Accessed 2nd Jun Guidelines in response to coronavirus disease for local governmentKorea Centers of Disease Control and Prevention2020 httpswwwcdcgokrboardboardesmida20507020000bid0019actviewlist_no367279tagnPage1 Accessed 2ndJun Disinfection guidelines to prevent the spread of COVID19 at public and multipurpose facilities Korea Centers of Disease Controland Prevention httpswwwcdcgokrboardboardesmida20507020000bid0019 Acessed 15th Jun Publisher™s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c"
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" 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 pearson™s 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 cancercenter™s 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 × bp the data wereprocessed according to the manufacturer™s protocol the mean coverage was × and × 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 ×candidate 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 pearson™s 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 695eˆ’ to 452eˆ’ 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 3k“80k 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 ia“iiia 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 a“f 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 iiib“iv 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 3k“80k 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 “× 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 œaccompanying 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 lyu™s 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 people™s republic of china for their contribution tothe st
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continuously increasing with development of the economy and the environment [“] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [“] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [“] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrna™e1e2abcbasepairinge3dguriche4™ecrichpretrnarna bindingproteinsrbp™™™™™™™™gnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the ™ hydroxyl of the upstream exon reacts with the ™ phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the ™ hydroxyl of the ™ intron interacts with the ™ phosphate of the ™intron to form an ecircrna following an interaction between the ™ hydroxyl of the ™ exon and the ™ phosphate of the ™ exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a ™“™ phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [“] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas progression of hcc circrnas may play an important role in the occurrence and development of tumors however the molecular mechanism underlying the relationship between circrnas and hcc has not been fully elucidated therefore indepth research is needed on the potential regulatory relationships and to uncover regulatory patterns between circrnas and hcc so that new diagnostic markers for hcc can be developedreferences bray f ferlay j soerjomataram i global cancer statistics globocan estimates of incidence and mortality worldwide for cancers in countries cancer j clin “ feng rm zong yn cao sm xu rh current cancer situation in china good or bad news from the global cancer statistics cancer commun lond jemal a bray f center mm global cancer statistics cancer j clin “ li r jiang j shi h circrna a rising star in gastric cancer cell mol life sci “ salzman j gawad c wang pl circular rnas are the predominant transcript isoform from hundreds of human genes in diverse cell types plos one e30733 lukiw wj circular rna circrna in alzheimer™s disease ad front genet liu y yang y wang z insights into the regulatory role of circrna in angiogenesis and clinical implications atherosclerosis “ zhao y alexandrov pn jaber v lukiw wj deficiency in the ubiquitin conjugating enzyme ube2a in alzheimer™s disease ad is linked to deficits in a natural circular mirna7 sponge circrna cirs7 genes basel shen f liu p xu z circrna_001569 promotes cell proliferation through absorbing mir“ in gastric cancer j biochem “ song t xu a zhang z circrna hsa_circrna_101996 increases cervical cancer proliferation and invasion through activating tpx2 expression by restraining mir8075 j cell physiol “ min l wang h zeng y circrna_104916 regulates migration apoptosis and epithelial“mesenchymal transition in colon cancer cells front biosci landmark ed “ verduci l strano s yarden y blandino g the circrnamicrorna code emerging implications for cancer diagnosis and treatment mol oncol “ wei j wei w xu h circular rna 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international cc byncnd e9238326indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832 yang y fan x mao m extensive translation of circular rnas driven by n6methyladenosine cell res “ yang y gao x zhang m novel role of fbxw7 circular rna in repressing glioma tumorigenesis j natl cancer inst “ abe n hiroshima m maruyama h rolling circle amplification in a prokaryotic translation system using small circular rna angew chem int ed engl “ zheng x chen l zhou y a novel protein encoded by a circular rna circppp1r12a promotes tumor pathogenesis and metastasis of colon cancer via hippo“yap signaling mol cancer holdt lm stahringer a sass k circular noncoding rna anril modulates ribosomal rna maturation and atherosclerosis in humans nat commun gokul s rajanikant gk circular rnas in brain physiology and disease 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“ yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun “ liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun “ li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle “ “ shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c'
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the ongoing pandemic coronavirus disease outbreak covid2019 stemming from the beta coronavirus class sarscov2 causing severe acute respiratory syndrome sars has created a global health emergency in countries around the globe huang nalla sarscov2 a novel coronavirus strain belonging to the sarbecovirus subgenus genus betacoronavirus family coronaviridae had first appeared in late in wuhan china infecting a large number of hosts million people with a mortality rate of ‰¥ neogi carter such putative etiopathogenic agents associated with the zoonotic viral transmission pathways are responsible for respiratory viral pneumonia and gastrointestinal infections leading to infected people or patients having multiple an failure in a b s t r a c t severe acute respiratory syndrome coronavirus sarscov2 a neoteric virus belonging to the beta coronavirus class has created a global health concern responsible for an outbreak of severe acute respiratory illness the covid19 pandemic infected hosts exhibit diverse clinical features ranging from asymptomatic to severe symptoms in their genital ans respiratory digestive and circulatory systems considering the high transmissibility r0 ‰ compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for the clinical development of suitable antiviral nanotherapeutics ntps is incessant we are presenting a systematic review of the literature published between and to validate the hypothesis that the pharmacokinetics collateral acutechronic side effects of nano drugs and spike proteins arrangement of coronaviruses can revolutionize the therapeutic approach to cure covid19 our aim is also to critically assess the slow release kinetics and specific target site chemical synthesis influenced competence of ntps and nanotoxicity based antiviral actions which are commonly exploited in the synthesis of modulated nanomedicines the pathogenesis of novel virulent pathogens at the cellular and molecular levels are also considered which is of utmost importance to characterize the emerging nanodrug agents as diagnostics or therapeutics or viral entry inhibitors such types of approaches trigger the scientists and policymakers in the development of a conceptual framework of nanobiotechnology by linking nanoscience and virology to present a smart molecular diagnosis treatment for pandemic viral infections comorbidities till date no therapeutic drug has been discovered for the treatment of sarscov2 infection though the importance of monoclonal antibodies protease and helicase inhibitors and interferons ifnsα treatments have been highlighted in a handful of literature farzin torchilin palmieri and papi development of drugresistant strains host cell toxicitytarget specific actions costs associated with the serological diagnosis and therapeutics limit the widespread application of synthetic drugs nucleoside analogs palmieri and papi torchilin harnessing the potential of nanobiotechnology in the biomedical science development of engineered nanostructured materialsnanomedicines may lead to better drug delivery advanced therapeutics and medical diagnostics at nanoscale in recent years with the advancement of clinical practices the use of metal gold silver zinc and copper corresponding author discipline of earth sciences room no 336a block indian institute of technology gandhinagar gujarat india email addresses manishenvgmailcom manishkumariitgnacin m kumar 101016jenvres2020110119 received may received in revised form july accepted august environmentalresearch1912020110119availableonline23august2020001393512020elsevierincallrightsreserved 0cs mukherjee nanops in magnetic immunoassay viral diagnostics and microfluidic technology has driven the researchers to explore the potential of nanotherapeutics makvandi farzin letko and ahlawat and narayan have investigated the multistrain inhibition of sars coronavirus sarscov herpes simplex virus hsv1 and human immunodeficiency virus hiv of tcells by sulfonated nanops binding they have reported that small p size tunable surface charges biomimetic properties faster encapsulation have made the engineered nanops smart and stable colloidal carriers for the delivery of genes and drugs the mode of action of functionalized nanops can be explained by their covalent linkages with biological substrates such as peptides proteins antibodies and nucleic acids some researchers have reported virusnanops electrostatic nonspecific interactions elsewhere ie influenza a h5n1 strain inhibition alizadeh and khodavandi rothan and byrareddy nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha bachmaier balakrishna such surface proteins often act as a prime inducer of neutralizing antibodies as observed by boulware and chaturvedi and shrivastava where antigenic determinants enable nucleocapsid to take entry into the host cells as reported for in vitro sarscov inhibition of virus replication and the fusion between the viral and host cell endosomal membrane are often facilitated by some emerging nanobased technologies ie silver nanorod array surface enhanced raman spectroscopy sers substrate interferometric biosensor immunoassay chauhan chhikara choudhary the development of such biomolecular detectors enable the molecular binding of virus ps to target specific antigen phase antibodies coated waveguide which help in the fast and reliable detection of viral infections kirchdoerfer and ward cojocaru das considering the high transmissibility r0 ‰ and low to moderate pathogenicity of sarscov2 compared to middle east respiratory syndrome coronavirus merscov and sarscov the quest for clinical development of suitable antiviral nanotherapeutics is need of table a summary of biocompatible nanomaterials and antiviral nanopharmaceuticals commonly used for biomedical drug delivery action as virucidal agents souce of data weiss udugama letko jamshidi gao dong nanocarriers aunps immobilized aunps tio2 nps modified tio2nps agnps engineered agnps sinps mesoporous si fullerenes modified fullerenes fenps engineered fenps acidbasic functionalized nanotube metal functionalized nanotube carbon dots graphene oxide go polystyrene nps virucidal action immunizationviral detection viral detection inactivation of virus by photolysis virus inhibitioninactivation viral entry inhibition viral replication deformation virus detection viral entry inhibition virus destabilization inhibition of virus entry host pathogen interaction viral detectionremoval virus inactivation immunization viral inhibition viral entry hindrance mucosal vaccine development target specific cell bhk21 helacdltr vero cells c636 balbc mice na mdck vero cells human rhabdomyosarcoma vero cells hek293t na supt1 na na ncih292 grass carp pk15 marc145 vero cells na virus types hin1 h3n2 hiv1 h3n2 dengue virus h5n1 h3n2 h5n1 h1n1 h7n3 feline calicivirus influenza papilloma virus hsv1 bacteriophage λ hiv1 wild and resistant type zika virus bacteriophage ms2 h5n2 h3n2 reovirus rsv pseudorabies virus porcine epidemic diarrhea virus hiv1 mode of antiviral activity binding with peroxidasemimic enzymes and viral gp120 antibody mediated inhibition viral capsid protein interaction viral surface protein interaction inhibit cd4based binding viral envelop rupture cell mediated immunenucleic acid inhibition hinder viral attachment viral capsidenvelop attachment and interaction impairing viral polyproteinhinder gag processing viral envelopprotein binding phosphatidylserine inhibit viral tropism photoactivated mediated viral inhibition destabilization vp7dna mediated inhibition type i interferon production inhibited negative single layered sharp edged p interaction with virus viral gp120 antigen binding and mannoselectin specific inhibition action immune system inhibition cd8 t cells inhibition antibodydependent cellmediated phagocytosiscytotoxicity viral transcriptase inhibition gene silencing action viral cell entry inhibition peroxidase inhibition viral entry inhibition coagulation results from virus surface protein interaction magnb mediated enzymeatic signaling inhibition cell cycle inhibition at g2m phase glycosaminoglycan binding affinity and in vitro replication inhibition chitosan coated nps peptide coated nps protein coated nps amide coated nps nanoliposomes nanomicelle aunpscarbon nanotubes polymeric micelle aunpsagnps aufenpscarbon nanotubes nanolipid carriers dendrimer na balbc strain balbc mice neuro 2a cell lines na apc49 huh75 na male wistar rats cell mdck na vk2e6e7 vero cells helfs rabies virus influenza a virus influenza a virus hiv na hepatitis c virus h3n2 hiv h1n1 h3n2 norovirus h1n1 papilloma virus hsv12 immunization nanoparticulate vaccine influenza vaccine antiviral therapy drug delivery immunomodulator antiviral activity and bioavailable vaccines viral detection by colorimetric assay oral bioavailable drugs viral inhibition and drug delivery action viral dna detection nontoxic viral inhibition mrna vaccine na not applicable aunpsgold nanops agnpssilver nanops fenpsiron nanops sinpssilica nanops tio2 titanium nanops gographene oxides environmentalresearch19120201101192 0ccome under anic nanocarrier systems based on the their function as therapeutic agents encapsulated chemical attachment adsorbed or dissolved grein neogi gacem fig demonstrates the comparative size range of nanops whereas for hybrid nanobased systems the molecular composition and target specific action of individual engineered nanoparticulate systems play a vital role in drug delivery actions table the mode of action of such nanobased therapeutic agents can be categorised based on the permeability of vasculature passive targeting for malignancy or attachment of bioactive ligands to the selective nanotherapeutics kirchdoerfer and ward gao gadade and pekamwar role of metallic nanops as in vivo and in vitro drug discharging agents s mukherjee the hour rosenberg nalla dung though it is noteworthy to mention that r0 is not data specific to the covid19 disease rather it depends on the place and the behavior of the population it is associated with social isolation hygienic habits among others and the variation must occur in different ways viceconte and petrosillo li the drug development for antiviral treatment of sarscov2 depends on several factors such as pharmacokinetics drug properties collateral acutechronic side effects and spike proteins s arrangement of coronaviruses virus properties these can act as therapeutic targets to prevent the fusion of the virus to host cell via binding with host cell receptors trimeric scaffolds such as nsp10 a viral transcription factor used along with secondary antigens in vaccines for lung and lower respiratory tract infections these are essential to nucleate and stabilize pseudosubdomains of protein and peptide antigens which are also responsible for cell infection and polycistronic expression that may help in the nanobased therapeutic vaccine development dong grein etman peginterferon α2a2b pegasys ifnα2b show excellent drug delivery actions against hepatitis virus c whereas cationiccrosslinked nanops biodegradable polymers such as mpolyethylene glycol mpeg€“pla clpm are found suitable for hepatitis b and c virus infections synthetic and natural polymeric nanops pnps are widely used since the last decade for the effective control of pathogenic viruses based on their individual biochemical properties immuno biocompatibility biodistribution factors table low to zero toxicity profile high stability against proteinase degradation improved safety efficacy profiles and good internalization properties make such nanozymes effective against chronic infectious diseases and also help in exerting their cytoprotective actions against cytopathic effect polymeric nanops in hybridized forms have found their way in biomedical sectors as therapeutics and diagnostics of human adenovirus influenza virus and hiv therefore the widespread application of hybridized nanoformulation systems have been documented by kerry and kim where functionalized dnazyme along with cellular peptides encoding viral envelopes help in the knockdown of hsv2 hvc and influenza a viruses for the first time the purpose of the present systematic review lays out the framework for a developing a critical understanding of selfassembling metal nanops targeting a variety of fusion proteins for vaccine development b the spatial geometry three fold symmetry axis and radial distributions that drive the rapid antigen processing and render virucidal activity c building up a deep insight for biomarkers research in both prophylactic and therapeutic approaches d the critical assessment of nanops as therapeutics pathways biodegradation this review also provides a guide map for the regulation of metabolic enzymes on selected nanopharmaceuticals through which the multigenerational effects can be evaluated this publication is designed to provide vital information on biocompatible nanocarriers active vs passive targeted drug delivery action of nanomedicines and critically analyze the possible hybrid nanobased therapy for sarscov2 inhibition our current review also highlights the stateofart molecular fingerprinting techniques of virus identification through advanced biosensing methods which critically explore integral surveillance and monitoring of novel viral genotypes mode of action and biomedical applications of biocompatible nanocarriers the presence of multiple surface binding sites in vivo clinical interaction with the targeted sites composition based multiple interactions shape luminescence and large surfacevolume ratio of inanic nanops render their multifarious biomedical applications ignatov dong the virucidal activity of silver nanops agnps allows its wider application in the annihilation or amelioration of several viral infections such as poliovirus type1 coxsackievirus b3 influenza a virus etc the mode of action of such nanops can be described either inhibition of cd4dependent cellular bindingpathogenesis or by covalent linking with sulfhydryl group virion surface hill neogi elsheekh the viral internalization can also be prevented by merging nanops with viral genomecore protein that hinders viral replicationattachment release of viral core into the cytoplasm and governing conformational changes in viruscoreceptor association letko hu this type of viral replication inhibitions are quite effective in curing of dsrna viruses infectious hsv2 and bursal disease viruses iravani jamshidi on the other hand gold nanops aunps may also exert their antiviral efficacy through hindering of gp120 fusion to cd4 that are surrounded by capping agents encapsulated aunps table hybridized and charged aunps render virucidal mechanism after associating with nanobased formulation and mimicking peroxidase enzymatic reaction where inhibited viral entry is facilitated by blocking of transcription within the host cell isida kumar 2020a 2020b 2020c kang on the other hand stabilization of magnetic nanops is often carried out by biocompatible polymers which helps in the translation of magneto responsive nanoparticulate systems in clinical diagnostics bio imaging bioresonanceimaging and cell separation the use of superparamagnetic iron nanops γfe2o3fe2o3fe3o4 is not directly involved in the therapeutics but their indirect application may inhibit viral multiplication even at postentry cellular level as seen for zika virus h5n2 and hcv zhou 2020ab dong photothermal nanotherapy high refractive index photocatalytic activities and high solubility properties of titanium nanops tinps enable them to find their wide antiviral application for bacteriophages and h3n2 viruses grein kar the clinical application of functionalized silica nanops sinps drives the diverse antiviral therapeutics or diagnostics application of nanocarriers in inhibition of hiv hepatitis b virus and other recombinant viruses kim nucleic acid hybridization and fluorescent based virus viral protein detection methods are getting popular based on the antiviral efficacy of sinps though their meso to nanoporous biocompatible surfaces allow hostpathogen interaction the emerging demand for novel antiviral nanotherapeutics triggers the researchers to ponder about immunomodulation and immunization of host cells these are essential to regulate premature drug release or inhibition of viral entry through in vivo biodistribution kalantarzadeh kerry the different forms of nanomaterials that are mostly used as antiviral agents depend largely on the pathway of drug delivery system which provide versatile forms of nanobased carriers starting from complex anic hybrid nanosystems to simple inanic metallic composites nanocapsules nanocages and nanospheres are categorised under inanic nanop based systems though nanocapsules can sometimes through and associated drug metabolism environmentalresearch19120201101193 0cs mukherjee fig schematic of the size range of nanops commonly applied in clinical practice as drug delivery agents for gene and drug delivery system quantum dots qd a type of nanosized crystals have excellent nanobased sensing which allow them to be used as antiviral therapeutics or in vitro diagnosis of virulent pathogens where strong chemical interactionsbonding render their biochemical conjugation with therapeutic molecules kostarelos kumar et al 2020a 2020b 2020c and zhou 2020ab have shown that different metallic composition pb cu ga zn hg based qd showed target specific actions against hiv1 human tlymphotropic virus1 after their binding with nh2 receptorbiotin acceptor some researchers have shown that nanoformulations based qds crossed the bbb in vitro model along with target dna and saquinavir antiviral agent which have been widely utilized as highly active antiretroviral therapy kumar 2020a 2020b 2020c lembo evaluation of anic and hybrid nanops as therapeutics and in drug delivery action anic nanopsnanocarriers onp play an important role in the drug delivery action if the therapeutic compounds are of large sized molecules €“ nm encapsulation of such nanoagents through environmentalresearch19120201101194 0cs mukherjee specific designstructure render offtarget toxicity which is required for target specific action lopez gacem slow release kinetics and specific target site chemical synthesis have direct influence on onps therapeutic competence kumar 2020a 2020b 2020c li polymeric nanops pnps have the clinical potential to carry the target drugs to its core or can coordinate with target molecules on its planar surface mainardes and diedrich nasrollahzadeh biocorona formation ease of biodegradation strong mechanicalthermal properties of carbonbased nanocarriers nanorods nanodots etc render their antiviral activities against respiratory syncytial virus rsv hiv1 ebola virus etc table in spite of having broad spectrum activities against antiviral infections the ratelimiting phase of these carbon materials limits their further biomedical applications nikaeen and nguyen have suggested that drug conjugated nanops having excellent antiviral activities can help in the development of an influenza vaccine with matrix protein m2e and hemagglutinin haamine functionalized gelatin surface coating though more research is required to validate the immunity and protection of cellular genes for such impeccable protein vaccines against infectious virulent pathogens like sarscov2 with the advancement of medical sciences the emergence of multifunctional nanobased lipid nanocarriers opens a new door in the field of novel antiviral nanotherapeutics table read and risitano have demonstrated that podophyllotoxin stearic acidglycol loaded lipid nanocarriers are useful in maintaining in vitro slow drug release nontoxic viral inhibition and hemocompatibility of vk2e6e7 hela cells such solidlipid nanops and other nanolipid carriers can fig a and b schematic of the internalization of nanodrugs through the plasma membrane and targeted drug release a and transcytosis of nanodrugs through cell barriers b nanoparticulate drug carriers through the cellular and mechanistic establishment crossed the membranes bloodbrain barrier and bloodtestis barrier and with the help of capping agents such as sulfate polysaccharidespolymers undergo multivalent bond interactions with virus glycoproteins ie hemagglutinin ha environmentalresearch19120201101195 0cs mukherjee become the exceptional antiviral drug discharging agents for infectious viral pathogens ie hpv hiv and hepatitis c virus nú˜nezdelgado prather hyperbranched monodispersed easily biodegradable anic dendrimers have gained importance for more than a decade in the field of nanomedicine because they act as targeted carriers for biological systems the antiviral activity of dendrimers is still under scientific investigations which may be facilitated through conjugated drug delivery mechanisms patil palestino the efficacy of such nanoparticulated dendrimer system can be observed for the inhibition of hsv1influenza virus where antibody mediated responsecd8 t cell activation and regulation of gene expressions are achieved through small rnas inactivation shang sportelli niosomes the nonionic surfactant based liposome alike anic nanovesicle are getting popular in the advanced biomedical sciences considering their nonimmunogenicity and stable optical properties which make them a suitable carrier of both hydrolipophilic drug molecules excellent bioavailability and controlled release of specific drugs at the targeted sites make such nanocarriers ideal antiviral agents hsv virus where nanoniosome was loaded with suitable antiviral drug acyclovir s´anchezl´opez et al improved drug delivery mechanism and suitable drug release kinetics may allow such nanocarriers to be used in infectious virus diseases nanomicelles fig supramolecular globular micelles exhibit colloidal stability and super encapsulation potential which help such polymeric micelles to show antiviral activity in vitro as observed from curcumin loaded bioavailable nanoformulations hepatitis c virus sivasankarapillai some researchers used graphene oxides conjugated adnps against infectious sars and bursal viruses where the drug resistance event after such antiviral efficacy was mediated by selenium nanops senps andor amantadine am arrangement te velthuis kumar 2020a 2020b 2020c diagnosis or detection of such virulent pathogens have been documented by viceconte and petrosillo and vazquezmunoz and lopez through thiolstabilized gold cluster or enteroviruses labeling with cysteine molecule a brief overview about antiviral nanomedicines rsv virus tremiliosi udugama the size and zeta potential of silver nanops can exert inhibition effects on different human parainfluenza three virus strains or on their replication event the nanocolloidal system of vivagel® is immensely used for the control of zika virus infection tkm130803 is widely used in the treatment of ebola virus utilizing the concept of rnaibased therapy for the lipidbased nanosystem it is well doccumented that for human norovirus treatment the employment of goldcopper sulfide coreshell capsid protein binding results into excellent virucidal activity ziaie on the other hand nanotrap ps are quite often used in the inhibition of infection of target cells by capturing viral rnaviral proteins ie influenza virus treatment zhou 2020ab zhang intrinsic in vivo instability poor immunogenicity and toxicity multiple therapeutic and prophylactic approaches can be overcome by nanovaccinology where cellular and humoral immune response drive the faster uptake of mucosagut associated lymphoid tissue slow controlled release of antigens is facilitated by surface modifications of nanovaccines with antibodiescarbohydrates which results in the target specific immune response by different immune cells additionally their small size and prolonged shelf life help in the faster recognition of the hostreceptor immune system ie hepatitis a virus hav and influenza virus where epaxalexapal is used with immuno targeting agents yu yang and wang nonresponsive immune systems high dose administration coldchain transport of parenteral vaccines limit their widespread application in drug therapy particularly for mucosally administered vaccines the chitosannanop embedded system might be useful for therapeutic proteins or antigens having negative charges which makes its wider application in vaccination against hbv virus through gene delivery systems table the utilization of mouse model employing humoral and mucosal immune responses helped in the liposomebased vaccine development in case of hepaxen used for hepatitis a c and e which further utilized the recombinant surface antigen as a prophylactic vaccine waris wang 2020a 2020b the usages of inflexal v and influvac as standard virosomal vaccine against influenza virus are getting quite popular considering its active biocompatible and immunogenicity wu weiss these types of licensed subunit nanovaccines are found quite useful for older infants and middleaged group people in terms of nanosafety issue as they mimic natural infections as seen in table a cysteineguanine rich oligonucleotide combination with extracellular m2egold conjugates renders molecular protection for pr8h1n1 influenza which was further activated by thiolgold interactions zhang zhou 2020ab from the discussion provided here it is clear that most of the research has been done for influenza virus vaccination but some scanty literature also report several nanomedicines including nanovaccines are under clinical trial or at least in the stage of commercialization for the cure of infectious viral diseases table in general the use of drugs for antiviral therapy is usually employed to target different life cycles of virulent pathogens ie hiv ebola virus or hsv1 valdiglesias and laffon alnrsv01 is a commonly used lipid nanop drug for lower tract respiratory disease which targets the nucleocapsid €œn€ gene of table commercial nanomedicines or under clinical trial for the antiviral therapytreatment souce of data neogi letko dong kalantarzadeh kang nanomedicines influvac plus tkmhbv cervisil doravirine dermavir inflexal v epaxal pegasys geovax novavax fluquit curevac peglntron vivagel a pepreclinical evaluation uceunder clinical evaluation hav hepatitis a virus hiv human immunodeficiency virus hbv hepatitis b virus hpv human papillomavirus hcv hepatitis c virus hsv herpes simplex virus mode of action presence of neuraminidase and hemagglutinin rnai therapeutics gene silencing reverse trancriptase inhibitor nonnucleoside dna immunogen with hiv specific t cell precursor antigens specific on speherical carriers surface natural process mimics peroxidases pegylation control stability of protein ankara€”virus alike drug therapy clinical stage antiviral nanobiotechnology gene silencing mrna technique pegylation control stability of protein dendrimer with sulphonic acid group interaction disease indication influenza hbv hpv hiv hiv influenza hav hbv hcv sarscov2 sarscov2 influenza sarscov2 hcv hsv hiv biomedical application virosome vaccine solidlipid nanop sirna therapeutic nanoparticulate formulation therapeutic vaccine liposome vaccine liposome vaccine pegylated interferon antiviral therapy nanoparticulate therapeutics sirna therapeutic infections virus vaccine pegylated interferon dendrimer yearstage of development uce preclinical evaluation ucea uce pe pe pea pe uce environmentalresearch19120201101196 0cs mukherjee vaccine research for rotanoroebola virus hpv rsv and others dung das metabolic pathway of nanotherapeutics and their limitations in clinical practice pulmonary and hepatotoxicity studies are required to build a safety profile of such nanodelivery therapeutic or diagnostic agents nanobased approach for sarscov2 infection inhibition nanops uptake cellular process in nanotherapeutics are governed by their physicochemical properties along with cellular membrane characteristics which may have direct influence on the rate of administered drug dosages and structure of engineered nanops it is hypothesized that nanops with optimum diameter of ‰ nm and high surface charge density are quite effective in crossing the cellular membranes for hivderived tat cell penetrating peptides weiss waris immunoliposomes and other carbon based nanotubesnanocarriers play an important role in the activation of the complement pathway of host immune systems to deregulate in vitro utilization of nps neogi kumar 2020a 2020b 2020c antibodies that are specifically targeted at polyethylene glycol pegmacrogol polymers and peglike nanostructures can show independent therapeutic efficacy based on their individual immunotoxicology and risk assessment strategies letko read experimental findings wang 2020a 2020b hill with nanobased therapeutic agents reveal the urgent requirement of more rigorous scientific investigations to prove their clinical efficiency in reversing the drug resistance event ie h1n1 virus through seam biodegradation process of nanotherapeutics has gained special attention considering uniform biodistribution kinetics and sustained drug release which are essential for improved drug design process distribution metabolism absorption excretion are important pharmacokinetic features which rate of biochemical features are directly governed by hydrophobichydrophilic profile and tacticity of the nano based formulations at in vitro level patil exocytosis process plays a very important role in the clearance of the foreign nanops out of the cell depending on administered nanocarriers it was hypothesized that ps with nm diameter can
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Hepatitis B virus HBV infection has been associated with the risk andprognosis of many malignancies Nevertheless the association between HBV and theprognosis of breast cancer is unclear The objectives of this study were to investigate theprognostic role of hepatitis B surface antigen HBsAg and to integrate HBsAg to establishnomograms for better prognostic prediction of very young patients with breast cancerMethods This analysis was performed retrospectively in a cohort of consecutivevery young ‰ at diagnosis patients who received curative resection for breastcancer The significance of HBsAg in the prognosis of these patients was investigatedUnivariate and multivariate analyses were used to identify independent variables fordiseasefree survival DFS and overall survival OS Nomograms were built based onthose identified variablesResults Overall of the patients were seropositive for HBsAgThe median followup was CI “ months forthe entirepopulation The HBsAgpositive cohort had significantly inferior DFS HR CI “ P and OS HR CI “ P as compared with the HBsAgnegative cohort The rates of 10year DFS andOS were and in the HBsAgpositive group and and in the HBsAgnegative group respectively In multivariable analysis HBsAg statuswas identified as an independent significant unfavorable prognostic factor for DFSP and OS P in very young patients with breast cancer Nomogramswere established and displayed good calibration and acceptable discrimination TheCindex values for DFS and OS were CI “ and CI “ respectively Based on the total prognostic scores TPSof the nomograms different prognosis groups were identified for DFS and OSEdited byImtiaz Ahmad SiddiquiUniversity of Colorado AnschutzMedical Campus United StatesReviewed byAbhinit NagarUMass Memorial Medical CenterUnited StatesNidhi JainBeckman Coulter IndiaCorrespondenceLu YangyanglusysucccnWeiDong Weiweiwdsysucccn These authors have contributedequally to this workSpecialty sectionThis was submitted toCancer Epidemiology and Preventiona section of the journalFrontiers in OncologyReceived March Accepted July Published August CitationLi N Zhong QQ Yang XRWang QC Zhang DT Zheng SYang L and Wei WD Prognostic Value of Hepatitis B VirusInfection in Very Young Patients WithCuratively Resected Breast CancerAnalyses From an Endemic Area inChina Front Oncol 103389fonc202001403Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerConclusions HBsAg is an independent unfavorable prognostic factor for DFS andOS in very young patients with curatively resected breast cancer and nomogramsintegrating HBsAg provide individual survival prediction to benefit prognosis evaluationand individualized therapyKeywords HBV young breast cancer prognosis nomogram survivalINTRODUCTIONGlobally breast cancer is the most common cancer and theleading cause of cancer death for women accounting for of total cancer cases and of total cancer deaths Breast cancer has also been the top one malignancy in termsof incidence in Chinese women constituting of newlydiagnosed cases and of all deaths from breast cancer in theworld Although breast cancer occurs at a lower incidence inChinese women than in western women this disease occurs at ayounger age in China than in highincome countries and China™scontribution to global breast cancer rate is increasing rapidly The disparities between young and old breast cancer include ahigher mortality rate higher risk of recurrence poorer treatmentresponse and more aggressive phenotypes “ Thereforeunderstanding the etiology and identifying novel prognosticfactors are essential for early diagnosis prognosis evaluationearly intervention and personalized therapy in young patientswith breast cancerHepatitis B virus HBV infection is a serious public healthdilemma with ˆ¼ million chronic carriers worldwide China account for about a third of infectionassociated cancerglobally driven by high prevalence of HBV and H pylori infection Although China has made tremendous eï¬orts in controllingHBV over the past years and the prevalence of HBV ininfants and children has remarkably declined the hepatitisB surface antigen HBsAg prevalence is still high in Chineseadults ranging from to “ HBV is the leading causeof hepatocellular carcinoma and cholangiocarcinoma Inaddition there is also accumulating evidence that HBV infectionis associated with many extrahepatic malignancies includingnonHodgkin™s lymphoma pancreatic cancer gastriccancer nasopharyngeal carcinoma lung cancer esophageal cancer and ovarian cancer Thus it seemsreasonable that HBV is an important factor in the developmentof extrahepatic malignancies in endemic areasDespite the facts that HBsAg status is one of the routineexaminations in patients with operable breast cancer and severalstudies have showed that HBV is not associated with therisk of breast cancer the impact of HBV on theclinicopathological characteristics and prognosis of very youngpatients with breast cancer remains to be determined Giventhat both early breast cancer and HBV are endemic in China itis possible that HBV infection is associated with the prognosisof early breast cancer even though the precise mechanismsare yet to be determined It is crucial to address this issuesince HBV has been reported to be found in breast cancertissue We therefore performed this study to investigate theHBsAg prevalence in very young breast cancer and the impactof HBsAg on the survival of these patients and to establishnomograms to better predict prognosis for very young patientswith breast cancerMATERIALS AND METHODSPatient SelectionA retrospective review was conducted in a cohort of consecutive breast tumor women who were aged ‰ years oldand received curative resection for breast cancer at Sun Yatsen University Cancer Center between May and July This study was conducted according to the ethicalstandards of the Declaration of Helsinki Institutional ReviewBoard approval was obtained from the Medical Ethics Committeeof this cancer center All patients were restaged by the eighthinternational classification system for breast cancer Dueto the retrospective nature of this studyinformed consentwas waivedInformation was collected from electronic patient recordsand survival data were obtained from the followup registryofthis center The information collected included HBsAgstatus laterality type of breast surgery type of axillary surgeryhistological type tumor grade tumornodemetastasis TNMstage dates of surgeryrelapsedeath status of estrogen receptorER progesterone receptor PR human epidermal growthfactor receptor HER2 and Ki67 Breast cancers wereclassified as luminal Alike ER PR‰¥ HER2“ andKi6715luminal Blike ER andor PR HER2“HER2enriched ER“ PR“ HER2 or triplenegative ER“PR“ HER2“ subtypesPotentially eligible patients had to have curatively resectedbreast cancer without previous therapy other than neoadjuvanttherapy be aged years old or below and have definiteinformation of HBsAg The main exclusion criteria includedbenign tumor not having surgery having incomplete resectionprevious malignant disease hepatitis viral infections other thanHBV men patients and insufficient data of survival or HBsAgStatistical AnalysisThe main objectives of this study were to compare diseasefreesurvival DFS and overall survival OS between HBsAgpositivepatients and HBsAgnegative patients DFS was defined as theinterval from the date of being diagnosed to the date of diseaserecurrencemetastasis or death from any cause OS was defined asthe interval from the date of being diagnosed to the date of deathfrom any cause Median followup was estimated by KaplanMeier analysis with reversed meaning of status indicator Frontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerforindependent variablesDFS and OS were estimated by the Kaplan“Meier methodand diï¬erences were compared by the logrank test Univariateand multivariate analyses with a Cox proportional hazardsmodel were used to testforDFS and OS Covariates included laterality left vs righttype of surgery breastconserving surgery vs mastectomytype of axillary surgery sentinellymph node dissection vsaxillary lymph node dissection histological type ductal vsotherstumor grade grade III vs grade III T stageT34 vs T12 N stage N23 vs N1 HER2statustriplenegativeHER2enriched vs luminal All variables reaching asignificance of in univariate analyses were included inmultivariate analysispositive vs negative molecularsubtypesThe nomograms for predicting and 10year DFSand OS were formulated based on the results of multivariateanalysis by the œrms package of R The discriminationofthe nomogram models was estimated by the Harrell™sconcordance index Cindex The value of the Cindex rangesfrom to with implying a random chance and indicating a perfect prediction Calibration curves of thenomogram models for DFS OS were plotted to assess thepredictive value of the model In addition patients weredivided into three diï¬erent risk groups highintermediatelow according to total prognostic scores TPS The totalprognostic scores of patients were transformed into categoricalvariables based on cutoï¬ points which were determined by theminimum Pvalue from logrank × statistics with the Xtileprogram Pearson™s chisquare test was used to compare categoricaldata All Pvalues were twosided and P was consideredstatistically significant Statistical analyses were performed by theSPSS software SPSS Inc version Chicago IL USA and Rfor Windows version httpwwwrprojectData AvailabilityThe authenticity of this has been validated by uploadingthe key raw data onto the Research Data Deposit public platformwwwresearchdatacn with the approval RDD numberas RDDA2020001410 The data that support the findings ofthe study are available from the corresponding authors uponreasonable requestRESULTSPatient CharacteristicsA total of very young ‰ at diagnosis breasttumor patients were screened of whom were excludedbecause of benign tumor n not having surgeryn not having R0 resection n or unknownHBsAg status n With further exclusions forinsufficientfollowup data a total of patients withcurative resection for breast cancer and aged years orbelow were included in this study Figure The patients™FIGURE The process of patient selectionFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerTABLE Patient characteristics by HBsAg statusCharacteristicsHBsAgpositiveN No HBsAgnegativeN No Pvalue FIGURE Number of recurrences by year of entire patientsLateralityLeftRightBilateralType of breast surgeryMastectomyBreastconserving surgeryType of axillary surgeryALNDSLNDHistological typeDuctalInvasive lobularOtherTumor gradeIIIIIIUnknownT StageT1T2T3T4N StageN0N1N2N3HER2 statusPositiveNegativeUnknownMolecular subtypesLuminal ALuminal BHER2enrichedTriple negativeUnknown HBV Hepatitis B Virus ALND axillary lymph node dissection SLND sentinelnode dissectionHER2 human epidermal growth factor receptor2lymphcharacteristics are shown in Table Overall ofthe patients were seropositive for HBsAg HBsAgpositive group and HBsAgnegative group were wellmatchedfor basic characteristics including laterality type of breast andaxillary surgery histological type tumor grade T stage Nstage and molecular subtypes About in patients receivedmastectomy and most patients received axillary lymph nodedissection ALNDAssociations Between the Status of HBsAgand SurvivalThe median followup was CI “ months forthe entire population By the time of analysis December instances of disease recurrence had occurred Thenumber of recurrences in each year of the followup is shown inFigure Of note although HBsAgpositive patients had a higherfrequency of extrahepatic metastasis vs P thanHBsAgnegative patients they had a comparable frequency ofliver metastasis vs P when compared withHBsAgnegative patientsDFS was significantly shorter among those who were HBsAgpositive than among those who were HBsAgnegative HR CI “ P The rates of 10yearDFS were in the HBsAgpositive group and inthe HBsAgnegative group respectively Figure 3A A totalof death events had occurred by the data cutoï¬ HBsAgpositive group had significantly inferior OS compared withHBsAgnegative group HR CI “ P with a 10year OS of and respectivelyFigure 3B The association of HBsAg status and survival ineach molecular subtype was further analyzed As expectedDFS and OS were significantly longer among those in theluminal A subgroup and the HER2enriched and triplenegativegroups had significantly shorter DFS and OS Figures 4ABNotably HBsAgpositive status was associated with shorterDFS P and OS P in the luminalB cohort HBsAgpositive status was also associated with aslightly shorter DFS in the triplenegative cohort and shorterOS in the HER2enriched cohort but statistical significancewas not reached Supplementary Figures There was nosignificant diï¬erence in DFS between HBsAgpositive patientsand HBsAgnegative patients in luminal A and HER2enrichedcohorts and in OS in luminal A and triplenegative cohortsSupplementary Figures In the univariate analysis type of breast surgery tumor gradeT stage N stage molecular subtype and HBsAg status wereFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerindividual patient Figures 6AB The model™s explanatorycovariables consisted of HBsAg status T stage N stage andmolecular subtype Patients with higher scores correspondedto inferior survival The scatter plots for the TPS of DFSand OS and percentage of patient number were presentedin Figures 6CD The Cindex values for DFS and OS were CI “ and CI “ respectively The calibration curves for the probabilityof DFS and OS at or year presented an optimalagreement between the prediction by nomogram and actualobservation Supplementary Figure Next we divided thepatients into the following groups based on the TPS ofthe nomogram modelfor DFS using the Xtile programlowrisk group TPS “ patients intermediateriskgroup TPS “ patients and highrisk group TPS patients The 10year DFS for lowrisk groupintermediaterisk group and highrisk group were and respectively Survival analyses for DFS demonstratedsignificant discrimination between these three groups P Figure 7A Same procedures were performed for OS in theentire population and patients were divided into the following groups based on the TPS of the nomogram model for OS withthe Xtile program lowrisk group TPS “ patientsintermediaterisk group TPS “ patients and highrisk group TPS patients OS diï¬erences were alsoobserved among three subgroups with a 10year OS of and for lowrisk intermediaterisk and highrisk groupsP Figure 7BDISCUSSIONIn this study we investigated the association of HBsAg statusand very young breast cancer and to our knowledge reportfor the firsttime that HBsAgpositive status is associatedwith inferior DFS and OS from a population with a highprevalence of both HBV infection and young breast canceridentified as a significant unfavorableHBsAg status wasprognostic predictor for DFS and OSindependent of anyother clinicopathological features of breast cancer includingT stage N stage and molecular subtype We also integratedHBsAg to build nomograms to better predict prognosis foryoung patients with breast cancer The results of our studydemonstrated that the prevalence of HBsAg in young patientswith breast cancer in southern China was which wasin accordance with the “ reported in the populationof this endemic area This result suggests that unlikecervical cancer young breast cancer is not correlatedwith an increased prevalence of HBV infection Indeed breastcancer patients with HBsAg did not demonstrate a diï¬erentpattern of characteristics It is noteworthy that in our studyHBsAg did not increase the rate of liver metastases for veryyoung patients with breast cancer This results are comparableto those reported in previous studies for esophageal cancerand colorectal cancer which suggested that HBVinfection is associated with decreased risk of liver metastasis inthese malignanciesFIGURE Kaplan“Meier curves for A diseasefree survival and B overallsurvival stratified by HBsAg status in very young patients with breast canceridentified as significant prognostic factors for DFS Figure 5AWhen those variables were further analyzed in the multivariateanalysis we found that T stage P N stage P molecular subtype P and HBsAg status P remained statistically significant indicating that theyare significant independent predictors for DFS Figure 5ABy the same methods for OSthe results showed that Tstage P N stage P molecular subtype and HBsAg status P were independentprognostic factors for OS Figure 5B HBsAgpositive status isan independent negative prognostic factor for survival in veryyoung breast cancerPrognostic Nomograms For Very YoungBreast Cancer PatientsTo better assess the DFS and OS of very young breastcancer patients prognostic nomograms for DFS and OS wereestablished respectively All the independent predictors of DFSand OS in the multivariate analysis were integrated into thenomogram models and and 10year survivals weregraphically computed according to the characteristics of anFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by molecular subtype in very young patients with breast cancerOur study shows that HBsAgpositive status is associatedwith inferior DFS and OS in young patients with curativelyresected breast cancer decreasing the 10year DFS and OS by and respectively The association between HBsAgpositive status and poor prognosis is in keeping with thatdemonstrated in nasopharyngeal carcinoma lung cancer and ovarian cancer However some studies regardingother cancers indicate that HBsAgpositive cancer patientshad a favorable survival as compared with HBsAgnegativepatients This discrepancy can be partly explainedby the diversity and heterogeneity of diï¬erent malignanciesThe genetic or biological mechanisms underlying the inferiorFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Univariate and multivariate analysis for diseasefree survival A and overall survival B for very young patients with breast cancerprognosis remain to be elucidated butthis may largelyrelate to the presence of hepatitis B Xinteracting proteinHBXIP which has been welldocumented to function asan oncoprotein in breast cancer HBXIP can act as atransactivator by activating certain genes including cMyc E2F1STAT4 and Sp1 to play a crucial role in the progression ofbreast cancer HBXIP is associated with controlling cellapoptosis and promoting cell proliferation by mTOC1 activation HBXIP can also act as a modulation factor of cellularoxidative stress by competitively binding KEAP1 to enhancethe progression of breast cancer Previously studies showedthat HBV is not associated with risk of breast cancer These results combined with the data of our study suggestthat HBV is not a risk factor but a prognostic factor forbreast cancerAnother reason for this might relate to the HBV reactivationin HBsAgpositive patients with breast cancer who werereceiving chemotherapy HBV reactivation occurs frequentlyin breast cancer patients who are HBV carriers while receivingcytotoxic chemotherapy HBV reactivation can result inliver failure and interruption of the chemotherapy scheduleOther potential mechanisms underlyingassociationtheFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE AB Nomograms predicting and 10year A diseasefree survival and B overall survival for very young patients with breast cancer CD Thescatter plots of percentage of patient number and groups of C total prognostic score for DFS and D total prognostic score for OSbetween the HBsAg and patient survival include the immunesuppression Chronic HBV infection is characterized by thefailure to elicit an eï¬ective adaptive immune response andthe immune modulation of key innate immune response Chronic HBV infection can lead to immune anergy andimpair the function of the immune system which has longbeen deemed to protect the host against the developmentof nonviral cancerstogether couldpartially explain the positive association between HBsAgpositive status and the poor prognosis in young patients withbreast cancer These reasonsInthisstudy wecharacteristicscombined HBsAgstatus withclinicopathologicaleï¬ectiveprognostic nomogram models of DFS and OS for very youngpatients with breast cancer Both nomograms showed goodcalibration and acceptable discrimination These nomogrammodels can be used for prognosis evaluation at diagnosis forvery young patients with breast cancer and may benefit patientestablishtocounseling and personalized therapy for these patients Weadopted Xtile program to divide these patients into three riskgroups based on TPS from nomograms for DFS and OS Thesurvival curves for DFS and OS separated very well Thusspecial attention should be paid to and active surveillanceshould be conducted over patients with high risk group for DFSand OSThis study nevertheless has certain limitations that shouldbe noted First this study was retrospective in nature andwe cannot rule outthe impact of selection bias Secondthe sample size was relatively small and the sample sizesof the two cohorts were unequal as only patients wereHBsAgpositive The small sample size may be insufficientto allow us to perform subgroup analysis by each molecularsubtype Anotherthat Cantonese constitutemost of our study population The monotonicity ofthestudy population confines the universality of our resultsFurthermore the information was insufficient to perform otherlimitation isFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast CancerFIGURE Kaplan“Meier curves for A diseasefree survival and B overall survival stratified by risk groups based on total prognostic scores fromnomogram modelsanalysis such as that of hepatic function and HBV DNAcopy number Nevertheless the results of our study providewhat to our knowledge is the first evidence of the impactof HBsAg on the prognosis of very young patients withbreast cancerIn conclusion we have demonstrated in a retrospectivestudy that HBsAg is an independent unfavorable prognosticfactor for patients with very young breast cancer Furtherprospective studies involving varied ethnic populations arewarranted to confirm the prognostic value of HBsAg status invery young breast cancer and simultaneously other potentialclinicopathologic factors for breast cancer and HBV infection arerequired to be taken into account The mechanisms of the impactof HBV infection on the progression of breast cancer also need tobe elucidatedDATA AVAILABILITY STATEMENTThe datasets generated for this study are available on request tothe corresponding authorETHICS STATEMENTThe studiesinvolving human participants were reviewedand approved by the Medical Ethics Committee of SunYatsen University Cancer Center WritteninformedFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancerconsentin accordance with the nationalinstitutional requirementsfor participation was not required for this studylegislation and theAUTHOR CONTRIBUTIONSNL QQZ LY and WDW designed the study NL QQZXRY QCW DTZ and SZ collected the data NL QQZ LYand WDW interpreted and analyzed the data All authors wereinvolved in writing the and approved the final versionREFERENCES Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Globalcancer statistics GLOBOCAN estimates of incidence and mortalityworldwide for cancers in countries CA Cancer J Clin “ 103322caac21492 Fan L StrasserWeippl K LiJJ St LouisJ Finkelstein DM Yu 15e279“KD et al Breast cancer 101016S1470204513705679in China Lancet Oncol Colleoni M Rotmensz N Robertson C Orlando L Viale G Renneet al 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107314APJCP201516135285lung cancer Asian Pacific J Cancer Prevent Zou J Chen J Xie X Liu Z Cai X Liu Q et al Hepatitis B virus infectionis a prognostic biomarker for better survival in operable esophageal canceranalysis of patients from an endemic area in China Cancer EpidemiolBiomarkers Prevent “ 10115810559965EPI181095 Wong L Cheung TH Yim SF Lao TT Prevalence and impact of hepatitis Bvirus infection in ovarian cancer patients in an endemic areaA retrospectivecohort study J Viral Hepat “ 101111jvh13250 Song C Lv J Liu Y Chen JG Ge Z Zhu J et al Associations between hepatitisB virus infection and risk of all cancer types JAMA Netw Open 2e195718 101001jamanetworkopen20195718 Su FH Chang SN Chen PC Sung FC Su CT Yeh CC Associationrisk abetween chronic viral hepatitisinfection and breast cancernationwide populationbased casecontrol study BMC Cancer Qin B Zhao K Wei J Wang X Xu M Lang J et al Novel evidence indicatesthe presence and replication of hepatitis B virus in breast cancer tissue OncolRep “ 103892or20197393 Te HS Jensen DM Epidemiology of hepatitis B and C viruses a globaloverview Clinics Liver Dis “ vii 101016jcld200911009 Siu SS Cheung TH Chan PK Lin CK Lo KW Patients with malignant or premalignant cervical lesion have increased risk of becoming hepatitis B carrierJ Exp Clin Cancer Res “in patients with colorectal Zhao Y Lin J Peng J Deng Y Zhao R Sui Q et al Hepatitis B virus infectionpredicts better survivalliveronly metastasesundergoing liver resection J Cancer “ 107150jca24544 Liu X Li X Jiang N Lei Y Tang LL Chen L et al Prognostic value ofchronic hepatitis B virus infection in patients with nasopharyngeal carcinomaanalysis of patients from an endemic area in China Cancer “ 101002cncr28377 Zhao Y Li H Zhang Y Li L Fang R Li Y et al Oncoprotein HBXIPmodulates abnormal lipid metabolism and growth of breast cancer cells byactivating the LXRsSREBP1cFAS signaling cascade Cancer Res “ 10115800085472CAN151734 Zhang Y Zhao Y Li H Li Y Cai X Shen Y et al The nuclear import ofoncoprotein hepatitis B Xinteracting protein depends on interacting with cFos and phosphorylation of both proteins in breast cancer cells J Biol Chem “ 101074jbcM113458638 BarPeled L Schweitzer LD Zoncu R Sabatini DM Ragulator is a GEFfor the rag GTPases that signal amino acid levels to mTORC1 Cell “ 101016jcell201207032 Zhou XL Zhu CY Wu ZG Guo X Zou W The oncoproteinHBXIP competitively binds KEAP1 to activate NRF2 and enhanceFrontiers in Oncology wwwfrontiersinAugust Volume 0cLi et alHepatitis B Virus and Breast Cancercancerbreast“ 101038s4138801906985growthcelland metastasis Oncogene Liu Z Jiang L Liang G Song E Jiang W Zheng Y et al Hepatitis B virusreactivation in breast cancer patients undergoing chemotherapy a reviewand metaanalysis of prophylaxis management J Viral Hepat “ 101111jvh12672 Yuen MF Chen DS Dusheiko GM Janssen HLA Lau DTY LocarniniSA et al Hepatitis B virus infection Nat Rev Dis Primers 101038nrdp201835 Dunn GP Old LJ Schreiber RD Theimmunobiology ofimmunosurveillance“ 101016jimmuni200407017immunoeditingandImmunitycancer Giuliano AE Edge SB Hortobagyi GN Eighth edition ofthe AJCCcancer staging manual breast cancer Ann Surg Oncol “ 101245s1043401864866 Schemper MSmith TL A note on quantifyingstudies 101016019724569600075Xfailuretime Control ClinofTrialsfollowup in“ Vickers AJ Elkin EB Decision curve analysis a novel method for “evaluating prediction models Med Decision Making 1011770272989X06295361 Camp RL DolledFilhart M Rimm DL Xtile a new bioinformatics toolfor biomarker assessment and outcomebased cutpoint optimization ClinCancer Res “ 10115810780432CCR040713Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Li Zhong Yang Wang Zhang Zheng Yang and Wei This is anopenaccess distributed under the terms of the Creative Commons AttributionLicense CC BY The use distribution or reproduction in other forums is permittedprovided the original authors and the copyright owners are credited and that theoriginal publication in this journal is cited in accordance with accepted academicpractice No use distribution or reproduction is permitted which does not complywith these termsFrontiers in Oncology wwwfrontiersinAugust Volume 0c'
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"protein protein interaction screen carried out to maphuman cellsurface receptorligandinteractions between proteins belongingto the immunoglobulin domainsuperfamily IgSF begins to unravel thecomplex network of cellsurfaceinteractions that allows cells to recognizeand respond to one another and theirdynamically changing environmentHighlightsd Human IgSF interactome reveals complex network of cellsurface protein interactionsd Phylogenetic homology analysis predicts proteinproteininteractionsd 18 previously unknown proteinprotein interactionsidentifiedd Deorphanization of receptors and new binding partners forwellstudied receptorsWojtowicz Cell “August ª Elsevier Inc101016jcell202007025ll 0cllResourceA Human IgSF CellSurface Interactome Reveals aComplex Network of ProteinProtein InteractionsWoj M Wojtowicz16 Jost Vielmetter26 Ricardo A Fernandes17 Dirk H Siepe137 Catharine L Eastman17Gregory B Chisholm2 Sarah Cox4 Heath Klock4 Paul W Anderson4 Sarah M Rue4 Jessica J Miller4 Scott M Glaser4Melisa L Bragstad4 Julie Vance4 Annie W Lam2 Scott A Lesley4 Kai Zinn2 and K Christopher Garcia13581Department of Molecular and Cellular Physiology Stanford University School of Medicine Stanford CA USA2Division of Biology and Biological Engineering California Institute of Technology Pasadena CA USA3Howard Hughes Medical Institute Stanford University School of Medicine Stanford CA USA4The Genomics Institute of the Novartis Research Foundation San Diego CA USA5Department of Structural Biology Stanford University School of Medicine Stanford CA USA6These authors contributed equally7These authors contributed equally8Lead ContactCorrespondence wojstanfordedu WMW kcgarciastanfordedu KCG101016jcell202007025SUMMARYCellsurface proteinprotein interactions PPIs mediate cellcell communication recognition and responses We executed an interactome screen of human cellsurface and secreted proteins most ofwhich are immunoglobulin superfamily IgSF proteins using a highthroughput automated ELISAbasedscreening platform employing a pooledprotein strategy to test all PPI combinations Screen resultsaugmented by phylogenetic homology analysis revealed 18 previously unreported PPIs We validated asubset using surface plasmon resonance and cell binding assays Observed PPIs reveal a large and complexnetwork of interactions both within and across biological systems We identified new PPIs for receptors withwellcharacterized ligands and binding partners for ˜˜orphan™™ receptors New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous systemdevelopment and function differentiationproliferation metabolism vascularization and reproductionThese PPIs provide a resource for further biological investigation into their functional relevance and may offernew therapeutic drug targetsINTRODUCTIONProteinprotein interactions PPIs at the cell surface allowcells to respond to one another and their environment in ahighly dynamic contextdependent and spatiotemporalmanner Wood and Wright The essential role playedby cellsurface PPIs is exemplified by estimates that 18 of genes in the human genome encode cellsurfaceproteins and 18 encode secreted proteins Fonseca At present a comprehensive human cellsurface interactomeis lacking Mapping extracellular PPIs has proved challengingbecause most cellsurface proteins are refractory to classicbiochemical screening techniques and cellsurface PPIs are underrepresented in affinity purificationmass spectrometrybaseddatasets Huttlin Additionally cell surfacePPIs often have fast binding kinetics spanning a broad rangeof affinities low nM to hundreds of mM van der Merwe and Barclay rendering them difficult to detect using standardbiochemical assaysIn recent years several assays have been developed thatallow detection of lowaffinity cellsurface PPIs by impartingavidity through clustering of secreted proteins and the extracellular domains ECDs of single transmembrane STM cellsurface proteins Clustering is achieved using multimerization domains and can increase binding signal up to 250fold Bushell Experimental platforms that utilize clusteringinclude several variations of ELISAbased binding assaysWojtowicz Bushell O¨ zkan BioPlex beads Li protein microarrays Sun cellsignaling assays Barrow cellsurface staining microarrays Turner and beadbasedassays Husain Multiple groups have shown thatELISAbased binding assays have a remarkably low falsepositive rate Wojtowicz Bushell So¨ llnerand Wright Martin Crosnier O¨ zkan Visser Ranaivoson Previously we conducted a screen for all 18 Drosophilacellsurface and secreted proteins containing three types of domains immunoglobulin Ig and Iglike fibronectin type III FN3Cell “ August ª Elsevier Inc 0clland leucinerich repeats LRRs O¨ zkan This screenreported over new PPIs including a previously unknownimmunoglobulin superfamily IgSF PPI network between members of the Dpr and DIP subfamilies Since we reported theDprDIP network functional studies have revealed that thisnetwork mediates neuronal wiring decisions in the fly brain andneuromuscular system for review see Honig and Shapiro Sanes and Zipursky In humans there are an estimated 18 secreted and STMproteins totaling 18 M putative PPIs Screening this vast number requires a highthroughput assay Here we developed ascreening platform that combines a highthroughput version ofthe ELISAbased extracellular interactome assay ECIA O¨ zkan with an automated pooledprotein strategy apECIA We performed a screen of human IgSF secreted andSTM cellsurface proteins excluding antibodies and T cell receptors along with other select proteins of interest The IgSFis the largest and most functionally diverse family in the cellsurface proteome Members include receptor tyrosine kinasesphosphatases costimulatory or coinhibitory immune receptors growth factor and adhesion receptors among many othersand are present in most if not all cell typesWe produced proteins and screened every possible PPI We observed PPIs of which are previously unreported New PPIs form a complexnetwork and include PPIs between phylogenetically related proteins within a subfamily different subfamilies and distantlyrelated proteins Screen results were combined with phylogenetic homology analysis PHA to predict additional PPIs between subfamily members using a nearestneighbor approachWe confirmed a subset of both screen and PHA predictedPPIs using surface plasmon resonance SPR and cell bindingassaysRESULTSSelection and Production of Proteins for PPI ScreeningTo identify human IgSF proteins we utilized the HUGO GeneNomenclature Committee HGNC Yates HumanProtein Atlas Uhle´ n and UniProt UniProt Consortium databases ECDs and secreted proteins for IgSF and nonIgSF proteins of interest were produced with˜˜bait™™ and ˜˜prey™™ multimerization domains into cell supernatantsFigures 1A and 1B Data S1 and S2 and expression wasconfirmed by western blot Data S3 Westerns revealed detectable levels of protein for of baits and of preys We andothers have observed that PPIs can be detected in the ECIA andother ELISAbased binding assays even when proteins are present at levels below the limit of detection by western O¨ zkan Visser Ranaivoson Assuch all bait and prey were included in the screen regardlessof whether protein was detectedDevelopment of an Automated PooledPrey ECIAPlatform apECIAECIA and other ELISAbased assays allow bait and preyproteins to be tested for binding directly from conditionedmedia B These assays test one baitprey pair per Cell “ August Resourcewell To increase throughput we pooled three preys andfollowing screening deconvoluted positive wells to identifyPPIs B Pooling experiments with a panel of knownPPIs showed binding for all 3fold diluted prey Figures 1CS1C and data not shown As baitprey pairs are testedin both orientations a falsenegative resulting from poolingcan be ˜˜rescued™™ by a positive resultin the converseorientation We reasoned thatthe advantages of poolingwhich reduces the number of binding reactions outweighedthe potentialincrease in false negatives To further improvethroughput we optimized a 384well format and developedan automated workflow using liquid handling robots TheapECIA platform allows testing of baitprey combinations per weekPPI Screen Reveals a Complex Network of PPIsFollowing screening deconvolution of positive wells R2 foldoverbackground was performed by retesting each preyindividually B Nine prey gave rise to large numbers ofwells with positive signals and were excluded as nonspecificbinders Figure S2 Following removal of nonspecific PPIs deconvolution revealed positive wells comprising uniquePPIs Data S4 In each case only one of the three deconvolutedprey yielded a positive signal To confirm binding the positiveprey was retested againstin triplicateEightyone percent of PPIs are between IgSF proteinsThe remaining include PPIs between IgSF and otherproteins present in the screen Figure S3its cognate baitAlmost half of the proteins tested were involved in a PPI Proteins not involved in PPIs may be misfolded have binding partners notincluded in the screenrequire coreceptors or fall outside the dynamic range of theassay false negatives which is determined by PPI affinityand bait and prey concentrations Figure S4F Confirming thesensitivity of the assay many bait or prey proteins expressedat very low levels still engaged in one or two PPIs FiguresS4A and S4B A small number of PPIs were observed withbait or prey proteins exhibiting undetectable levels in conditioned media Figures S4C“S4E To interrogate the dynamicrange of the assay we plotted prey AP levels for PPIs with reported affinities Figure S4F These data suggest thatforvery poorly expressed prey proteins Figures S1A S4A andS4B PPIs with KD mM are likely to be missed false negatives Figure S4FOf the PPIs are previously unreported basedon literature and PPI databases Data S4 The majority of PPIsform one large network comprising proteinsD Only proteins involving PPIs are not connectedto the network Different regions of the network are shown inFigure Ninetyeight proteins had one PPI had two to five PPIs and had PPIs EBecause of proteins exhibit binding we calculated theexpected frequency with which each protein will bind at least˜˜x™™ number of proteins up to the maximally observed PPIsFigure S2B and compared the expected and observed frequencies F The observed number of binding partnersis significantly greater than predicted by random chance ofPPIs for a network of this size 0cResourcellABCEDFFigure apECIA Screen Details and Overview of ResultsA Schematic depiction of a subset of proteins in library Pie graph of library distribution Full protein domain names at martemblde Sun B Left flow chart of screen HT highthroughput Right example plate from screen Schematic of ECIA and pooledprey strategy Illustration of screen matrixC ECIA of undiluted prey single prey versus 3fold diluted prey pooled prey Background subtracted data are represented as ±SD Bkgd backgroundD Network of PPIs observed in screen Inset the PPIs that are not connected to the network Node size is proportional to number of PPIs Siglec subfamilynodes are highlighted in colorE Pie graph of distribution of the number of binding partners observed in screen overlaid on the network in a degreesorted circular layoutF Observed versus expected frequency with which each protein will bind at least ˜˜x™™ number of proteins up to the maximally observed PPIs assuming randomchance of interactions p KolmogorovSmirnov [KS] testSee also Figures S1 S2 S3 and S4 and Data S1 S2 S3 and S4Cell “ August 0cllResource Cell “ August legend on next page 0cResourcellPhylogenetic Homology Analysis PHA to Predict PPIsPPIs often occur between phylogenetically related proteins bothwithin and between subfamilies We performed multiplesequence alignments to identify subfamily members within ourlibrary and generated phylogenetic trees Using a combinedapproach which we call apECIAPHA we analyzed screendata alongside the phylogenetic trees to predict additionalPPIs between subfamily members that may have been missedin the screenPPI Validation by Surface Plasmon ResonanceWe selected a subset of screen and PHA predicted PPIs to validate by SPR Bona fide PPIs are expected to display distinct association and dissociation which can be observed with highsensitivity by SPR To increase avidity and therefore sensitivityFc dimerized ECD analytes and ligands were used in most experiments Figure S5 This increase in sensitivity prevented us fromdetermining monomeric KD constants Binding profiles characteristic of PPIs exhibiting clear resonance signals above background negative control ligand and receptor and concentrationdependent binding curves were deemed indicative of aspecific ligandanalyte PPI Nonspecific PPIs generally exhibitdeviations from this behavior such as high background andnonlinear concentration responsesTwentyfour newly identified PPIs observed in our screen weretested by SPR Of these we observed specific ligandanalyteinteractions We additionally tested PHA predicted PPIs andobserved PPIs for Three additional PPIs were observed between homologous proteins in mouse and crossspecies humanmouse In total we SPR validated newly identifiedPPIs Table Data S5Combined apECIAPHA Approach Reveals Multiple DCCSubfamily PPIsThe netrin1 receptor DCC has wellcharacterized functions inthe nervous system Finci DCC is a dependence receptor and is implicated in colorectal and other cancers but itsroles in these cancers are not well understood Goldschneiderand Mehlen We observed DCC binding to insulinlikegrowth factorbinding proteinlike IGFBPL1 but not to insulinlike growth factorbinding protein IGFBP7 Figures 2I and3A Our phylogenetic tree revealed IGFBPL1 and IGFBP7 residein a cluster and share the highest amino acid sequence similarity among subfamily members As such we examined binding of DCC to both IGFBPL1 and IGFBP7 by SPR and observedbinding for both Figure 3CPHA pointed us to four proteins that cluster with DCC PUNCPUNC e11 neogenin NEO1 and protogenin PRTG Figure 3ATogetherthese proteins play roles in diverse processesincluding nervous system development myogenesis and angiogenesis inflammation and tissue regeneration leukocyte migration neural tube and mammary gland formation development ofbone and connective tissues and stem cell differentiation Salbaum Wilson and Key Takahashi Schievenbusch DakouaneGiudicelli PUNC is an ˜˜orphan™™ receptor expressed in the developing nervous system Salbaum PUNC e11 and PRTG bound intercellular adhesion molecule ICAM5Figure 3A a proteinexclusively expressed in the brain that functions in synapse formation stabilization and refinement Gahmberg Cleaved ICAM5 ECD exhibits immunosuppressive functionsthrough cytokine regulation and may play important roles inregulation of brain inflammation We confirmed binding ofPUNC e11 and PRTG to ICAM5 by SPR Figure 3C AlthoughPPIs with ICAM5 were not detected in the screen for the remaining DCC subfamily members we tested them by SPR andobserved binding for all three Figure 3CIn our screen one or more DCC subfamily members alsobound to WFIKKN2 a secreted protein that binds transforming growth factorbeta subfamily members and modulates theirpresentation to cells Monestier and Blanquet lactotransferrin LTF an ironbinding protein with antimicrobial activity Hao interleukin6 receptor subunit alpha IL6Ra a cytokine receptor Schaper and RoseJohn and ISLR2LINX which functions in nervous system developmentMandai Panza Abudureyimu We confirmed binding of all DCC subfamily members tothese proteins by SPR and to other proteins observed in ourscreen Figures 3C 3D and 3F These results demonstratethe value of using a combined apECIAPHA approach to identifyadditional PPIs beyond screen data resulting in the elucidationof a more complete network Figure 3FLARPTPR Subfamily PPIs with SALMsThe LARfamily of type IIA protein tyrosine phosphatase receptors LARPTPRs comprises PTPRF also known as LARFigure Select Regions of the Complex PPI NetworkA Select PPIs including four proteins not connected to the network CD146 CNTN1 NFASC and NrCAMB Region largely comprised of immune system proteinsC Region comprising PPIs both within and across biological systemsD Two regions highlighting subfamilyspecific type IIA and type IIB PTPR PPIs Type IIA PPIs with SALMs and IL1APs include PPIs observed in screen and PHApredicted PPIs validated by SPR Figures and S6E Region highlighting PPIs for ˜˜orphan™™ receptors ILDR1 and PUNCF Region showing a subset of LILR subfamily PPIsG Region showing a subset of Siglec PPIs with nonSiglecs CD33Siglec3H Region showing SiglecSiglec PPIs CD33Siglec3 MAGSiglec4a SNSiglec1I Region highlighting PPIs between nervous system proteins and with proteins in immune and reproductive systems Within this region multiple additional PHApredicted PPIs were validated by SPR Figure Table Because a network is composed of interconnected nodes some linkage proteins are present in more than one panel Colored nodes denote proteins and PPIsvalidated by additional experiments Green line previously reported PPI gray line previously unreported PPISee also Data S4Cell “ August 0cllResourceTable SPRValidated apECIA Screen and PHA Predicted PPIsTable ContinuedLigandFcAnalyteFcapECIATIE1hISLR2mIslr2SALM1SALM2SALM3SALM4SALM5IL1RAPL1IL1RAPL2DSCAMCTDSCAML1CTKIR2DL5IL6RaPSG7PSG9ICAM5WFIKKN2PDL1PDL2LTFTrkAhLEPamLepahLEPamLepaPTPRDPTPRFPTPRSPTPRDPTPRFPTPRSPTPRDPTPRFPTPRSPTPRDPTPRSPTPRDPTPRFPTPRSPTPRSFGFR3PTPRSPTPRMPTPRTPTPRMPTPRTPVRISLR2DCCDSCAMDSCAMDCCIL6RaISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2PRTGPUNCPUNC e11PDL1PDL1DCCIL6RaNANANANANANAknownknownknownNANAknownknownknownknownNANANANANANANANANAknownNANAPHANANANANANANANANANANANANANANANANANANANANANANANANANANANANANAContinued on next page Cell “ August LigandFcAnalyteFcapECIAPHAIGFBPL1IGFBP7ISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2NEO1PRTGPUNCPUNC e11DCCIL6RaISLR2NEO1PRTGPUNCPUNC e11NANANANANANANANANANANANANANANANANANANAECDFc ligand and ECDFc analyte PPIs observed by SPR apECIA denotes PPI was observed in screen PHA denotes PPI was predicted byphylogenetic homology analysis SPR conditions are included in DataS5 See also Figure S6ahLEP and mLEP monomer proteinPTPRD and PTPRS Figures 4A and 4B LARPTPRs playimportant roles in synaptic anization and function Nam Presynaptic LARPTPRs mediate transsynapticadhesion through PPIs with multiple postsynaptic ligandsMouse mutants in LARPTPRs and their ligands exhibit defectsin synapse structure and function Lie Several PPIs are known between specific LARPTPRs andSALM235 Li Choi GotoIto Figure 4A We observed these as well as additionalPPIs in our screen Figures 2D 4A and 4C No LARPTPR binding to SALM1 or SALM4 has been reported and we did notobserve binding in our screen PHA led us to test binding of allLARPTPRs to all SALMs by SPR With the exception ofPTPRFSALM4 we observed binding of all LARPTPRs to allSALMs Figures 4E and S6A PTPRSALM pairs exhibited differences in maximum response units RU a relative comparison ofbinding strength suggesting a spectrum of binding affinities mayexist among LARPTPRs and SALMs Figure 4FLARPTPR Subfamily PPIs with Interleukin1 ReceptorAccessory Proteins IL1APsTranssynaptic LARPTPR interactions with IL1RAP and IL1RAPL1 induce bidirectional pre and postsynaptic differentiation Yoshida IL1RAP is known to interactwith all three LARPTPRs and IL1RAPL1 with PTPRD Weobserved these PPIs and additionally binding of IL1RAPL1 toPTPRS Figure 2D IL1RAPL1 shares sequence similarity 0cllBResourceACDEFlegend on next pageCell “ August 0cllResourcewith IL1RAPL2 an orphan receptor expressed in the brain withno known biological function Boraschi In ourscreen we detected binding of IL1RAPL2 to PTPRD and PTPRSFigure 2D We tested both known and previously unknown PPIsby SPR and observed binding for all Figure S6B IL1RAPL2additionally interacted with fibroblast growth factor receptor FGFR3 in the screen and by SPR Figure S6BType IIB PTPR Subfamily PPIs with DSCAMsType IIB PTPRs are expressed in most tissues and regulatediverse processes including cell growth migration and differentiation immune cell development endothelial cell adhesion andmigration neuronal development and synapse formation andoncogenic transformation Lee Stoker The typeIIB subfamily is composed of PTPRK PTPRM and PTPRT Figures 4A and 4B and multiple binding partners are known Weobserved binding of all type IIB PTPRs with Down syndromecell adhesion molecule DSCAM and Down syndrome cell adhesion molecule like1 DSCAML1 Figures 4A and 4C proteinsthat play various roles in nervous system development Sanesand Zipursky The ECD of DSCAMs contains nine Iglike domains followedby four FN3 domains one Iglike domain and two FN3 domainsFigure 4B The Nterminal region engages in homophilic bindingand mediates binding to secreted chemoattractant and chemorepellent ligands netrin1 and slit1 respectively Wojtowicz Ly Alavi No binding partnerhas been reported for the Cterminal ECD region In our screenwe observed binding between the Cterminal regions of bothDSCAMs DSCAMCT and DSCAML1CT and all three typeIIA PTPRs Figure 4C We confirmed binding of DSCAMCTand DSCAML1CT to PTPRT and PTPRM by SPR PTPRK wasnot expressed at sufficient levels Figure 4D vascular endothelial growth factorNew PPIs between Immune System ProteinsNew PPIs were observed for wellstudied immune systemproteins as well as orphan receptors Figure 2B Two PPIswere detected for the orphan granulocyte receptor CEACAM4receptor VEGFR3 and programmed cell death ligand PDL2 CEACAM4 is a member of the carcinoembryonic antigenrelated cell adhesion molecule subfamily expressed inthe immune system epithelial and endothelial cells and brainWakabayashiNakao Zinn and O¨ zkan Inthe immune system CEACAMs play roles in immunity anddevelopment We also observed binding between CEACAM1and the immune checkpoint receptor programmed cell deathprotein PD1Interactions of PD1 with its ligands PDL1 and PDL2 inhibitT cell proliferation cytokine production and cytotoxic activityBardhan PDL2 competes with PDL1 for bindingto PD1 and can be expressed on tumor cells where it mayplay a role in tumor evasion Ghiotto Cheng Bardhan Using SPR we confirmed the knownPPI between PDL1 and PDL2 Lee and observedhomophilic binding of PDL1 a previously unknown PPI Figure S6C By comparison with PDL1 binding to PD1 which approaches saturation at 18 nM we infer that the affinity of PDL1 homophilic binding is lower than binding to PD1 This mayexplain why this PPI within the wellstudied PD1PDL1 axishas not been identified previously and highlights the value of using multimerized proteins to detect lowaffinity PPIsHomophilic and Heterophilic Siglec Subfamily PPIsEight of the proteins with the highest number of PPIs in ourscreen are members of the Siglec subfamily members proteins highly restricted to the immune system that have immunemodulatory effects on Tolllike receptor TLR signaling andplay important roles in self versus nonself discrimination Macauley Siglecs display differential expression on cellsand exhibit a broad spectrum of Siglecspecific recognition ofsialylated glycan profiles present on healthy cells inflamed ormalignant cells and pathogens We observed a network of homophilic and heterophilic PPIs among Siglecs Figure 2H DataS4 as well as PPIs with distantly related proteins a subset areshown in Figure 2G Data S4PPIs between Immune and Nervous System ProteinsThe signaling lymphocytic activation molecule subfamily SLAM members is expressed on most immune cells Dragovich andMor SLAMs function as both costimulatory and coinhibitory molecules in innate and adaptive immunity and playan integral role in autoimmune disorders SLAMF9 is an orphanreceptorWe observed SLAMF9 binding to bactericidal permeabilityincreasing protein BPI and IGSF10 Figure 2C BPI is a neutrophilderived antibiotic protein that participates in bacteria killingthrough its highly cationic Nterminal region Bu¨ low The Cterminal region of BPI exhibits no bactericidal activity andis believed to interact with other proteins and function in differentprocesses IgSF10 is an orphan receptor involved in the migration of gonadotropinreleasing hormone expressing GnRH neurons Howard IgSF10 has no known function in theFigure SPR Validation of DCC Subfamily PPIs Identified by apECIAPHA ApproachA Dendrograms of a subset of DCC subfamily PPIs showing the number of SPRvalidated PPIs observed in the apECIA screen turquoise and predicted by PHAmagentaB Dendrogram of a subset of PSG PPIsC SPR sensrams for DCC subfamily IL6Ra and ISLR2 analytes 2fold dilutions “ nM nM for PRTGWFIKKN2 due to incomplete chipregeneration at higher concentrations binding to various ligands TrkA and TIE1 negative controlsD SPR sensram for IL6Ra analyte 2fold dilutions “ nM binding to ISLR2 ligand PUNC negative controlE SPR sensrams for DSCAM and DCC analytes 2fold dilutions “ nM binding to PSG ligands TIE1 negative controlF Subnetwork of SPRvalidated PPIsRU resonance unitsSee also Figure S5 and Data S5 Cell “ August 0cResourcellABCEDFlegend on next pageCell “ August 0cllResourceimmune system and SLAMF9 function in the nervous system isunknown revealing a PPI between two orphan receptors fromdifferent biological systemsFigure 2F These PPIs present MOG and MPZ PPIs with LILRsas new candidates for neuronal regeneration studiesPPIs with PregnancySpecific Glycoproteins PSGsDuring pregnancy PSGs members are the most abundanttrophoblastic proteins in maternal blood and serve as markersfor trophoblast quality and embryo viability Moore and Dveksler The mechanisms of PSG action in pregnancy are not wellunderstood Studies suggest PSGs also have immunoregulatory proangiogenic and antiplatelet aggregation functionsWe observed several PSG binding partners includingplateletderived growth factor receptor alpha PDGFRAfibroblast growth factor receptor FGFR4 Ctype lectindomain family member A CLEC4A DCC and DSCAMFigures 2I and 3BObserved PSG interactions occur selectively and differentiallyto these binding partners Figure 2I Data S4 Using SPR weexamined binding of DCC and DSCAM to PSG7 and PSG9 Figure 3E We observed binding for DSCAM with PSG9 and bothDCC and DSCAM with PSG7 Interestingly DCC is expressedin human placenta DakouaneGiudicelli ThesePPIs present new candidate receptors for studying the role ofPSGs in pregnancy immunoregulation and angiogenesisLILR Subfamily PPIs with BTNL8 and MyelinationProteinsLeukocyte immunoglobulinlike receptors LILRs membersare a subfamily of activating LILRA and inhibitory LILRB receptors that exhibit immunomodulatory activity and function ininflammation regulation tolerance and differentiation Burshtynand Morcos We observed binding of multiple activatingand inhibitory LILRs to butyrophilin8 BTNL8 Figure 2F BTNLsare members of the extended B7 family of molecules and function as costimulatory or coinhibitory signals for T cell activationRhodes LILRs are also expressed on neurons and function in the regulation of development synaptic plasticity and axonal regeneration Hirayasu and Arase Myelin the protective insulatinglayer around axons inhibits neuronal regeneration following spinal injury Monje Three myelin proteins Nogo MAG andOMgp are known to interact with PirB in mouse one of only twomouse LILRB orthologs Atwal We observed bindingof multiple LILRs to two additional myelin proteins myelinoligodendrocyte glycoprotein MOG and myelin protein P0 MPZPVR Selectively Interacts with a KillerCellImmunoglobulinlike Receptor KIRThe polygenic Killercellimmunoglobulinlike receptors KIRsare a highly polymorphic subfamily of activating and inhibitoryproteins expressed on natural killer NK cells that regulate development maturation and activation Pende NK cellsinitially express a stochastic combination of KIRs that is refinedduring maturation to tune killing response threshold and ensureoptimal discrimination of target cells from healthy cells Weobserved binding of KIR2DL5 to poliovirus receptor PVR Figures 2C and 5A which validates a recently reported PPI Husain We confirmed KIR2DL5 binding to PVR by ECIAtitration analysis and SPR Figures 5C and 5DTo examine binding at the cell surface fluorescent tetramersof PVRFc and KIR2DL5Fc ECDFcSA647 were incubatedwith fulllength KIR2DL5 and PVRtransfected cells respectively Flow cytometry analysis revealed concentrationdependent binding of both ligands to cells expressing cognate fulllength receptor but not control cells Figure 5E PVRFc tetramers did not bind cells transfected with fulllength KIR2DL4and KIR2DL1 which share and sequence similaritywith KIR2DL5 respectively Figures 5B and 5F To furtherexamine the specificity of PVR for KIR2DL5 ECIA was performed using KIRs PVR bound specifically to KIR2DL5 Figure 5G Because KIR2DL5 has been associated with increasedvirus susceptibility and reduced antiviral response to therapythis specificity may have implications for the role of KIR2DL5 inimmunityTrkA Selectively Interacts with TIE1Highaffinity nerve growth factor receptor NTRK1 also knownas TrkA has multiple wellstudied functions in the nervous system Amatu In the immune system where its function is not well understood TrkA is expressed on monocytesmacrophages dendritic cells resting and activated B cells andneutrophils and erythroblasts Minnone In ourscreen we observed TrkA binding to tyrosineprotein kinase receptor TIE1 but not to TIE2 Figures 2A and 6A TIE1 is expressed on endothelial cells immature hematopoietic cells andplatelets and functions in complex with the angiopoietinTIE2pathway to inhibit angiogenesis Eklund We performed SPR and observed TrkA binding to TIE1 but not toFigure SPR Validation of Type IIA and Type IIB PTPRs with SALMs and DSCAMsA Left dendrogram highlighting phylogenetic clustering of SALMs DSCAMs type IIA PTPRs PTPRFSD and type IIB PTPRs PTPRTMK Right SPRvalidated PPIs green line previously known PPI turquoise line previously unknown PPI observed in screen magenta line previously unknown PHA predictedPPI Gray line screen PPI not SPR testedB Protein domain structures Ig immunoglobulinlike domain FN fibronectin type III domain MAM meprin A5 protein domain LRR leucinerich repeat TMtransmembrane NT Nterminal region of ECD CT Cterminal region of ECDC Screen data showing PPI specificity of PTPRFSD and PTPRTMK subfamilies Background subtracted OD nm data are represented as mean oftriplicate wellsD SPR sensrams for DSCAML1CT and DSCAMCT analytes 2fold dilutions “ nM binding to PTPRM and PTPRT ligandsE SPR sensrams for PTPRS analyte 2fold dilutions “ nM binding to SALMs and DSCAMCT ligandsF SPR max RU values for every pairwise combination of PTPRFSD analytes with SALM ligandsRU resonance unitsSee also Figures S5 and S6 and Data S5 Cell “ August 0cResourcellABCEDFGlegend on next pageCell “ August 0cllResourceTIE2 Figures 6B and S7A As a positive control for TIE2 weconfirmed binding to monomer angiopoietin1 Figure S7A Davis We next investigated whether TrkA can bind NGF and TIE1simultaneously We preincubated TrkA with NGF and comparedbinding of T
2
LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on progressionfree survival in uveal melanoma a randomized clinical trial JAMA Shain AH Bagger MM Yu R Chang D Liu SS Vemula S Weier JF Wadt K Heegaard S Bastian BC Kiilgaard JF The genetic evolution of metastatic uveal melanoma Nat Genet Bagger M SmidtNielsen I Andersen MK Jensen PK Heegaard S Andersen KK Friis S Kiilgaard JF Longterm metastatic risk after biopsy of posterior uveal melanoma Ophthalmology Kujala E Mäkitie T Kivelä T Very longterm prognosis of patients with malignant uveal melanoma Invest Ophthalmol Vis Sci Chandran SS Somerville RPT Yang JC Sherry RM Klebanoff CA Goff SL Wunderlich JR Danforth DN Zlott D Paria BC Sabesan AC Srivastava AK Xi LQ Pham TH Raffeld M White DE Toomey MA Rosenberg SA Kammula US Treatment of metastatic uveal melanoma with adoptive transfer of tumourinfiltrating lymphocytes a singlecentre twostage singlearm phase study Lancet Oncol Mendell JT Targeting a long noncoding RNA in breast cancer N Engl J Med Lan Y Xiao XW He ZC Luo Y Wu CF Li L Song X Long noncoding RNA OCC1 suppresses cell growth through destabilizing HuR protein in colorectal cancer Nucleic Acids Res Cao CH Sun JY Zhang DY Guo XJ Xie LW Li X Wu DH Liu L The long intergenic noncoding RNA UFC1 a target of microRNA 34a interacts with the mRNA stabilizing protein HuR to increase levels of βcatenin in HCC cells Gastroenterology 20151482415426e18 Wang P Xue YQ Han YM Lin L Wu C Xu S Jiang ZP Xu JF Liu QY Cao XT The STAT3binding long noncoding RNA lncDC controls human dendritic cell differentiation Science Zheng XL Tang HW Zhao XF Sun YM Jiang YF Liu YH Long noncoding RNA FTH1P3 facilitates uveal melanoma cell growth and invasion through miR2245p PLoS One 20171211e0184746 Lu QK Zhao N Zha GP Wang HY Tong QH Xin SH LncRNA HOXA11AS exerts oncogenic functions by repressing p21 and miR in uveal melanoma DNA Cell Biol Lu LN Yu XY Zhang LL Ding X Pan H Wen XY Xu SQ Xing Y Fan JY Ge SF Zhang H Jia RB Fan XQ The long noncoding RNA RHPN1AS1 promotes uveal melanoma progression Int J Mol Sci Wu S Chen H Han N Zhang CX Yan HT Long noncoding RNA PVT1 silencing prevents the development of uveal melanoma by impairing MicroRNA173pdependent MDM2 upregulation Invest Ophthalmol Vis Sci Li P He J Yang Z Ge SF Zhang H Zhong Q Fan XQ ZNNT1 long noncoding RNA induces autophagy to inhibit tumorigenesis of uveal melanoma by regulating key autophagy gene expression Autophagy Dong YZ Meng XM Li GS Long noncoding RNA SNHG15 indicates poor prognosis of nonsmall cell lung cancer and promotes 0ccell proliferation and invasion Eur Rev Med Pharmacol Sci SNHG15 serves as an oncogene and predicts poor prognosis in epithelial ovarian cancer Onco Targets Ther Liu K Hou Y Liu YK Zheng J LncRNA SNHG15 contributes to proliferation invasion and autophagy in osteosarcoma cells by sponging miR141 J Biomed Sci Wu DM Wang S Wen X Han XR Wang YJ Shen M Fan SH Zhang ZF Shan Q Li MQ Hu B Lu J Chen GQ Zheng YL LncRNA SNHG15 acts as a ceRNA to regulate YAP1Hippo signaling pathway by sponging miR200a3p in papillary thyroid carcinoma Cell Death Dis Guo XB Yin HS Wang JY Evaluating the diagnostic and prognostic value of long noncoding RNA SNHG15 in pancreatic ductal adenocarcinoma Eur Rev Med Pharmacol Sci Sun XT Bai Y Yang C Hu SY Hou ZL Wang GX Long noncoding RNA SNHG15 enhances the development of colorectal carcinoma via functioning as a ceRNA through miR141SIRT1Wntβcatenin axis Artif Cells Nanomed Biotechnol Ye JF Tan LD Fu Y Xu HJ Wen LJ Deng Y Liu K LncRNA SNHG15 promotes hepatocellular carcinoma progression by sponging miR1413p J Cell Biochem Zhang JH Wei HW Yang HG Long noncoding RNA SNHG15 a potential prognostic biomarker for hepatocellular carcinoma Eur Rev Med Pharmacol Sci Zhang YL Zhang DH Lv J Wang S Zhang Q LncRNA SNHG15 Acts as an oncogene in prostate cancer by regulating miR3383pFKBP1A axis Gene Kong QL Qiu M Long noncoding RNA SNHG15 promotes human breast cancer proliferation migration and invasion by sponging miR2113p Biochem Biophys Res Commun Wang TQ Sun HB Bao Y En R Tian YJ Zhao W Jia LZ POM121 overexpression is related to a poor prognosis in colorectal cancer Expert Rev Mol Diagn Shuai Y Ma ZH Lu JW Feng JF LncRNA SNHG15 a new budding star in human cancers Cell Prolif 2020531e12716 Qu C Dai CM Guo YH Qin R Liu JB Long noncoding RNA Ma YW Xue YX Liu XB Qu CB Cai H Wang P Li ZQ Li Z Liu YH SNHG15 affects the growth of glioma microvascular endothelial cells by negatively regulating miR153 Oncol Rep Li M Bian ZH Jin GY Zhang J Yao SR Feng YY Wang X Yin Y Fei BJ You QJ Huang ZH LncRNASNHG15 enhances cell proliferation in colorectal cancer by inhibiting miR3383p Cancer Med Liu YC Li JL Li F Li M Shao Y Wu LP SNHG15 functions as a tumor suppressor in thyroid cancer J Cell Biochem Liu YC Li JL Li M Li F Shao Y Wu LP microRNA5105p promotes thyroid cancer cell proliferation migration and invasion through suppressing SNHG15 J Cell Biochem Li YW Guo HY Jin CJ Qiu CP Gao M Zhang L Liu ZJ Kong BH Spliceosomeassociated factor CTNNBL1 promotes proliferation and invasion in ovarian cancer Exp Cell Res Williams GH Stoeber K The cell cycle and cancer J Pathol Yu B Ding YM Liao XF Wang CH Wang B Chen XY Overexpression of PARPBP correlates with tumor progression and poor prognosis in hepatocellular carcinoma Dig Dis Sci Maiuri AR Peng M Podicheti R Sriramkumar S Kamplain CM Rusch DB DeStefano Shields CE Sears CL O€™Hagan HM Mismatch repair proteins initiate epigenetic alterations during inflammationdriven tumorigenesis Cancer Res Berry D Seider M Stinnett S Mruthyunjaya P Schefler AC Ocular Oncology Study Consortium Relationship of clinical features and baseline tumor size with gene expression profile status in uveal melanoma a Multiinstitutional study Retina Jiang ZM Yu FH Li M Upregulation of BCL2 kD proteininteracting protein BNIP3 is predictive of unfavorable prognosis in uveal melanoma Med Sci Monit Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0c'
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" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ‚ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an ‚amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the efficacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInflammatory 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 fistulas 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Ž Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot 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 financial 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 tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy 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 efficacy 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 findings 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 reflecting 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 aspecificity 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 first of onset areassociated with a poor prognosis at the first 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 efficient at reflecting disease activity []Some studies have also investigated the efficacy 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 efficacy 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 efficacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta “ Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for “ daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at ˆ’ °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 specificity 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 first evidence of the efficacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the first and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow confirmed and complemented the findings of this study In another study published in AUC values of CI “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI “ for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity 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 specificity 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 specificity [] Caviglia et al in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren “ yearsChildren “ yearsAdultsOver years “ “ “ “ “Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in Crohn's disease [] Given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity 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 efficacy 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 andspecificity 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 andspecificity 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 findings 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 firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting findings 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 SpecificityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and specificity 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 findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity 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 diseaseidentification 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 modifiedBaron 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 specificity 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¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta “Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity 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 specificity 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 specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed 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 specificity 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 thedifficulty 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 isRobert™s 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 Robert™s scoring system [] Theede et al also used themodified 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 Specificity 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 specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes
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colorectal carcinoma crc as one of the most commongastrointestinal malignancies has developed the world™sfourth most deadly cancer with a high rate of incidence andmortality [ ] liver metastasis which is the most commonform for crc metastasis is the leading cause of high mortality for this severe malignancy however few clinical prevention and treatment measures could be available for tumormetastasis therefore it is really urgent to develop new biomarkers and explore the underlying mechanism for crcmetastasis and eventually develop new therapeutic strategiesfor crc patientsduring cancer progression metabolic reprograming withincreasing glucose utilization is termed as warburg aï¬ectwhich is accompanied by altered pyruvate and mitochondrialmetabolism the fate of pyruvate is the core manifestationto distinguish normal cells and tumor cells through metabolism in normal cells pyruvate was used for efficient atpproduction directly into mitochondria however pyruvatewas converted into lactate in cytosol despite of normoxicand hypoxic conditions in cancer cells this may be dueto the impaired process of pyruvate from the cytosol intothe mitochondrial matrix which is a critical metabolic steplinking glycolysis and mitochondrial oxidative phosphorylation the mitochondrial pyruvate carriermpc a 0c of immunology researchmultimeric complex containing two distinct proteins mpc1and mpc2 which is located in the inner mitochondrialmembrane is responsible for efficient mitochondrial pyruvate uptake loss of mpc expression or activity blockspyruvate entry into the tca cycle which results in a metabolism switch to increase glycolysis and the compensatoryusage of glutamine [ ]existed studies have reported that mpc1 was related withimmunoregulation stemness metabolism cellular morphology etc [ “] currently the important role of mpc1was uncovered in several tumors in crc and esophagealsquamous cell carcinoma decreased mpc1 results in accelerated aerobic glycolysis and malignant progression [ ] inlung adenocarcinoma mpc1 deficiency accelerates lung adenocarcinoma progression through the stat3 pathway in prostate cancer mpc1 was reported to be involved instemness and metabolism which regulated by couptfii[ ] in renal cell carcinoma hypoxiainduced loss ofmpc1 enhanced the expression of mmp7 and mmp9 to promote cell invasion collectively these data suggestedthat mpc1 maybe serves as a suppressor to disrupt tumormalignancy however whether mpc1 is involved in crcmetastasis and the underlying mechanisms remain to beillustratedin the present study we figured out the relationshipbetween the mpc1 expression and crc liver metastasiswe identified that decreased mpc1 was closely correlatedwith patient™s metastasis as well as led to poor prognosisfunctionally mpc1 overexpression could attenuate themigration and invasion capacities of crc cells both in vitroand in vivo mechanically mpc1 suppressed crc metastasisthrough mediating the wntcatenin signaling thus ourfinding firstly revealed a critical role of mpc1 in crc livermetastasis materials and methods data mining seven geo datasets gse21510 gse5206gse20916 gse9348 gse89393 gse67675 and gse4183and tcga were used to analyze the mpc1 expression pattern in crc the primary data for tcga datasets weredownloaded wwwcancergov the primary data feo datasets were downloaded at wwwncbinlmnihgovgeo the oncolnc database httpwwwoncolnc was used to detect the prognostic value of mpc1 inthe tcga cohort patients and clinical specimens in our study a total of patients containing paired crc tissues and adjacentnontumor tissues were enrolled from the department of gastrointestinal surgery renji hospital school of medicineshanghai jiao tong university among them cases ofliver metastases were collected and only cases wereavailable with complete followup data for survival analysisinformed consents were signed by all patients the researchwas approved by the research ethics committee of renjihospital and carried out in accordance with ethical standardsas formulated in the helsinki declarationtable sequences of primers used for realtime pcrprimermmp7 forwardmmp7 reverseecadherin forwardecadherin reversesnail1 forwardsnail1 reversemyc forwardmyc reversegapdh forwardgapdh reversesequence ²²gagtgagctacagtgggaacactatgacgcgggagtttaacatatgagtgtcccccggtatcttcacgagcagagaatcataaggcggtatccagagctgtttggaaacattttcctcccaggccatcacagccctcactcacacagattccacaaggtgcctgggctacactgagcaccaagtggtcgttgagggcaatg cell culture and cell transduction human crc celllines hct116 ht29 sw620 rko sw480 and lovoand mouse crc cell line mc38 were gained from the cellbank of the chinese academy of sciences shanghai chinaall cells were cultured in dmem medium supplemented°with fetal bovine serum and antibiotics at c in ahumidified incubator with co2mc38 cells were transfected with lentivirus containing aluciferase reporter plasmid stable transfection cells werescreened with μgml blastisidin for days which termedmc38luc in addition one short hairpin rna shrnasequence against mpc1 was packaged as lentivirus andtransfected into mc38luc cells lovo and sw480 were transfected with lentivirus containing fulllength human mpc1cdna or empty vehicle control stable transfection cells werescreened under μgml puromycin for days and verifiedby western blot in those assays all lentiviral transfectionswere performed in the presence of μgml polybrene immunohistochemical ihc the protocol of this assayand quantify the mpc1 protein expression level were performed according to previously reported primary antibodies used as follows mmp7 yt2663 immunoway ecadherin cst snail1 ab53519 abcam and mycyp0861 immunoway cellular immunofluorescence assays were performedaccording to the previous description briefly cells wereincubated with antibodies against catenin ab32572abcam and incubated with alexa 488conjugated secondaryantibody the nuclei were stained with ²6diamidino2phenylindole dapi western blotting wholecell lysates or nuclear proteinwas extracted using a protein extraction buï¬er beyotimeshanghai china or nucleoprotein extraction kit sangonbiotech c500009 respectively proteins were resolved bysdspage and transferred onto nitrocellulose nc membranes using standard methods primary antibodies used asfollows mpc1 ab74871 abcam catenin ab32572abcamlamin ac ab8984 abcam and gapdhab9485 abcam speciesspecific secondary antibodies used 0c of immunology researchtable related enzyme and carrier of pyruvate analyzed in this studygenepdha1pdhbpdk4pdhxmpc2pdk2pdk1pdk3mpc1pcpdp1pdprpdha2pdp2pklrdescriptionpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase lipoamide betapyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase complex component xmitochondrial pyruvate carrier pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme pyruvate dehydrogenase kinase isozyme mitochondrial pyruvate carrier pyruvate carboxylasepyruvate dehydrogenase phosphatase catalytic subunit pyruvate dehydrogenase phosphatase regulatory subunitpyruvate dehydrogenase lipoamide alpha pyruvate dehydrogenase phosphatase catalytic subunit pyruvate kinase liver and rbclocationmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion inner membranemitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrion matrixmitochondrionas follows irdye goat antimouse igg licor andirdye goat antirabbit igg licorttest was used for comparison between groups p wasconsidered as statistically significantsynthesized using the primescript realtime pcr total rna was extracted using trizoltakara according to the manufacturer™sinstructionscdna wasreversetranscriptionpolymerasechain reaction rtpcr kittakara the qpcr was performed using sybr green masˆ’–ct method was used to analyze theter mix roche the data and gapdh was used as a loading control the primersequences are listed in table liver metastasis model this study was performed inaccordance with the recommendations in the guide for thecare and use of laboratory animals and relevant chineselaws and regulations the protocol was approved by theinstitutional animal care and use committee iacucof shanghai jiao tong university the mc38 cells transplanting luciferaseexpressing were injected into the spleensof c57bl6n mice n with a concentration of cellsmouse two weeks later the mice were killed andthe liver metastasis tissues were harvested luciferase reporter assay the protocols of this assaywere operated in accordance with previous reported ng top reporter plasmid wntcatenin signalingor ng fop reporter plasmid negative control ofwntcatenin signaling and ng renilla were mixedand transfected into crc cells using lipofectamine dualluciferase reporter assay system promega was usedto detect the firefly and renilla luciferase activities statistical analysesdata are shown as means ± sd spss chicago il usaand graphpad prism software were used to manipulate statistical analyses kaplanmeier method was used to calculatecumulative survival time the chisquare test or student™s results mpc1 expression was aberrantly decreased in crcpyruvate is a pivotal intermediate in the process of cellmetabolism which connects glycolysis and the tca cycleto determine the potential maladjustment genes involvedin pyruvate metabolism which is located in mitochondriaas shown in table we analyzed the tcga dataset containing crc and their normal counterparts the resultsshowed that multiple genes are significantly upregulated ordownregulated in crc t tissues compared to normal colonn notably mpc1 had a log2 fold change less than figures 1a and 1b as known to us pyruvate translocation from the cytoplasm to the mitochondria is the first stepinto the tca cycle which needs mpc1mpc2 heterodimerin the analysis no significant change was found in mpc2therefore mpc1 was selected for further study the expression pattern of mpc1 was further analyzed in five independent geo datasetsgse21510 gse5206 gse20916gse9348 and gse4183 consistently we found thatmpc1 was downregulated with statistical diï¬erence in crctissues figure 1c and inflammatory tissue supplementary figure in comparison to their normal counterpartsmeanwhile we found decreased mpc1 expression inhuman crc tissues compared to their normal counterpartsfigure 1d in addition a similar phenomenon wasrevealed in aomdss induced mouse crc modelsfigure 1e then we evaluated the protein level of mpc1used a tissue microarray containing matching cancerand corresponding adjacent nontumortissues whichsubjected ihc staining the expression of mpc1 wasscored as œ  based on the staining area andintensity figure 1f we found that more lower mpc1expression score as œ and œ was presented in crc 0c of immunology researcheulavp gol“tcga t nmpc1“mpc2“log2 fold changenoisserpxe cpm evitalertcgaŽŽŽn normaltumornoisserpxe cpm evitalergeo datasetsŽŽŽŽŽŽŽŽŽŽŽŽgse21510 gse5206 gse20916 gse9348normaltumorabhuman samplecrcnormalaom dssnormalccrcesacesac“esacesacdempc1 lower expressionmpc1 higher expressionfŽŽ egatnecrepromutlamron“gfigure expression pattern of mpc1 in crc a volcano plot showed fold changes xaxis and corresponding p values log10 yaxis ofpyruvate metabolismrelated genes located in mitochondria analyzed in the tcga dataset between paired normal and crc samplesstudent™s ttest b the comparison of mpc1 expression in tumor and matched normal tissues using the tcga dataset student™s ttest ˆ—ˆ—ˆ—p c expression analysis of mpc1 in tumors and corresponding normal tissue using four independent geo datasetsgse21510 gse5206 gse20916 and gse9348 student™s ttest ˆ—ˆ—ˆ—p d ihc staining of mpc1 expression in matched crctumor and nontumor tissues scale bar μm e ihc staining of mpc1 expression in the aomdss induced crc mouse models andcontrol animal scale bar μm f representative ihc staining of mpc1 expression in tissue microarray from the ren ji cohortwhich contained crc patients and paired adjacent normal tissue n the tumor tissues were divided into two groups based on theexpression level which scored as œ  scale bar μm g the percentage of tissue displaying diï¬erent expression level ofmpc1 in crc tumor and adjacent nontumor tissues fisher™s exact test ˆ—ˆ—p tissues figure 1g overall these results revealed thatmpc1 was disrupted during crc decreased mpc1 enhanced tumor metastasis capabilityand predicated poor prognosis in crc it is well known thatcrc is one of the most malignant tumors given its strongmetastasis ability so we tried to figure out whether thempc1 expression was correlated with metastasis we foundthat lower mpc1 expression was closely correlated withmetastasis p lymph node invasion p and tnm stage p which revealed by the analysisbetween the clinical significance and mpc1 expression incrc table next to illuminate the expression patternof mpc1 in diï¬erent process of crc data mining was carried out by two independent geo datasets gse21510 andgse89393 which contained normal tissue primary crcand metastatic lesion in the liver mcrc as shown infigures 2a and 2b mpc1 expression was gradually downregulated in patients with an increase in metastasis ability asimilar result was found in mice cells ct26 with high livermetastasis hmct26 or poor liver metastasis pmct26figure 2c which isolated by in vivo selection in an orthotopic mouse model of colon cancer metastasis to the liverfurther analysis showed that mpc1 protein expression was 0c of immunology researchtable clinicopathological correlation of mpc1 expression in theren ji crc cohortclinicopathological featureexpression ofmpc1lowhighp value χ2testage years‰¥gendermalefemalemetastasisyesnolymph node invasionyesnotumor size cm‰¥ cmtnm stageiiiiiiivkras mutationmutationno mutationgradually downregulated in normal tissue primary crc andliver metastasis crc crlm tissues figure 2d furthermore survival analysis showed that patients with lowermpc1 expression had a worse outcome compared to thepatients with higher mpc1 expression using the tcgacohort figure 2e and ren ji cohort figure 2f additionally among patients with metastasis worse prognosiswas emerged in the mpc1 low cases figure 2g while nosignificant association was observed in patients withoutmetastasis figure 2h mpc1 overexpression impaired crc cells motility bothin vitro and in vivo to evaluate the role of mpc1 on themotility of crc cells the transwell assay was performedfirstly we examined low mpc1 protein expression in humanand high mpc1 protein expression in mouse mc38 crccells by western blot figure 3a mpc1overexpressingstable cell lines were established using a lentivirus carrying the mpc1 gene in lovo and sw480 cells and theoverexpression efficiency was confirmed by immunoblotsfigure 3b mpc1 knockdown in lucmc38 cells wereestablished and verified by wb figure 3c mpc1 overexpression exhibited significantly weaker migration and invasion ability than the control cells in both lovo figure 3dand sw480 figure 3e cells following the liver metastasismodel of crc was established by spleen orthotopicallyinjecting mc38 cells transplanting luciferaseexpressingwhich would simulate mc38 metastasis to the liver throughsplenic veinportal vein the results revealed that the mpc1knockdown promoted mc38 cells metastasis to the liverthrough detecting the luminescence intensity monitoredby bioluminescence imaging figure 3f notablythenumber of metastatic liver nodules in the mpc1 silencinggroup wasin the control groupfigure 3g histological examination also proved thatmpc1 knockdown decreased the metastatic potential ofcrc in vivo figure 3gthan thatsmaller decreased mpc1 activated the wntcatenin pathwayby promoting nuclear translocation of catenin to furtherexplore the underlying mechanism of mpc1mediated inhibition of crc metastasis the tcga database was used toperform gsea analysis the results indicated that mpc1was involved in the wntcatenin signaling when set themrna expression median as a cutoï¬ figure 4a anddualluciferase reporter gene assay revealed that mpc1 overexpression obviously inhibited the activity of wntcateninpathway figure 4b which confirmed the result abovewe then tested the distribution changes of catenin innuclear and cytoplasmic which was a crucial step inwntcatenin pathway as shown in figure 4c no significant diï¬erence was emerged in the total amount ofcatenin between mpc1 overexpression cells and the control cells figure 4c however obviously decreased distribution of nuclear catenin was presented in mpc1overexpression cells compared to that in the control cellsas revealed by the stronger gray corresponding to cateninfigures 4c and 4d consistently immunofluorescenceif staining showed that mpc1 overexpression weakenednuclear catenin localization in both lovo and sw480 cellswhen compared to control cells figures 4e and 4f asimilar phenomenon was observed in mouse liver metastasistissues by ihc figure 4g following qpcr analyses displayed some downstream target genes of catenin such asmmp7 ecadherin snail1 and myc obviously increasedin ecadherin and decreased in mmp7 snail1 and mycwere observed after the mpc1 overexpression in both lovoand sw480 cells compared to that in the control cellsfigures 4h and 4i meanwhilethe opposite trendwas observed in mc38 cell after mpc1 knockdownfigure 4j and similar results were observed in mouseliver tissue detected by ihc figures 4k“4n takentogether the data above indicated that mpc1 mediatedcrc cell metastasis through the wntcatenin pathway discussionaccumulating evidences have shown that reprogrammedenergy metabolism conduces to the tumor malignant propertiesincluding enhanced crc liver metastatic capacity[“] mitochondria as the primary site of energy production regulate the pyruvate metabolism under both physiologic and pathologic conditions the first step of the tcacycle is mediated by mpc which transports pyruvate into 0cnoisserpxe cpm evitalergse21510ŽŽŽnormal crc mcrcnoisserpxe cpm evitalernormalacrccrlmesacesac of immunology researchgse67675ŽŽŽpmct26 hmct26ctcgan n p hr time daysgse89393ŽŽnormal crcmcrcbnoisserpxe cpm evitaler““Ž egatnecreplavivrus llarevolamroncrcmlrc“lavivrus llarevoren ji cohortn p hr n time monthsfdlavivrus llarevolavivrus llarevocrc with metastasis n n p hr n time monthsmpc1 higher expressionmpc1 lower expressiongecrc without metastasis n n n p hr time monthshfigure decreased mpc1 enhances tumor metastasis and predicts poor prognosis in crc a b data mining showed mpc1 expressionwas gradually decreased in normal tissue primary crc and liver lesion of metastatic crc mcrc from two independent geo datasetsoneway anova a gse21510 ˆ—ˆ—ˆ—p b gse89393 ˆ—ˆ—p c data from gse67675 revealed that mpc1 expressionwas lower in high liver metastasis ct26 cells hmct26 than that in poor liver metastasis ct26 cells pmct26 student™s ttestˆ—ˆ—ˆ—p d gradually decreased mpc1 expression was presented in normal tissue primary crc and liver metastasis crccrlm tissue scale bar μm n fisher™s exact test ˆ—p e kaplanmeier overall survival os curves in the tcgadataset of crc patients according to the mrna expression of mpc1 the lower quartile value of expression was utilized as a cutoï¬logrank test p f kaplanmeier os curve for the mpc1 expression in the ren ji cohort logrank test p gkaplanmeier os curve for the mpc1 expression in patients with metastasis logrank test p h kaplanmeier os curve forthe mpc1 expression in patients without metastasis logrank test p mitochondrial from cytoplasm in the beginning tcgawas used to analyze the relative genes involved in pyruvatemetabolism which is located in mitochondria the resultsrevealed that mpc1 expression was significantly downregulated in crc tissues meanwhile the geo datasets analysisas well as ihc staining on crc patients™ tissue and mousemodels confirmed this trend these phenomena may indicate that loss of mpc activity enhanced tumorigenic glucoseutilization by blocking mitochondrial pyruvate uptake andoxidation interestingly in the course of data analysis wefound that the expression of mpc1 was decreased at thestage of intestinal inflammation which was not diï¬erentfrom that in tumor tissue mpc1 has been reported to beinvolved in immune regulation of peripheral t cell homeostasis through metabolic regulation and a decrease inmpc1 was found at the earliest stages of crc hence 0c of immunology researchhumanmouselovosw480lovosw480mpc1gapdhmpc1““mpc1gapdhmpc1““mpc1gapdhwsthwsovolokrcmtchablovoŽŽrotcevitnelcpmitnelrotcevitnelcpmitneldlefi rep sllec edavnidlefi rep sllec edavnirotcevitnelcpmitneldsw480rotcevitnelcpmitnelcncpmŽcncpmdlefi rep sllec detargimdlefi rep sllec detargimcŽŽŽcncpmŽŽcncpmmc38shnc shmpc1¨¯ŽŽ sp xufl latotcnhscpmhscnhscpmhsradiancepseccm2srfemc38ŽŽsuledon sisatsatemcnhscpmhsgfigure mpc1 overexpression inhibits the motility of crc cells in vitro and in vivo a mpc1 expression in human and mouse crc celllines examined by western blot gapdh serves as loading control b mpc1 overexpression in lovo and sw480 cells c mpc1 silencing by shrnampc1 in mouse mc38 cells d e transwell assays showed that upregulated mpc1 suppressed the invasion and migration ability of lovod and sw480 e cells quantification of invaded and migrated cells was performed for five randomly selected fields values are means ± sd frepresentative bioluminescence photograph of mice spleen implanted with luciferaseexpressing mc38 cells treated with shmpc1 or controlvector total flux was quantified by the ivis system to verify the ability of liver metastasis g representative image of liver metastases andquantified by the nodules in mice inoculated with mc38 cells treated with shmpc1 or control vector as well as representative images ofhe staining of the liver metastatic lesions scale bar μm student™s ttest ˆ—p ˆ—ˆ—p ˆ—ˆ—ˆ—p 0c of immunology researchse erocs tnemhcirnewnt signaling pathwaynes “p mpc1 highmpc1 lowoitar pof pot evitaler𝛽cateninlamin ac𝛽cateningapdhmpc1alovosw480““csuelcunlatot icanmalnnetac𝛽iŽŽŽŽlovoncmpc1bŽsw480Žlovosw480ncmpc1d𝛽catenindapimerge𝛽catenindapimerge𝛽catenincpmcnovolnoisserpxe evitaleregnahc doflelovoŽŽŽŽlianscymŽpmmncmpc1nirehdacehcpmcnwsfsw480ŽŽŽlianscymŽŽpmmncmpc1nirehdaceifigure continuedcmcnhscpmhsmc38gŽŽlianscymŽŽpmmŽnirehdaceshncshmpc1jnoisserpxe evitaleregnahc doflnoisserpxe evitaleregnahc dofl 0c of immunology researchmmp7ecadherinsnail1myccmcnhscpmhscmcnhscpmhscmcnhscpmhscmcnhscpmhsklmnfigure decreased mpc1 activates the wntcatenin pathway by promoting nuclear translocation of catenin a gsea analysis ofmpc1 expression in crc using the tcga dataset nes normalized enrichment score b luciferase reporter gene assay of crc cellstreated with mpc1 overexpression or not c the expression of total catenin and nuclear catenin was detected in control and mpc1overexpression crc cells respectively gapdh and lamin ac were used as the loading control of total and nuclear protein respectivelyd the gray value analysis of nuclear catenin in mpc1overexpression cells and control cells e f mpc1 overexpression could inhibitthe nuclear translocation of catenin in crc cells scale bar μm g ihc staining of catenin in mouse liver metastatic lesionsinoculated with mc38 cells treatment with shmpc1 or control vector scale bar μm h i relative mrna expression level of catenin target genes in crc cells with mpc1 overexpression or control vector j relative mrna expression level of catenin targetgenes in mc38 cells with shmpc1 or control vector k“n relative protein expression level of catenin target genes in mouse livertissue detected by ihc scale bar μm student™s ttest ˆ—p ˆ—ˆ—p we suspect that mpc1 is involved in bowel inflammation totumorigenesis and more studies need to be devised to illustrate this processfollowing in the analysis between the clinical significance and mpc1 expression in crc we found that mpc1expression was especially correlated with metastasis inspiredby this we detected the mpc1 expression pattern in normaltissue primary crc and metastasis crc by geo datasetsand patients™ tissue all results revealed that gradually downregulated mpc1 was in patients with an increase in metastasis ability survival analysis indicated that worse outcome waspresented in patients with lower mpc1 expression especiallyin patients with metastasis additionally function assays verified that mpc1 overexpression could attenuate the migration and invasion capacities of crc cells in vitro andmpc1 knockdown could enhance the metastasis capacityin vivo and existing studies have revealed that the mpc1was participated in metastatic dissemination of pgc1αtransduced cholangiocarcinoma through elevating reactiveoxygen species ros production besides mpc inhibitor uk5099 treatment could trigger strong invasive capacitythrough blocking pyruvate translocation into the mitochondria so as to attenuate mitochondrial oxidative phosphorylation and trigger aerobic glycolysis these phenomenatogether with our results indicate that mpc1 could act as atumor suppressor through inhibiting tumor metastasis andexisting studies have shown that mpc1 could alter the maintenance and fate of stem cells through regulating cancermetabolism in crc however the relationship betweenthe metabolism and tumor metastasis regulated by mpc1was not being mentioned thus further evidence should bereceived to confirm thissubsequently the underlying mechanism of mpc1 inregulating metastasis was explored for this purpose gseaanalysis was performed the results indicated that mpc1was involved in the wntcatenin signaling the next seriesof experiments also confirmed that mpc1 could mediate thewntcatenin pathway by redistribution of catenin previous reports showed that cytoplasmic catenin phosphorylated in nterminally localized to sites of cellcell contact isassociated with ecadherin and was required for intact cellcell adhesions without any change detected in the levels oftotal catenin [“] simultaneously cell“cell adhesionbased on cadherin binding with catenin limited wnt signals in addition catenin was reported to interact withusp9x to inhibit the degradation of catenin through thedeubiquitination of catenin in breast cancer a constitutive irs1 and catenin protein interaction activated mycexpression in acute lymphoblastic leukemia cells inhcc catenin was reported to interact with yap1 to leadto rapid tumorigenesis hence it is reasonable to guessthat accumulated cytoplasmic catenin maybe crosstalkwith other genes or involved in other biological processeswhat is more some downstream target genes of cateninsuch as mmp7 ecadherin snail1 and myc were changedin expression as known to us mmp7 is a member of theproteolytic enzyme family which promotes the invasionand metastasis of tumor cells by degrading the basementmembrane and extracellular matrix and previous studies had evidenced for involvement of mmp7 activation incolorectal cancer liver metastases [ ] ecadherin andsnail1 were considered as the epithelialmesenchymal transition emt marker which was involved in metastasis ofmalignant tumor moreover ecadherin was reportedto be involved in cellcell junction to regulate cancer invasionand metastasis [ ] and the gsea analysis also revealedthat mpc1 could aï¬ect the cellcell contacts data notshown as described previously mmp7 could facilitatemorphological transition by cleaving ecadherin thecommunication between the cells is disrupted when ecadherin was shredded leading to destructed cell adhesionand induction of emt followed by increased cell migration inspired by this we assumed that mpc1 could mediatetumor cell motility through aï¬ecting mmp7 activity cellcell 0c of immunology researchcontacts and emt however further studies need to be performed to clarify the detailed underlying mechanisms conclusionsin conclusion we firstly demonstrated that decreased mpc1was closely correlated with patient™s metastasis as well as ledto poor outcome moreover mpc1driven nuclear translocation of catenin contributed to crc cell motility thismeans that mpc1 has the potential to be a diagnostic biomarker and therapeutic target for metastasis patientsabbreviationscrc colorectal carcinomampc mitochondrial pyruvate carrieremt epithelialmesenchymal transitiongsea gene set enrichment analysisdata availabilitythe data used to support the findings of this study are available from the corresponding author upon requestconflicts of interestall authors declare no conflicts of interestauthors™ contributionsyahui wang and guangang tian conceived and designedthe study chunjie xu and kaixia zhou obtained and anized the data guangang tian analyzed the data zhigangzhang and jianren gu contributed reagentsmaterialsanalysis tools xueli zhang and guangang tian wrote themanuscript guangang tian chunjie xu and kaixiazhou contributed equally to this workacknowledgmentsthis work was supported by the national natural sciencefoundation of china no to zhigang zhangand the natural science foundation of shanghai no18zr1436900 to xuelili zhangsupplementary materialsœsupplementary figure expression analysis of mpc1 innormal ibd and tumor tissues using the gse4183 datasetoneway anova was used to analyze the statistical diï¬erences between ibd adenoma and crc tissues ns no significance student™s ttest ˆ—ˆ—ˆ—p ˆ—ˆ—p ˆ—p supplementary materialsreferences h brody œcolorectal cancer nature vol no r l siegel k d miller s a fedewa et al œcolorectal cancerstatistics  ca a cancer for clinicians vol no pp “ a j rauckhorst and e b taylor œmitochondrial pyruvatecarrier function and cancer metabolism current opinion ingenetics development vol pp “ m g vander heiden l c cantley and c b thompsonœunderstanding the warburg eï¬ect the metabolic requirements of cell proliferation science vol no pp “ s herzig e raemy s montessuit et al œidentification andfunctional expression of the mitochondrial pyruvate carrierscience vol no pp “ c yang b ko c t hensley et al œglutamine oxidationmaintains the tca cycle and cell survival during impairedmitochondrial pyruvate transport molecular cell vol no pp “ t bender g pena and j c martinou œregulation of mitochondrial pyruvate uptake by alternative pyruvate carrier complexes the embo vol no pp “ a g ramstead j a wallace sh lee et al œmitochondrialpyruvate carrier promotes peripheral t cell homeostasisthrough metabolic regulation of thymic development cellreports vol no pp “2899e6 x zhou z j xiong s m xiao et al œoverexpression ofmpc1 inhibits the proliferation migration invasion and stemcelllike properties of gastric cancer cells oncotargets andtherapy vol pp “ d li c wang p ma et al œpgc1α promotes cholangiocarcinoma metastasis by upregulating pdha1 and mpc1 expression to reverse the warburg eï¬ect cell death diseasevol no j c schell k a olson l jiang et al œa role for the mitochondrial pyruvate carrier as a repressor of the warburg eï¬ectand colon cancer cell growth molecular cell vol no pp “ y li x li q kan et al œmitochondrial pyruvate carrierfunction is negatively linked to warburg phenotype in vitroand malignant features in esophageal squamous cell carcinomas oncotarget vol no pp “ h zou q chen a zhang et al œmpc1 deficiency accelerateslung adenocarcinoma progression through the stat3 pathway cell death disease vol no l wang m xu j qin et al œmpc1 a key gene in cancermetabolism is regulated by couptfii in human prostatecancer oncotarget vol no pp “ y zhong x li d yu et al œapplication of mitochondrialpyruvate carrier blocker uk5099 creates metabolic reprogramand greater stemlike properties in lncap prostate cancer cellsin vitro oncotarget vol no pp “ x p tang q chen y li et al œmitochondrial pyruvatecarrier functions as a tumor suppressor and predicts theprognosis of human renal cell carcinoma laboratory investigation vol no pp “ g a tian c c zhu x x zhang et al œccbe1 promotesgist developmentthrough enhancing angiogenesis andmediating resistance to imatinib scientific reports vol no article c xu g tian c jiang et al œnptx2 promotes colorectalcancer growth and live
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" tumor microenvironment tme plays an important role in malignant tumors our study aimed toinvestigate the effect of the tme and related genes in osteosarcoma patientsmethods gene expression profiles and clinical data of osteosarcoma patients were downloaded from the targetdataset estimate algorithm was used to quantify the immune score then the association between immune scoreand prognosis was studied afterward a differential analysis was performed based on the high and lowimmunescores to determine tmerelated genes additionally cox analyses were performed to construct two prognosticsignatures for overall survival os and diseasefree survival dfs respectively two datasets obtained from the geodatabase were used to validate signaturesresults eightyfive patients were included in our research the survival analysis indicated that patients with higherimmune score have a favorable os and dfs moreover genes were determined as tmerelated genes theunsupervised clustering analysis revealed two clusters were significantly related to immune score and t cells cd4memory fraction in addition two signatures were generated based on three and two tmerelated genesrespectively both two signatures can significantly divide patients into low and highrisk groups and were validatedin two geo datasets afterward the risk score and metastatic status were identified as independent prognosticfactors for both os and dfs and two nomograms were generated the cindexes of os nomogram and dfsnomogram were and respectively tme was associated with the prognosis of osteosarcoma patients prognostic models based on tmerelated genes can effectively predict os and dfs of osteosarcoma patientskeywords tumor microenvironment osteosarcoma prognosis immune features nomogram osteosarcoma is the most common bone tumor especiallyin children and adolescents it was reported that approximately of patients are between and yearsold and osteosarcoma is considered as the second leadingcause of death in this age group currently surgery and correspondence 407404159qqcom4wenzhou medical university wenzhou chinafull list of author information is available at the end of the chemotherapy are still major treatments for osteosarcomapatients and these therapies are constantly improving inrecent years however due to the susceptibility of localaggressiveness and lung metastasis in osteosarcoma patients the prognosis of osteosarcoma remains unfavorable previous studies indicated that the 5years survivalrates were and in metastatic and nonmetastaticpatients respectively thereforeit is necessary to the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chu bmc cancer page of investigate the mechanism of pathogenesis and progressionof osteosarcoma and accurately classify the risk of patientsrecently an increasing number of diagnostic and prognostic biomarkers of osteosarcoma patients have beenidentified for example chen reported that tumorsuppressor p27 is a novel biomarker for the metastasis andsurvival status in osteosarcoma patients moreover huang discovered that dysregulated circrnas serve asprognostic and diagnostic biomarkers in osteosarcomapatients and the relative potential mechanism mainly attributes to the regulation of downstream signaling pathwaysby sponging microrna in addition lncrna microrna and many clinical data were also identified asprognostic biomarkers for osteosarcoma patients however osteosarcoma is one of the malignant cancers entitiescharacterized by the high level of heterogeneity in humanstherefore it is necessary to find accurate biomarkers forosteosarcomain recent years researchers have paid more and moreattention to the role of the tumor microenvironmenttme in malignant tumors the function of tme inthe tumorigenesis progression and therapy of tumorshave been initially understood [ ] more importantly estimation of stromal and immune cells in malignant tumor tissues using expression data estimate an algorithm to quantify the score of immune cellsand stromal cells by analyzing the gene expression datawas developed in based on the algorithm theprognostic value of immune and stromal cells in bladdercancer acute myeloid leukemia gastric cancer cervicalsquamous cell carcinoma adrenocortical carcinomaclear cell renal cell carcinoma hepatocellular carcinomathyroid cancer and cutaneous melanoma have beenreported [“] generally the above research indicatedthat tme can serve as the prognostic biomarker in tumorsand many tmerelated genes were determined as the prognostic genes however the role of tme and tmerelatedgenes in osteosarcoma patients remains unclearin the present study gene expression data and corresponding clinicopathologic data were obtained from thetherapeutically applicable research to generate effectivetreatments target dataset then the estimatealgorithm was performed to quantify the immune score ofosteosarcoma and the tmerelated genes were identifiedby the differential expression analysis subsequently theprognostic value of tme and tmerelated genes weredetermined by a series of bioinformatics methodsmethodsgene expression datasetslevel data of gene expression profiles and correspondingclinical data of osteosarcoma patients were downloadedfrom target dataset ocgcancergovprogramstarget accessed on oct the correspondingclinicopathologic data included in the present study wereage gender race ethnicity tumor site and metastaticstatus after data were extracted from the public domainthe estimate an algorithm inferring tumor puritystromal score and immune cell admixture from expression data was performed to evaluate the immune score byusing the estimate package in r software version meanwhile the messenger rnamrna expressionprofiles and clinical data ofincludinggse21257 and gse39055 were obtained fromthe gene expression omnibus as external validationcohortstwo cohortssurvival analysis and correlation analysisafter scores were obtained patients were divided intohighscore group and lowscore group according to themedian of the immune score the kaplanmeier survivalanalysis with logrank test was performed to estimatethe differences of overall survival os and diseasefreesurvival dfs between high and lowscore cohorts inaddition the association between clinicopathologic dataand tme score was also studied mannwhitney signedrank test was performed to compare the differences ofimmune score between each clinical group all statisticalanalyses in the present study were performed using rsoftware except for the special instructions p value twoside was identified as statistically significantin the present studydegexpressed genedifferentially expressed gene analysisdifferentiallyanalysis wasperformed by comparing the proteincoding genesexpression between the lowimmune score group andthe highimmune score group the limma package in rsoftware was used to perform the differential analysisand genes with log fc and adjusted pvalue qvalue were identified as degs to further understand the function of degs identifiedin the present study gene ontology goincludingbiological processes bp molecular functions mf andcellular componentscc and kyoto encyclopedia ofgenes and genomes kegg analysis were performedby clusterprofiler package in r software evaluation of association with immune cellsto further investigate the association between degs andimmune cells the cibersort package was used toestimate the relative proportions of types of immunecells meanwhile the œconsensusclusterplus 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 ˆ’ 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 œphagosomeœautoimmune thyroid disease œantigen processing andpresentation œb cell receptor signaling pathway œintestinal immune network for iga production œinflammatorybowel disease œprimary immunodeficiency œth1 andth2 cell differentiation œth17 cell differentiation œnatural killer cell mediated cytotoxicity and œnfˆ’kappa 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 didn™t 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 martínez e vegesna r kim h torresgarcia wtreviño v shen h laird pw levine da inferring tumour purity and stromaland immune cell admixture from expression data nat commun yang s liu t nan h wang y chen h zhang x zhang y shen b qian pxu s comprehensive analysis of prognostic immunerelated genes inthe tumor microenvironment of cutaneous melanoma j cell physiol “ deng z wang j xu b jin z wu g zeng j peng m guo y wen z miningtcga database for tumor microenvironmentrelated genes of prognosticvalue in hepatocellular carcinoma biomed res int zhao k yang h kang h wu a identification of key genes in thyroid cancermicroenvironment med sci monit xu wh xu y wang j wan fn wang hk cao dl shi gh qu yyzhang hl ye dw prognostic value and immune infiltration of novelsignatures in clear cell renal cell carcinoma microenvironment agingalbany ny chen b chen w jin j wang x cao y he y data mining of prognosticmicroenvironmentrelated genes in clear cell renal cell carcinoma a studywith tcga database dis markers li x gao y xu z zhang z zheng y qi f identification of prognostic genesin adrenocortical carcinoma microenvironment based on bioinformaticmethods cancer med “ pan xb lu y huang jl long y yao ds prognostic genes in the tumormicroenvironment in cervical squamous cell carcinoma aging albany ny wang h wu x chen y stromalimmune scorebased gene signature aprognosis stratification tool in gastric cancer front oncol huang s zhang b fan w zhao q yang l xin w fu d identification ofprognostic genes in the acute myeloid leukemia microenvironment agingalbany ny yan h qu j cao w liu y zheng g zhang e cai z identification ofprognostic genes in the acute myeloid leukemia immunemicroenvironment based on tcga data a
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the molecular heterogeneity of renal cell carcinoma rcc complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge this study investigates the role of thelncrna cdkn2bas1 and mir1413p interactions in the progression and metastasis of kidney cancer human renalcancer cell lines achn and caki1 normal rptec cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study an overexpression of cdkn2bas1 was observed in rcc compared to normal samplesin tcga and our inhouse sfvamc tissue cohorts reciprocally we observed reduced expression of mir141 in rcccompared to normal in the same cohorts cdkn2bas1 shares regulatory mir141 binding sites with ccnd1 andccnd2 genes direct interactions of cdkn2bas1mir141cyclin d1“d2 were confirmed by rna immunoprecipitationand luciferase reporter assays indicating that cdkn2bas1mir141cyclin d1“d2 acts as a cerna network in rccfunctionally attenuation of cdkn2bas1 andor overexpression of mir141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model furtheroverexpression of cdkn2bas1 is positively correlated with poor overall survival of rcc patients expression of mir141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal rptec cells induced procancerous characteristics cdkn2bas1 attenuation or mir141 overexpression decreased ccnd1ccnd2 expressionresulting in reduced rac1ppxn that are involved in migration invasion and epithelial“mesenchymal transition thisstudy for the first time deciphered the role of cdkn2bas1mir141cyclin d axis in rcc and highlights this networkas a promising therapeutic target for the regulation of emt driven metastasis in rccintroductionrenal cell carcinoma rcc is one of the most commoncancers in the usa accounting for nearly deathsand new cases in surgery is the first line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of morecorrespondence rajvir dahiya rdahiyaucsfedu1department of urology veterans affairs medical center san francisco anduniversity of california san francisco san francisco ca usa2department of surgery university of miami miller school of medicine miamifl usafull list of author information is available at the end of the these authors contributed equally pritha dasgupta priyanka kulkarniedited by e candithan however in approximately of localizedrcc cases recurrence occurs with distant metastasis2the obstinate nature of rcc to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence lack of sensitivity to both chemotherapytherapeuticoptions difficult3“ it is therefore of utmost importanceto improve our understanding of rcc pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive rcc6and immunotherapy makesemerging lines of evidence suggest that cancer aggressiveness is associated with epithelial“mesenchymal transition emt7 it is a wellorchestrated process involved in the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cdasgupta cell death and disease page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8emt changes in cellular morphology and migratoryproperties are governed by numerous factors9 increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like ncadherbronectinvimentin and matrix metalloproteinasemmps and decreased expression of epithelial markerslike ecadherin αecatenin claudin etc10“ are common emt phenomena often the progression of cancerthrough emt is significantly induced by the interaction ofcyclind with its binding partner cdk4 which act astranscriptional regulators controlling cell proliferationand migration14“ it is well known that cyclind regulates the ratelimiting step in cell cycle progression fromg1 to s phase accumulating evidence also suggest thatabnormal cyclindcdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodreports show that noncoding rnas are involved in thefactors involved in emt18 micrornasregulation ofmirnas a naturally occurring class of small noncodingrna molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mrna degradation20 whereas long noncoding rnaslncrnas with more than nucleotides can also actas regulators for tumorsuppressive mirnas in differentcancers21“ recently a class oflncrnas have beencategorized as competing endogenous rna cernawhich involves crosstalk among lncrnas mrnas andtheir shared mirnas thus a novel regulatory mechanismis hypothesized suggesting that lncrnas and mrnascommunicate with each other by competing for commonmirna response elements24“in this context we describe the novel role of lncrnacdkn2bas1 and mir1413p mir141 in the regulation of cyclind to govern the metastatic progression ofrcc to our knowledge this is the first report to directlydemonstrate that cdkn2bas1mir141 interaction is acrucial component in rcc progression and metastasisthrough the cyclindrac paxillin pathwaymaterials and methodscell lines and cell culturethe normal rptec atcc number crl4031 andrenalcancer achn atcc number crl1611and caki1 atcc number htb46 cell lines were purchased from the atcc manassas va these humanderived celllines were authenticated by dna shorttandem repeat analysis by atcc cell line experimentswere performed within “ months of their procurementresuscitation achn cells were cultured in mem mediaofficial of the cell death differentiation associationcaki1 cells in and mccoy 5a medium and rptec cellsin dmemf12 medium atcc® „¢ all mediawere supplemented with fbs and 1x antibioticspenicillin and streptomycin cell lines were maintainedat °c and humidified atmosphere of co2mirnasirna transfectionsto induce overexpression or knockdown cells were transiently transfected with either mirvana mirna mimic nmoll or antimir mirna inhibitor nmollthermo fisher scientific and nmoll of sirna sigmaaldrich using lipofectamine rnai max thermo fisherscientific according to the manufacturers™ protocol toverify transfection efficiency mirvana mirna mimicnegative control mirna inhibitor control and sirnacontrol were used respectively in each transfection experiment at the same concentration all transfection experimentswere carried out for hclinical specimensformaldehydefixedparaffinembedded ffpe tissue specimens from patients undergoing radical nephrectomy wereobtained from the san francisco veterans affairs medicalcenter sfvamc written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines irb approval no allpatient samples were pathologically confirmed for clear cellrcc ccrcc and slides were reviewed by a boardcertifiedpathologist for the identification of tumor foci and adjacentnormal tissue apart from sfvamc cohort tcgakirctcgakich tcgakirp icgc and geo cohorts forrcc from online databases were also used to check theexpression levelsrnamirna extraction and quantitative realtime pcrqrtpcrtotal rna was extracted from microdissected ffpetissues and cell lines using mirneasy ffpe and mirneasymini kits qiagen respectively in accordance to manufacturer™s instructions mature mirna and mrnas wereassayed by qrtpcr using quantstudio flexreal timepcr system applied biosystem using fast sybr®green master mixtaqman universal pcr master mixprobes and primers applied biosystems inc foster cityca usa following manufacturer™s protocol humangapdh and rnu48 were used as endogenous controlsand relative expression of rnamirna were calculatedusing comparative ct threshold cycle primer sequencesare provided in supplementary table t1dna methylation analysis insilico in cell lines and 5azacdr treatmentdna hypermethylation of the mir141 promoter innormal and rcc samples was first confirmed in the 0cdasgupta cell death and disease page of tcga database using wanderer software27 in order toconfirm the methylation status of the mir141 promoterin rcc cell lines we extracted dna from achn andcaki1 using dneasy tissue kit qiagen sodium bisulphite modification was done using ez dna methylationgold kit zymo research orange ca usa followingthe manufacturer™s protocol bisulfitetreated dna wasanalyzed by methylationspecific quantitative polymerasechain reaction msqpcr with primer pairs specific formethylated and unmethylated regions of the mir141promoter msqpcr was performed as described earlier28 for each sample the percent of methylation wascalculated by the difference of ct in methylated samplectm and ct in unmethylated sample ctu the primers sequences are mentioned in supplementary table achn and caki1 cells were treated daily with μmoll5azadeoxycytidine 5azacdrfor h29 and total rna was isolated using a mirneasy minikit qiagen to check mir141 expressionsigmaaldrichcell viability clonability migratory invasion andapoptosis assayscell viability was measured at and h using acelltiter aqueous solution cell proliferation assay kitpromega madison wi following the manufacturer™sinstructions for colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed plates were then stained with giemsafollowed by crystal violet and colonies were countedculture inserts of 8µm pore size transwell costar wereused for migration and invasion assay inserts were coatedwith matrigel bd biosciences µgwell for invasionbriefly h posttransfection cells were counted andplaced on inserts at × cellsml for migration and × for invasion in serumfree medium and wereallowed to migrateinvade for “ h at °c cellsmigrated or invaded through the pores were fixed stainedwith crystal violet crystal violet was solubilizedwith methanol and quantified at nm by a kineticmicroplate reader spectra max molecular devicesfacs analysis for apoptosis was done h posttransfection using annexin vfitc and 7aad kit beckmanin accordance with the manufacturer™scoulter incinstructions cold pbs washed cells were resuspended in1x binding buffer and stained with annexin vfitc7aad viability dye after min of incubation at roomtemperature in the dark stained cells were analyzed usingbd facsverse bd pharmingendualluciferase reporter assaythe wild type wt and offtarget ot luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingofficial of the cell death differentiation associationsites and corresponding nontarget mutant sites into thepmirglo reporter vector luciferase constructs µgwere cotransfected into achn and caki1 cells along with nmoll mir141 mimic or controlmir using transfection reagent jetprime polyplustransfection illkirchfrance luciferase activities were measured using thedualluciferase assay promega madison wi h posttransfection relative luciferase activity was calculated bynormalizing firefly luciferase to renilla luminescencerna immunoprecipitation rip assaysimultaneous binding of mir141 to lncrna andmrna was confirmed by rip assay an imprint rip kitwas used following the manufacturer™s protocol sigmaaldrich st louis mo usa igg control and ago2antibodies were used forimmunoprecipitation theimmunoprecipitated rna fraction was reverse transcribed to cdna using high capacity cdna reversetranscription kit thermo fisher fold enrichment oflncrna and mrna to ago2 with respect to igg wascalculated using quantitative rtpcrwestern blot and immunofluorescence analysistotal protein extraction was performed as describedpreviously18 proteins were then separated by nupage“ bistris protein gels invitrogen and subsequentlytransferred onto nitrocellulose membraneresulting blots were blocked using odyssey blockingbuffer licor and subsequently probed with primaryand secondary antibodies blots were scanned using anodyssey infrared imaging system scan and quantificationwas carried out with the licor odyssey® scanner andsoftware licor biosciences the primary antibodiesused are listed in supplementary table for immunofluorescencetransfected achn andcaki1 cells were fixed in paraformaldehyde for minfollowed by blocking 1x pbs5 normal goat serum triton x100 for h at room temperature cellswere then incubated overnight in fold diluted primary antibody at °c cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoledapi for min cells were then mounted on a slideusing prolong gold antifade reagent images were captured using zeiss microscope model axio imagerd2transientlyrenal cancer xenograftswe studied the antitumorigenic effects of mir141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 male nude mice“ weekold n charles river lab were subcutaneously injected with × caki1 cells oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups five in 0cdasgupta cell death and disease page of each synthetic mirna mir141 mimicmircon of μg was complexed with μl siportamine transfection reagent ambion in μl pbs and deliveredintratumorally in 3day intervals tumor volume wascalculated according to the formula x2 y2 where x yx width y length experiments were terminated days after the last treatment day tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups all animal care was in accordance withinstitutional guidelines iacuc approval no statistical analysisall quantified data represents an average of at leastthree independent experiments or as indicated statisticalanalyses were performed using graphpad prism andmedcalc error bars represent ± standard error meansem the mann“whitney u test was used to assessthe difference between mirna expressions in tumornormal adjacent tissue significant differences betweenthe groups were determined using the student™s ttest alltests were performed either one tailed or two tailed andresults were considered statistically significant at p ‰¤ receiver operating curves roc were performed toevaluate the potential of mir141 to differentiate betweenmalignant and nonmalignant samples using medcalcsoftware showing area under curve auc and confidence interval kaplan“meier analyses for overall survival with respectto mir141 methylation levels intcgakirc cohort were generated using softwareœezrhttpsdoi101038bmt2012244 tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsresultslncrna cdkn2bas1 is oncogenic and is a direct target ofmir141initially we found cdkn2bas1 is an oncogeniclncrna in rcc based on tcga fig 1a icgc andgeo databases fig s1 tcga cohort also revealed thathigh cdkn2bas1 expression increases from lower gradeand stage to higher grade and stage fig 1b moreoverhigher expression is significantly p correlated tooverall survival fig 1c in agreement with these datacohorts significantly higher cdkn2bas1 expression wasalso seen in rcc cell lines achn caki1 as compared tonormal rptec fig 1d and sfvamc cohort fig 1epatient and tumor characteristics are summarized insupplementary table t3 roc analysis shows an auc of p ci “ fig 1f suggesting the diagnostic potential of cdkn2bas1 to discriminate between normal and tumor tissues we usedcomputational algorithms and identified putative mir141binding sites in the cdkn2bas1 sequence fig 1g toofficial of the cell death differentiation associationexamine potential mir141cdkn2bas1 interactionexperimentally we performed luciferase reporter assayboth achn and caki1 cells cotransfected with mir141and cdkn2bas1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmir141 directly interacts and regulates cdkn2bas1fig 1h thus all these data suggest that clinicallyimportant cdkn2bas1 is an oncogenic lncrna in rccand is a novel target of mir141cdkn2bas1 inhibition by sirna suppressestumorigenicity in rcctransient transfection of achn and caki1 cells withcdkn2bas1 sirnas for h showed significant reduction in cdkn2bas1 expression fig 2a cdkn2bas1knockdown in both cell lines significantly inhibited cellproliferation figs 2b s2a and clonogenic survivalfigs 2c s2b with a significant increase in apoptosis fig2d e decreased cell migrationinvasion figs 2f s2c dwith simultaneous changes in emt markers such as anincrease in epithelial markers αecatenin and claudinand decrease in mesenchymal markers vimentin andfibronectin were also observed figs 2g s3expression of mir141 and its clinical importance in renalcarcinomaand samples lowersince our results confirmed cdkn2bas1 as a directtarget of mir141 we examined mir141 status andclinical importance in rcc expression of mir141 wassignificantly downregulated in rcc cell lines fig 3aand in tumor samples fig 3b“e compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors fig 3c“e patientand tumor characteristics are summarized in supplementary table t3 roc analysis showed an auc of p ci “ fig 3f suggesting thatmir141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsepigenetic regulation of the mir141 locuswe identified a genomic site rich in cpg island locatedupstream of the mir141 in chromosome 12p13 in thetcga cohort we observed hypermethylation of mir141promoter in tumor tissues as compared to normalfigs 3g s4a which is significantly associated with poorpatient survival fig 3h similarly rcc cell lines achnand caki1 also showed hypermethylation compared tonormal rptec cells fig 3i further we treated achnand caki1 cell lines with demethylating agent 5azacdrand observed decrease in methylation fig s4b withconcomitant increase in mir141 expression fig 3jindicating possible epigenetic regulation a significantdecrease in the expression of methylation regulatory 0cdasgupta cell death and disease page of fig lncrna cdkn2bas1 is oncogenic in renal cancer and is a direct target of mir141 a expression levels of cdkn2bas1 among kircnormal tumor kich normal tumor and kirp normal tumor patient samples in tcga cohort using wanderersoftware pvalue calculated by mann“whitney twotailed test b expression of cdkn2bas1 in tcgakirc cohort among different grades normal grade “ grade “ and stages normal stage i“ii and stage iii“iv c overall survival in tcgakirc cohort as performedby kaplan“meier analysis using ualcan software d relative expression levels of lncrna cdkn2bas1 in rcc cell lines achn and caki1 e expressioncdkn2bas1 in matched pairs of rcc tissue samples from sfvamc cohort pvalue calculated by mann“whitney twotailed test f receiver operatingcurve roc analysis on sfvamc cohort showing ability of lncrna cdkn2bas1 to differentiate between malignant and nonmalignant samples sfvamccohort g predicted binding sites of mir141 in cdkn2bas1 sequence h luciferase assays showing decreased reporter activity after cotransfection witheither wildtype wt offtarget ot cdkn2bas1 or luciferase control constructs ev with mirconmir141 in achn and caki1 cellsgenes such as dnmtl dnmt3a and dnmt3b werealso noted after 5azacdr treatment compared to control dmso in both achn and caki1 cell lines31mir141 overexpression phenocopies functional effectsobtained with cdkn2bas1 inhibition in vitro andsuppresses tumorigenicity and in vivowe sought to determine if cdkn2bas1 causes itsantitumorigenic effects through mir141 we checkedthe effect of mir141 overexpression in rcc cellstransient transfection of mir141 mimic in achn andcaki1 cells for h led to over expression of mir141compared to control mircon fig 4a also overexpression of mir141 significantly reduced cdkn2bas1 expression fig 4b indicating a reciprocal correlation between mir141 and cdkn2bas1 a significant decrease in cell proliferation over time fig 4cand marked decreasefig 4din clonogenicityofficial of the cell death differentiation association 0cdasgupta cell death and disease page of fig knockdown of lncrna cdkn2bas1 reduces tumorigenicity in renal cancer a relative expression of cdkn2bas1 in achn and caki1 cellstransfected with cdkn2bas1 sirnas b cell proliferation assessed by mts assay after knockdown of cdkn2bas1 in both cell lines with sirna2 cgraphical representation showing knockdown of cdkn2bas1 with sirna2 significantly decreased colony formation in achn and caki1 cellsd e achn and caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of cdkn2bas1f reduced migration and invasion in cdkn2bas1 sirna transfected cells compared to control treatment g changes in emt related proteins in bothachn and caki1after knockdown of cdkn2bas1compared to controls were also observed further westudied the therapeutic potential of mir141 in amouse xenograft model a significant decrease intumor growth was observed by intratumoral delivery ofmir141 mimic compared to control over the course ofexperiment average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received mir141 mimic fig 4e in additionmir141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both rcc celllines compared to controlfig 5a this proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyadpribose polymerase parp an increase in bax and adecrease in bcl2 at protein levels fig 5b a significantdecrease in migration fig 5c and invasion fig 5dwas also observed in both rcc cell lines with mir141overexpression we also examined emt markers aschange in migration and invasion are directly associated with emt our results showed an increase inepithelial markers αecatenin and claudin with concomitant decrease in mesenchymal markers fibronectinand vimentin at both protein fig 5e and mrnaofficial of the cell death differentiation associationfig s5 levels taken together overexpression of mir phenocopies the functional effects of cdkn2bas1 inhibition in vitro and tumor growth suppressioneffects in vivolike cdkn2bas1aremir141cdkn2bas1 interaction negatively regulatescyclind and its downstream effectors in rcccyclind1cyclind2alsooncogenic in rcc fig s6 and are direct targets of mir as discussed earlier lncrnas can act as cernas tocarry out their regulatory functions32“ we observedthat cdkn2bas1 shared regulatory mir141 bindingsites with cyclind1cyclind2 fig 6a and therebysponges mir141 allowing cyclind1d2 to be expressedin tumors to determine potential mir141cdkn2bas1cyclind interaction experimentally we performedrip assay both achn and caki1 cells over expressingmir141 revealed significant enrichment of cyclind1cyclind2 and cdkn2bas1 with ago2 as compared toigg control fig 6b moreover decreased luciferaseactivity also confirmed direct binding of mir141 tocyclind in mir141overexpressing achn andcaki1 cells compared to controls fig 6c we also found 0cdasgupta cell death and disease page of fig expression clinical significance and epigenetic regulation of mir141 in renal cancer a mir141 expression levels in achn caki1 andrptec cells b expression levels of mir141 in kirctcga cohort normal and tumor c expression levels of mir141 in normal n nonmetastatic n metastatic n kirc patient samples in tcga cohort d expression levels of mir141 in kirctcga cohort amongdifferent grades normal grade “ grade “ and stages normal stage i“ii and stage iii“iv e relative mir expression in rcc tissue vs matched adjacent normal regions n among different grades normal grade grade “ andin different stages normal stage i“ii stage iii“iv as assessed by qrtpcr sfvamc cohort f receiver operating curve roc analysisshowing ability of mir141 to differentiate between malignant and nonmalignant samples g methylation for kirc patient samples in tcgakirccohort for probe cg02624246 h overall survival with tcgakirc methylation as performed by kaplan“meier analysis i methylation status of mir141promoter in rcc cell lines compared to nonmalignant rptec as assessed by msqpcr j expression of mir141 in 5azacdr treated and untreatedachn and caki1 cell lines results are representative of three independent experiments p value calculated by student t test bar mean ± standarderror mean semthat overexpression of mir141 or inhibition of cdkn2bas1 significantly decreased cyclind1d2 expression atboth the mrna fig 6d e and protein levels figs 6f“hs8a b this effect was significantly attenuated by mir inhibitor fig s7 indicating that cyclind expressionis dependent on the interaction between mir141 andcdkn2bas1 we further observed a decrease in rac1 asmall gtpase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mrna levelsfigs s3 s5 and protein levels figs 6g i“j s8c“f inboth mir141 overexpressed and cdkn2bas1 inhibitedrcc cell lines which in turn are involved in regulatingcellular migrationinvasion cumulatively these resultsindicate that suppression of cdkn2bas1 by mir141inhibits renal cell proliferation invasion and migration byinhibiting cyclind rac1 and phosphorylation of paxillinofficial of the cell death differentiation association 0cdasgupta cell death and disease page of fig mir141 overexpression mimics the knockdown effect of lncrna cdkn2bas1 in vitro and reduces tumorigenicity in vivo a relativeexpression of mir141 in achn and caki1 cells transfected with mir141 mimic and control b significant decrease in cdkn2bas1 expressioncompared to control in both cell lines overexpressed with mir141 c rcc cell proliferation after transfecting with mir141 mimic and control asassessed by mts assay d colony formation and its graphical representation in mir141 overexpressing achn and caki1 cells compared to controlse pictures of excised tumors are taken at the termination of experiment day graph represents tumor volume after intratumoral injection ofcontrol or mir141 mimic into established tumors injection was started at day and was followed for days each mouse in both groups mirconand mir1413pmimic received a total of eleven injections intermittently data represent the mean of each group and error bars are sem results arerepresentative of three independent experiments p value calculated by student t test bar mean ± standard error mean semattenuation of mir141 exerts tumorigenic attributes innormal rptec cellswe next determined whether attenuation of mir141induces tumorigenic characteristics in normal rpteccells by targeting cdkn2bas1 and cyclind transienttransfection of mir141 inhibitor indeed showed a significant decrease in mir141 expression fig 7a and anincrease in cdkn2bas1 expression fig 7b along withother procancerous phenotypes such as increased cellproliferation fig 7c colony formation fig 7d migration and invasion fig 7e as compared to controlsadditionally a significant increase in cyclind1 cyclind2 rac1 and paxillin pxn expressions were observed inmir141 inhibited rptec cells fig 7f a noticeableincrease in prometastatic fibronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαecatenin genes were also observed in mir141 inhibited rptec cells compared to controls fig 7gdiscussionprior studies have shown the regulatory role of noncoding rnas in tumorigenesis especially in the emtofficial of the cell death differentiation associationpathway leading to cancer aggressiveness cdkn2bas1also known as anril is located at chromosome 9p21cdkn2bas1 is reported to be upregulated in tumortissues and function as an oncogenic lncrna in pancreatic ovarian and laryngeal squamous cell carcinoma36“ human mir141 is located at chromosome12p1331 and is transcribed from a mir200 familyclusterinterestingly expression of mir141 is controversial since it exhibits either oncogenic39“ or tumorsuppressive roles42“ in specific types of cancer theprime goal of the present study was to understand therole of cdkn2bas1mir141 interactions in regulatingrcc progression and metastasisin this study we identify cdkn2bas1 to be a crucialoncogenic lncrna that plays an important role in renalcarcinogenesis cdkn2bas1significantly overexpressed in rcc and the expression increases fromlower to higher grades and stages lncrna cdkn2bas1directly interacted with mir141 as it was found to be anovel target of mir141 in contrast to cdkn2bas1 weobserved significant attenuation of mir141 expression inrcc cell lines and tumor samples compared to normalis 0cdasgupta cell death and disease page of fig ectopic mir141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a apoptosis assessed by flowcytometric analysis of annexinvfitc 7aad“stained achn and caki1 cells transfected with mirconmir141mimic graph represents totalapoptosis early late b immunoblots showing apoptotic proteins in mirconmir141mimic treated achn and caki1 cells with gapdh asendogenous control c migration assay and d invasion assay as seen in pictures and graphical representation of both achn and caki1 cells after mir overexpression compared to control e immunoblot assay showing emt related proteins in mirconmir141mimic treated achn andcaki1 cells with βactin as endogenous control graphs are average of three independent experiments p value calculated by student t testbar mean ± standard error mean semcell line or matched normal samples as it is known thatextensive dna hypermethylation of cpg islands is highlycorrelated to activation of cancerspecific genes45 wechecked the methylation status of mir141 in normal andrcc tissues interestingly insilico analysis showed thepresence of cpg island in the promoter region of mir and we also found hypermethylation of mir141 intcga samples as compared to normal this hypermethylation is also found to be significantly associatedwith poor survival of patients similar results were alsoobserved in rcc cell lines compared to a normal rpteccell line functionally inhibition of cdkn2bas1 andoroverexpression of mir141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in rcc in vitro in vivodata show suppression of tumor growth by mir141overexpression conversely attenuation of mir141 innormal rptec cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionfrom a clinical point of view noncoding rnas signatures are powerful tools for early cancer diagnosisofficial of the cell death differentiation associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 our results revealed thathigher expression of cdkn2bas1 is positively correlatedwith poor overall survival probability of rcc patientsindicating its prognostic capability cdkn2bas1 canalso discriminate normal from tumor samples showing itsdiagnostic potential similarly mir141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be a diagnostic biomarker for rcc collectivelythese resultshighlight the biomarker potential of cdkn2bas1mir expression in rcc although it needs to be confirmedin a larger independent sample cohortinterestingly we found tha
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to machine learning with python a guide for data scientists o™reilly 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 œp  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 university™s 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 wwwnaturecomreprintspublisher™s 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'
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recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm
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"There are two kinds of localizing procedures: marking with thoracoscopically directly visible materials and marking with radio-opaque materials. Examples of directly visible materials are hook wire methylene blue and indocyanine green. Ethiodized oil (lipiodol) barium and iodine contrast agents are used for radio-opaque markers. Each marking method has strong and weak points. Localization with a hook wire is easy to perform but carries a high risk of pneumothorax and a propensity to dislodge during transport and surgical preparation (6 7). Methylene blue and indigo carmine have a tendency to diffuse over a large area by the time the operation is done and render localization features inadequate (8 9). The use of a radio-opaque marker (such as barium or lipiodol) 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"
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"A decrease of 50% or more from baseline urinary prostaglandin E2 metabolite after a 5-day open-label run-in period was used to select eligible patients. One hundred twenty patients (median age 64 years) were randomized (78 to AP/E and 42 to P/E). Overall median TTP was 1.8 months in the AP/E group and 2.1 months in the P/E group with a 12% objective response rate in both groups (intent-to-treat analysis). A subgroup analysis in patients aged 65 years or younger demonstrated a statistically significant TTP benefit for AP/E (hazard ratio 0.5 [95% confidence interval: not applicable“0.9]; p=0.018) and overall survival advantage at minimum 1-year follow-up (median 12.2 versus 4.0 months; hazard ratio=0.5; p=0.021). The most common adverse events were rash diarrhea fatigue and nausea. Toxicity contributed to early discontinuations in patients aged more than 65 years treated with AP/E. This is the first randomized placebo-controlled study of a COX-2 inhibitor in NSCLC to use a prospective patient-selection strategy. Although AP/E seemed to improve TTP and overall survival in a subset of patients aged 65 years or younger the primary endpoint of the trial was not met. Non“small-cell lung cancer Apricoxib Erlotinib Cyclooxygenase-2 inhibitor Prostaglandin E2 metabolite 0413066 2830 Cell Cell Cell 0092-8674 1097-4172 24630729 4040459 10.1016/j.cell.2014.02.031 NIHMS573682 Genetic and Clonal Dissection of Murine Small Cell Lung Carcinoma Progression by Genome Sequencing McFadden David G. 1 5 Papagiannakopoulos Thales 1 5 Taylor-Weiner Amaro 3 5 Stewart Chip 3 5 Carter Scott L. 3 5 Cibulskis Kristian 3 Bhutkar Arjun 1 McKenna Aaron 3 Dooley Alison 1 Vernon Amanda 1 Sougnez Carrie 3 Malstrom Scott 1 Heimann Megan 1 Park Jennifer 1 Chen Frances 1 Farago Anna F. 1 Dayton Talya 1 Shefler Erica 3 Gabriel Stacey 3 Getz Gad 3 4 * Jacks Tyler 1 2 * 1Koch Institute for Integrative Cancer Research and Department of Biology Massachusetts Institute of Technology Cambridge MA 02142 USA 2Howard Hughes Medical Institute Massachusetts Institute of Technology Cambridge MA 02142 USA 3Cancer Program Broad Institute of MIT and Harvard Cambridge MA 02142 USA 4Cancer Center and Department of Pathology Massachusetts General Hospital Boston MA 02114 USA *Correspondence: gadgetzbroadinstitute. (G.G.) tjacksmit.edu (T.J.) 5 Co-first author 6 5 2014 13 3 2014 13 3 2015 156 6 1298 1311 2014 Elsevier Inc. 2014 Summary Small cell lung carcinoma (SCLC) is a highly lethal smoking-associated cancer with few known targetable genetic alterations. Using genome sequencing we characterized the somatic evolution of a genetically engineered mouse model (GEMM) of SCLC initiated by loss of Trp53 and Rb1. We identified alterations in DNA copy number and complex genomic rearrangements and demonstrated a low somatic point mutation frequency in the absence of tobacco mutagens. Alterations targeting the tumor suppressor Pten occurred in the majority of murine SCLC studied and engineered Pten deletion accelerated murine SCLC and abrogated loss of Chr19 in Trp53; Rb1; Pten compound mutant tumors. Finally we found evidence for polyclonal and sequential metastatic spread of murine SCLC by comparative sequencing of families of related primary tumors and metastases. We propose a temporal model of SCLC tumorigenesis with implications for human SCLC therapeutics and the nature of cancer-genome evolution in GEMMs. J Natl Cancer Inst J. Natl. Cancer Inst jnci jnci.j JNCI Journal of the National Cancer Institute 0027-8874 1460-2105 Oxford University Press US 24317180 3906987 10.1093/jnci/djt338 Brief Communication Novel Germline Mutation in the Transmembrane Domain of HER2 in Familial Lung Adenocarcinomas Yamamoto Hiromasa Higasa Koichiro Sakaguchi Masakiyo Shien Kazuhiko Soh Junichi Ichimura Koichi Furukawa Masashi Hashida Shinsuke Tsukuda Kazunori Takigawa Nagio Matsuo Keitaro Kiura Katsuyuki Miyoshi Shinichiro Matsuda Fumihiko Toyooka Shinichi Affiliations of authors:Department of Thoracic Breast and Endocrinological Surgery (HY KS JS MF SH KT SM ST) Department of Clinical Genomic Medicine (KS ST) Department of Cell Biology (MS) Department of Pathology (KI) and Department of Hematology Oncology and Respiratory Medicine (KK) Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences Okayama Japan; Center for Genomic Medicine Kyoto University School of Medicine Kyoto Japan (KH FM); Department of General Internal Medicine 4 Kawasaki Medical School Okayama Japan (NT); Department of Preventive Medicine Kyushu University Faculty of Medical Sciences Fukuoka Japan (KM). Correspondence to: Shinichi Toyooka MD PhD Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences Clinical Genomic Medicine/Thoracic Breast and Endocrinological Surgery 2-5-1 Shikata-cho Kita-ku Okayama Okayama 700“8558 Japan (e-mail: toyookamd.okayama-u.ac.jp). 1 2014 7 12 2013 7 12 2013 106 1 djt338 7 7 2013 14 10 2013 16 10 2013 The Author 2013. Published by Oxford University Press. 2013 This is an Open Access distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons./licenses/by-nc-nd/3.0/) which permits non-commercial reproduction and distribution of the work in any medium provided the original work is not altered or transformed in any way and that the work is properly cited. For commercial re-use please contact journals.permissionsoup.com We encountered a family of Japanese descent in which multiple members developed lung cancer. Using whole-exome sequencing we identified a novel germline mutation in the transmembrane domain of the human epidermal growth factor receptor 2 (HER2) gene (G660D). A novel somatic mutation (V659E) was also detected in the transmembrane domain of HER2 in one of 253 sporadic lung adenocarcinomas. Because the transmembrane domain of HER2 is considered to be responsible for the dimerization and subsequent activation of the HER family and downstream signaling pathways we performed functional analyses of these HER2 mutants. Mutant HER2 G660D and V659E proteins were more stable than wild-type protein. Both the G660D and V659E mutants activated Akt. In addition they activated p38 which is thought to promote cell proliferation in lung adenocarcinoma. Our findings strongly suggest that mutations in the transmembrane domain of HER2 may be oncogenic causing hereditary and sporadic lung adenocarcinomas. Familial lung cancers are rare among human malignancies. Recent studies have reported that germline mutations in the epidermal growth factor receptor (EGFR) gene predispose the development of lung cancer. Reported familial lung adenocarcinomas with a germline EGFR mutation such as T790M carry secondary somatic EGFR mutations including exon 19 deletion and exon 21 L858R mutation (1“4). We encountered a family of Japanese descent in which multiple members developed lung cancer (). The proband (III-4) was a 53-year-old woman with multiple lung adenocarcinomas in bilateral lungs. She was a light smoker with a 1.2-pack-year history of smoking. She had undergone a left lower lobectomy for multiple lung adenocarcinomas at the age of 44 years. Her mother (II-4) a never smoker also had multiple lung adenocarcinomas. Partial pulmonary resections of two tumors were performed for II-4 for the purpose of diagnosis after pleural dissemination was found during surgery and multiple lesions were removed in a lobectomy or partial resections in III-4. A histological examination of the resected tumors in II-4 revealed nonmucinous adenocarcinoma in situ and nonmucinous minimally invasive adenocarcinoma whereas the histological findings of pleural dissemination indicated mucus-containing adenocarcinoma. Those of III-4 contained various subtypes of adenocarcinoma including nonmucinous and mucinous adenocarcinoma in situ and invasive mucinous adenocarcinoma. In addition normal-appearing lung parenchyma obtained from a lobectomy in III-4 revealed innumerable small preinvasive lesions implying the presence of precancerous changes throughout the lung (Supplementary available online). Sequencing analyses of EGFR exons 18 to 21 and KRAS as well as an immunohistochemical staining for ALK protein in the resected tumors indicated no genetic alterations in these genes. The pedigree chart suggested that lung cancer was inherited in an autosomal dominant manner. . Pedigree chart of a Japanese family in which multiple members developed lung cancer. The boxes and circles indicate men and women respectively. The numbers at the bottom of each member indicate the age at the time of death or the time of the analysis. An oblique line shows deceased family members. The proband (III-4) had multiple lung adenocarcinomas (arrow). "
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"Single agent and combination treatment protocols were well tolerated by mice with no weight loss or other signs of acute or delayed toxicity (A-C). Antitumor activity of AZD6244 and/or BEZ235 in mouse xenograft models of human tumors. Nude mice-bearing NCI-H1993 (A) NCI-H1975 (B) and NCI-H460 (C) tumors were administered 25 mg/kg AZD6244 twice daily and/or 20 mg/kg BEZ235 once daily up to 21 days. Tumors were resected from nude mice on day 21 (D). Tumor volume was measured using calipers on the indicated days. Mean?±?SD n?=?10. * P?<?0.05 vs control group. ** P?<?0.01 vs control group. Changes in body weight of mice treated with control group or AZD6244 and/or BEZ235. Nude mice-bearing NCI-H1993 (A) NCI-H1975 (B) and NCI-H460 (C) tumors were administered 25 mg/kg AZD6244 twice daily and/or 20 mg/kg BEZ235 once daily up to 21 days. Body weight was measured on the indicated days. Mean?±?SD n?=?10. Effect of MEK and PIK3/mTOR inhibitors on signaling transduction pathways in gefitinib-resistant NSCLC tumor models To assess the impact of both compounds on downstream molecules of the MEK and PI3K pathways we used Western blot analysis to observe phosphorylation status and total protein expression in tumor tissues. The results showed that p-MEK1/2 p-ERK1/2 p-AKT p-S6 and p-4E-BP1 appeared to be inhibited by AZD6244 and BEZ235 combination treatment whereas the total protein levels of MEK1/2 ERK1/2 AKT S6 and 4E-BP1 remained unchanged in each tumor model (). Western blot analysis of downstream signals also showed treatment with AZD6244 or BEZ235 inhibited the phosphorylation of ERK1/2 or AKT in all three tumor models respectively. In addition combined treatment with AZD6244 and BEZ235 showed greater inhibition of p-ERK1/2 and p-AKT than observed in control group or mice treated with each compound alone in vivo (). Interestingly the impact from both inhibitors on p-S6 and p-4E-BP1 levels was alternatively tumor model specific. For example AZD6244 and BEZ235 alone and in combination markedly inhibited p-S6 and p-4E-BP1 expression levels in NCI-H1993 tumor models compared with the minimal suppression observed in NCI-H460 tumor model (). Neither AZD6244 nor BEZ235 alone suppressed p-S6 and p-4E-BP1 in NCI-H1975 tumor model. We also found that the expression of MMP-9 was significantly inhibited by AZD6244 and BEZ235 combination treatment whereas the expression of MMP-2 was not affected by the treatment. Effects of AZD6244-BEZ235 combination therapy on PI3K/AKT and MEK/ERK pathways. All three gefitinib-resistant tumor xenograft models were treated with the AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study tumor tissues were then harvested to detect p-AKT (S473)/AKT p-ERK (T202/Y204)/ERK p-S6 (S240/244)/S6 and p-4E-BP1 (S65)/4E-BP1. Effect of MEK and PIK3/mTOR inhibitors on the expressions of Ki-67 and CD31 in gefitinib-resistant NSCLC tumor models To characterize the mechanism of tumor growth inhibition observed in our gefitinib-resistant NSCLC tumor models by AZD6244 and BEZ235 lung tumor tissues were assessed by evaluating Ki-67 expression using immunohistochemical analyses. We observed active cell proliferation in NCI-H1993 tumor model with a 56% proliferation index (A). Monotherapy with AZD6244 or BEZ235 slightly decreased the percentage of Ki-67-positive proliferating tumor tissues with proliferation indices of 42% and 39% respectively (A). Combined treatment with AZD6244 and BEZ235 markedly decreased the percentage of Ki-67-positive proliferating tumor tissues to 10% consistent with the marked inhibition of ERK1/2 and AKT phosphorylation. We also found the similar results in NCI-H1975 and NCI-H460 tumor models (A).To evaluate the potential antiangiogenic mechanism of AZD6244 and BEZ235 gefitinib-resistant NSCLC tumor tissues were analyzed by immunostaining for CD31 (platelet endothelial cell adhesion molecule 1). The results showed BEZ235 significantly decreased the vascular density of all three gefitinib-resistant NSCLC tumors whereas AZD6244 monotherapy had only a mildly effect upon lung tumor angiogenesis. The antiangiogenic effects AZD6244 and BEZ235 were markedly increased when they were combined (B). Effects of AZD6244 and/or BEZ235 on the expressions of Ki-67 (A) and CD31 (B) in NCI-1993 NCI-1975 and NCI-H460 xenograft models. All three gefitinib-resistant tumor xenograft models were treated with the AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study tumor tissues in each group were resected and immunostained with anti-Ki67 and anti-CD31 antibodies. N?=?10 * P?<?0.05 vs vehicle group. ** P?<?0.01 vs vehicle group. MEK and PIK3/mTOR inhibitors had no effect on caspase-3 8 and 9 activities in gefitinib-resistant NSCLC tumor models In order to investigate whether AZD6244 and/or BEZ235 would induce apoptosis in gefitinib-resistant NSCLC tumor models activity of caspase-3 8 and 9 were measured by the colorimetric assay. The results showed that AZD6244 and/or BEZ235 had no effect on caspase-3 8 and 9 activities in all three gefitinib-resistant NSCLC tumor models (). Effects of AZD6244 and/or BEZ235 on the activities of caspase-3 8 and 9 in NCI-1993 NCI-1975 and NCI-H460 xenograft models. All three gefitinib-resistant tumor xenograft models were treated with the AZD6244 and/or BEZ235 for 2 h on Day 21 of the efficacy study tumor tissues in each group were resected and measured by caspase colorimetric protease kits. N?=?10. Discussion Although advances have been made in cancer treatment with the development of selective molecular targeted therapies several relevant issues for their optimal and effective use remain unsolved. Recent studies have demonstrated that the EGFR-TKI gefitinib and erlotinib are associated with a high response rate and prolong progression-free survival in patients with EGFR mutant lung cancer [22]. Responders to these agents however later relapse after acquiring EGFR-TKI resistance making it urgent to develop novel therapeutic agents that can overcome acquired resistance to EGFR-TKI. Current clinical approaches to combat resistance in lung adenocarcinoma include irreversible and mutant-selective inhibitors of EGFR combination of cetuximab and afatinib [23] and combination of an EGFR inhibitor with a drug targeting a resistance pathway such as the combination of gefitinib and a MET inhibitor [24]. However alternative RTK pathways that are activated following EGFR inhibition are another area for investigation. These alternative pathways may bypass or evade inhibition of EGFR signaling thereby enabling combinations of agents to simultaneously attack multiple molecular targets for cancer growth inhibition. One potential solution to overcome multiple mechanisms of resistance is to target downstream pathways. In this study we show that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor is effective against tumor cell lines refractory to gefitinib by three different mechanisms: an EGFR gatekeeper T790M mutation MET amplification and KRAS/PIK3CA mutation. To our knowledge this is the first report of the effects of MEK TKI with PI3K/mTOR TKI therapy in gefitinib-resistant models of NSCLC. MEK is a potentially relevant molecular therapeutic target since it is the activated downstream of the axis RAS/RAF proteins and in turn activates ERK to induce cell proliferation. Therefore several selective MEK inhibitors have been developed [25] and more than ten MEK inhibitors have entered early clinical trial evaluation [26]. Unfortunately clinical activity as single agent has been rarely observed with MEK inhibitors in gefitinib-resistant NSCLC patients [27]. The benefit of blocking an individual pathway has been largely limited by the presence of a compensatory feedback loop between PI3K and MEK. For example inhibition of the MEK pathway results in activation of the PI3K pathway [28] and PI3K activation mediates resistance to MEK inhibition [29]. In our study in order to circumvent this compensatory feedback concurrent blockade of the two pathways has been tested and synergy in antitumor effects was detected providing the rationale for phase I clinical trials. Moreover early signs of clinical benefit have been reported in advanced cancer by a retrospective analysis on patients receiving agents that target both pathways [30]. Gatekeeper mutations the T790M mutation in EGFR associated with resistance to gefitinib [31] are common mechanisms by which tumor cells acquire resistance to molecularly targeted drugs. Although irreversible EGFR-TKIs including BIBW2992 have been developed to overcome T790M-mediated resistance to gefitinib [32] recent clinical trials have failed to show that monotherapy with irreversible EGFR-TKIs has benefits in patients with NSCLC refractory to gefitinib [33]. This may be due at least in part to the low selectivity of this class of compounds to wild-type and mutant EGFR. In addition because HGF overexpression was frequently observed in tumors with the gatekeeper T790M mutation [34] monotherapy with a mutant-selective EGFR-TKI may not be sufficient to inhibit the growth of tumors with acquired resistance to gefitinib. Our findings suggest that the combination of a MEK-TKI and a PIK3/mTOR-TKI may be effective in controlling these resistant tumors. Many KRAS-mutant cancer cells have been shown to be sensitive to MEK inhibitors [35] and KRAS mutations can be detected in up to 30% of lung cancers dependent upon histology and ethnicity [36-38] suggesting that a subset of lung cancers would likely be highly sensitive to AZD6244. Our finding that AZD6244 was effective in one distinct KRAS mutant human lung cancer NCI-H460 models supports and validates this hypothesis. Although monotherapy with AZD6244 resulted in antitumor and some antiangiogenic effects in all of our lung cancer models the antitumor effects were more apparent in the NCI-H1993 lung adenocarcinoma model. The increased antitumor efficacy observed in this model is associated with differences in the inhibitory effect of p-AKT signaling pathway in NCI-H1975 and NCI-H460 lung tumors. However additional studies are needed to elucidate this phenomenon. In this study we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. MEK or PI3K/mTOR inhibition resulted in antitumor effects for these gefitinib-resistance NSCLC models by blocking key intracellular pathways controlling cell proliferation and survival as demonstrated both in vitro and in vivo. Surprisingly PI3K/mTOR inhibition by BEZ235 also suppressed lung tumor angiogenesis and targeted both MEK and PI3K/mTOR activation in lung tumors resulting in substantial antiangiogenic effects which may due to the significantly reduced expression of MMP-9 in tumors. "
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the european commission asked efsa for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food this risk assessment coversedible parts of potato plants and other food plants containing gastomato andaubergine in humans acute toxic effects of potato gas asolanine and achaconine includegastrointestinal symptoms such as nausea vomiting and diarrhoea for these effects the contampanel identified a lowestobservedadverseeffect level of mg total potato gaskg body weight bwper day as a reference point for the risk characterisation following acute exposure in humans noevidence of health problems associated with repeated or longterm intake of gas via potatoes hasbeen identified no reference point for chronic exposure could be identified from the experimentalanimal studies occurrence data were available only for asolanine and achaconine mostly forpotatoes the acute dietary exposure to potato gas was estimated using a probabilistic approach andapplying processing factors for food due to the limited data available a margin of exposure moeapproach was applied the moes for the younger age groups indicate a health concern for the foodconsumption surveys with the highest mean exposure as well as for the p95 exposure in all surveysfor adult age groups the moes indicate a health concern only for the food consumption surveys withthe highest p95 exposures for tomato and aubergine gas the risk to human health could not becharacterised due to the lack of occurrence data and the limited toxicity data for horses farm andcompanion animals no risk characterisation for potato gas could be performed due to insufficient dataon occurrence in feed and on potential adverse effects of gas in these species european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritykeywords glycoalkaloids gas solanine chaconine potato margin of exposure moe food feedrequestor european commissionquestion number efsaq201600811correspondence contamefsaeuropaeu leon brimer was a member of the working group on glycoalkaloids in food and feed until august wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodpanel members margherita bignami laurent bodin james kevin chipman jes 13us del mazo bettinagraslkraupp christer hogstrand laurentius ron hoogenboom jeancharles leblanc carlo stefanonebbia elsa nielsen evangelia ntzani annette petersen salomon sand dieter schrenk tanjaschwerdtle christiane vleminckx and heather wallaceacknowledgements the panel wishes to thank the following for the support provided to thisscientific output kelly niermans the panel wishes to acknowledge all european competentinstitutions member state bodies and other anisations that provided consumption and occurrencedata for this scientific outputsuggested citation efsa contam panel efsa panel on contaminants in the food chain schrenk dbignami m bodin l chipman jk del mazo j hogstrand c hoogenboom lr leblanc jc nebbia csnielsen e ntzani e petersen a sand s schwerdtle t vleminckx c wallace h brimer l cottrill bdusemund b mulder p vollmer g binaglia m ramos bordajandi l riolo f rold 13antorres r and graslkraupp b scientific opinion “ risk assessment of glycoalkaloids in feed and food in particular inpotatoes and potatoderived products efsa pp 102903jefsa20206222issn european food safety authority efsa published by john wiley and sons ltd on behalfof european food safety authoritythis is an open access under the terms of the creative commons attributionnoderivs licensewhich permits use and distribution in any medium provided the original work is properly cited and nomodifications or adaptations are madereproduction of the images listed below is prohibited and permission must be sought directly from thecopyright holderfigure elsevier figure springer figure american chemical society springerthe efsa is a publication of the european foodsafety authority an agency of the european unionwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodsummarythe european commission asked efsa for a scientific opinion on the risks for animal and humanhealth related to the presence of glycoalkaloids gas in feed and food in particular in potatoes andpotatoderived products this risk assessment covers edible parts of potato plants and other foodplants containing gas in particular tomato and aubergine nonedible parts of ga containing plantshave not been considered with the exception of potato sprouts the panel developed the draftscientific opinion which underwent a public consultation from february to april thecomments received and how they were taken into account when finalising the scientific opinion werepublished in an efsa technical report efsa gas are present in many plants of the family of solanaceae and contribute to plant resistanceagainst pests and pathogens gas are composed of a steroidal aglycone and an oligosaccharide sidechain in commercial potato cultivars s tuberosum the main gas are achaconine and asolanineconsisting of the aglycone solanidine and chacotriose and solatriose as oligosaccharide side chainsrespectively the aubergine fruit s melongena contains primarily the gas asolamargine and asolasonine composed of the aglycone solasodine and chacotriose and solatriose respectively inlycopersicum atomatine and adehydrotomatine are the major gas withtomato fruitlycotetraose coupled to the aglycones tomatidine and tomatidenol respectivelyshuman risk assessmentin experimental animals the potato gas asolanine and achaconine show a relatively low oralbioavailability with differences between species hamsters exhibit higher absorption and slowerexcretion rates for both substances when compared to rats due to the limited information themetabolic profiles of potato gas in experimental animals could not be characterisedin humans asolanine and achaconine are systemically absorbed following ingestion for bothsubstances relatively long serum halflives were reported suggesting a possible accumulation the bloodclearance of the respective aglycone solanidine appears to be slow accordingly levels of solanidine wereregularly detected in the blood of human volunteers in several studies suggesting hydrolysis of gas nofurther information is available on metabolism and excretion of potato gas in humansthere are no toxicokinetic data on tomato and aubergine gas and their aglycones in experimentalanimals and humansin acute oral toxicity studies no adverse effects of asolanine were observed at doses of mgkgbody weight bw per day in rats and mgkg bw per day in mice reliable data on other potatogas or tomato and aubergine gas and their aglycones are missingin repeated oral dose studies on potato gas rodents showed nonspecific effects such as reducedbody weight and relative liver weight with indication of similar potencies of asolanine and achaconine hamsters exhibited these symptoms after a 5day treatment with mg of asolanine ora chaconinekg bw per day while mice showed these effects after one week of daily treatments with mg of asolanine or mg of achaconinekg bw solanidine however increased the absoluteand relative liver weight at mgkg bw per day in mice suggesting a different effect of theaglycone compared to the gasthe tomato ga atomatine and its aglycone tomatidine exerted no effects in rats when applied at mgkg bw per day for a period of day at higher doses atomatine reduced the cholesterol uptakeand increased fecal sterol and coprostanol excretion in hamsters and rats in mice a to 2weektreatment with the aubergine ga asolasonine increased the body weight gain at mgkg bw perday while its aglycone solasodine decreased body weight gain and caused gastric gland degenerationand liver toxicity at mgkg bw per daydevelopmental studies have been performed mainly in hamsters treated with potato gas and theiraglycones for only one day or for a short very restricted time period during gestation outcomes weremainly analysed in late gestational embryos and comprised effects in the central nervous systempredominantly exencephaly encephalocele and anophthalmia these malformations occurred at dosesof mgkg bw per day and above for gas and of mgkg bw per day and above for theaglycones no noobservedadverseeffectlevelloael could be identified from these studies reduced postnatal survival of pups due to insufficientmilk production was reported when pregnant holtzman rats had been exposed to mg of asolaninekg bw per day studies on the male fertility in dogs have been performed only with theaubergine aglycone solasodine decreased epididymal weight and cauda epididymal epithelial heightand also an epididymal lumen depleted of sperm occurred in dogs after mgkg bw per day givenlowestobservedadverseeffectnoael orlevelwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodfor month similar effects were observed in rhesus monkeys exposed to mgkg bw per day for monthsfrom the limited number of studies available there was no evidence for genotoxicity of the potatogas asolanine and achaconine and the aglycone solanidine as well as for the aubergine ga asolamargine however there is not sufficient information to conclude on the genotoxic potential ofthese gasno longterm chronic toxicitycarcinogencity study for potato tomato or aubergine gas or for therespective aglycones could be identifiedin humans acute toxic effects following ingestion of potato gas include gastrointestinal symptomsof varying severity such as vomiting diarrhoea and abdominal pain which may occur from a totalpotato gas potato tga intake of mgkg bw or more further symptoms including drowsinessapathy confusion weakness vision disturbances rapid and weak pulse and low blood pressure maybe the consequence of dehydration following vomiting and diarrhoeain severe cases paralysis respiratory insufficiency cardiac failure coma and death have beenreported doses in the range of “ mg potato tgaskg bw are considered to be potentially lethal forhumans results from limited volunteer studies suggest possible differences in the human populationwith respect to the individual susceptibility towards adverse effects associated with the intake ofpotato gasregarding the mode of action adverse effects of gas may be due to their ability to complex withmembrane 3bhydroxy sterols thereby causing disruption and loss of integrity of cell membranesafter oral exposure these effects may affect the mucosa of the gastrointestinal tract and cause thesymptoms observed in intoxicated humans such as nausea vomiting and diarrhoeagas inhibit acetylcholinesterase ache and serum butyrylcholinesterase buche by a reversiblecompetitive mode of action the relative potency of inhibition of asolanine and achaconine appearsto be similar the aglycones exert weak or no inhibitory effects the excess of acetylcholine at theneuronal and neuromuscular junctions upon inhibition of the enzymes might also contribute to thesymptoms described for intoxications with gasat high doses atomatine may form a nonabsorbable complex with cholesterol and other sterols inthe enteral lumen which may impair the absorption of cholesterol as a consequence blood cholesterollevels were lowered in rodentsthe contam panel considered that the use of rodent data on acute toxicity was not appropriate toestablish a reference point for acute exposure to potato gas in humans the contam panel selectedthe loael of mg potato tgakg bw per day as the reference point for acute risk characterisationbased on human data from case reports outbreaks and studies in volunteers the available data onacute toxicity were considered insufficient to establish a healthbased guidance value instead thepanel used the margin of exposure moe approach to assess a possible health concern from acuteexposure to potato tgas via foodassuming the main symptoms to be mainly due to localirritation of the gastrointestinal mucosarather than inhibition of ache activity the panel considered that the possible interindividual variabilityin toxicodynamics is more relevant than the interindividual variability in toxicokinetics accordingly anmoe higher than indicates that there is no health concern this moe of takes into account theextrapolation from a loael to a noael a factor of and the interindividual variability intoxicodynamics a factor of the experimental data available for repeated dose toxicity are not sufficient to identify a referencepoint for chronic exposure to potato gas in humans no evidence of health problems associated withrepeated or longterm intake of gas via potatoes has been identifiedregarding gas or aglycones occurring in edible parts of food plants other than s tuberosum nosuitable study for determining a reference point for tomato or aubergine gas or aglycones wasidentifiedoccurrence data were only available for asolanine and achaconine and mostly for ˜maincroppotatoes™ and ˜new potatoes™ few data were available for processed food no data on the occurrenceof tomato and aubergine gas and their aglycones were submitted to efsasince the occurrence data on potato gas did not cover all the food categories containing potatoesin the consumption database it was decided that the best approach for the exposure assessmentwould be to use the occurrence data in the raw primary commodities rpc maincrop potatoes andnew potatoes and the rpc consumption database the panel decided to combine the occurrence of˜new potatoes™ with that of ˜maincrop potatoes™ and the mean upper bound ub occurrence sum ofwwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodasolanine and achaconine for these two groups was mgkg and the p95 occurrence was mgkg the minimum and maximum reported concentrations were and mgkg respectivelythe acute dietary exposure to potato tgas was estimated using a probabilistic approach includingonly days in which there was consumption of maincrop potatoes as no occurrence data wereavailable for gas in tomato and aubergine these foods were not included in the exposure assessmentprocessing of potatoes has been reported to reduce the content of gas in the final processedproduct in general and according to the literature the peeling of potatoes reduced the ga contentby “ boiling in water and blanching of peeled potatoes by “ and frying in oil of peeledpotatoes by “ microwave and oven baking of unpeeled potatoes may cause a reduction in thega content by “ and by “ respectively no information has been found about thechemical nature of the ga degradation products for the exposure assessment processing factors forthe major food processing steps comprising peeling and heat processing boiling frying bakingwere applied to the occurrence data as follows processing factors between and wereattributed to the peeling of potatoes between and for frying and deep frying and between and for all other cooking methodsinformation about the peeling of potatoes was not available in the consumption database but itwas assumed that of the potatoes are consumed as peeled where information of the cookingmethod was not available a cooking method was randomly attributed to the eating event based onthe relative frequency of cooking methods reportedthe mean ub exposure to potato tgas across surveys ranged from lgkg bw per day inadults to lgkg bw per day in toddlers the 95th percentile exposure ranged from lgkgbw per day in adults to lgkg bw per day in toddlers up to lgkg bw per day in theupper limit of the confidence intervalcomparing the loael for potato tgas of mgkg bw per day with the acute exposure estimatesthe moes for the younger age groups indicate a health concern for the food consumption surveys withthe highest mean exposure as well as for the p95 exposure in all surveys for adult age groups themoes indicate a health concern only for the food consumption surveys with the highest p95exposuresthe contam panel calculated the mean percentage of days with potato consumption acrosssurveys per age group on which the potato tga intake may be below the moe of the highestnumber of survey days with intake of potatoes below the moe of was estimated for toddlers followed by children for the other age groups the estimated tga intake was below the moeof in up to “ of the survey daysfor tomato and aubergine gas the risk to human health could not be characterised due to the lackof occurrence data in food and the limited information on the adverse effects in experimental animalsand humansthe contam panel considered that the impact of the uncertainties on the risk assessment of acuteexposure to potato gas in food is moderate and that overall the identified uncertainties may eithercause an over or underestimation of the riskfarm animals horses and companion animals risk assessmentinformation on the toxicokinetics of gas was limited to ruminants for which the data suggest anextensive conversion of asolanine and achaconine to aglycones in rumen and a low potential ofsolanidine to transfer into cows™ milkno data on the potential adverse effects of potato gas in horses companion animals cats anddogs or fur animals were identified due to an insufficient database on the adverse effects of gas inruminants pigs poultry rabbits and fish an acute reference dose could not be derivedpotatoes are not grown specifically as feed for livestock but when supply exceeds marketrequirements for human consumption whole raw potatoes may be used as feed for ruminants andpigs some byproducts of potato processing and starch extraction are used as feeds for farmedlivestock principally nonruminants and for companion animalsdata on potato gas in feed were insufficient to perform an exposure assessmentthus no risk characterisation could be performed due to insufficient occurrence data of gas forfeed and the lack of or limited data on the adverse effects of gas in farm animals horses orcompanion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodrecommendationsthe following needs have been identified to improve the risk assessment for humans and reducethe uncertaintiescid129research on the occurrence of gas and their aglycones and other potentially toxicologicallyrelevant secondary plant metabolites in the potato cultivars available on the market and onnew potato cultivars resulting from breeding experimentscid129 occurrence data on gas and their aglycones in potato processed products including foods forinfantscid129 occurrence data on gas and their aglycones in tomato and aubergine and products thereofcid129 data on the toxicokinetics of potato tomato and aubergine gas and aglycones in experimentalanimals and humanscid129 data on repeated dose toxicity including reproductive and developmental toxicity of potatotomato and aubergine gas and aglycones in experimental animalsstudies in humans linking dietary exposure biomarkers of exposure and adverse effectscid129the following needs have been identified to improve the risk assessment for farm animals horsesand companion animals and reduce the uncertaintiescid129 occurrence data on potato gas and their aglycones in feedcid129studies on the kinetics and the potential adverse effects from feed material containing gas ofpotato gas in farm animals horses and companion animalswwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodtable of contentsabstractsummaryintroduction background and terms of reference as provided by the requestor interpretation of the terms of reference supporting information for the assessment chemistry analytical methods sources potatoes tomatoes aubergine previous risk assessments legislation and other standards data and methodologies methodology for data collection selection of evidence and study appraisal food and feed occurrence data submitted to efsa data collection and validation data analysis food and feed consumption data food consumption data feed consumption data food classification methodology for exposure assessment methodology for risk characterisation assessment hazard identification and characterisation toxicokinetics experimental animals asolanine achaconine humans mixtures of asolanine and achaconine solanidine biomarkers of exposure farm animals horses and companion animals summary on toxicokinetics toxicity in experimental animals acute toxicity studies gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum summary on acute toxicity studies repeated dose toxicity studies gas and aglycones from edible parts of s tuberosum gas and aglycones from edible parts of food plants other than s tuberosum developmental and reproductive toxicity studies developmental effects reproductive effects immunotoxicity studies studies on cardiovascular effects neurotoxicity studies genotoxicity gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum carcinogenicity studies studies on metabolic effects gas from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum observations in humans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and food gas from s tuberosum reports on intoxications studies in human volunteers epidemiological studies summary gas from food plants other than s tuberosum case reports adverse effects in farm animals horses and companion animals ruminants pigs poultry rabbits fish horses companion animals cats and dogs fur animals reports on intoxications mode of action membrane effects with implications for the gastrointestinal tract inhibition of cholinesterases ches comparative determination of inhibition of ches in vitro determination of inhibitory constants ki for gas on inhibition of ches in vitro inhibition of ches in vivo developmental and reproductive effects of gas and their aglycones inhibition of cholinesterases and effects in the immune system interference with metabolism considerations of critical effects and doseresponse analysis for the human risk assessment gas from edible parts of s tuberosum considerations of critical effects and doseresponse analysis derivation of a healthbased guidance value hbgv or margin of exposure moe approach gas from edible parts of food plants other than s tuberosum considerations of critical effects and doseresponse analysis consideration of critical effects and doseresponse analysis for the farm animal horses andcompanion animals risk assessment occurrence data occurrence data submitted to efsa previously reported occurrence data in the open literature literature on occurrence data on food occurrence data on gas in potatoes occurrence data on gas in tomatoes occurrence data on gas in aubergines occurrence data on gas in other food products literature occurrence data in feed influence of storage and processing on the content of gas gas from s tuberosum storage of potatoes processing of potatoes for food consumption processing of potatoes for feed gas from food plants other than s tuberosum summary on the influence of storage and processing on the levels of gas exposure assessment current acute dietary exposure assessment for humans previously reported dietary exposure assessments current dietary exposure assessment for farm animals horses and companion animals risk characterisation human health risk characterisation ga from edible parts of s tuberosum gas from edible parts of food plants other than s tuberosum farm animals horses and companion animal risk characterisation uncertainty analysis assessment objectives exposure scenarioexposure model wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodhazard identification and characterisation summary of uncertainties conclusions hazard identification and characterisation toxicokinetics toxicity in experimental animals observations in humans adverse effects in farm animals horses and companion animals mode of action margin of exposure moe approach occurrence and exposure food feed risk characterisation human health risk characterisation farm animals horses and companion animal health risk characterisation recommendations documentation provided to efsa references abbreviations appendix a “ major glycoalkaloids and their aglycones present in solanum species appendix b “ identification and selection of evidence relevant for the risk assessment of glycoalkaloids infeed and food appendix c “ details of the study design of the toxicokinetic studies appendix d “ comparison of developmental toxicity of single dose studies appendix e “ inhibition of cholinesterases by gas appendix f “ rapid alert system for food and feed rasff reports on the presence of solanum nigrum infood products appendix g “ studies on the toxicity of glycoalkaloids not considered in the risk assessment appendix h “ additional scenario for the human risk characterisation annex a “ occurrence data in food and feed submitted to efsa and dietary exposure assessment forhumans wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodintroductionbackground and terms of reference as provided by the requestorbackgroundmany plants in the family solanaceae contain glycoalkaloids and they are considered to be naturaltoxins the plant glycoalkaloids are toxic steroidal glycosides and the commonest types found in foodplants are asolanine and achaconine their natural function is probably to serve as stress metabolitesor phytoalexins for the protection of the plant when attacked by insects fungi etcamongst the most widely cultivated food crops aubergines tomatoes and potatoes are in thesolanaceae family but the levels of glycoalkaloids in tomatoes and aubergines are generally quite lowthe glycoalkaloids of most relevance to food safety are those occurring in the potato thepredominant toxic steroidal glycosides in potato are asolanine and achaconine they occur in potatotubers peel sprouts berries leaves and blossoms and their concentration in tubers depends on anumber offactors concentrations ofglycoalkaloids are “ times greater in the peel than in the flesh there is considerable variation inglycoalkaloid content among potato cultivars storage conditions especially light and temperature aremainly responsible for increases in solanine although the glycoalkaloid content can increase in thedark the rate of formation is only about the rate of formation in light increases of solanine inthe potato peel are closely associated with greening synthesis of chlorophyll of the peel thesebiochemical processes are independent of each other but are both activated by lightsuch as cultivar maturity and environmentalfactorsbitter or burning sensation in the mouth are sensory impressions which may accompanyglycoalkaloid poisoning symptoms from potatoes that include flulike symptoms such as nauseavomiting stomach and abdominal cramps and diarrhoea more severe cases of glycoalkaloid poisoningmay be accompanied by a variety of neurological effects ie drowsiness apathy restlessnessshaking confusion weakness and disturbed vision there are a few reports of deaths beingattributed to glycoalkaloid exposure from the consumption of potatoes potato leaves and potatoberriespotatoes and potatoderived products are listed in the catalogue of feed materials1terms of referencein accordance with art of regulation ec no the european commission asks theeuropean food safety authority for a scientific opinion on the risks for animal and human healthrelated to the presence of glycoalkaloids in feed and food in particular in potatoes and potatoderivedproductsinterpretation of the terms of referencethe contam panel considered that the opinion should cover edible parts of potato plants and alsoof other food plants containing glycoalkaloids gas eg tomato and aubergine nonedible parts ofga containing plants have not been considered with the exception of potato sprouts in particular thecontam panel concluded this opinion should comprise thea evaluation of the toxicity of gas in feed and food in particular in potatoes and potatoderivedproducts for farm and companion animals and humans considering all relevant toxicologicalend pointsb evaluation of the alkaloid profile ie composition of the alkaloids and their concentration ofthe food and feed samples submitted to efsac estimation of the dietary exposure of the european population to gas in food in particular inpotatoes and potatoderived products including the consumption patterns of specific groupsof the population if appropriated estimation of the dietary exposure offarm and companion animals to gas in feedinparticular in potatoes and potatoderived productse assessment of the human health risks for the european population including specific groupsof the population if appropriate as the consequence of the estimated dietary exposure commission regulation eu no of january on the catalogue of feed materials ojl p wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodf assessment of the farm and companion animal health risks in europe as the consequence ofthe estimated dietary exposure exposure to gas from weeds containing ga is only addressedin this opinion in the context of accidental intake by farm animalswhen referring to gas in potatoes the term total gas tga refers to a material comprising asolanineand achaconine as major fraction with no specification on the occurrence of minor gas as well as band cforms of solanine and chaconine similarly when referring to tomato and aubergine the termtga refers to the gas from the corresponding species and forms thereofsupporting information for the assessment chemistrysolanine is one of the first alkaloids that has been isolated from nature by desfosses in friedman et al in zwenger and kind reported that solanine contains a glycoside sidechain zwenger and kind only in it was shown that solanine extracted from potato is infact a mixture of two glycoalkaloids gas asolanine and achaconine that share the same solanidineaglycone kuhn and l‚¬ow since then at least different gas have been isolated and fullystructurally elucidated from over species of the solanaceae family s 13anchezmata et al alsinani and eltayeb the chemical structures and some physical properties of the most importantones are listed in appendix agas are composed of a steroidal aglycone and an oligosaccharide sidechain attached to the 3bhydroxy group of the aglycone see figure friedman et al friedman milner et al the gas of relevance can be divided into the i solanidane group with solanidine as thesteroid backbone and the ii spirosolane group with either the solasodine or the tomatidenoltomatidine backbone gas often contain a double bond between c5 and c6 but the corresponding 5a6hydrogenated forms are also common and in some species eg tomato they constitute the majorcomponents the stereochemistry at carbons c22 and c25 is well definedtheconfiguration is 22r 25stheitconfiguration is 22s 25s friedman et al in solanidineis 22r 25r and in tomatidenoltomatidinein solasodinefurther diversification is generated by the composition of the glycoside sidechain most gascontain either a trisaccharide chacotriose or solatriose or a tetrasaccharide lycotetraose ascarbohydrate in commercial potato cultivars solanum tuberosum mostly achaconine and asolaninecomposed ofthe solanidine aglycone and chacotriose and solatriose respectively are presentfigure wild s tuberosum varieties may contain a much wider range of gas friedman et al distl and wink the aubergine fruit derived from s melongena contains primarily asolamargine and asolasonine composed of the solasodine aglycone and chacotriose and solatrioselycopersicum varieties atomatine and arespectivelydehydrotomatine are the major compounds composed of the aglycones tomatidine and tomatidenolrespectively coupled to lycotetraose friedman derived from sin tomato fruitthe prefix alpha a refers to the intact glycoside while the prefixes beta b gamma c anddelta d refer to the corresponding gas with progressively truncated carbohydrate sidechains due tothe action of enzymatic or acidic hydrolysis friedman milner et al wwwefsaeuropaeuefsa efsa 0cglycoalkaloids in feed and foodohoooohohoohohooohohohsolatrioseohohooohohohsolatrioseohoohoohhh22rnhsolanidine25shhhohsolanine22r 25rnhohsolasodinehhhhooohsolasonineohohoohohohoooohoohoohohoohchacotrioseohohohchacotrioseoohoohohohoooohohohoooohohohohlycotetraoseoohohhhhoohoohoohohhohohoooohoh25sohoh22snhohtomatidinelycotetraoseoohohoohoohooohhhhhhnhsolanidinechaconinehnhohsolasodinehhhsolamarginehhhhnhohtomatidenoltomatinedehydrotomatinefigure s
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 benefit 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 profiles ofHCCš contributing to the evolution of strategies for preventionsurveillance early diagnosis and treatment23 Liver resectionablation and liver transplantation are potentially curative butrequire diagnosis at a sufficiently 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 AsiaPacific trial promoted the approvalof sorafenib as firstline targeted therapy for advanced HCC5“ushering in the era of systemic treatment Subsequently virtuallyall trials were centered around sorafenib and it was used as acontrol with which novel firstline 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 predefined primary end points Fig In addition during the decade when these agents were investigated the median overall survival OS of sorafenib monotherapy asfirstline 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 AsiaPacific trials found sorafenib was effective and safeirrespective of disease etiology disease burden ECOG EasternCooperative Oncology Group performance status status etc15“The 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 firstline 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 benefit 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 benefits 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 survivalbenefit in patients with unresectable HCCs who had alreadyresponded to TACE41 Contrasting with these findings 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 thefindings 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 redefined PFS not conventional PFS buttime until œunTACEable 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 benefit 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 benefit 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 modification 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 Classification BCLCguidelines and although the results are difficult 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 benefited 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 stratification would enable the identification of thosepatients who will benefit 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 firstlinetargeted agent for HCC emerged Lenvatinib was approved for thefirstline 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 œrealworld conditions confirmed the efficacy 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 asufficient 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 beneficial effects and an urgent need for secondline therapyRegorafenibInitial attempts to discover effective secondline agents wereunsuccessful and mirrored attempts to develop firstline 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 finallyconfirmed the potential of secondline agents and ushered inthe era of secondline and sequential therapy97 Regorafenibprovided survival benefitthe rate of diseaseprogression during prior sorafenib treatment or since the lastsorafenib dose98 This was confirmed by Yoo et al99 in aretrospective study of safety and efficacy 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 theefficacy 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 identified 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 andconfirms 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 benefit in patientswith AFP of ngml or higher118“ This was later confirmed inthe REACH2 trial122 which led to the approval of ramucirumab assecondline treatment for advanced HCC REACH2 is the firstpositive phase trialin patients with HCC performed in abiomarkerselected patient cohort and more recent findingsdemonstrated that AFPenriched HCCs displayed significantactivation of VEGF which suggests the underlying mechanism ofaction and confirms 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 andefficacy 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 efficacy of nivolumab in Asian cohort126 Inan internationalICIs have showedpromising efficacy and safety in advanced HCCs as systemicfirstsecondthirdfourthline 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 prespecified statistical significance 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 firstline treatment foradvanced HCC failed to achieve its endpoints ESMO butnivolumab did prolong OS vs months and achievelongtime disease control less adverse events AEs and survivalbenefit 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 firstline 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 efficacy 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 first 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 confirmed 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 firstline 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 firstline 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 first155 or secondline inadvanced HCCs156“ but unfortunately the heterogeneousnature of the results precludes firm 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 sufficiently 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 nameidentifierPatient 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 nameidentifierPatient 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 excellentsafetyprofile169 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 beneficially employed to produce a therapeutic œdualhitapproach in selected patients170 Donafenib is a novel multikinaseinhibitor which is similar to sorafenib displaying comparable orbetter safety and efficacy 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 fibroblast growth factorreceptor FGFR plateletderived growth factor receptors PDGFRSignal Transduction and Targeted Therapy 0cTargeted therapy for hepatocellular carcinomaHuang et alTable Molecular classification of HCCResearcherBoyault et alHoshida et alSchulze et alSia et alKurebayashi et alShinata et alJiang et alYearClassificationG1“G6S1“S3Msig “iC1“iC3 HCCs with adaptive or exhausted immune responsesImmunehigh mid and “lowMS1 ˆ’ ˆ’SI SII and SIIITypeTranscriptomeTranscriptomeExome sequencingMultiomocisImmune cell profilingImmunomicroenvironmentTranscriptome 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 prespecified 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 identified to be able to predict the effect of or responseto treatment with sorafenib172“ Although the molecularclassification of HCC has been widely reported Table to date itremains unclear whether this basic genomic and proteomic datawill prove valuable in guiding targeted therapies183“The 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
" IBDFecal calprotectinEndoscopic activityIBD noninvasive managementThe term IBD is usually used for referring to a group of ‚ammatory gastrointestinal diseases mainly Crohn'sdisease and ulcerative colitis Accordingly IBD arises as a result of inappropriate immune response to intestinalcommensal anisms among genetically susceptible individuals Performing colonoscopy and histopathologicevaluation on an ‚amed bowel biopsy specimen are currently considered as gold standards for diagnosis andmanagement of IBD Correspondingly these techniques are known to be invasive and costly In recent decadesfecal calprotectin as a biomarker has received much attention for the diagnosis and noninvasive managementof IBD Up to now many studies have investigated the efficacy of fecal calprotectin in the areas of IBD diï¬erentiation from IBS prediction of endoscopic and histologic activities of IBD and prediction of disease recurrenceAlthough some of these studies have reported promising results some others have shown significant limitationsTherefore in this paper we reviewed the most interesting ones of these studies after a brief discussion of thelaboratory measurement of fecal calprotectin Moreover we attempted to provide an answer for the question ofwhether fecalcalprotectin could be considered as a potential surrogate marker for colonoscopy IntroductionInflammatory 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 fistulas 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Ž Corresponding author at Department of Clinical Biochemistry and Laboratory Medicine Faculty of Medicine Tabriz University of Medical Sciences DaneshgahStreet PO Box Tabriz IranEmail address vagharimtbzmedacir M VaghariTabari101016jcca202008025Received July Received in revised form August Accepted August Available online August Elsevier BV All rights reserved 0cF KhakiKhatibi et alTable1Clinical endoscopic and histological features of CD and UCClinical FeaturesFeaturesRectal bleedingAbdominal painFeverMucus defectionIntestinal obstructionPerineal diseasePostoperative recurrenceASCA positiveANCA positiveEndoscopic FeaturesCDOccasionallyFrequentlyFrequentlyOccasionallyYESYESYESFrequentlyNot commonUCFrequentlyOccasionallyNot commonFrequentlyNONONONot commonFrequentlyFeaturesCDUCLocationMucosal involvementDepth of ulcerationfistulaCobblestone appearanceAphthous ulcerationMucosal friabilityHistological featuresFeaturesGranulomasCrypt abscessesPatchinessAny part of GI tractDiscontinuousDeepYesYESFrequentlyNot commonCDFrequentlyNot commonFrequentlyColon and rectumContinuoussuperficialNONOOccasionallyFrequentlyUCRareFrequentlyNot 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 financial 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 tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofIBD A high proportion of these studies have investigated the efficacy 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 efficacy 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 findings 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 reflecting 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 aspecificity 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 first of onset areassociated with a poor prognosis at the first 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 efficient at reflecting disease activity []Some studies have also investigated the efficacy 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 efficacy 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 efficacy of salivary calprotectin in the diagnosisof IBD has also been studied which showed that salivary calprotectinsignificantly increased in patients with IBD compared to healthy individuals In this study AUC values for unstimulated saliva and stimulated saliva to distinguish IBD patients from healthy individualswere reported to be and respectively [] However thepopularity of calprotectin is mainly due to the use of fecal calprotectinin the diagnosis and management of IBD that is discussed in the following 0cF KhakiKhatibi et alClinica Chimica Acta “ Laboratory measurement and reference intervalFecal calprotectin is a stable protein that remains stable for “ daysat room temperature [] This property is an excellent advantage for alaboratory marker Also it seems that keeping the specimen at refrigerated temperature °C can increase the stability of fecal calprotectin [] However evidence has been obtained regarding thatthe stability of this protein decreases after staying for three days atroom temperature On the other hand it is not also recommended tokeep samples in the refrigerator for more than days [] It seemsthat fecal calprotectin remains stable up to one year at ˆ’ °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 specificity 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 first evidence of the efficacy of fecal calprotectin inthe diagnosis of IBD was obtained in the 1990s Røseth et al in proposed a method for measuring Calprotectin in stool specimens []One of the first and most interesting studies regarding fecal calprotectinutility in IBD diagnosis was the study by Røseth et al published in In this study patients with ulcerative colitis were studied and according to their results fecal calprotectin levels are higher in patientswith ulcerative colitis compared to healthy controls This study havealso shown that even patients with low disease activities had higherlevels of fecal calprotectin compared to healthy individuals []Subsequent studies somehow confirmed and complemented the findings of this study In another study published in AUC values of CI “ were reported for fecal calprotectin in thediagnosis of colorectal ‚ammation [] Moreover in a study onchildren with IBD it was shown that the level of fecal calprotectin washigher in these patients compared to healthy children so it can beconcluded that it is also directly correlated with ESR levels [] In astudy published in Kolho et al reported AUC values of CI “ for fecal calprotectin in the diagnosis of pediatric IBD [] In a study on patients with Crohn disease a sensitivity of and a specificity of at cutoï¬ of μgg have been reportedfor fecal calprotectin in diagnosis of the disease [] The results of ourrecent study along with other studies showed that fecal calprotectin ispreferred over traditional ‚ammatory biomarkers such as CRP andESR in the diagnosis of IBD [][] Diamanti et al reported a sensitivity of and a specificity of for fecal calprotectin at a cutoï¬ of μgg in IBD diagnosis [] In our recent study a sensitivityof and a specificity 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 specificity 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 specificity [] Caviglia et al in their study reported a sensitivity of and a specificity of at a cutoï¬ of μgg for fecalcalprotectin in diï¬erentiating between IBS and IBD [] Howeversome studies have reported significantly lower values Accordingly in astudy on patients with ulcerative colitis Kalantari et al reported asensitivity of and a specificity of at a cutoï¬ of μgg []Besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with Crohn's disease Asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting CE findings anddiagnosis of Crohn's disease [] In another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard [] Furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity ofTable Reported median levels of fecal calprotectin in healthy individuals of diï¬erent agesAgesMedian levels of fecal calprotectin range µggNumber of subjectsUsed kitUp to monthChildren “ yearsChildren “ yearsAdultsOver years “ “ “ “ “Bühlmann ELISABühlmann ELISACALPRO® Calprotectin ELISA Test ALPPhiCalPhicalReference[][][][][] 0cF KhakiKhatibi et al were reported for this biomarker at μgg in the diagnosis ofsmall bowel ‚ammation in Crohn's disease [] Given these findings it seems that fecal calprotectin has no ideal sensitivity and specificity 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 efficacy 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 andspecificity 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 andspecificity 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 findings 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 firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s In one of the first studiesRoseth et al found a significant correlation between fecal calprotectinlevels and endoscopic and histologic activities in patients with ulcerative colitis [] Furthermore in another study they observed that IBDpatients who were in remission clinically and had normal fecal calprotectin levels less than mgL had normal colonoscopy results[] These interesting findings 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 SpecificityReferences[][][][][]mucosal healing in IBD patients Also these studies were the startingpoint of extensive studies that have been conducted up to now In astudy conducted on patients with Crohn's disease Sipponen et alinvestigated the sensitivity and specificity 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 findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of Crohn's disease with sensitivity and specificity 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 diseaseidentification 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 modifiedBaron 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 specificity 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¬SensitivitySpecificity CD and UC CD and UC CD and UC and unclassified68CD and UC CD and UC and unclassified CD and UC CD and UC UC CD UCAdultsAdultsAdultsBoth adult and pediatricpediatricAdultspediatricAdultsAdultsBoth adult and pediatricTaiwanChinaItalySpainFinlandIranItalyIranDenmarkIndia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggAUCReferences[][][]SPSrefidbib60[][][][][][][] 0cF KhakiKhatibi et alClinica Chimica Acta “Table Summary of the results of some studies regarding the correlation of fecal calprotectin with endoscopic activity in IBD patientsAge groupStudylocationUsedendoscopicactivity indexCorrelationcoefficientrReferenceNumberof IBDpatients CD UC UC CD UCAdultsAdultsAdultsAdultsAdultsFinlandIranSwitzerlandSwitzerlandSwitzerland ModifiedCDEISUCEISRachmilewitzSESCD UC CDAdultsAdults UC CD UC CD CD UC UCAdultsAdultsAdultsAdultsAdultsAdultsAdultsBaron ScoreRachmilewitzSESCDGermanyUSA andCanadaJapanItalyItalyBrazilFranceFranceSouth Korea UCEISMattsSESCDMayo scoreSESCDCDEISMayo score[][][][][][][][][][][][][][]sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactiveMES or from MES or [] In another study the sensitivityand specificity 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 specificity 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 specificity between and in the assessment of IBD clinical activity at diï¬erent stages ofpregnancy [] A recently published systematic review has also confirmed 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 specificity 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 thedifficulty 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 isRobert™s 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 Robert™s scoring system [] Theede et al also used themodified 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 Specificity 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 specificity the sensitivity of fecal calprotectin in theprediction of score of histological activity was achieved as at mgkg [] In a recent study the Geboes
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Growing evidence has demonstrated that glutathione peroxidases GPXs family genes play critical roles in onset and progression of human cancer However a systematic study regarding expression diagnostic and prognostic values and function of GPXs family genes in breast cancer remains absentMaterials and methods Several databases were employed to perform in silico analyses for GPXs family genes qRTPCR western blot and immunohistochemistry staining were introduced to validate GPX3 expression in breast cancer The functions of GPX3 in breast cancer cells were successively determinedResults By combination of receiver operating characteristic ROC curve analysis survival analysis and expression analysis GPX3 was considered as a potential tumor suppressor and a promising diagnosticprognostic biomarker in breast cancer Next low expression of GPX3 was confirmed in breast cancer cells and tissues when compared with corresponding normal controls Overexpression of GPX3 markedly suppressed proliferation colony formation migration and invasion of breast cancer in vitro Moreover two potential mechanisms responsible for GPX3 downregulation in breast cancer including hypermethylation of GPX3 promoter and release of hsamiR3245p inhibitionConclusions Collectively we demonstrate that GPX3 is markedly downregulated in breast cancer possesses significant diagnostic and prognostic values and attenuated in vitro growth and metastasis of breast cancerKeywords Glutathione peroxidase GPX3 Breast cancer Diagnosis Prognosis BiomarkerBackgroundBreast cancer is the most common diagnosed women™s malignant tumor and also the second leading cause of cancerrelated deaths in women worldwide [ ] Despite a variety of advancements have been achieved in diagnosis and therapy the total outcome of patients with breast cancer remains unsatisfactory Thus developing effective therapeutic targets and promising biomarkers for Correspondence 11718264zjueducn Peifen_Fu163comDepartment of Breast Surgery First Affiliated Hospital College of Medicine Zhejiang University QingChun Road Hangzhou Zhejiang Chinadiagnosis and prognosis prediction is very meaningful to improve prognosis of breast cancerGlutathione peroxidases GPXs consisting of eight members GPX18 are ubiquitously expressed proteins that catalyze the reduction of hydrogen peroxides and anic hydroperoxides by glutathione [] GPX family members have been well demonstrated to be frequently aberrantly expressed and are also closely linked to progression of diverse types of human cancer including kidney cancer [] pancreatic cancer [] hepatocellular carcinoma [] cervical cancer [] and gastric cancer [] However a comprehensive study about expression 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 0cLou a0et a0al Cancer Cell Int Page of function diagnostic and prognostic values of GPXs family in breast cancer remain absentIn this study we first assessed the roles of GPXs family genes in predicting diagnosis and prognosis of breast cancer and then determined the mRNA and protein expression of GPXs family genes in breast cancer using bioinformatic analysis Next the low expression of GPX3 was detected in breast cancer cells and tissues Subsequently the function of GPX3 in breast cancer cell growth and metastasis was also investigated Finally we explored the potential detailed mechanisms responsible for GPX3 downregulation in breast cancerMaterials and a0methodsROC curve analysisUsing TCGA breast cancer and normal breast expression data the diagnostic values of GPXs family genes were evaluated by ROC curve as we previously described [] Pvalue was considered as statistically significantKaplan“Meier‘plotter database analysisKaplan“Meierplotter database httpkmplo tcomanaly sis which is capable to access the effect of genes on survival in cancer types including breast cancer was employed to perform survival analysis for GPXs family genes and miRNAs in breast cancer [] Logrank Pvalue was considered as significantGEPIA database analysisGEPIA database httpgepia cance rpkucnindex html a newly developed interactive web server for analyzing the RNA sequencing expression data of tumors and normal samples from the TCGA and GTEx projects was used to determine mRNA expression profile of GPXs family genes in breast cancer [] Pvalue was considered as statistical significanceOncomine database analysisOncomine database wwwoncom ine which is a cancer microarray database and integrated datamining platform was also utilized to analyze mRNA expression of GPXs family genes in breast cancer [ ] Fold change FC Pvalue and a gene rank in top were set as the thresholds for selecting the included datasetsUALCAN database analysisThe protein expression levels of GPXs family genes in breast cancer were assessed using UALCAN database httpualca npathuabeduindex html which is a comprehensive userfriendly and interactive web resource for analyzing cancer OMICS data [] UALCAN database was also introduced to determine the promoter methylation level of GPX3 in breast cancer Pvalue of statistical analysis was considered to have significant differencesstarBase database analysisstarBase database tarb asesysueducnindex php an source platform for investigating miRNAassociated studies was used to predict the upstream binding miRNAs of GPX3 [ ] The correlation of GPX3 with miRNA in breast cancer and miRNA expression level in breast cancer were also assessed by starBase database Pvalue was considered as statistical significanceCell lines and a0clinical tissuesThe human breast cancer cell lines MCF7 and MDAMB231 and normal breast cell line MCF10A were purchased from Shanghai Institute of Biological Science Chinese Academy of Sciences Shanghai China breast cancer tissues and matched normal tissues were obtained from patients with breast cancer who received surgical resection in the First Affiliated Hospital of Zhejiang University College of Medicine Hangzhou China This study was approved by the ethics committee Table Correlation of a0 GPX3 expression with a0 various clinicopathological features in a0breast cancerFeaturesCasesBreast cancerLow expressionHigh expressionP‘valueAge ‰ Tumor size ‰ Lymph node metastasis Present AbsentHistopathological grade I“II IIIER status Positive NegativePR status Positive NegativeHER2 status Positive Negative 0cLou a0et a0al Cancer Cell Int Page of of the First Affiliated Hospital of Zhejiang University College of MedicineRNA isolation and a0qRT‘PCRTotal RNA was isolated from breast cancer cells and tissues by Trizol reagent Invitrogen USA qRTPCR was employed to detect GPX3 mRNA expression in breast cancer as we previously described [] GPX3 expression was normalized to GAPDH by the method of ˆ’ddCt The sequences of primers used in this study GPX3 forward primer ²GAG CTT GCA CCA TTC GGT CT3² GPX3 reverse primer ²GGG TAG GAA GGA TCT CTG AGTTC3² GAPDH forward primer ²AAT GGA CAA CTG GTC GTG GAC3² GAPDH reverse primer ²CCC TCC AGG GGA TCT GTT TG3²Protein extraction and a0western blotProtein of breast cancer cells was extracted using RIPA buffer Beyotime China supplemented with protease and phosphatase inhibitors Thermo Scientific USA Western blot was performed as previously described [] The primary antibodies of GPX3 and GAPDH were purchased from Abcam and antirabbit peroxidase conjugated secondary antibody was purchased from Sigma GPX3 band density was normalized to GAPDH and quantified by ImageJ softwareImmunohistochemistry IHC analysisIHC was utilized to analyze the protein expression of GPX3 in breast cancer tissues and matched normal breast tissues as we previously reported []Establishment of a0stably‘overexpressed cellFull length of GPX3 was first amplified after which the PCR product was cloned into pcDNA31PURO vector digested with BamH1 and XhoI GPX3overexpressed Lipofectamine„¢ Invitrogen USA according to the plasmid was transfected into breast cancer cells using manufactures™ instruction Then stablyoverexpressed cell was screened using puromycin a0μgmLCCK‘ assay stablyoverexpressed cells were seeded into 96well plates and cultured for varied period and a0h At the culture end of each time point a0μl CCK8 solution was added into each well and incubated for another a0 h at a0 °C Finally the optical density OD value at a0nm of each well was determined by a microplate readerColony formation assay stablyoverexpressed cells were seeded into sixwell plates and cultured for a0weeks At the end of culture the plates were washed using phosphate buffered saline PBS for two times Next the plates were fixed in methanol for a0min and stained with crystal violet solution for another a0 min Finally the visible colonies of each well were countedWound healing assayWound healing assay was introduced to detect the migrated ability of breast cancer cells × stablyoverexpressed cells were seeded into sixwell plates When the cells were grown to confluence a wound cross was made using a micropipette tip Photographs were then taken through a microscopy immediately or a0h after woundingTranswell invasion assayCell invasion was determined by Transwell invasion assay Briefly transwell inserts were firstly coated with Matrigel BD USA Then × stablyoverexpressed cells suspended in a0 mL serumfree medium were added into inserts And a0mL medium containing FBS was added to the lower compartment as a chemoattractant After culturing for a0h the cells on the upper membrane were carefully removed using a cotton bud and cells on the lower surface were fixed with methanol for a0 min and successively stained with crystal violet solution for a0min Photographs were then taken through a microscopyStatistical analysisStatistical analysis of bioinformatic analysis was performed by online databases as mentioned above The results of experimental data were shown as mean ± SD Student™s ttest was used to assess differences between two groups The diagnostic value was determined by ROC curve analysis A twotailed value of P was considered as statistically significantResultsThe diagnostic and a0prognostic values of a0GPXs family genes in a0breast cancerTo explore if the expression of GPXs family genes possesses significant diagnostic values in patients with breast cancer receiver operating characteristic ROC curve analysis was employed based on breast cancer data from TCGA database Fig a0 As shown in Fig a0 four GPXs family genes had the significant ability to distinguish breast cancer tissues from normal breast tissues including GPX2 GPX3 GPX4 and GPX8 However the other four GPXs family genes GPX1 GPX5 GPX6 and GPX7 showed no statistical diagnostic values in breast cancer Notably these findings suggested that GPX3 was the most potential diagnostic biomarker for patients 0cLou a0et a0al Cancer Cell Int Page of Fig The diagnostic values of GPXs family genes in breast cancer using ROC curve analysis a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8with breast cancer with the Area Under Curve AUC value being equal to Next we investigated the prognostic values of GPXs family genes in breast cancer using Kaplan“Meierplotter database Fig a0 Increased expression of GPX1 Fig a02a indicated poor prognosis of breast cancer Breast cancer patients with higher expression of GPX2 Fig a02b GPX3 Fig a02c or GPX5 Fig a02e had better prognosis GPX4 GPX6 and GPX7 had no significant predictive values for prognosis of breast cancer All these findings together indicated that only GPX2 and Fig The prognostic values of GPXs family genes in breast cancer determined by Kaplan“Meier plotter database a GPX1 b GPX2 c GPX3 d GPX4 e GPX5 f GPX6 g GPX7 h GPX8 0cLou a0et a0al Cancer Cell Int Page of GPX3 possessed significant diagnostic and prognostic values for breast cancerThe expression levels of a0GPXs family genes in a0breast cancerNext we further studied the expression levels of GPXs family genes in breast cancer First of all TCGA and GTEx databases were introduced to mine the mRNA expression of GPXs family genes in breast cancer The mRNA expression profile of GPXs family was shown in Fig a03a TCGA tumor tissues compared with TCGA normal tissues and Fig a03b TCGA tumor tissues compared with TCGA normal tissues and GTEx normal tissues We found that GPX2 and GPX3 were significantly downregulated in breast cancer Fig a0 3c“f Next Oncomine database was used to further analyze mRNA expression of GPXs family genes in breast cancer Fig a04a We performed metaanalysis for included studies about GPX3 and found that GPX3 mRNA expression was markedly decreased in breast cancer Fig a04b The downregulation of GPX3 mRNA expression in breast cancer of the GPX3associated studies was presented in Fig a04c“q However we found that GPX2 was not significantly downregulated in breast cancer Subsequently CPTAC database was utilized to assess the protein expression of GPXs family genes in breast cancer Fig a0 The results revealed that GPX1 GPX2 GPX3 and GPX4 protein levels were markedly decreased in breast cancer when compared with normal controls GPX7 protein expression in breast cancer was significantly increased GPX8 showed no statistical difference between breast cancer tissues and normal tissues And GPX5 and GPX6 were not found in CPTAC Taken together GPX3 was the most potential one among all GPXs family genes in breast cancer and was selected for following research Fig a0The expression level of a0GPX3 was a0confirmed in a0breast cancer and a0negatively correlated with a0tumor progressionTo further validate the results from in silico analysis we detected the mRNA and protein expression levels of GPX3 in breast cancer cells and tissues As presented in Fig a0 7a b GPX3 mRNA and protein were significantly downregulated in two breast cancer cells MCF7 and MDAMB231 when compared with normal cell MCF10A We also found that GPX3 mRNA expression in breast cancer tissues was much lower than that in adjacent matched normal tissues Fig a07c The protein expression of GPX3 was also detected using immunohistochemistry IHC analysis The results showed that GPX3 protein expression was significantly decreased in breast cancer tissues Fig a07d Collectively GPX3 mRNA and protein expression levels were significantly downregulated in breast cancer which was identical with the bioinformatic analytic results Furthermore Chi square test revealed that low expression of GPX3 was significantly negatively correlated with ERPR expression and positively linked to tumor size histopathological grade and lymph node metastasis Table a0 All these findings showed that GPX3 was negatively correlated with progression of breast cancer and might function as a tumor suppressor in breast cancerGPX3 overexpression suppressed proliferation and a0colony formation of a0breast cancer cellsGiven the low expression of GPX3 in breast cancer overexpression technology was used to study GPX3²s functions We then constructed the overexpressed plasmid of GPX3 After transfection of GPX3overexpressed plasmid GPX3 mRNA and protein expression levels were significantly upregulated in breast cancer cells Fig a0 8a b Firstly we explored the effect of GPX3 on growth of breast cancer cells CCK8 assay demonstrated that overexpression of GPX3 markedly suppressed in a0vitro proliferation of breast cancer cells MCF7 and MDAMB231 Fig a0 8c d Furthermore colony formation assay also revealed that GPX3 upregulation led to the inhibition of clonogenic capacity of breast cancer cells Fig a08e f These findings indicated that GPX3 overexpression significantly suppressed in a0 vitro proliferation and colony formation of breast cancer cellsGPX3 overexpression inhibited migration and a0invasion of a0breast cancer cellsMetastasis is another hallmark of malignant tumors including breast cancer We intended to ascertain if GPX3 affects metastasis of breast cancer Wound healing assay was first employed to investigate GPX3²s function in controlling migration of breast cancer cells and the result demonstrated that overexpression of GPX3 obviously attenuated the migrated ability of breast cancer cells Fig a09a b Moreover increased expression of GPX3 could also suppressed invasion of breast cancer cells which was detected by transwell invasion assay Fig a09c“f Taken together overexpression of GPX3 suppressed in a0vitro migration and invasion of breast cancer cellsThe potential mechanisms responsible for a0GPX3 downregulation in a0breast cancerFinally we preliminarily probed the possible molecular mechanisms that accounted for GPX3 downregulation in breast cancer Promoter hypermethylation may be responsible for expression suppression of tumor suppressors Intriguingly we found that the promoter methylation level of GPX3 was significantly upregulated in breast cancer tissues compared with normal controls Fig a010a Gene expression was also frequently negatively regulated by miRNAs at posttranscriptional 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by GEPIA database a The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA normal breast tissues b The mRNA expression profile of GPXs family genes in breast cancer tissues compared with TCGA and GTEx normal breast tissues c d GPX2 was significantly downregulated in breast cancer e f GPX3 was significantly downregulated in breast cancer P level The miRNAs that potentially bind to GPX3 were predicted by starBase database and miRNAs were finally found For better visualization miRNAGPX3 network was established Fig a0 10b Based on the action mechanism of miRNA there should be negative correlation between miRNA and target gene We performed expression correlation analysis for miRNAGPX3 pairs As listed in Table a0 four potential miRNAs hsamiR3245p hsamiR3283p hsalet7a5p and hsamiR449b5p which were inversely associated 0cLou a0et a0al Cancer Cell Int Page of Fig The mRNA expression of GPXs family genes in breast cancer determined by Oncomine database a The mRNA expression of GPXs family genes in breast cancer b Metaanalysis for the included GPX3associated datasets in breast cancer c“q The mRNA expression of GPX3 was markedly downregulated in breast cancer in included GPX3assocaited datasets 0cLou a0et a0al Cancer Cell Int Page of Fig The protein expression of GPXs family genes in breast cancer detected by UALCAN database a GPX1 b GPX2 c GPX3 d GPX4 e GPX7 f GPX8 P 0cLou a0et a0al Cancer Cell Int Page of Fig The visual flowprocess diagram of this studywith GPX3 expression in breast cancer were identified The prognostic values of the four miRNAs in breast cancer were also evaluated by Kaplan“Meierplotter database Fig a0 10c d Survival analysis revealed that among the four miRNAs only high expression of hsamiR3245p indicated poor prognosis for patients with breast cancer Fig a0 10c The expression levels of four miRNAs in breast cancer was subsequently determined by starBase Fig a010g“j and showed that miR3245p and hsamiR449b5p were significantly upregulated whereas hsamiR3283p and hsalet7a5p were markedly downregulated in breast cancer compared with normal controls By combination of survival and expression analysis miR3245p was considered as the most potential upstream miRNA of GPX3 in breast cancer The above results implied that promoter hypermethylation and miR3245pmediated suppression were two potential mechanisms that may be responsible for GPX3 downregulation in breast cancer Fig a010lDiscussionBreast cancer is the most common cancer type in women The molecular mechanism of carcinogenesis of breast cancer is still unclear and need to be further investigated Increasing findings have showed that GPXs are critical regulators in onset and progression of human cancer However the knowledge of GPXs in breast cancer is still limitedROC curve and survival analysis for GPXs family revealed that some of them might serve as promising diagnostic and prognostic biomarkers for breast cancer especially GPX2 and GPX3 Expression analysis demonstrated the significant low expression of GPX3 in breast cancer GPX3 was reported to act as a tumor suppressor 0cLou a0et a0al Cancer Cell Int Page of Fig The expression levels of GPX3 in breast cancer cells and tissues The mRNA a and protein b expression of GPX3 in breast cancer cells was significantly lower than that in normal breast cell c The mRNA expression of GPX3 was markedly decreased in breast cancer tissues compared with matched normal breast tissues d IHC analysis of GPX3 expression levels in normal breast tissues and breast cancer tissues Bar scale um P in human cancer For example Cai et a0al indicated that GPX3 prevented migration and invasion of gastric cancer by targeting NFkBWnt5aJNK signaling [] Lee et a0al suggested that GPX3 arrested cell cycle and functioned as a tumor suppressor in lung cancer [] Hua et a0 al showed that silencing GPX3 expression promoted tumor metastasis in human thyroid cancer [] Caitlyn et a0 al revealed that plasma GPX3 limited the development of colitis associated carcinoma [] However the function and mechanism of GPX3 in breast cancer have not been reported and need to be further elucidatedNext we confirmed the low expression of GPX3 in breast cancer cells and tissues using qRTPCR western blot and IHC which supported the results of bioinformatic analysis Functional experiments revealed that overexpression of GPX3 significantly inhibited in a0 vitro proliferation colony formation migration and invasion of breast cancer cellsPrevious studies have showed the effect of promoter methylation level in regulating gene expression [] Thus we preliminarily evaluated the promoter methylation level of GPX3 in breast cancer and found that it was significantly upregulated in breast cancer compared with normal breast tissues Moreover Mohamed et a0 al also demonstrated the link between promoter hypermethylation of GPX3 and inflammatory breast carcinogenesis [] The report together with our finding revealed that hypermethylation of GPX3 promoter might be a potential mechanism responsible for GPX3 downregulation in breast cancermiRNAs are involved in multiple biological processes by suppressing gene expression [ “] We also explored the upstream regulatory miRNAs of GPX3 By combination of correlation analysis survival analysis and expression analysis for these miRNAs miR3245p was regarded as the most potential miRNA which was overexpressed negatively correlated with GPX3 expression and possessed poor prognosis in breast cancer Numerous studies have demonstrated that miR3245p served as an oncogenic miRNA in human cancer For example miR3245p promoted progression of papillary thyroid carcinoma via microenvironment alteration [] miR3245p facilitated progression of colon cancer by activating Wntbetacatenin pathway [] Moreover the 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 inhibited proliferation and colony formation of breast cancer cells in vitro a“b The overexpression effect of GPX3overexpressed plasmid in breast cancer cells c“d Overexpression of GPX3 inhibited proliferation of MCF7 and MDAMB231 cells e“f Overexpression of GPX3 inhibited colony formation of MCF7 and MDAMB231 cells P relationship between GPX3 and miR3245p has already been reported in lung cancer [] Thus overexpressed miR3244p might be another mechanism that accounted for GPX3 downregulation in breast cancer In the future the oncogenic roles of miR3245p need to be further investigated by in a0vitro and in a0vivo assaysConclusionsIn summary our current findings indicate that GPX3 is markedly downregulated in breast cancer promotes in a0 vitro growth and metastasis of breast cancer cells and servers as a promising diagnostic or prognostic biomarker for patients with breast cancer Moreover we also elucidate that promoter hypermethylation and miR3245pmediated suppression may be two 0cLou a0et a0al Cancer Cell Int Page of Fig Overexpression of GPX3 suppressed migration and invasion of breast cancer cells in vitro a b Increased expression of GPX3 attenuated migration of MCF7 and MDAMB231 cells c d Increased expression of GPX3 attenuated invasion of MCF7 cell e f Increased expression of GPX3 attenuated invasion of MDAMB231 cell Bar scale um P See figure on next pageFig The potential mechanisms responsible for GPX3 downregulation in breast cancer a The promoter methylation level of GPX3 was increased in breast cancer compared with normal controls b The miRNAGPX3 network c“f The prognostic values of four miRNAs in breast cancer g“j The expression levels of four miRNAs in breast cancer k The intersection analysis of survival analysis and expression analysis l The model of GPX3²s function and dysregulated mechanism in breast cancer P was considered as statistically significant 0cLou a0et a0al Cancer Cell Int Page of 0cLou a0et a0al Cancer Cell Int Page of Table The expression correlation of a0GPX3 with a0predicted miRNAs using TCGA breast cancer datamiRNAhsamiR3245phsamiR3283phsalet7a5phsamiR449b5phsamiR6295phsamiR47565phsamiR642a5phsalet7d5phsamiR449ahsamiR5895phsamiR181d5phsamiR21145phsamiR34a5phsamiR449c5phsamiR23a3phsamiR3150a3phsamiR47315phsamiR23b3phsamiR4915phsamiR4739hsamiR181c5phsamiR3612hsamiR5823phsamiR650hsalet7b5phsamiR3383phsamiR2278hsamiR1225phsamiR181a5phsamiR181b5phsamiR3139hsamiR4644hsamiR5013phsamiR26825phsamiR4306hsamiR95phsamiR620hsamiR1321hsamiR985phsalet7e5phsamiR1855phsamiR1270hsalet7 g5phsamiR2055phsamiR371a5phsamiR8735phsamiR34b5phsamiR5325phsamiR2963pRˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ ˆ’ P‘valueTable continuedmiRNAhsamiR7085phsamiR35295phsamiR5745phsamiR8765phsamiR2965phsamiR5023phsamiR1365phsamiR285phsamiR3615phsamiR520 hhsamiR6683phsamiR520 g3phsamiR376b3phsamiR6753phsalet7f5phsamiR34c5phsamiR665hsamiR1385phsamiR146a5phsamiR376a3phsamiR223phsamiR2995phsalet7i5phsamiR4953phsamiR1433phsamiR8893phsamiR146b5phsamiR3795phsamiR2233phsalet7c5pRP‘valuepotential mechanisms responsible for GPX3 downregulation in breast cancer These results provide key clues for developing effective therapeutic targets and biomarkers for breast cancerAcknowledgementsNot applicableAuthors™ contributionsWL and PF designed this work performed experiments analyzed data and draft the manuscript BD performed some experiments SW revised the manuscript All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe data in this work are available from the corresponding author on reasonable request 0cLou a0et a0al Cancer Cell Int Page of Lou W Ding B Fan W High expression of pseudogene PTTG3P indicates a poor prognosis in human breast cancer Mol Therapy Oncolytics “ Lou W Liu J Ding B Jin L Xu L Li X Chen J Fan W Five miRNAsmediated PIEZO2 downregulation accompanied with activation of Hedgehog signaling pathway predicts poor prognosis of breast cancer Aging “ Chen D Si W Shen J Du C Lou W Bao C Zheng H Pan J Zhong G Xu L et al miR27b3p inhibits proliferation and potentially reverses multichemoresistance by targeting CBLBGRB2 in breast cancer cells Cell Death Dis Cai M Sikong Y Wang Q Zhu S Pang F Cui X Gpx3 prevents migration and invasion in gastric cancer by targeting NFsmall ka CyrillicBWnt5aJNK signaling Int J Clin Exp Pathol “ An BC Choi YD Oh IJ GPx3mediated redox signaling arrests the cell cycle and acts as a tumor suppressor in lung cancer cell lines Plos One 2018139e0204170 Zhao H Li J Li X Han C Zhang Y Zheng L Guo M Silencing GPX3 expression promotes tumor metastasis in human thyroid cancer Curr Prot Peptide Sci “ Barrett CW Ning W Chen X Smith JJ Washington MK Hill KE Coburn LA Peek RM Chaturvedi R Wilson KT et al Tumor suppressor function of the plasma glutathione peroxidase gpx3 in colitisassociated carcinoma Cancer Res “ Kulis M Esteller M DNA methylation and cancer Adv Genet “ Mohamed MM Sabet S Peng DF Nouh MA ElShinawi M Promoter hypermethylation and suppression of glutathione peroxidase are associated with inflammatory breast carcinogenesis Oxid Med Cell Lou W Liu J Ding B Chen D Xu L Ding J Jiang D Zhou L Zheng S Fan W Identification of potential miRNAmRNA regulatory network contributing to pathogenesis of HBVrelated HCC J Transl Med Lou W Liu J Gao Y Zhong G Chen D Shen J Bao C Xu L Pan J Cheng J et al MicroRNAs in cancer metastasis and angiogenesis Oncotarget “ Lou W Liu J Gao Y Zhong G Ding B Xu L Fan W MicroRNA regulation of liver cancer stem cells Am J Cancer Res “ Yang Y Xia S Zhang L Wang W Chen L Zhan W MiR3245pPTPRDCEBPD axis promotes papillary thyroid carcinoma progression via microenvironment alteration Cancer Biol Therapy “ Yan D Liu W Liu Y Luo M LINC00261 suppresses human colon cancer progression via sponging miR3243p and inactivating the Wntbetacatenin pathway J Cell Physiol “ Lin MH Chen YZ Lee MY Weng KP Chang HT Yu SY Dong BJ Kuo FR Hung LT Liu LF et al Comprehensive identification of microRNA arm selection preference in lung cancer miR3245p and3p serve oncogenic functions in lung cancer Oncol Lett “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsEthics approval and consent to participateThis study was approved by the ethics committee of the First Affiliated Hospital of Zhejiang University College of MedicineConsent for publicationNot applicableCompeting interestsThe authors state that they have no conflicts of interestReceived June Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “Lou W Liu J Ding B Xu L Fan W Identification of chemoresistanceassociated miRNAs in breast cancer Cancer Manag Res “ Matouskova P Hanouskova B Skalova L MicroRNAs as potential regulators of glutathione peroxidases expression and their role in obesity and related pathologie
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" Based on the above analysis we speculated that there may be some interaction between p120ctn isoform 3A and snail which plays a role in suppressing EMT in lung cancer cells expressing cytoplasmic E-cadherin but this hypothesis requires further study. Importantly we also found that knockdown of p120ctn-1A in SPC cells with cytoplasmic E-cadherin resulted in decreased twist expression (B). Meanwhile transfection of LK2 cells which also showed cytoplasmic localization of E-cadherin with the p120ctn isoform 1A plasmid resulted in increased twist expression (B). However no changes in twist expression were observed in the rest of the experiments (A 4A). As a transcription factor and master gene regulator of EMT [39] [40] twist can downregulate E-cadherin expression [41] and upregulate N-cadherin and other mesenchymal biomarkers [42]. Increased twist expression in LK2 cells transfected with the p120ctn isoform 1A plasmid indicated that transcriptional activation took place and further suggested that the p120ctn isoform 1A may have translocated into the nucleus upon binding of E-cad/CTF2 in the cytoplasm consequently activating the Wnt signaling pathway to promote EMT. Decreased twist expression in SPC cells transfected with p120ctn-1A-siRNA indicated that transcriptional activity was downregulated and suggested that ablation of p120ctn isoform 1A resulted in the inhibtion of EMT by removing the stimulatory effect of the Wnt signaling activity by p120ctn isoform 1A. In the H460 and H1299 cells with E-cadherin localized in the membrane the unchanged twist expression confirmed that p120ctn isoforms 1A and 3A could bind to E-cadherin and maintain effective cell-cell adhesion in order to suppress EMT instead of affecting the Wnt/twist pathway. Intriguingly overexpression of p120ctn isoform 3A did not change twist expression in LK2 cells expressing cytoplasmic E-cadherin indicating that p120ctn isoform 3A did not activate transcription. Therefore we firmly believe in the above hypothesis that p120ctn isoform 3A may interact with snail in some manner to influence E-cadherin expression and suppress EMT in lung cancer cells carrying cytoplasmic E-cadherin. Previous studies have observed that p120ctn-1A restored the cytoplasmic expression of E-cadherin whereas p120ctn-3A could not [20] which seems to be contradictory with the results of this study. However the method in previous studies of knocking down p120ctn expression and then transfecting p120ctn isoforms 1A and 3A plasmids into cells is different from that in the current study in which cells were only transiently transfected with p120ctn isoforms 1A and 3A plasmids. Therefore the different research methods may have led to different effects on E-cadherin. We also noted that in previous studies decreased and almost undetectable levels of E-cadherin by ablation of p120ctn resulted in the failure of exogenous p120ctn-1A to translocate into the nucleus to activate the Wnt/b-catenin pathway and decrease E-cadherin expression due to the deletion of the binding partner E-cad/CTF2. However the LK2 and H1299 cell lines used in these experiments expressed E-cadherin in the present study. E-cadherin binds primarily to unphosphorylated p120ctn isoform 3A whereas tyrosine-phosphorylated p120ctn isoform 1A interacts exclusively with N-cadherin [23]. In the previous studies exogenous p120ctn isoform 3A was prevented from binding and stabilizing E-cadherin after its ablation while in the present study the exogenous p120ctn isoform 3A could stabilize E-cadherin expression directly on the membrane or indirectly by increasing its cytoplasmic expression via regulation of snail expression. Of course all of these findings will need to be further investigated. In we for the first time found that p120ctn isoforms 1A and 3A to have different functions in EMT of lung cancer cells with E-cadherin expressed in different subcellular locations. When E-cadherin was localized on the cell membrane p120ctn isoforms 1A and 3A both could inhibit EMT and reduce the cell invasiveness phenotype. When E-cadherin was localized in the cytoplasm p120ctn isoform 1A promoted EMT and enhanced cell invasion while p120ctn isoform 3A inhibited EMT and reduced cell invasiveness. We thank Dr. Albert B. Reynolds (Department of Cancer Biology Vanderbilt University School of Medicine TN USA) for kindly providing p120ctn isoforms 1A and 3A cDNA plasmids. References 1 ThieryJP SleemanJP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. 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Semin Cell Dev Biol15: 657“66315561585 27 CheungLW LeungPC WongAS (2010) Cadherin switching and activation of p120 catenin signaling are mediators of gonadotropin-releasing hormone to promote tumor cell migration and invasion in ovarian cancer. Oncogene29: 2427“244020118984 28 ThoresonMA AnastasiadisPZ DanielJM IretonRC WheelockMJ et al (2000) Selective uncoupling of p120(ctn) from E-cadherin disrupts strong adhesion. J Cell Biol148: 189“20210629228 29 BukholmIK NeslandJM Borresen-DaleAL (2000) Re-expression of E-cadherin alpha-catenin and beta-catenin but not of gamma-catenin in metastatic tissue from breast cancer patients [seecomments]. J Pathol190: 15“1910640987 30 PeiferM YapAS (2003) Traffic control: p120-catenin acts as a gatekeeper to control the fate of classical cadherins in mammalian cells. J Cell Biol163: 437“44014610049 31 DavisMA IretonRC ReynoldsAB (2003) A core function for p120-catenin in cadherin turnover. 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Mol Biol Cell20: 2121“212919193763 Stat Med Stat Med sim Statistics in Medicine 0277-6715 1097-0258 BlackWell Publishing Ltd Oxford UK 24027094 4098103 10.1002/sim.5963 Research Articles Modeling exposure“lag“response associations with distributed lag non-linear models Gasparrini Antonio *   Medical Statistics Department London School of Hygiene and Tropical Medicine London U.K. *Correspondence to: Antonio Gasparrini London School of Hygiene and Tropical Medicine Keppel Street London WC1E 7HT U.K.  E-mail: [email protected] 28 2 2014 12 9 2013 33 5 881 899 29 3 2012 10 8 2013 © 2013 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. 2013 This is an open access article under the terms of the Creative Commons Attribution License which permits use distribution and reproduction in any medium provided the original work is properly cited. In biomedical research a health effect is frequently associated with protracted exposures of varying intensity sustained in the past. The main complexity of modeling and interpreting such phenomena lies in the additional temporal dimension needed to express the association as the risk depends on both intensity and timing of past exposures. This type of dependency is defined here as exposure“lag“response association. In this contribution I illustrate a general statistical framework for such associations established through the extension of distributed lag non-linear models originally developed in time series analysis. This modeling class is based on the definition of a cross-basis obtained by the combination of two functions to flexibly model linear or nonlinear exposure-responses and the lag structure of the relationship respectively. The methodology is illustrated with an example application to cohort data and validated through a simulation study. This modeling framework generalizes to various study designs and regression models and can be applied to study the health effects of protracted exposures to environmental factors drugs or carcinogenic agents among others. © 2013 The Authors. Statistics in Medicine published by John Wiley & Sons Ltd. latency distributed lag models exposure“lag“response delayed effects splines 1. Introduction In biomedical research it is commonly appreciated that an exposure event produces effects lasting well beyond the exposure period with an increase in risk occurring from few hours to many years later depending on the physiological processes linking the exposure and the health outcome. The problem is made even more complicated in the presence of protracted time-varying exposures when the health effect measured at a given time can be described as the result of multiple exposure events of different intensities sustained in the past. This phenomenon common to various research fields has been associated for example with peak 1 or chronic exposures 2 to environmental stressors drug intake 34 or occupational exposures to carcinogenic substances 5. The main complexity of modeling and interpreting such dependencies lies in the additional temporal dimension needed to express the association beyond the usual exposure“response relationship as the risk depends on both intensity and timing of past exposures. Nonetheless the appropriate representation of the temporal pattern of risks may provide further insights on the association of interest in particular regarding the underlying pathophysiological mechanisms and prevent biases in estimates and predictions. Revising previous terminology 6 I define these dependencies as exposure“lag“response associations. In particular this issue has been debated in cancer epidemiology 7“9. Analytical approaches extend simple indices such as cumulative exposure in order to accommodate the temporal variation in risk because of protracted exposures. In particular the pioneering work by Thomas 106 helped develop sophisticated statistical methods on the basis of weighting past exposures through specific functions whose parameters are estimated by the data. Vacek 11 Langholz and colleagues 12 and Richardson 13 provided interesting applications in case-control studies with weights represented through simple parametric functions. The methodology was improved by Hauptmann and colleagues in a series of papers 14“16 by using flexible and smooth spline functions. Sylvestre and Abrahamowicz 17 and Abrahamowicz and colleagues 18 extended the spline methods to the analysis of time-to-event data with a cohort design and presented their applications in pharmaco-epidemiology. The main limitation of the statistical techniques described in these papers is the assumption of a linear exposure“response relationship. Models for nonlinear dependencies introduce further nontrivial complexities from both statistical and interpretational perspectives as the problem becomes inherently bidimensional. Abrahamowicz and Mackenzie 19 proposed a model for analyzing the nonlinear time-dependent effects of fixed exposures while Vacek 11 and Berhane and colleagues 20 extended this scheme to the case of protracted time-varying exposures. However the modeling techniques illustrated in these other papers still face some limitations as they are based on complex estimation routines with convergence issues and problems in producing uncertainty measures such as standard errors and confidence intervals. Interestingly equivalent approaches were previously established in time series analysis on the basis of distributed lag models (DLMs) a methodology originally formulated in econometrics 21 then applied in epidemiological research 22. These models involve the definition of a distributed lag function analogous to the weighting function described before. In particular Armstrong 23 generalized the method to distributed lag non-linear models (DLNMs) a class of models with different options for the functions applied to model nonlinearity and distributed lag effects. The theory of DLMs and DLNMs have been recently re-evaluated 24 offering a well-grounded statistical tool and a comprehensive scheme for interpretation. In this paper I aim to establish a general conceptual and statistical framework for modeling exposure“lag“response associations built upon the paradigm of DLMs and DLNMs. This modeling class extended beyond time series analysis provides a unified methodology applicable in different study designs data structures and regression models including most of the previous methods as specific cases. Also the statistical framework is defined by completely parametric functions and fitted through standard regression methods with measures of uncertainty and fit statistics easily available. The R package dlnm originally developed for time series data 25 is extended in parallel offering a easy-to-use implementation of the modeling approach. The manuscript is structured as follows. The development and algebraic definition of the modeling framework is described in Section 2. As an illustrative example in I apply the method for investigating the relationship between occupational exposure to radon and lung cancer mortality by using the data from the Colorado Plateau miners cohort. The modeling framework is then validated in a simulation study n. A final discussion is provided in. Information on data and software implementation is included in. The R code and data are included in the supporting information together with additional details making the results of the illustrative example and of the simulation study entirely reproducible. 2. Modeling framework The modeling skeleton is derived by extending the class of DLNMs beyond the time series context. This extension provides a neat algebraic representation and a comprehensive statistical definition. The focus is on a function here defined s(xt) which describes the dependency in terms of the exposure history to x evaluated at time t. The function s(xt) is commonly included in regression models in order to estimate the association while controlling for potential confounders. Although the regression model varies depending on the study design and the type of data the definition of s(xt) provided later and the related modeling framework generally apply. 2.1. Models for linear exposure“response relationships Previous studies on the topic have defined the function s(xt) by using slightly different algebraic formulae 1026111417. Assuming a linear exposure“response relationship a general notation can be given by (1a) (1b) (1c) In (1a) the increase in risk at time t is defined as the integral of the instantaneous exposure intensity xu over the period ?t = [t0t1] with t0 and t1 representing the times of the first and last relevant exposures. Here w(t ? u) is the weighting function previously described in which assigns weights to past exposures experienced at time t ? u on the basis of their contribution to the risk at time t. The model can be reparameterized as in (1b) where the risk is now expressed along the lag with ? ? [?0L]. Here L ? ?0 = t1 ? t0 is interpreted as the lag period over which an exposure to x is assumed to affect the risk at time t usually with ?0 = 0. This parameterization offers the advantage that the function w is now directly defined in the new dimension of lag ? and it is independent of the time axis chosen for t which may represent different time scales depending on the study design. The function w(?) termed from here on as the lag“response function models the lag“response curve associated with exposure x. Finally for computational purposes the integral is approximated in (1c) by a sum of terms derived by partitioning the lag interval in equally spaced discrete units and assuming the protracted exposure as a sequence of exposure events xt ? ? at lags ? = ?0 ¦ L. A statistical model for (1) can be defined by expressing the lag“response function w(?) as a linear combination of terms obtained through basis transformation with related parameters. By using matrix notation let the vector qxt of exposure history be defined by (2) Such exposure history changes along time depending on the time t at which the vector qxt is computed. Given (2) the cumulative function s(xt) in (1) can be written using a compact and general matrix notation as (3) The (L ? ?0 + 1) × v? matrix C is obtained from the transformation of the lag vector ? = [?0 ¦ ? ¦ L]T by choosing a specific basis with dimension v? for w(?) which defines the related basis functions. In this parameterization the function s(xt) representing the integral of x · w(?) over the interval [?0L] is defined as a lag“basis function with parameters ?. Interestingly the equation in (3) is almost identical to that defining DLMs 24 Eq. (4). The different indexing in the original version reflects the specific application in time series where the data are perfectly ordered in time and the matrix Q has a structure such that qt? ? qt + 1? + 1. However this is a specific case of the general representation in (2)“(3). The theory and software already developed for DLMs can be therefore extended in parallel. Alternative lag“basis functions for representing s(xt) are derived through different lag“response functions w(?) in (1). In particular the traditional index of unweighted cumulative exposure is a specific case of (3) where reduces to with w(?) equal to a constant c. This is obtained by specifying C as an (L ? ?0 + 1)-dimensional vector of 1's with v? = 1."
1
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 People™s 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 manufacturer™s 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] Children™s 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 patient™s 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 manufacturer™s 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 patient™s 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 × 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 Agilent™s 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 × 109L with blasts a hemoglobin count of a0 gL and a platelet count of × 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 French“American“Britishh 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 People™s 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 People™s 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 “ DíazBeyá 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 Styczyński 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 “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations 0c'
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"‚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 fistulas 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Ž 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 ulcerationfistulacobblestone appearanceaphthous ulcerationmucosal friabilityhistological featuresfeaturesgranulomascrypt abscessespatchinessany part of gi tractdiscontinuousdeepyesyesfrequentlynot commoncdfrequentlynot commonfrequentlycolon and rectumcontinuoussuperficialnonooccasionallyfrequentlyucrarefrequentlynot 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 financial 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 tofind a suitable laboratory marker with sufficient sensitivity and specificity for the purpose of diagnosing and noninvasive management ofibd a high proportion of these studies have investigated the efficacy 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 efficacy 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 findings 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 reflecting 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 aspecificity 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 first of onset areassociated with a poor prognosis at the first 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 efficient at reflecting disease activity some studies have also investigated the efficacy 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 efficacy 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 efficacy 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 ˆ’ °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 specificity 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 first evidence of the efficacy 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 first 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 confirmed and complemented the findings 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 specificity 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 specificity of for fecal calprotectin at a cutoï¬ of μgg in ibd diagnosis in our recent study a sensitivityof and a specificity 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 specificity 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 specificity caviglia in their study reported a sensitivity of and a specificity 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 specificity of at a cutoï¬ of μgg besides there is a considerable agreement between fecal calprotectinand capsule endoscopy findings in patients with crohn's disease asensitivity of and a specificity of have also been reported at acutoï¬ of mgkg for fecal calprotectin in predicting ce findings anddiagnosis of crohn's disease in another study lower sensitivityand specificity rates sensitivity specificity were reportedfor fecal calprotectin in this regard furthermore in one studythat examined the efficacy of fecal calprotectin in predicting wirelesscapsule endoscopy findings a sensitivity of and a specificity 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 findings it seems that fecal calprotectin has no ideal sensitivity and specificity 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 efficacy 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 andspecificity 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 andspecificity 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 findings 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 firstevidence of a link between fecal calprotectin and disease endoscopicactivity was obtained in the late 1990s in one of the first 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 findings 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 specificityreferences 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 specificity 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 findings of this study demonstrated that fecal calprotectin at µgg cutoï¬ can predict theendoscopic activity of crohn's disease with sensitivity and specificity 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 diseaseidentification 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 modifiedbaron 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 specificity 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¬sensitivityspecificity cd and uc cd and uc cd and uc and unclassified68cd and uc cd and uc and unclassified cd and uc cd and uc uc cd ucadultsadultsadultsboth adult and pediatricpediatricadultspediatricadultsadultsboth adult and pediatrictaiwanchinaitalyspainfinlandiranitalyirandenmarkindia48µgg µgg150µgg150µgg595µgg784µgg160µgg164µgg150µgg188µggaucreferences spsrefidbib60 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 indexcorrelationcoefficientrreferencenumberof ibdpatients cd uc uc cd ucadultsadultsadultsadultsadultsfinlandiranswitzerlandswitzerlandswitzerland modifiedcdeisuceisrachmilewitzsescd uc cdadultsadults uc cd uc cd cd uc ucadultsadultsadultsadultsadultsadultsadultsbaron scorerachmilewitzsescdgermanyusa andcanadajapanitalyitalybrazilfrancefrancesouth korea uceismattssescdmayo scoresescdcdeismayo score sensitivity of and a specificity of were reported for fecalcalprotectin at µgg to diï¬erentiate active endoscopic from inactivemes or from mes or in another study the sensitivityand specificity 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 specificity 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 specificity between and in the assessment of ibd clinical activity at diï¬erent stages ofpregnancy a recently published systematic review has also confirmed 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 specificity 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 thedifficulty 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 isrobert™s 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 robert™s scoring system theede also used themodified 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 specificity 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 specificity 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 specificity for fecal calprotectin in the prediction of geboesscore sensitivity specificity 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 specificity were finally reported for fecalcalprotectin at a cutoï¬ of µgg in the prediction of histologic activity however some studies have reported both high sensitivityand specificity for fecal calprotectin in the prediction of histologicalremission for example one study reported a sensitivity of aswell as a specificity of for fecal calprotectin at a cutoï¬ of µggin the prediction of gs it seems that the cause of theseconflicts 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 first evidence of the efficacy offecal calprotectin to predict recurrence dates back to the early 2000saccordingly a study published in by tibble is one of the firststudies 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 specificity of at a cutoï¬ of mgl to predictibd recurrence in a study published in costa showedthat the sensitivity and specificity of fecal calprotectin to predict ulcerative colitis recurrence are more than that of crohn's disease asensitivity of and a specificity of versus a sensitivity of and a specificity at a cutoï¬ of μgg another studyconducted on patients with ulcerative colitis has also reported appropriate sensitivity and specificity rates for fecal calprotectin in the prediction of relapse a sensitivity of and a specificity at a cutoï¬of μgg however another study was conducted on patientswith ulcerative colitis who have been followedup for year and finally a lower sensitivity was reported this study have shown that fecalcalprotectin at cutoï¬ of μgg could predict diseas
0
"We therefore envision that the effects of phenothiazines per se as well as its potential chemosensitizing properties can help improve the outcome for relapsed SCLC patients who no longer respond to conventional treatment. In summary the present work uncovered a novel activity of phenothiazines as agents capable of inhibiting survival and inducing cell death in human SCLC () a tumor type that is notoriously difficult to treat with conventional CT because of its ability to rapidly acquire resistance. Moreover we provide evidence showing that phenothiazine-induced disruption of lysosomal functions is critically required for phenothiazine-induced cell death effects with SCLC especially susceptible because of their inherently low lysosomal number and/or pH. Finally our data suggest that analysis of intrinsic lysosomal functions may identify SCLC cases that are most likely to respond to phenothiazine-based regimens however extended cell line analyses as well as in vivo studies are needed to formulate such . On the basis of these findings we conclude that phenothiazines are suitable lead compounds for further development of lysosome-targeted treatment to combat SCLC and which merit further analysis for antitumor efficacy in patient-derived xenograft models and/or genetically modified mice models of SCLC. Materials and Methods Cells lines and culture conditions The following human LC cell lines were used: H69 H82 H592 U-1258 U-1568 U-1690 U-1906 and U-2020 (all SCLC); A549 H125 H1299 H157 H23 H661 U-1752 and U-1810 (all NSCLC). Cell lines whose name begin with the letter ˜U' were established at the University of Uppsala Sweden while others were obtained from American Type Culture Collection (ATCC Manassas VA USA) or Coriell Cell Repositories (Camden NJ USA). The characteristics of these cell lines as well as for some of them their p53 status is indicated in Supplementary Table S1. For comparison we also used the non-cancerous cell line WI-38 (primary fetal lung fibroblasts).32 WI-38 was grown in MEM medium containing L-glutamine and supplemented with 15% fetal bovine serum (FBS). All other cell lines were maintained in RPMI-1640 medium containing L-glutamine and supplemented with 10% FBS. Chemicals and reagents TFP FPZ CPZ TFPZ PZ (all from Sigma-Aldrich Stockholm Sweden) and cis-FPX (H Lundbeck A/S Copenhangen Denmark) were prepared in dimethyl sulfoxide and used at a final concentration of 0“20??M. TMX CQ diphosphate BafA1 NH4Cl N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7) L-741626 acridine orange fluorescein di-?-D-galactopyranoside (FDG) and carboxyfluorescein diacetate N-succinimidyl ester (CFSE) were from Sigma-Aldrich. z-VAD-fmk E-64d and CHX were from BD PharMingen (San Jose CA USA) Enzo Life Sciences (Ann Arbor MI USA) and Sigma-Aldrich respectively. PI tetramethylrhodamine ethyl ester perchlorate (TMRE) and LysoTracker Green DND-26 were from Invitrogen Stockholm Sweden. Cell viability analysis Cell viability was analyzed with MTT (3-(45-dimethylthiazol-2-yl)-25-diphenyl-tetrazolium bromide) assay as previously described.33 Briefly 5000“10?000 cells per well were seeded in 96-well plates. Twenty-four hours after seeding cells were subjected to a concentration range of phenothiazines alone as indicated in the figures or were treated with cisplatin gemcitabine or etoposide. Cell viability was determined after 72?h of continuous treatment where the viability of untreated cells was arbitrary set to 1. Alternatively area under the curve was calculated in the GraphPad Prism software (GraphPad Prism Software Inc. La Jolla CA USA). Cytotoxicity values reported in and Supplementary Figure S1 were calculated by the following formula: cytotoxicity=100%?viability (%). Flow cytometry Fluorescent probe-based analyses of intracellular anelles were performed in non-fixed cells. Labeling of lysosomes was performed using LysoTracker Green DND-26 (50?nM 1?h). Lysosome-associated ?-gal activity was assayed by enzymatic cleavage of FDG (50??M 30?min). Mitochondrial transmembrane potential was determined by retention of the potentiometric dye TMRE (50?nM 30?min). Cellular proliferative capacity was assessed using the CFSE-labeling method as previously described.34 DNA content analysis was performed in ethanol-fixed cells by PI staining. At least 10?000 events were recorded on a Becton-Dickinson FACSCalibur flow cytometer (BD Biosciences San Jose CA USA). Data analysis was conducted using the built-in Cell Quest software of FACSCalibur flow cytometer (BD Biosciences). Cell cycle distribution was assigned using ModFit (Verity Software House Topsham ME USA). Microscopy Gross morphology of cells was examined under a Nikon Eclipse TS100 light microscope (Nikon Instruments Europe B.V. Amsterdam The Netherlands) at — 10 magnification. Immunoblotting Whole-cell lysate (WCL) was obtained by lysing cells in radio-immunoprecipitation assay buffer (50?mM Tris-HCl pH 7.4 150?mM NaCl 1?mM ethylenediaminetetraacetic acid 0.1% Na-deoxycholate 1% NP-40) supplemented with protease and phosphatase inhibitor cocktail tablets (Roche Diagnostics AB Stockholm Sweden). "
1
"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 RP11‘284F219 in lung cell lines and tissues was measured using reverse transcription‘quantitative 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 RP11‘284F219 was identified in both lung carcinoma tissues and cells Knockdown of RP11‘284F219 in lung carcinoma cells inhibited cell proliferation and invasion but promoted cell apoptosis The present study identified the existence of a direct interaction between RP11‘284F219 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 RP11‘284F219 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 non‘small 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 carcinoma‘associated 0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONis significantly upregulated in NSCLC and may induce resistance to treatment of EGFR‘tyrosine kinase inhibitors by activating the AKTmTOR pathway lncRNA RP11284F219 was primarily discovered in a Pancancer transcriptomic analysis lncRNA RP11‘284F219 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 RP11‘284F219miR‘‘3pCCAR1 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 RP11‘284F219miR‘‘3pCCAR1 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 flash‘frozen in liquid nitrogen and transferred to ‘ËšC 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 Penicillin‘streptomycin 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 NCI‘H460 and NCI‘H1299 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 Penicillin‘streptomycin Thermo Fisher Scientific Inc All cells were culture at ˚C with CO2RNA extraction and reverse transcription‘quantitative PCR RT‘qPCR 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 first‘strand cDNA using the PrimeScript RT reagent kit Takara Bio InccDNA samples were diluted times to perform the RT‘qPCR 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‘' RP11‘284F219 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 ˚C for h followed by ˚C for min Thermal conditions of PCR reactions were Initial denaturation at ˚C for min followed by cycles for sec at ˚C and sec at ˚C The mRNA expression levels were determined using the 2ΔΔCq method Oligonucleotides and cell transfection The small interfering RNA siRNA synthetic negative control siNC RP11284F219 siRNAs siRP11284F219 miRNC miR‘‘3p mimics and miR‘‘3p 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 prolif‘erating 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 ˚C 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 Beckman‘Coulter 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 ˚C for h A total of 1x105 cells in µl serum‘free 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 si‘NC si‘RP11‘284F219 miR‘NC miR‘‘3p mimics miR‘‘3p 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 ˚C 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 co‘transfected with miR‘NC 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 Sigma‘Aldrich 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 SDS‘PAGE transferred to a PVDF membrane and blocked with non‘fat milk at room temperature for h The membranes were then incubated with CCAR1 primary antibody cat no ab70243 Abcam overnight at ˚C followed by incubation with goat anti‘mouse or goat anti‘rabbit IgG‘horseradish 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 ˚C with a h lightdark cycle The mice were maintained in an individually ventilated cage system under specific pathogen‘free conditions temperature ˚C humidity and fed with sterile food and water free access To evaluate the effect of RP11‘284F219 knockdown on the growth of lung carcinoma in vivo 5x106 siNC or siRP11284F219 treated NCI‘H1299 cells in µl serum‘free 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 body‘weight 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 miR‘‘3p 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 two‘tailed unpaired Student's t‘test or one‘way ANOVA with Tukey's post‘hoc analysis were performed to evaluate the statistical significance P005 was considered to indicate a statistically significant difference\x0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11‘284F219 expression is upregulated in LC tissues and cell lines A Expression of RP11‘284F219 in LC tissues in comparison with adjacent healthy tissues was analyzed using RTqPCR P0001 vs adjacent tissues n13 B Expression of RP11‘284F219 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 BEAS‘2B n3 LC lung carcinoma RT‘qPCR reverse transcription‘quantitative PCRResultsExpression of RP11‘284F219 is upregulated in lung carci‘noma 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 RP11‘284F219 was significantly upregulated in tumor tissues compared with healthy tissues Fig 1A The expression of RP11‘284F219 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 RP11‘284F219 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 RP11‘284F219 exerts anti‘oncogenic 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 2B‘D The results suggested that knocking down RP11‘284F219 significantly reduced the proliferation of lung carcinoma cells compared with the NC group Fig 2BD The invasiveness of si‘RP11‘284F219 transfected cells also significantly decreased as indicated by the data from the Transwell assay Fig 2F To further validate the invasive capability a RT‘qPCR assay was performed to detect the expression levels of invasion‘related genes and the results identified that both MMP2 and MMP9 were significantly decreased when RP11‘284F219 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 RP11‘284F219 knockdown in lung carcinoma cells indicating an oncogenic role of RP11284F219RP11‘284F219 directly interacts with miR‘‘3p Based on the prediction of the online tool lncBase v2 from DIANA Prediction module httpcarolinaimisathena‘innovationgrdiana_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 miR‘‘3p was partially complementary with the '‘UTR of RP11‘284F219 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 miR‘‘3p mimics Results from the luciferase reporter assay suggested that miR6273p mimics significantly decrease the signal of RP11‘284F219‘wt transfected cells but not the RP11‘284F219‘mut transfected cells indicating a direct interaction between the two non‘coding RNAs Fig 3A Furthermore transfection of siRP11284F219 into NCIH1299 and NCIH460 cells resulted in the suppression of endogenous RP11‘284F219 leading to a significant increase in miR‘‘3p expression Fig 3B Thus these findings suggested an inhibitory effect of RP11‘284F219 on the expression of miR‘‘3p in lung carcinoma cellsThe expression of miR‘‘3p was detected in both lung carcinoma tissues and cell lines It was demonstrated that miR‘‘3p 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 miR‘‘3pRP11‘284F219 regulates the proliferation and invasiveness of lung carcinoma cells via miR‘‘3p To rescue the antitumor effects of siRP11284F219 in lung carcinoma cells the miR‘‘3p inhibitor which specifically downregulates the expression of miR‘‘3p was transfected into NCI‘H1299 and NCI‘H460 cells Fig 4A The results from the CCK‘ and EdU assays demonstrated that treatment with si‘RP11‘284F219 0cONCOLOGY REPORTS Figure RP11‘284F219 knockdown inhibits lung carcinoma cell proliferation and invasion and promotes cell apoptosis A RP11‘284F219 knockdown was achieved via RP11‘284F219 siRNA and the knockdown efficiency was verified using reverse transcription‘quantitative 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 si‘NC group n5 D An EdU assay was performed to measure the proliferation of NCI‘H1299 and NCI‘H460 cells after transfection with si‘NC and si‘RP11‘284F219 Magnification x200 E Flow cytometry analysis was performed to determine the effects of RP11‘284F219 knockdown on apoptotic rates in NCI‘H1299 and NCI‘H460 cells n3 F Transwell assay was performed to determine the effects of RP11‘284F219 knockdown on NCI‘H1299 and NCI‘H460 cell invasion n3 Magnification x200 P005 P001 vs control group NC negative control siRNA small interfering RNA OD optical density and miR‘NC significantly decrease the proliferation of both NCIH1299 and NCIH460 cells Fig 4BD However the administration of miR‘‘3p 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 RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure RP11‘284F219 directly interacts with miR‘‘3p A Binding site between RP11‘284F219 and miR‘‘3p 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 miR‘NC n3 P005 vs miR‘NC B Expression of miR‘‘3p in NCI‘H1299 and NCI‘H460 cells transfected with si‘RP11‘284F219 was analyzed using RTqPCR P001 vs si‘NC n3 miR‘‘3p expression in C LC tissues and D NCI‘H460 NCI‘H1299 and A549 cells compared with adjacent healthy tissues and normal lung epithelial cells was analyzed using RT‘qPCR n3 P005 P001 vs adjacent tissue or BEAS‘2B cells NC negative control siRNA small interfering RNA wt wild‘type mut mutant miR microRNA LC lung carcinoma miR‘‘3p inhibitor restored the reduction in the number of NCIH1299 and NCIH460 cells that migrated through the Transwell membrane induced by si‘RP11‘284F219 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 miR‘NC group Fig S3 In addition transfection with miR‘‘3p inhibitor also diminished the pro‘apoptosis effect of si‘RP11‘284F219 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 miR‘‘3pRP11‘284F219 regulates CCAR1 via targeting miR‘‘3p To further evaluate how RP11‘284F219 exerts an oncogenic role via miR‘‘3p 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 miR‘‘3p 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 miR‘‘3p 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 CCAR1‘wt treated cells but not the CCAR1‘mut treated cells suggesting a direct binding of miR‘‘3p 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 miR‘‘3p in lung carcinoma cells and tissuesRP11‘284F219 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 RP11‘284F219 regulates proliferation and invasiveness in lung carcinoma cells via miR‘‘3p A Expression of miR‘‘3p in NCI‘H1299 and NCI‘H460 cells transfected with miR‘NC or miR‘‘3p inhibitor was detected using RT‘qPCR analysis n3 P005 vs miR‘NC Cell Counting Kit‘ assay was performed in B NCI‘H1299 and C NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor n5 D EdU assay was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor Magnification x200 E Flow cytometry analysis was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor n3 F Transwell assay was performed in NCI‘H1299 and NCI‘H460 cells stably transfected with si‘RP11‘284F219 in the presence of miR‘NC or miR‘‘3p inhibitor Magnification x200 n3 P005 vs si‘NC NC negative control siRNA small interfering RNA OD optical density miR microRNA 0cLI RP11‘284F219 PROMOTES LUNG CARCINOMA PROLIFERATION AND INVASIONFigure miR‘‘3p directly targets CCAR1 A Bioinformatic analysis of the predicted binding sites between the CCAR1 '‘untranslated region and miR‘‘3p B Luciferase reporter assay in CCAR1‘wt or CCAR1‘mut treated cells in the presence of miR‘NC or miR‘‘3p mimic n3 P005 vs miR‘NC C CCAR1 expression in LC tissues compared with adjacent healthy tissues was analyzed using RT‘qPCR n13 P001 vs adjacent tissue Expression of CCAR1 in NCI‘H1299 and NCI‘H460 cells transfected with miR‘NC or miR‘‘3p mimics was detected using D RT‘qPCR and E western blotting n3 P005 vs miR‘NC miR microRNA NC negative control wt wild‘type mut mutant RT‘qPCR reverse transcription‘quantitative 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 si‘RP11‘284F219 group compared with the control group Fig 6A and B RTqPCR analysis also demonstrated that compared with the siNC group the tumors in the si‘RP11‘284F219 group expressed higher levels of miR6273p Fig 6C and lower levels of CCAR1 Fig 6D providing further evidence to the existence of the RP11‘284F219miR‘‘3pCCAR1 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 RP11‘284F219 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 miR‘‘3p directly interacts with RP11‘284F219 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 D‘enhanced efficacy of irinotecan via inhibition of the cytochrome P450 enzyme‘mediated intratumoral drug metabolism miR‘ is also reported to be a potential non‘invasive diagnostic marker in gastric and breast cancer types In pulmonary diseases miR627 is downregulated in patients with chronic obstructive pulmonary disease and targets the high‘mobility group box protein to inhibit its expression thus improving transforming growth factorβ1‘induced pulmonary fibrosis The present results demonstrated the inhibitory effect of RP11‘284F219 on the expression of miR‘‘3p In addition it was identified that the miR‘‘3p inhibitor can neutralize the anti‘tumor effects of RP11‘284F219 knockdown indicating that RP11‘284F219 promotes the proliferation and invasiveness of lung carcinoma cells partially by regulating miR‘‘3p This anti‘tumor 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 RP11‘284F219 knockdown inhibits tumor growth in vivo A Macroscopic image of xenografted tumors B Tumor volume in nude mice injected with NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 measured over weeks n5 C Weight of tumors in nude mice at weeks after injection of NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 n5 Expression levels of D miR‘‘3p and E CCAR1 in the tumor tissues from nude mice injected with NCI‘H1299 cells transfected with si‘NC or si‘RP11‘284F219 for weeks were detected using reverse transcription‘quantitative PCR n5 P005 P001 P0001 vs si‘NC 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 RP11‘284F219 Furthermore the regulatory axis of RP11‘284F219miR‘‘3pCCAR1 exists in the lung carcinoma cells both in vitro and in vivo in the tumor growth model The interaction between RP11‘284F219 and miR‘‘3p and the interaction between miR‘‘3p and CCAR1 were demonstrated by the dual‘luciferase assay Although this method has been used to validate RNA‘RNA 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 RNA‘protein 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 Doxorubicin‘induced 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 miR‘‘3p the expression of CCA
2
" new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease In addition to the personto person transmission dynamic of the novel respiratory virus it has been recently studied the role of environmental factors in accelerate SARSCoV2 spread and its lethality The time being air pollution has been identified as the largest environmental cause of disease and premature death in the world It affects body™s immunity making people more vulnerable to pathogens The hypothesis that air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography can acts both as a carrier of the infection and as a worsening factor of the health impact of COVID19 disease has been raised recently With this review we want to provide an update state of art relating the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality The Authors who first investigated this association often used different research methods or not all include confounding factors whenever possible In addition to date incidence data are underestimated in all countries and to a lesser extent also mortality data For this reason the cases included in the reviewed studies cannot be considered conclusive Although it determines important limitations for direct comparison of results and more studies are needed to strengthen scientific evidences and support firm s major findings are consistent highlighting the important contribution of PM25 and NO2 as triggering of the COVID19 spread and lethality and with a less extent also PM10 although the potential effect of airborne virus exposure it has not been still demonstrated Introduction A new coronavirus SARSCoV2 has determined a pneumonia outbreak in China Wuhan Hubei Province in December called COVID19 disease The scientific community has come together to implement pharmaceutical and nonpharmaceutical intervention measures designed to contain SARSCoV2 global spread Nevertheless on March 11th WHO™s Directeneral announced that COVID19 can be characterized as a pandemic SARSCoV2 is primarily transmitted from persontoperson through close contact approximately m by aerosol respiratory droplets smaller than μm in diameter wwwwhoint Indoor environments might be especially hazardous because of their reduced ventilation Morawska lack ultraviolet light which rapidly inactivates the virus and because it can become less diluted than it would in outdoor environments Schuit It is also known how the virus can survive and being infectious in aerosols for hours and on surface up to days van Doremalen et al similarly with transmission dynamics known for SARSCoV1 associated with nosocomial spread and superspreading events Chen et al 2020ab Beyond the causality it is uncertain even if certain demographics of the population are more vulnerable to SARSCoV2 infection Based on recent reports male gender advancing age and comorbidities seem to be correlated with death and severe illness Harris et al Furthermore COVID19 seems to be associated with an increasing rate of thromboembolic events in hospitalized patients Llitjos Mechanisms of social and economic interactions are additionally supposed to be involved in the diffusion dynamic of COVID19 in the diverse parts of the world or of the same country such as the living conditions the healthrelated behaviour KhalatbariSoltani et al Corresponding author Email address ccopatunictit C Copat 101016jenvres2020110129 Received July Received in revised form August Accepted August EnvironmentalResearch1912020110129Availableonline24August2020001393512020ElsevierIncAllrightsreserved 0cC Copat the commercial exchanges Bontempi 2020a or the migration scale index H Chen It seems that these diffusion dynamics have particularly affected the COVID19 spread at the early stage Among the environmental parameters some climate condition such as temperature humidity sunlight and wind revealed a reduction of the COVID19 spread S Chen Coccia 2020a and air pollution seems to have a role in airborne transmission of SARSCoV2 and severity of COVID19 Domingo Nevertheless to better understand COVID19™s diffusion patterns an interdisciplinary multidimensional approach should be encourage in order to develop firm s Bontempi Air pollution has been identified as the largest environmental cause of disease and premature death in the world GBD Ambient particulate matter PM induces its proinflammatory and thrombogenic effects through the generation of oxidative stress by its chemical compounds and metals Li Signorelli The recent identification of environmentally persistent free radicals EPFRs in the PM resulting from a mixture of combustion sources theorize its role in the increase of disease severity of lower respiratory tract infections LRTI Jaligama Scientific evidences support that short and longterm exposures to ambient air pollutants are associated with a broad of adverse health outcomes Ferrante and Conti Fiore such as higher mortality rates greater hospital admissions and increased outpatient visits Bremner Cohen Dehghani Dockery It has markedly detrimental consequences on asthma bronchitis pneumonia and COPD Dick Perng and Chen Raji Vignal Yarahmadi et al where bacteria and viruses are the most accepted causative factors that harm airway stability driving to infection exacerbation Furthermore air pollution represents an aggravating factor for infection diseases caused by some viral infections Domingo and Rovira such as respiratory syncytial virus RSV influenza A and B para influenza virus type pneumonia and influenzalike illness Carugno Croft Fukuda Huang Huh Liang Lin Silva Somayaji It determines an increase in the rate of hospitalizations and access to emergency department visits Studies related to the epidemic of SARSCoV coronavirus identified in November from the Guangdong province of southern China reported similar associations Cai Cui Kan Several Authors suggest that outdoor air pollution resulting from a combination of factors such as meteorological data level of industrialization as well as regional topography could operate both as a carrier of the infection and as a worsening factor of the COVID19 severity Conticini Frontera Isaifan Martelletti and Martelletti This association is getting stronger thanks to the results of the numerous studies that have been launched all over the world and summarized with this review Most of the reviewed studies support that chronic exposure to air pollution might led people more susceptible to COVID19 disease leading to widespread COVID19 spread and lethality Nevertheless as suggested by Bontempi 2020b the potential effect of airborne virus exposure due to PM10 remain unclear With this review we want to provide an updated state of art of the recently epidemiological studies dealing with understanding the role of air pollution in particular PM25 PM10 and NO2 in COVID19 spread and lethality Fig PRISMA Flow Diagram of identification screening and inclusion of studies EnvironmentalResearch19120201101292 0cC Copat Method We have conducted a systematic review of the literature concerning the relationship between some air pollutants PM25 PM10 and NO2 and COVID19 outbreak The research was performed in compliance with the PRISMA criteria Preferred Reporting Items for Systematic Reviews and MetaAnalyses and the Flow Diagram is showed in Fig The research was conducted between April and July 6th in PubMed database It was used the Advanced Search Builder and the keywords were searched in [Title OR Abstract] We have filtered only research articles published in English language and selected the following keywords Air pollution and COVID19 or SARSCOV2 Particulate matter or PM and COVID19 or SARSCOV2 Nitrogen dioxide or NO2 and COVID19 or SARSCOV2 We choose as inclusion criteria all the available epidemiological studies aimed to identify any temporal and spatial association between reported COVID19 cases andor deaths and air pollution data related to PM25 PM10 and NO2 thus excluding any Letter Opinion Commentary Review or nonrelevant articles We obtained a total of eligible published research articles in their final version and paper in its preprint version For some of them we chose to include only principal findings that clearly fit the aim this review Particulate Matter and COVID19 Atmospheric particulate matter PM is originated by a wide range of anthropogenic and natural sources Kim It consists of a heterogeneous mixture of solid and liquid particles suspended in air that varies continuously in size and chemical composition including nitrates sulphates elemental and anic carbon anic compounds biological compounds and metals WHO It has been associated with increased respiratory morbidity and mortality Liu especially in susceptible people due to cardiorespiratory events including asthma chronic obstructive pulmonary disease and thrombosis Li Rhee In vitro and in vivo studies highlighted its role in the exacerbation of respiratory viral infections Becker and Soukup Recently the research group of Setti gave first preliminary evidence that SARSCoV2 RNA can be present on outdoor particulate matter thus suggesting that in conditions of atmospheric stability and high concentrations of PM it could represent a potential early indicator of COVID19 although it does not give information regarding COVID19 progression or severity Several observations report a significant association between ambient concentrations of PM25 Adhikari and Yin Bashir Fattorini and Regoli Frontera Jiang Li VasquezApestegui Wu Yao Zhu Zoran 2020a and PM10 Bashir Coccia 2020b Fattorini and Regoli Jiang Li Yao Zhu Zoran 2020a with COVID19 pandemic across the most affected countries China Italy and USA see Table First evidences on the temporal association between air pollution and COVID19 were reported in China where the outbreak was first identified Zhu explored the relationship between particulate matter and the viral infection caused by the novel coronavirus in cities in China The Authors included over of dailyconfirmed new cases in the whole of China between January 23rd and February 29th They applied a Generalized Additive Model GAM to examine the effects of meteorological factors and air pollution on COVID19 incidence applying a movingaverage approach to capture the cumulative lag effect of ambient air pollution and considering population size and density as potential confounders They observed that the effect of PM25 on daily confirmed cases was greater than PM10 In particular they found that a 10μgm3 increase lag0“ in PM25 and PM10 was associated with a CI to and CI to increase in the daily counts of COVID19 confirmed cases respectively Jiang focused their attention on three most affected cities of China Wuhan XiaoGan and HuangGang collecting data of daily cases and ambient air pollutant from Jan 25th to Feb 29th The Authors by applying a multivariate Poisson regression revealed a significant temporal association between PM25 increased and COVID19 incidence in all the considered cities especially in HuangGang Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Conversely an increase in PM10 concentrations was associated with a decrease of COVID19 incidence These results were partially confirmed by findings of Li who conducted a simple linear regression to compare COVID19 incidence with PM concentrations in Wuhan and XiaoGan from Jan 26th to Feb 29th in They found that an increase in PM25 was correlated with an increase of COVID19 incidence in both cities Wuhan R2 p XiaoGan R2 p while for PM10 only in XiaoGan R2 p The spatial distribution of particulate matter and case fatality rate CFR of COVID19 was studied by Yao in cities of China including Wuhan collecting data up to March 22nd First they found a significantly positive global spatial autocorrelation of COVID19 CFR Global Moran™s index I p highlighting a high CFR clustering located in Hubei Province With a multiple linear regression they adjusted their results for several effect modifiers and confounder factors such temperature relative humidity gross domestic product GDP per capita hospital beds per capita local indicators of spatial association LISA map values city size and population or proportion of people older than years It was found that for every μgm3 increase in PM25 and PM10 the CFR increased by “ and “ respectively and the risk estimates increased to “ and “ with every μgm3 increase in average concentrations of PM25 and PM10 in “ respectively Some studies describe the association between air pollution and COVID19 across Italy the second country of the world where the infection spread significantly at the beginning of the pandemic and suddenly has reached many other European countries The 28th of July Italy recorded more than total confirmed cases and deaths WHO most of which were distributed in the regions of Northern Italy especially the Lombardy It is recognized as one the most air polluted areas of Europe EEA where the frequent PM10 annual exceedances of the WHO threshold of μgm3 are responsible for attributable deaths per year corresponding to attributable community rates of deaths per inhabitants per year Baccini Bontempi 2020bfocused the attention on two of the most affected regions of Northern Italy Lombardy and Piedmont The Authors based on PM10 daily exceedances and COVID19 confirmed cases on March 12th thus before the Italian sanitary crisis observed that PM10 concentration was exceeded only few times among the Lombard cities that at the beginning of the epidemic were most affected On the contrary among some Piedmont cities suffering of severe PM10 pollution events COVID19 incidence was lower Based on their results the Authors concluded that COVID19 diffusion by airborne PM10 is hard to demonstrate nevertheless several research article revealed how PM in particular PM25 could had a role in accelerate and vast diffusion of COVID19 in Northern Italy For example Coccia 2020b by analyzed data on Italian province capitals and data of infected individuals up to April 7th revealed a relationship between air pollution of cities measured with days exceeding the limits set for PM10 in previous years and COVID19 diffusion In particular cities with more than days of PM10 exceedances showed a very high average number of infected individual about infected individuals on 7th April whereas cities having less than days of PM10 exceedances showed a lower average number of infected about infected individuals Frontera gave also evidences on the role of PM25 as a contributing factor of COVID19 outbreak in Northern Italy where EnvironmentalResearch19120201101293 0cC Copat Table Summary table reporting reviewed results on the association between COVID19 casesdeaths and air pollution PM25 PM10 and NO2 References Zhu Data analysis Generalized Additive Model GAM Aim Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Temporal association between daily confirmed cases and air pollution PM25 PM10 and NO2 Spatial association between fatality rate and air pollution PM25 and PM10 Spatial association between deaths counts and air pollution NO2 Temporal association between total cases daily confirmed cases and total deaths and air pollution PM25 and PM10 Temporal association between total cases daily confirmed cases and total deaths and air pollution NO2 Spatial description of PM10 exceedances versus COVID19 cases Multivariate Poisson regression Simple linear regression Multiple linear regression Descriptive analysis percentage of deaths in three NO2 μmol m2concentration range “ “ “ Pearson coefficient correlation Pearson coefficient correlation Descriptive analysis Number of days of PM10 exceeding μgm3 and COVID19 incidence Area of Study cities of China Period From Jan 23rd to Feb 29th Jiang Li Yao Ogen Zoran 2020a Zoran 2020b Bontempi 2020b From Jan 25th to Feb 29th From Jan 26th to Feb 29th in Data up to March 22nd Data up to the end of Feb From Jan 1st to Apr 30th From Jan 1st to Apr 30th From Feb 10th to March 12th Wuhan XiaoGan and HuangGang China Wuhan and XiaoGan cities of China administrative regions in Italy Spain France and Germany Milan Italy Milan Italy provinces of Lombardy Italy provinces of Piedmont Italy Coccia 2020b Data up to April 7th Italian Provinces Fattorini and Regoli Data up to April 27th Italian provinces PM25 A 10μgm3 PM25 increase lag0“ was associated with a increase of daily confirmed new cases PM10 A 10μgm3 PM10 increase lag0“ was associated with a increase of daily confirmed new cases Wuhan RR CI1032“ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p Wuhan RR CI “ XiaoGan RR CI “ HuangGang RR CI “ Wuhan R2 p XiaoGan R2 p χ2 p A μgm3 increase in PM25 was associated with a “ increase in fatality rate χ2 p A μgm3 increase in PM10 was associated with a “ increase in fatality rate NO2 A 10μgm3 NO2 increase lag0“ was associated with a increase in daily confirmed new cases Wuhan RR CI “ XiaoGan RR CI “ HuangGang no association found Wuhan R2 p XiaoGan R2 p of fatality cases are associated with NO2 μmolm2 R cid0 R R cid0 R cid0 R R cid0 R cid0 R cid0 R cid0 Lombardy PM10 exceeding between and COVID19 incidence between and Piedmont PM10 exceeding between and COVID19 incidence between and COVID19 in North Italy has a high association with air pollution of cities measured with days exceeding the limits set for PM10 R2 p R2 p continued on next page Hierarchical multiple regression model Pearson regression coefficient analysis R2 p Spatial association between confirmed cases and air pollution PM10 Spatial association between total confirmed cases and air pollution PM25 PM10 and NO2 EnvironmentalResearch19120201101294 0cC Copat Table continued References Frontera Frontera Wu Adhikari and Yin Bashir Bashir VasquezApestegui VasquezApestegui VasquezApestegui Period Data up to 31st March Data up to 31st March Data up to April 04th From March 1st to Apr 20th From March 4th to April 24th From March 4th to April 24th Data up to June 12th Data up to June 12th Data up to June 12th Area of Study Italian regions Italian regions counties in the USA Queens county New York USA California California districts of Lima Perù districts of Lima Perù districts of Lima Perù Aim Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Prediction of risk of COVID19 deaths in the long term average exposure to fine particulate matter PM25 Temporal association between daily confirmed cases and total deaths and air pollution PM25 Association between confirmed cases and air pollution PM25 PM10 and NO2 Association between deaths and air pollution PM25 PM10 and NO2 Spatial association between total confirmed cases and air pollution PM25 Spatial association between deaths and air pollution PM25 Spatial association between case fatality rate and air pollution PM25 Data analysis Pearson regression coefficient analysis PM25 R2 p PM10 Pearson regression coefficient analysis R2 p Longterm exposure increase of μgm3 in PM25 is associated with a increase in the COVID19 death rate Estimate on cases values cid0 CI “ Estimate on deaths value cid0 CI “ Kendall r cid0 Spearman r cid0 Zeroinflated negative binomia models Negative binomial regression model Spearman and Kendall correlation tests Spearman and Kendall correlation tests NO2 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Kendall r cid0 Spearman r cid0 Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient p Multivariate regression model Crude coefficient cid0 p mortality was found significantly higher than less polluted Italian regions By collecting data up to March 31st for all Italian regions and performing a Pearson correlation analysis they found a strong positive association both with the total number of confirmed cases R and deaths R other than with hospitalized cases R The Italian situation was further highlighted by the study of Fattorini and Regoli in Italian provinces They explored the spatial association between air pollution and COVID19 cases with data up to April 27th By applying the Pearson regression coefficient analysis they revealed a positive association both with PM25 and PM10 R2 p and R2 p respectively A focus on the most affected city of Italy Milan was conducted by Zoran 2020a This city is located in the Po valley basin known hotspot for atmospheric pollution at the continental scale EEA The Authors performed a temporal association between COVID19 Total cases Daily New positive cases and Total Deaths and particulate matter from Jan 1st and Apr 30th by applying a Person correlation In accordance with other studied they found a positive association between daily confirmed cases and PM25 R and PM10 R although they did not consider any delay time from infection to COVID19 onset Nevertheless they found a negative association between total cases and total deaths and particulate matter but the assumption of a temporal linear correlation may be inaccurate because the above mentioned variables could have more complex nonlinear relationships To date the USA have more than million confirmed cases and thousand deaths WHO Here ambient concentrations of PM and O3 were found responsible to cause between and premature deaths Fann The association between air pollutants and COVID19 cases and deaths was studied by Bashir in the state of California from March 4th to April 24th corresponding to the beginning of the COVID19 outbreak in USA Based on their significant correlation found the Authors state that a limited human exposure to these pollutants will contribute to defeating COVID19 This seems unclear because they found a negative correlation with PM25 and PM10 EnvironmentalResearch19120201101295 0cC Copat by applying both the Kendall rank correlation and Spearman™s one and it is not clear if they normalized COVID19 cases by population size and if they performed a day by day association or a spatial association across the country A focus on the Queen county New York USA was provided by Adhikari and Yin They retrieved data of PM daily concentrations from two ground monitoring stations and collected data of confirmed COVID19 cases and numbers of related deaths from USAFacts in the period from March to April The Authors elaborated their data with a negative binomial regression model and considered the cumulative lag effect of PM25 on disease outcomes over the past days They found a significant negative association among PM25 and new daily confirmed COVID19 cases cid0 CI “ and deaths cid0 CI “ Low PM concentrations in this area of study mean μgm3 are likely to have played a less central role in the spread of infection than in other areas such as Italy where PM25 monthly concentrations reached values higher than μgm3 Fattorini and Regoli Frontera or in China where PM25 monthly concentrations reached values higher than μgm3 Zhu Jiang As said by the Authors other gaseous pollutants such as NO2 and SO2 could have influenced transmission and pathogenesis of COVID19 In the United States Wu investigated whether longterm average exposure to fine particulate matter PM25 increases the risk of COVID19 deaths by considering approximately counties in the United States of the population With an exposure prediction model the Authors calculated the county level longterm exposure to PM25 averaged for to and collected COVID19 deaths counts up to April 04th They conducted a strong and robust statistical analysis with zeroinflated negative binomial mixed models adjusting their results by several potential confounders such as sociodemographic socioeconomic behavioural and meteorological factors They found that a small longterm exposure increase of only μgm3 in PM25 is associated with a increase in the COVID19 death rate confidence interval CI VasquezApestegui recently reported first evidences on the spatial relationship between particulate matter and COVID19 outbreak from Latin America The Authors described the situation occurred in districts of Lima located in the second most affected country of Latin America Peru In particular by applying a multivariate regression model they evaluated the association between the population exposure to PM25 concentrations in the previous years “ and cases deaths and casefatality rates of COVID19 with data up to June 12th A significant association has been found both with cases and deaths Crude coefficient with p and with p respectively but not with case fatality rate All these studies highlight the role of PM in triggers of the COVID19 disease and how government measures targeting to sustainable growth such as the reduction of urban and industrial emissions could have a positive impact on the prevention of health outcomes reducing mortality rate as well the burden on health care systems Nitrogen dioxide NO2 and COVID19 induced lung damage Hence viral infection becomes more common after exposure to NO2 Zhu Furthermore NO2 is associated with other several health effects such as elevated risks for asthma allergic rhinitis and eczema in children To increase of outpatient visits and hospitalizations due to bronchitis and asthma exacerbation Bahrami Asl Kowalska increase of chronic obstructive pulmonary disease COPD Ghanbari Ghozikali Pfeffer and increase of pulmonary heart disease related mortality Chen A recent study explored the possible role of NO2 in interference in Angiotensin converting enzyme ACE2 The expression of ACE2 is high on lung alveolar epithelial cells and it is the human cell receptor of virus agent of COVID19 Alifano First observations report an association between ambient concentrations of NO2 and COVID19 pandemic across Europe China and USA Bashir Fattorini and Regoli Jiang Li et al Ogen Zhu et al Zoran et al 2020b Conversely to the other papers findings of Zoran 2020b and Bashir provides different findings reporting no association or a negative one between NO2 and daily deaths counts In China Zhu by applying the same method explained for PM observed that a 10μgm3 increase lag0“ in NO2 is associated with a CI “ increase in the daily counts of COVID19 confirmed cases in cities of China These findings are confirmed by Jiang and Li et a who applied the same method described for PM Jiang revealed a significant positive association between NO2 and COVID19 both in Wuhan and XiaoGan Wuhan RR CI1053“ XiaoGan RR CI “ but did not found any significant association in HuangGang Li found a significant linear correlation both in Wuhan R2 p and XiaoGan R2 p Ogen presented evidences on the relationship between exposure to NO2 including the months of January and February shortly before the COVID19 spread in Europe and novel coronavirus fatality in the most affected European countries concluding that longterm exposure to NO2 may be a potential contributor to mortality caused by SARSCoV2 He collected data concerning the number of fatality cases from administrative regions in Italy Spain France and Germany and correlated mortality with tropospheric NO2 concentrations measured by the Sentinel5 Precursor spaceborne satellite The major tropospheric NO2 hotspot identified was located in the Northern Italy In all European regions considered gas concentrations ranged between and μmolm2 with airflows directed downwards Results showed that out of the fatality cases by March were in five regions located in north Italy and central Spain Furthermore by analysing mortality trends it was revealed that the highest percentage of deaths occurred in regions where the maximum NO2 concentration was above μmolm2 with a significant decrease where the maximum concentration was between and μmolm2 and below μmolm2 The methodology used by Ogen cannot support a longterm exposure investigation Surely a validation of the satellite measure with those of the ground ™ones the adjustment of the results according to the different population size of each country could have made their results more robust Nevertheless the study provide new insights for future investigation The Italian situation was further studied by Fattorini and Regoli who collected data of COVID19 incidence up to April 27th from Italian provinces They revealed a strong spatial corr
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glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research “ du 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 wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city people™s hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city people™s hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage years‰gendermalefemalegradei“iiiii“ivtumor size cm‰low n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturer™s instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq„¢ ii takara japan gapdh was the normalized control relative expression was calculated through the ˆ’δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturer™s instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturer™s instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using student™s ttest or oneway anova survival rate was analyzed by the kaplan“meier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells b“e proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4c“f therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearson™s correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1“ v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387“e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med “ 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci “ 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun “ 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther “ 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther “ 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys “ 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene “ to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem ““ 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer “ 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell “ xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett “ 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers “ 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med “ 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med “ 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci “ 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res “ yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer “ 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep “ 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open 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 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" 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 model™s components and links 0crakhshanderou bmc medical education page of in thisstudy using the sample size formula ¾ z¾2δ2d2 in which δ2 α n ¼ °zˆd 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 cronbach™s 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 tool™s 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 cronbach™s 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 bartlett™s 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 truefalsedon™t know items 5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagree items5point likert scalestrongly disagree to stronglyagreescoring range˜correct™ response ˜don™t know™response ˜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 individual™s perception of his orher competence to successfully perform a behavior behavioral intention refers to a person™s perceived probability orœsubjective 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 χ2 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 bartlett™s 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 education““femalemalediplomaassociate 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 ± ˆ’ ± ± ± ± ˆ’ ± ± ˆ’ ± ˆ’ ± ± ± ˆ’ ± ± ˆ’ ± ± ˆ’ ± 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 one™sintention 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 œresearcherdesired 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 publisher™s 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 winter239“ghaffari 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 rimer“janz nk becker mh the health belief model a decade later health educ q“lp 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"
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" microwave ablation mwa is widely used to treat unresectable primary and secondary malignanciesof the liver and a limited number of studies indicate that ablation can cause not only necrosis at the in situ site butalso an immunoreaction of the whole body this study aimed to investigate the effects of mwa on cytokines inpatients who underwent mwa for a hepatic malignancymethods patients admitted to the oncology department in the first affiliated hospital of soochow universitybetween june and february were selected peripheral blood was collected from patients with a hepaticmalignancy treated with mwa the levels of cytokines il2 ifnÎ tnfα il12 p40 il12 p70 il4 il6 il8 il10and vascular endothelial growth factor vegf were detected with a milliplex® map kit the comparison times wereas follows before ablation h after ablation days after ablation and days after ablation data were analyzedusing a paired sample ttests and spearman™s correlation analysisresults a total of patients with hepatic malignancies were assessed there were significant differences in il2il12 p40 il12 p70 il1 il8 and tnfα at h after mwa significant increases 2fold vs before ablation wereobserved in il2 il1 il6 il8 il10 and tnfα after mwa elevated il2 and il6 levels after ablation werepositively correlated with energy output during the mwa procedures wa treatment for hepatic malignancies can alter the serum levels of several cytokines such as il2 and il6keywords microwave ablation hepatic malignancy cytokines il2 il6 immunoregulation primary and secondary malignancies of the liver have asubstantial impact on morbidity and mortality worldwidein china hepatocellular carcinoma hcc has the secondhighest mortality rate of malignancies the treatmentof primary and secondary hepatic malignancies via correspondence lengbengsudaeducn jing zhao qiang li and merlin muktiali contributed equally to this work2department of oncology the first affiliated hospital of soochow universitysuzhou china5division of neurosurgery city of hope beckman research institute duartecalifornia usafull list of author information is available at the end of the interventional imaging therapy is undertaken by investigators in the field of interventional radiology and possibly bya smaller group of practitioners known as interventionaloncologists whose major focus is cancer care via minimally invasive approaches [ ] recently percutaneous ablation therapy has been widely accepted as a radicaltreatment method for hcc and its fiveyear survival rateis similar to that of resection microwave ablationmwa is widely used to treat unresectable hcc and recurrent hcc and has the advantages of minimal invasiona good curative effect and no side effects due to radiationor chemotherapy immune checkpoint inhibitors icis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhao bmc cancer page of such as pd1pdl1 and ctla4 antibodies have beenwidely applied in several cancers and studies have indicated that ici treatment could enhance the effect of ablation evidence hasindicated that hyperthermicdestruction causes the release of a large population of heterogeneous tumor antigens and inflammatory cytokinesmay play crucial roles in this process cytokines aremediators that regulate a broad range of processes involved in the pathogenesis of cancer several cytokineswhich can arise from either tumor cells or immunocytes such as tumor necrosis factor tnfα interleukinil1 il6 il8 il10 and vascular endothelial growthfactor vegf have been linked with cancers and can either promote or inhibit tumor development the serumlevels of cytokines differ during cancer development although cytokines have been found to be altered after anticancer treatment such as chemotherapy and radiotherapy[ ] few investigations have focused on cytokines beforeand after mwa it is still unknown whether the above cytokines changed before andor after mwa in patientswith hepatic malignancies in this study we investigatedthe effects of mwa on the serum levels of cytokines inpatients with hepatic malignanciesmethodspatients and samplesthe patient population examined in this study was derivedfrom the first affiliated hospital of soochow universitypatients were admitted to the oncology department between june and february the total number ofpatients was with liver metastases and primaryliver cancers the inclusion criterion was a tumor locatedat a hepatic site either primary or metastases all patients with metastatic hepatic malignances should be givensystematic treatments chemotherapy or target therapyand get at least stable disease sd or partial responsepr for more than days informed consent for blooddraw and the relevant therapy was obtained from all patients the protocol was approved by the human ethicscommittee of the first affiliated hospital of soochowuniversity and was conducted in accordance with thedeclaration of helsinki all written informed consent wasobtained from all participants and clearly stated wholeblood ml was drawn into edta anticoagulant tubeson days ˆ’ to before and h days and days afterablation mostly on the last day of the course for cytometry and cytokine analysesablation procedurethe ablation procedure used in this research was mwathe puncture site and pathway were determined underthe guidance of a computed tomography ct scanlocal infiltration anesthesia was achieved by using lidocaine the placement of microwave ablation probeswas guided by a ct scan or ultrasonic device and allprobes were placed at the maximum diameter layerdouble probes were employed when the maximumdiameter of the tumor was up to cm the power andtime of ablation were designed for each patient in therange of w and min respectively basedon the size number and position of the tumor theboundaries of ablation zones were designed as extended cm upon the tumor sitecytokine detectiona milliplex map kit with human cytokinechemokinepanels that measured ifnÎ il2 il6 il8 il10 il12p40 il12 p70 il1 tnfα and vegf was utilized according to the manufacturer™s instructions briefly chemically dyed antibodybound beads were mixed withstandard or sample incubated overnight at °c washedand then incubated with a biotinylated detection antibodyafter the beads were washed they were incubated with astreptavidin phycoerythrin complex and the mean fluorescent intensities were quantified on a luminex analyzer luminex corporation all samples were measured in duplicate standard curves of known concentrations of recombinant human cytokineschemokines wereused to convert fluorescence units to cytokine concentration units pgml the minimum detectable concentrations were as follows ifnÎ pgml il2 pgmlil12 p40 pgml il12 p70 pgml il1 pgml il6 pgml il8 pgml il10 pgml tnfα pgml and vegf pgml all resultsbelow the minimum concentrations were processed as theminimum concentrationsstatistical analysisibm spss statistics software was used for the statistical analysis along with graphpad prism for figurecreations normally distributed numerical data areexpressed as the mean ± standard deviation and nonnormally distributed numerical data are expressed as themedian and confidence interval ci cytokinesat different times were compared using a onetailedpaired ttest spearman™s correlation analysis was executed to determine the correlation between clinical indexes and cytokine levels p indicates a significantdifferenceresultsclinical characteristics of the enrolled patientsas shown in table a total of patients with tumorslocated on the liver liver metastases primary livercancers were analyzed the patients™ cytokine levelswere compared according to time before treatment h after treatment days after treatment and daysafter treatment 0czhao bmc cancer page of table clinical characteristics of the patients enrolled n characteristicsexmalefemaleagepathogenesisprimarysecondaryprimary site for metastatic hepatic malignancescolon rectalpancreasstomachebreastothersmaximum tumor length mmablation probe usedablation time minaverage power per probe w ± ± ± ± average energy time × power time × power–¼–¼ time and power indicate the time and power respectively ofdifferent probes used during the operation ± ifnÎ il12 p40 and il12 p70 were slightly increasedafter mwa treatmentas shown in table and fig the median level ofifnÎ before the mwa treatment was pgml ci “ pgml at days and days after themwa treatment there was a slight increase comparedto that premwa with median levels of pgml ci “ pgml and pgml ci“ pgml respectively the median level of il p40 before the mwa treatment was pgml ci “ pgml there was a slight increase to pgml ci “ pgml days postmwathe median il12 p70 level before the mwa treatmentwas pgml ci “ pgml and increasedto pgml ci “ pgml days afterthe mwa treatment and to pgml ci “ pgml days postmwa no significant alteration in the vegf median level was detected after themwa treatmentil2 il1 il6 il8 and il10 were elevated over 2foldafter the mwa treatmentas shown in table fig and fig the median levelof il2 before the mwa treatment was pgml ci “ pgml there was a significant increase at h postmwa with a median level of pgml ci “ pgml the median level ofil1 before the mwa treatment was pgml ci “ pgml and a significantincrease wasnoted days after the mwa treatment pgml ci “ pgml the median level of il6before the mwa treatment was pgml ci“ pgml and significantly increased daysafter the mwa treatment pgml ci “ pgml the median level ofil8 before themwa treatment was pgml ci “ pgml and increased significantly to pgml ci“ pgml days after the mwa treatmentthe median level of il10 before the mwa treatmentwas pgml ci “ pgml and increasedsignificantly days after the mwa treatment pgml ci “ pgml the median level oftnfα before the mwa treatment was pgml ci “ pgml and increased significantlyto pgml ci “ pgml days afterthe mwa treatmentlevelselevated il2 and il6 levels after ablation were positivelycorrelated with energy output during mwato further evaluate the relationship between the increased cytokineand mwa treatment weemployed the concept of œenergy time × power time × power time and power indicated thetime and power of different probes used in the operation to reflect total hyperthermic damage to hepatictissues during the mwa procedure as shown in table and fig the il2 levels at h postmwa and the il levels at days postmwa illustrated significant correlations with energy the relative indexes were and respectivelydiscussionas technology continues to develop other types of localtherapy such as radiotherapy chemical ablation andhyperthermal ablation for primary and metastatic livercancer are increasingly being used mwa for liver malignances is reserved for patients who cannot undergosurgical removal or for whom other treatments havefailed a consensus guideline was recently developed to address indications for mwa in these patientsthermal ablation is a process that heats the target tissueto a temperature that causes immediate coagulative necrosis usually over °c terminal treatment requiresthat a necrotic area surrounds the target site with anadditional “10mm margins however in the liverhigh tissue perfusion and large blood vessels can cause aœheat sink effect around the ablation zone making itdifficult to achieve terminal ablation the heat sink 0czhao bmc cancer page of table median levels of cytokines before and after mwacytokineifnÎil2premwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ h postmwa pgml ci “ ci “ –¼ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “il12 p40il12 p70il1il6il8il10tnfαvegf p vs premwa –¼ 2fold vs premwa days postmwa pgml ci “ ci “ ci “ ci “ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ –¼ ci “ days postmwa pgml ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “ ci “effect can lead to sublethal temperatures and the retention of malignant cells thereby increasing the likelihoodof local tumor progression ltp however an incompletely ablated zone containing immune cells andcancer cells as well as functional vessels could establisha serious inflammatory site that may provide tumorspecific antigens cytokines and activated immune cellsin our study significant increases in the secretion ofchemokines il8 proinflammatory cytokines il1il12 ifnÎ and tnfα and antiinflammatory cytokines il10 were observed after mwa il8 is mainlyproduced by macrophages the classical biological activity of il8 is to attract and activate neutrophils whichcan lead to a local inflammatory response however recent studies have indicated that il8 both macrophageand cancer cellderived can recruit myeloidderivedsuppressor cells mdscs into the tumor microenvironment eventually inhibiting antitumor immunity andpromoting cancer progression [ ] il1 is mainlyproduced by macrophages b cells and nk cells couldproduce il1 under certain circumstances generallycells can only synthesize and secrete il1 after beingstimulated by foreign antigens or mitogens il1 couldpromote the th1 response promoting the activation ofdendritic cells dcs and cytotoxic t lymphocytesctls il12 is mainly produced by b cells and macrophages human il12 is a heterodimer with two subunits p40 kd and p35 kd which areinactivated in isolated form in general il12 functionsas a combination of two subunits il12 p70 while p40alone possesses partial functions of il12 p70 it™s mentionable that il12 p40 and p35 are not expressed inequal proportions so the amounts of il12 p40 and il p70 are different in one cell il12 can stimulate theproliferation of activated t cells and promote the differentiation of th0 cells into th1 cells moreover il12could induce the cytotoxic activity of ctls and nk cellsand promote the secretion of several cytokines such asifnÎ and tnfα previous research indicatedthat tnfα may play a crucial role in mwa in combination with immunotherapy notably our data illustrated that the il12 results were consistent with thoseof ifnÎ after the ablation operation but not with thoseof tnfα this result indicated that upregulation ofifnÎ may be a major effect of the il12 increase aftermwa on the other handan antiinflammatory and immunosuppressive cytokine wasevaluated after mwa il10 is a multicellularderivedmultifunctional cytokine that regulates cell growth anddifferentiation and could participate in inflammatoryand immune responses il10 was reported to increaseafter thermal ablation in the literature [ ] strategiesto inhibit il10induced immunosuppression after thermal ablation treatment would be of interestil10asablation therapy can mediate antitumor immunity astumor tissue necrosis caused by ablation may release various antigens that eventually form a kind of œin situ vaccination moreover ablative therapy can not onlydirectly kill cancer cells in situ but also regulate immunecells and promote the immune function of patients withliver cancer [ ] many immunoregulatory cytokineswere released or expressed after thermal ablation notablythe cytokines released after thermal ablation can regulatethe positive and negative aspects of the cancer immunecycle previously researchers demonstrated that proinflammatory cytokines such as il1 il6 il8 il18 andtnfα were increased several hours or days after thermalablation [ ] to our knowledge terminal tumorthermal ablation may not only cause local heat injury intissues surrounding the tumor site but also induce a systemic reaction this systemic reaction would becaused by different mechanisms first interventional operation may result in trauma to the liver although this procedure is very minimally invasive the healing process maycause alteration of some cytokines second heat injurycould cause acute thermal necrosis in liver and tumor 0czhao bmc cancer page of fig levels of cytokines before and after mwa treatment slightly increased ifnÎ il12 p40 and il12 p70 levels after mwa treatment over fold enhancement of il2 h postmwa and of il1 il6 il8 il10 and tnfα d postmwa p 0czhao bmc cancer page of fig trends in cytokines significantly altered after mwa treatment the levels of il2 at h postmwa il1 at d postmwa il6 at dpostmwa il8 at d postmwa and il10 at d postmwa were elevated over 2fold compared to the levels premwatable correlation between the ablation energy and significantly elevated cytokinesenergyvsil2 h postmwaenergyvsil1 d postmwaˆ’energyvsil6 d postmwaenergyvsil8 d postmwaenergyvsil10 d postmwaenergyvstnfα d postmwaspearman™s rp value onetailed p 0czhao bmc cancer page of fig correlation between the ablation energy and the serum levels of il2 and il6 the serum levels of il2 at h postmwa and il6 at dpostmwa were positively correlated with energy output during the mwa procedureand nonspecifictissues and release of necrotic tissue fragments into bloodcould cause immunological reactions including nonspecific and specific reactions generally cytokines affectedby wound healingimmunologicalreactions do not last longer than those affected by specificimmunologicalreactions ablation treatmentinducedspecific immunological reactions are more complicatedand could affect more immunocytes [ ] which wouldmake this process last longer than other reactions theseexplanations may be the reason why the cytokine changeslasted different durations moreover cytokines affected bythe second manner would be positively correlated withthe ablation scale which is why we employed the œenergyindex in our ablation operation design to receive a terminal ablation larger tumor would cost higher energy including higher power and longer duration time terminaltumorthermal ablation would release tumorrelatedneoantigen to blood circulation eventually induce a systemic reaction this reaction is dependent on the scale ofthermal injury and the local immunological microenvironment of the tumor our findings indicated that il2 andil6 were significantly altered after the ablation procedureand positively correlated with mwa energy il2 is commonly derived from activated t cells primarily th1 cellsil2 can stimulate t cells to proliferate and differentiateactivate natural killer nk cells and macrophages and enhance the functions of cytotoxic t lymphocytes ctls our data illustrated that il2 is significantly increased at h after mwa indicating that il2 may induce a nonspecific immune response after mwa but il decreased after h postmwa in our study suggesting that the il2induced immune response may not belong lasting mentionable many cytokines detected il8il1 il12 were mainly derived from macrophagewhich was a widely distributed antigen presenting cellthis result support the theory that mwa could releasefragment of cancer cells into blood as neoantigen macrophages could response to this proceed and cause a systemic immunoreaction additional cytokines alterationsuch as il6 after ablation may be no anspecific inliver evidences indicate that increase of il6 was not onlyoccurred in liver ablation researches focus on lung cancerincluding primary lung cancer and pulmonary metastasesdemonstrated that serum il8 il1 il6 il10 il12and tnfα were significantly raised after radiofrequencythermal ablation moreover joseph found that imageguided thermal ablation of tumors located in lung liver orsoft tissues increases plasma levels of il6 and il10 another question remain unveiled was if our result wasœcancerspecific we checked literature about cytokinemodulation after thermal ablation in benign diseases andonly got limit evidences based on benign thyroid nodules and adenomyosis according to these literatureil6 levels did not show any significant difference aftertreatment compared with pretreatment values indicatingthat elevation of il6 may be caused by tumour antigenreleased by ablation treatment however the ablationenergy used in thyroid nodules was much lower thanliver and lung which would lead to a false negativein cytokine detection to the research about adenomyosis on the other hand experiment design was determined to followup the il6 at months afterhifu ablation as our data demonstrated mostly cytokines were return to premwa level after monthdetection after months may miss the modulation ofil6 overall few evidences support that some of thecytokines were altered in a œcancerspecific mannerwhile no solid results could confirm that further animal experiments were required to make a clarifieddata and answer this question 0czhao bmc cancer page of thetumorassociated immunein recent years ablationinduced systemic effects suchasresponse haveattracted increased attention de baere t first reported two cases of spontaneous regression of multiplepulmonary metastases occurring after radiofrequencyablation of a single lung metastasis although growing evidence suggests that thermal ablation can inducespontaneous regression of the socalled œabscopal effecton distant tumors the incidence rate of such an effect israre probably due to uncontested immunological activation caused by one ablation treatment and the lack ofimmuneamplification management in it was described that in situ tumor destruction can provide a useful antigen source forthe induction of antitumorimmunity however clinical studies could not sufficiently utilize such an effect until the development ofimmune checkpoint inhibitors icis [ ] icis suchas pd1pdl1 and ctla4 antibodies are widely applied in several cancers and studies have indicated thatici treatment could enhance the effect of ablation evidence indicates that hyperthermic destruction causesthe release of a large population of heterogeneous tumorantigens and inflammatory cytokines may play crucialroles in this process however opposite evidence indicated that incomplete radiofrequency ablation couldinduce inflammation which may accelerates tumor progression and hinders pd1 immunotherapy suggesting that ablation treatment may promote tumorprogression our data demonstrated that il6 was significantly increased after mwa treatment il6 is derived from monocytes macrophages dcs th2 cells andsometimes cancer cells and it plays a key role in t cellproliferation and survival the role of il6 appearsto be rather complex korn classified il6 as œdifferentiation factor which could involve in differentiation ofth17 cells however il6 does not direct the commitment to the th1 or th2 cell lineage but controls theproliferation and survival of immunocytes cooperatingwith other cytokines such as tgf tnf or il21 for instance il6 activated stat3 pathway in naivecd4 t cells in the presence of the morphogen tgfbpromotes the population expansion of th17 cells recent evidence indicates that il6 plays an indispensable role in t cellinfiltration to the tumor sitewhich could benefit immunomodulatory therapy incontrast il6 can increase mdscs inhibit the development and maturation of dendritic cells dcs and inhibit the polarization of th1 cells eventuallyresulting in negative immunomodulatory effects according to muneeb ahmed™s work the adjuvant uses ofa nanop smallinterfering rna sirna can besuccessfully used to target the il6mediated locoregional and systemic effects of thermal ablation il6 knockout via a nanop antiil6 sirna in mice coulddecrease the local vegf level at the ablation site therefore how to utilize the positive effect of il6 whileavoiding the negative effect after mwa needs further investigation preclinical research indicated that il6 andpdl1 blockade combination therapy reduced tumorprogression in animal models [ ] thus an antiil strategy after ablation should be considered whencombined with ici therapy previous studies and ourshave demonstrated that most cytokine levels returned topretreatment levels days after ablation this resultsuggests that h to days after ablation may be optimal timing for additional immunomodulatory therapysour results reported here support the evidence for terminal tumor thermal ablation could cause heat injury totissues surrounding the tumor site and release neoantigento blood circulation eventually induce a systemic reactionthis reaction could lead to a detectable alteration of cytokine levels further investigation is required to revealwhether the cytokines altered by mwa treatment couldaffect cancer progression whether positive or negativeabbreviationsmwa microwave ablation hcc hepatocellular carcinoma icis immunecheckpoint inhibitors tnf tumor necrosis factor il interleukinvegf vascular endothelial growth factor sd stable disease pr partialresponse ct computed tomography ci confidence interval ltp likelihoodof local tumor progression mdscs myeloidderived suppressor cellsctls cytotoxic t lymphocytes nk natural killer sirna small interfering rnaacknowledgementsnot applicableauthors™ contributionsjz conceptualization data curation writing“original draft and writing“review and editing ql conceptualization and writing“review and editingmm conceptualization and writing“review and editing brconceptualization and writing“review and editing and collect samples yhexecute milliplex assay and collect data dpl patient enrollment executemwa ablation and collect samples zl execute mwa ablation and collectsamples dml patient enrollment execute mwa ablation and collectsamples yx execute milliplex assay and collect data mt conceptualizationand writing“review and editing rl conceptualization data curation formalanalysis visualization writing“original draft and writing“review and editingall authors have read and approved the manuscriptfundingthis work was supported by the national natural science foundation ofchina the natural science foundation ofjiangsu province of china bk20140295 the jiangsu governmentscholarship for oversea studies js2018179 and the œsix one projects forhighlevel health personnel in jiangsu province lgy2018077availability of data and materialsthe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestethics approval and consent to participatethe protocol was approved by the human ethics committee of the firstaffiliated hospital of soochow university and was conducted in accordancewith the declaration of helsinki patients were informed that the bloodsamples were stored by the hospital and potentially used for scientific 0czhao bmc cancer page of research and that their privacy would be maintained all written informedconsent was obtained from all participants and clearly statedconsent for publicationnot applicablecompeting intereststhere is no financial or personal relationship with other people oranizations that could inappropriately influence bias this workauthor details1department of radiation oncology the first affiliated hospital of soochowuniversity suzhou china 2department of oncology the first affiliatedhospital of soochow university suzhou china 3department of lymphatichematologic oncology jiangxi cancer hospital nanchang china4department of interventional radiology the first affiliated hospital ofsoochow university suzhou china 5division of neurosurgery city of hopebeckman research institute duarte california usareceived january accepted august referencesfu j wang h precision diagnosis and treatment of liver cancer in chinacancer lett “bruix j han kh gores g llovet jm mazzaferro v liver cancer approachinga personalized care j hepatol suppls144“rognoni c ciani o sommariva s bargellini i bhoori s cioni r facciorussoa golfieri r gramenzi a mazzaferro v transarterial radioembolizationfor intermediateadvanced hepatocellular carcinoma a budget impactanalysis bmc cancer nault jc sutter o nahon p gannecarrie n seror o percutaneoustreatment of hepatocellular carcinoma state of the art and innovations jhepatol “yin j dong j gao w wang y a case report of remarkable response toassociation of radiofrequency ablation with subsequent atezolizumab instage iv nonsmall cell lung cancer medicine baltimore 20189744e13112shi l chen l wu c zhu y xu b zheng x sun m wen w dai x yang m pd1 blockade boosts radiofrequency ablationelicited adaptiveimmune responses against tumor clin cancer res “lippitz be cytokine patterns in patients with cancer a systematic reviewlancet oncol 2013146e218“jin yb zhang gy lin kr chen xp cui jh wang yj luo w changes ofplasma cytokines and chemokines expression level in nasopharyngealcarcinoma patients after treatment with definitive intensitymodulatedradiotherapy imrt plos one 2017122e0172264kim mj jang jw oh bs kwon jh chung kw jung hs jekarl dw lee schange in inflammatory cytokine profiles after transarterial chemotherapy inpatients with hepatocellular carcinoma cytokine “ gillams a goldberg n ahmed m bale r breen d callstrom m chen mhchoi bi de baere t dupuy d thermal ablation of colorectal livermetastases a position paper by an international panel of ablation expertsthe interventional oncology sans frontieres meeting eur radiol “ ahmed m solbiati l brace cl breen dj callstrom mr charboneau jwchen mh choi bi de baere t dodd gd 3rd imageguided tumorablation standardization of terminology and reporting criteriaa 10yearupdate radiology “ chiang j hynes k brace cl flowdependent vascular heat transfer duringmicrowave thermal ablation conf proc ieee eng med biol soc “ huang hw influence of blood vessel on the thermal lesion formationduring radiofrequency ablation for liver tumors med phys najjar yg rayman p jia x pavicic pg jr rini bi tannenbaum c ko jhaywood s cohen p hamilton t myeloidderived suppressor cellsubset accumulation in renal cell carcinoma parenchyma is associated withintratumoral expression of il1beta il8 cxcl5 and mip1alpha clin cancerres “ alfaro c teijeira a onate c perez g sanmamed mf andueza mp alignanid labiano s azpilikueta a rodriguezpaulete a tumorproducedinterleukin8 attracts human myeloidderived suppressor cells and elicitsextrusion of neutrophil extracellular traps nets clin cancer res “kundu m roy a pahan k selective neutralization of il12 p40 monomerinduces death in prostate cancer cells via il12ifngamma proc natl acadsci u s a “ onishi h kuroki h matsumoto k baba e sasaki n kuga h tanaka mkatano m morisaki t monocytederived dendritic cells that capture deadtumor cells secrete il12 and tnfalpha through il12tnfalphanfkappabautocrine loop cancer immunol immunother “ yu z geng j zhang m zhou y fan q chen j treatment of osteosarcomawith microwave thermal ablation to induce immunogenic cell deathoncotarget “ yang w wang w liu b zhu b li j xu d ni y bai l liu gimmunomodulation characteristics by thermal ablation therapy in cancerpatients asia pac j clin oncol 2018145e490“erinjeri jp thomas ct samoilia a fleisher m gonen m sofocleous ctthornton rh siegelbaum rh covey am brody la imageguidedthermal ablation of tumors increases the plasma level of interleukin6 andinterleukin10 j vasc interv radiol “ den brok mh sutmuller rp van der voort r bennink ej figdor cg ruerstj adema gj in situ tumor ablation creates an antigen source for thegeneration of antitumor immunity cancer res “ zerbini a pilli m laccabue d pelosi g molinari a negri e cerioni sfagnoni f soliani p ferrari c radiofrequency thermal ablation forhepatocellular carcinoma stimulates autologous nkcell responsegastroenterology “ zhang h hou x cai h zhuang x effects of microwave ablation on tcellsubsets and cytokines of patients with hepatocellular carcinoma minim
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EpidemiologyEPIDEMIOLOGICAL SCIENCERisk factors for hospital admissions related to COVID19 in patients with autoimmune inflammatory rheumatic diseasesDalifer D Freites Nu±ez1 Leticia Leon Arkaitz Mucientes1 Luis Rodriguez Rodriguez Judit Font Urgelles3 Alfredo Madrid Garc­a1 Jose I Colomer1 Juan A Jover34 Benjam­n Fernandez Gutierrez3 Lydia Abasolo1Handling editor Josef S Smolen1Rheumatology Department and IDISSC La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain2Department of Health and Education Universidad Camilo Jose Cela Villafranca del Castillo Madrid Spain3Rheumatology Department Hospital Clinico San Carlos Madrid Spain4Medicine Department Universidad Complutense de Madrid Madrid Comunidad de Madrid SpainCorrespondence toDr Leticia Leon IdISSC and Rheumatology La Fundacion para la Investigacion Biomedica del Hospital Clinico San Carlos Madrid Spain lleon hcsc salud madrid Received May Revised July Accepted July Objectives To describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 disease to compare patients who required hospital admission with those who did not and assess risk factors for hospital admission related to COVID19Methods An observational longitudinal study was conducted during the pandemic peak of severe acute respiratory syndrome coronavirus March to April All patients attended at the rheumatology outpatient clinic of a tertiary hospital in Madrid Spain with a medical diagnosis of AIRD and with symptomatic COVID19 were included The main outcome was hospital admission related to COVID19 The covariates were sociodemographic clinical and treatments We ran a multivariable logistic regression model to assess risk factors for the hospital admissionResults The study population included patients with AIRD and COVID19 Of these patients required hospital admission related to COVID19 The mean age on admission was years and the median time from onset of symptoms to hospital admission was “ days The median length of stay was “ days A total of patients died during admission Compared with outpatients the factors independently associated with hospital admission were older age OR p000 and autoimmune systemic condition vs chronic inflammatory arthritis OR p001 No statistically significant findings for exposure to disease modifying antirheumatic drugs were found in the final modelConclusion Our results suggest that age and having a systemic autoimmune condition increased the risk of hospital admission whereas disease modifying antirheumatic drugs were not associated with hospital admission Authors or their employers No commercial re use See rights and permissions Published by BMJTo cite Freites Nu±ez a0DD Leon a0L Mucientes a0A et a0al Ann Rheum Dis Epub ahead of print [please include Day Month Year] 101136annrheumdis2020217984INTRODUCTIONSevere acute respiratory syndrome coronavirus SARS CoV2 causes a myriad of clinical signs and symptoms together with typical laboratory abnormalities that manifest as the disease COVID191Since the confirmation of the first patient infected with SARS CoV2 in Spain in January the current COVID19 outbreak has had a considerable impact especially in the Madrid region where the highest incidence of COVID19 cases has been Key messagesWhat is already known about this subject –º The epidemiological scenario is changing daily There is little evidence for risk factors of poor outcome with COVID19 specific to autoimmune inflammatory rheumatic diseasesWhat does this study add –º Patients with an autoimmune systemic condition have a higher risk of hospital admission related to COVID19 compared with those with chronic inflammatory arthritis –º Disease modifying agents were not associated with a higher risk of hospital admission related to COVID19How might this impact on clinical practice or future developments –º Our data show that in a real world setting a high percentage of patients with autoimmune inflammatory rheumatic diseases and COVID19 required hospital admission The patients were mainly elderly with comorbidities and a systemic autoimmune conditionrecorded with more than patients admitted to the hospital until the first week of May2The incidence and severity of COVID19 disease seem to be higher in patients with risk factors such as advanced age and associated comorbidities mainly hypertension diabetes heart disease and previous respiratory diseases3 It is not clear whether patients with rheumatic diseases are more susceptible to SARS CoV2 infection or when they are infected whether they have more severe disease or a poorer outcome Previous outbreaks caused by coronaviruses did not yield overwhelming evidence that patients with rheumatic diseases are at an increased risk4 although some patients are candidates for a higher number of infections owing to their rheumatic disease predominantly systemic or the treatment they are receiving for rheumatic diseases5 Preliminary experiences in patients with COVID19 show that those with chronic arthritis treated with synthetic conventional or targeted syntheticbiologic disease modifying antirheumatic Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologydrugs DMARDs do not seem to be at a greater risk of respiratory or life threatening complications from SARS CoV2 than the general population6 The epidemiological scenario is changing and evidence on the risk factors of poor outcome with COVID19 specific to inflammatory rheumatic disease is scarce In addition there are little data on how the hospital admissions of these patients with severe COVID19 infection have evolved8The aim of our study was to describe patients with autoimmune inflammatory rheumatic diseases AIRD who had COVID19 during the pandemic peak We also explored possible risk factors associated with hospital admission related to COVID19 disease in patients with AIRD from a tertiary hospital in Madrid SpainMETHODSSetting study design and patientsThe study was performed in a public tertiary hospital Hospital Cl­nico San Carlos HCSC in Madrid Spain The catchment area is home to almost peopleWe performed a prospective observational study from March when our health area had the first hospital admission related to COVID19 to April We preselected all patients attended at the rheumatology outpatient clinic of our centre during the study period whose data were recorded in the electronic clinical history of our department HCR Penelope The inclusion criteria were age years a medical diagnosis according to International Classification of Diseases ICD10 of inflammatory rheumatic disease and symptomatic COVID19 disease assessed by medical diagnosis or confirmed with a positive SARS CoV2 PCR diagnostic testPatient data were obtained during routine clinical practice The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice and was approved by the HCSC Ethics Committee approval number E BSVariablesThe primary outcome was admission to hospital with a medical diagnosis of COVID19 andor a positive PCR result between March and April compared with outpatients with symptomatic COVID19 diseaseThe covariables recorded were as follows sociodemographic baseline characteristics including sex age and rheumatic disease duration Type of AIRD including systemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud phenomenon polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus and chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uveitis and inflammatory bowel disease Baseline comorbid conditions including hypertension dyslipidaemia depression diabetes mellitus smoking habit kidney disease chronic liver disease respiratory diseases chronic obstructive pulmonary disease and interstitial lung disease thyroid disease heart disease valve disease arrythmias cardiomyopathy heart failure and pericarditis ischaemic vascular disease stroke cardiovascular and peripheral vascular disease venous thrombosislung embolism and cancer Treatment for inflammatory rheumatic disease a glucocorticoids b non steroidal anti inflammatory drugs NSAIDs c conventional synthetic disease modifying antirheumatic drugs csDMARDs antimalarials hydroxychloroquine and chloroquine azathioprine cyclophosphamide cyclosporine colchicine leflunomide methotrexate mycophenolate mofetilmycophenolic acid and sulfasalazine d targeted syntheticbiologic DMARDs tsbDMARDs including antitumour necrosis factor TNF alpha drugs infliximab adalimumab etanercept certolizumab and golimumab other biologics anti interleukin IL6 tocilizumab and sarilumab rituximab abatacept belimumab anti IL1723 anti IL17 ustekinumab ixekizumab and secukinumab Janus kinase JAK inhibitors tofacitinib and baricitinibTreatment had to start at least month before the beginning of the study and continue during the study period until the end of the study or hospital admission for antimalarial therapy glucocorticoids sulfasalazine NSAIDs or colchicine Regarding csDMARDs and tsbDMARDs treatment had to start at least month before the beginning of the study and continue until at least 21st March the end of the study or hospital admission In the case of rituximab the last infusion had to be at least in JanuaryData sourcesPatient sociodemographic clinical laboratory and data on treatment of rheumatic disease were obtained through HCR PenelopePatients with COVID19 were detected by warning calls to our rheumatologists or nurses or via routine telephone consultation Other infected patients were detected through their sick leave forms for COVID19 The results of SARS CoV2 PCR diagnostic assays were obtained from the microbiologyinfectious service of HCSC In addition our Hospital Central Services registered all medical admissions to HCSC This information was provided from March to AprilThe researchers carried out an exhaustive review of the clinical histories of admitted patients to identify COVID19 cases and rule out patients admitted for other reasons Once the COVID19 cases were identified we collected clinical laboratory and treatment data during admission until the end of admission either discharge or death in order to describe the progress of the disease The review was performed until 24th April in order to include follow up data from patients admitted to the hospital with COVID19Statistical analysisPatient characteristics are expressed as mean and SD or median and IQR for continuous variables categorical variables are expressed as percentages Statistical tests were performed to compare characteristics between patients admitted with COVID19 and those without hospital admissions Continuous variables were analysed using the Mann Whitney test or t test and discrete variables were analysed using the χ2 or Fisher exact test Univariable logistic regression analyses were performed to assess differences between hospital admissions related to COVID19 risk and covariates Multivariable logistic regression models adjusted for age sex and comorbidity were run in a stepwise manner to examine the possible effect of sociodemographic clinical and therapeutic factors on hospital admissions related to COVID19 The model also included csDMARDs and all other variables with a p02 from the simple regression analysis The results were expressed as the OR with its respective CIAll analyses were performed in Stata V13 statistical software Stata Corp A two tailed p value was considered to indicate statistical significanceFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cRESULTSA total of patients with AIRD with symptomatic COVID19 disease were included in the study table The tests were performed as an exploratory measure of the association between a variable and the outcomeMost of the patients were women with a mean age of years and a mean time since diagnosis of years The main diagnosis was rheumatoid arthritis followed by axial spondyloarthritis Many patients had at least one baseline comorbid condition the most prevalent being hypertension dyslipidaemia and lung disease Most patients were taking csDMARDs Half of the patients were taking glucocorticoids a quarter were taking NSAIDs and were taking tsbDMARDs of which adalimumab was the most frequently prescribed followed by rituximab Only one patient was taking a JAK inhibitor Interestingly of the patients taking tsbDMARDs were taking the drug in combination with a csDMARDA total of patients had to be admitted to the hospital because of COVID19 Of these were evaluated in the HCSC Emergency Department were admitted to HCSC and were transferred to the Institucion Ferial de Madrid IFEMA support hospital owing to the lack of capacity in our hospital at that time The remaining three patients were evaluated and admitted to other hospitals in the Autonomous Community of Madrid Table presents data for the patients admitted to HCSCOf the patients admitted to our hospital were women with a mean age at admission of years and median lag time from the onset of symptoms to the admission of “ days The median length of stay was “ days table At admission the median haemoglobin was “ gdL and the median total lymphocyte count was “ ngmL The median D dimer value was “ ngmL In of patients median interleukin IL6 levels were “ pgmL Patients received various antibiotics mainly azithromycin levofloxacin and third generation cephalosporinsMost patients were treated with hydroxychloroquine during admission About half received glucocorticoids Eighteen were treated with lopinavirritonavir and received the anti IL 6R antibody tocilizumab table 2FEDERA total of patients developed relevant complications during admission the most frequent being myocarditis thrombosis and kidney failure Only two patients were admitted to the intensive care unit during admission The first was a patient in 50s with mixed connective tissue disease and associated comorbidities who developed acute respiratory insufficiency and bilateral pneumonia The patient was treated with antibiotic therapy lopinavirritonavir hydroxychloroquine and βinterferon Finally the patient was extubated days later and is recovering The other was a young adult patient with systemic lupus erythematosus treated with methotrexate rituximab hydroxychloroquine and glucocorticoids who days after being diagnosed with COVID19 PCR developed an erythematous rash and generalised urticaria requiring hospitalisation in the intensive care unit owing to general clinical and laboratory worsening elevated D dimer values The patient was treated with methylprednisolone heparin and a cephalosporin A few days later the patient™s condition improved and he recovered completely at dischargeOf the patients admitted to HCSC were sent to another care centre converted hotel hospitalIFEMA support hospital when their condition improved A further patients Epidemiologywere discharged home to continue self isolation after improvement At the end of the study five patients remained in hospital A total of patients died during admission men and women with a median age of “ years Of the patients who died had relevant comorbidity diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease and or liver disease The main diagnoses were rheumatoid arthritis followed by spondyloarthritis polymyalgia rheumatica vasculitis and Sjogren™s syndrome The results of the univariable analysis are shown in table Older age systemic autoimmune conditions vs chronic inflammatory arthritis OR CI “ p0014 hypertension diabetes mellitus lung disease heart disease and glucocorticoids were associated with statistically significant greater risk of admission to the hospital Female sex NSAIDs and anti TNF drugs vs non use were associated with a statistically significant lower risk The differences reported for the remaining variables did not reach statistical significanceThe multivariable analysis was adjusted for gender age and comorbidities related to COVID19 These comorbidities were diabetes mellitus pulmonary disease ischaemic vascular disease hypertension venous thrombosislung embolism lung disease andor liver disease table Age and systemic autoimmune conditions had more probability of hospital admissions regardless of other factors Differences in exposure to glucocorticoids were not statistically significant The type of exposed DMARDs did not reach statistical significance in the multivariate model In fact long term treatment with antimalarials OR CI “ p066 other csDMARDs including methotrexate leflunomide and azathioprine OR CI “ p09 and NSAIDs OR CI “ p05 dropped from the final model The variable tsbDMARDs was also eliminated from the final model anti TNF vs none OR CI “ p016 and non anti TNF vs none OR CI “ p03DISCUSSIONOur study aims to shed light on rheumatologists™ concerns regarding their patients We found that in a real world setting of patients with AIRD and COVID19 required hospital admission These were mainly elderly patients with more comorbidities and systemic autoimmune conditions Our data show that patients exposed to disease modifying agents do not seem to be at higher risk of hospital admission related to COVID19Of the patients included in the study with COVID19 required hospital admission Comparison of the characteristics of patients admitted to hospital because of COVID19 and those who did not require hospital admission were as follows admitted patients had a median age close to years that is more than years older than patients who were not admitted Moreover those who were admitted more frequently had baseline comorbidities and systemic autoimmune conditions As for therapy admitted patients were less frequently exposed to antimalarial and anti TNF alpha agentsThe median lag time from onset of symptoms to admission was days and almost of patients had pneumonia at admission The baseline laboratory results for admitted patients in our study are consistent with those published elsewhere9“ and are characterised by lymphopenia and elevated acute phase reactants In fact of the patients had elevated D dimers normal and elevated IL6 normal pgmL Treatment during admission varied widely as the disease proved Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Baseline demographic and clinical characteristics of patients with AIRD and with COVID19 admitted vs no admitted at the hospitalAIRD“COVID19 patientsAIRD“COVID non admitted patientsAIRD“COVID admitted patientsVariableN123N69N54P value Positive Negative Not performed Women n Age years mean SDTime since diagnosis years mean SDPCR test n Smoking habit active vs noneDiagnosis AIRD n Rheumatoid arthritis Axial spondyloarthritis Polymyalgia rheumatica Psoriatic arthritis Systemic lupus erythematosus Mixed connective tissue disease Sjogren™s syndrome Vasculitis Uveitis Systemic sclerosis Inflammatory polyarthritis Polychondritis Polymyositis Raynaud phenomenon OtherComorbidities n NSAIDs n Glucocorticoids n csDMARDs n TsbDMARDs n JAKi n Others inflammatory bowel disease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatory syndromes and sarcoidosis Heart disease arrhythmiasvalve disease cardiomyopathy and heart failure Ischaemic vascular disease stroke cardiovascular and peripheral vascular diseaseAIRD autoimmune inflammatory rheumatic disease Anti TNF tumour necrosis factor alpha COPD chronic obstructive pulmonary disease csDMARD conventional synthetic disease modifying antirheumatic drug ILD interstitial lung disease JAKi JAK inhibitor tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid disease Anti TNF alpha agent Other biologics Abatacept Tocilizumab Belimumab Rituximab Methotrexate“leflunomide“azathioprine Sulfasalazine AntimalarialsFreites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cTable Hospital admissions related to COVID19 among patients with AIRDVariableValueTable OR of hospital admission related to COVID19 in patients with AIRD univariable analysisVariable CIORPEpidemiology Haemoglobin gdL D dimer ngmL Neutrophil count —109L Lymphocyte count —109L CRP mgdL LDH UL Platelet count —109L Creatinine mgdL Ferritine ngmLAdmissions nLag time from onset of symptoms to admission days median IQRPneumonia at admission n Systemic autoimmune conditions n Laboratory data at admission median IQR COVID19 related treatments during admission n Admitted by intensive care unit during hospital admission Length of stay days median IQRDischarge reason n Azithromycin Other antibiotics Glucocorticoids Lopinavirritonavir Remdesivir Darunavircobicistat Tocilizumab Interferon HCQ Immunoglobulin Improvement home isolation Other care centre medicalised hotelIFEMA hospital Death End of study no discharge No Yes “ Data for patients patients were treated in other support centres after referral or admission in other centresCRP C reactive protein HCQ hydroxychloroquine LDH lactate dehydrogenase challenging for specialists who prescribed various combinations of drugs based on little published evidence In this sense the anti IL 6R antibody tocilizumab has proven to be beneficial in patients with COVID1912 Treatment may also be successful in the early stages of cytokine release syndrome if it can effectively block the signal transduction pathway of IL6 therefore tocilizumab and sarilumab are likely to emerge as effective drugs for patients with moderate to severe COVID1913 In our study almost of the patients were treated with tocilizumabThe patients who eventually died had a median age of years This finding is in line with data for the general population where over of deaths occurred in persons years and more than of all deaths were in people aged ‰¥ years7The multivariable regression model showed that only age increasing by per year and systemic autoimmune conditions continued to be risk factors for hospital admission related to COVID19 “ “ “ “ “ “ “ “ “ ““““““““““““ Rheumatoid arthritis Inflammatory polyarthritis Systemic lupus erythematosus Psoriatic arthritis Spondyloarthritis MTCD Sjogren syndrome Hypertension Dyslipidaemia Depression Diabetes mellitus Heart disease Vascular disease Liver disease Kidney disease Lung disease ILDCOPD Cancer Venous thrombosislung embolism Thyroid diseaseGender womenAge yearsDiagnosis AIRD one category vs the rest Disease durationSmoking habit active vs noneComorbidities yes NSAIDsGlucocorticoidscsDMARDSs TsbDMARDs JAKisOther biologics anti IL6 tocilizumab sarilumab rituximab Rtx anti IL1723 anti IL17Othercategories could not be represented polymalgia rheumatica systemicsclerosis vasculitis Raynaud phenomenon polychondritis Beh§et diseasepolymyositis uveitis inflammatory boweldisease antiphospholipid syndrome juvenile idiopathic arthritis autoinflammatorysyndromes and sarcoidosisAIRD autoimmune inflammatory rheumatic disease anti TNF tumour necrosis factor csDMARD Conventional synthetic disease modifying antirheumatic drug IL6 interleukin6 JAKi JAK inhibitors tsbDMARDs target syntheticbiologic disease modifying antirheumatic drug““““““““““““ ““ Methotrexate“leflunomide“azathioprine Sulfasalazine Antimalarial agents““““““““““ None Anti TNF agents Other biologics““As for the association between sex and risk of hospital admission we did not find a higher risk of admission in women despite the fact that rheumatic diseases are more prevalent in this group The type of diagnosis seems to play an important role in the probability of hospital admission and patients with systemic autoimmune conditions seem to have the highest risk compared with chronic inflammatory arthritisAs it has been reported elsewhere comorbidities play an important role in the risk of hospital admission15 Clinical outcomes are poorer in patients with COVID19 with a comorbid condition than in those without and a greater number of comorbidities correlates with poorer clinical outcomes16 Diabetes is a major comorbidity in COVID19 and patient™s history of diabetes is an independent risk factor for morbidity and mortality in this condition17 Diabetes has been associated with admissions to Freites Nu±ez DD et a0al Ann Rheum Dis “ 101136annrheumdis2020217984 0cEpidemiologyTable Multivariable analysis risk factors for hospital admission related to COVID19 in patients with AIRDVariableOR CIP value“““““Gender womenAge yearsAIRD systemic autoimmune conditions vs chronic inflammatory arthritisCOVID comorbidities yesGlucocorticoidsSystemic autoimmune conditions polymyalgia rheumatica mixed connective tissue disease systemic sclerosis Sjogren™s syndrome vasculitis Raynaud polymyositis polychondritis sarcoidosis antiphospholipid syndrome autoinflammatory syndromes and systemic lupus erythematosus vs chronic inflammatory arthritis rheumatoid arthritis inflammatory polyarthritis juvenile idiopathic arthritis psoriatic arthritis axial spondyloarthritis uvetis and inflammatory bowel disease Comorbidities including the presence of at least one of the follows hypertension heart disease vascular disease diabetes mellitus venous thrombosislung embolism chronic kidney disease liver disease and lung disease ILDCOPDAIRD autoimmune inflammatory rheumatic disease COPD chronic obstructive pulmonary disease ILD interstitial lung diseasethe intensive care unit due to COVID19 in recent series19 and has been shown to increase mortality6 Therefore we adjusted for comorbidity in the multivariable analysisTreatment with glucocorticoids lost its statistical significance in the multiple regression model However the dose was not reported in our data and in the case of these agents the risk could be dose dependent In a recent publication from a European registry the authors found that exposure to mgday was associated with a greater probability of hospitalisation21The exposure to DMARDs regardless of whether they were biological or synthetic does not seem to be associated with a higher hospital admission related to COVID19 Although we have to consider the limited number of patients in our study our results are in concordance with data reported elsewhere8 Our results should be interpreted taking into account other limitations First patients were included from a single centre Second of all the patients with COVID19 who did not require admission one third contacted the rheumatology service to report the disease and the remainder were detected through the COVID19 discharge reports sent to their primary care physician Elderly persons and homemakers who did not contact us can be considered missing Consequently there may be some selection bias between those admitted and those not admitted although this problem was addressed by adjusting for confounders in the multivariable analysis Third while it is acknowledged that clinical suspicion must be confirmed by PCR assay almost of patients admitted did not undergo PCR owing to the lack of tests or the extreme healthcare overload Nevertheless all cases included were clinically compatible and managed as COVID19The key strength of our study is that it was performed in real life conditions during then pandemic peak with access to complete sociodemographic and clinical data from our rheumatology electronic clinical history including thorough hospital admission data such as laboratory abnormalities and COVID19 treatment data from the hospital computer services Consequently this has allowed us to analyse the risk of hospital admission related to COVID19 adjusted for confounders thus avoiding possible biasAlthough we are unable to modify the factors reported here knowing them can help rheumatologists to treat and advise their patients during this new and challenging period Results provided by our study are preliminary and should be corroborated with other real life studies but we consider our findings helpful to increase the knowledge in the management of patients with AIRD and COVID19Twitter Benjam­n Fernandez Gutierrez Fergutbe2001Acknowledgements The authors would like to thank Ana M Perez for her help with data collection They would like to say a special thanks to all the rheumatologists and nurses who contributed to the care of the patients in such an innovative and conscientious wayContributors BF G LL JAJ LR R and LA conceived and designed the study DDFN JFU AMG JIC and LL collected data LA and LL performed the data analysis and interpreted the data All of the authors were involved in the drafting andor revising of the manuscriptFunding This work was supported by the Instituto de Salud Carlos III ISCIII Ministry of Health Spain CP1600916 PI1801188 and RD1600120014 and cofunded by el Fondo Europeo de Desarrollo Regional FEDER The funders had no role in study design data collection analysis manuscript preparation or decision to publishCompeting interests None declaredPatient and public involvement Patients andor the public were not involved in the design or conduct or reporting or dissemination plans of this researchPatient consent for publication Not requiredEthics approval The study was approved by the Hospital Cl­nico San Carlos institutional ethics committee approval number E BS This study was conducted according to the principles of the Declaration of HelsinkiProvenance and peer review Not commissioned externally peer reviewedData availability statement Data are available upon reasonable requestThis article is made freely available for use in accordance with BMJ™s website terms and conditions for the duration of the covid19 pandemic or until otherwise determined by BMJ You may use download and print the article for any lawful non commercial purpose including text and data mining provided that all copyright notices and trade marks are retainedORCID iDsLeticia a0Leon http orcid Luis a0Rodriguez Rodriguez http orcid REFERENCES Fernandez Gutierrez B COVID19 with pulmonary involvement An autoimmune disease of known cause Reumatol Clin “ COVID19 Situaci³n actual en La Comunidad de Madrid Informe de situaci³n del de Mayo Available httpswww comunidad madrid sites default files doc sanidad 200508_ cam_ covid19 pdf [Accessed May ] Chen N Zhou M Dong X et a0al Epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in Wuhan China a descriptive study Lancet “ Figueroa Parra G Aguirre Garcia GM Gamboa Alonso CM et a0al Are my patients with rheumatic diseases at higher risk of COVID19 Ann Rheum Dis “ Favalli EG Ingegnoli F De Lucia O et a0al COVID19 infection and rheumatoid arthritis Faraway so close Autoimmun Rev Zhou F Yu T Du R et a0al Clinical course and risk factors for mortality of adult inpatients with COVID19 in Wuhan China a retrospective cohort study Lancet “ Monti S Balduzzi S Delvino P et a0al Clinical course of COVID19 in a series of patients with chronic art
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"Cellular recognition of microbial DNA is an evolutionarily conserved mechanism by which the innate immunesystem detects pathogens Cyclic GMPAMP synthase cGAS and its downstream effector stimulator of interferongenes STING are involved in mediating fundamental innate antimicrobial immunity by promoting the release oftype I interferons IFNs and other inflammatory cytokines Accumulating evidence suggests that the activation ofthe cGASSTING axis is critical for antitumor immunity The downstream cytokines regulated by cGASSTINGespecially type I IFNs serve as bridges connecting innate immunity with adaptive immunity Accordingly a growingnumber of studies have focused on the synthesis and screening of STING pathway agonists However chronicSTING activation may lead to a protumor phenotype in certain malignancies Hence the cGASSTING signalingpathway must be orchestrated properly when STING agonists are used alone or in combination In this review wediscuss the dichotomous roles of the cGASSTING pathway in tumor development and the latest advances in theuse of STING agonistsKeywords cGASSTING Innate immunity Type I interferon STING agonists Antitumor response CancerdevelopmentIntroductionThe discovery of phagocytosis in advanced the understanding of innate immunity the first line of host defenses[]Protection againston patternrecognition receptors PRRs which recognize microbialproducts coordinate antimicrobial defenses and activateinfection dependsagainstinfection byvarious pathogens Correspondence zqliucsueducn Juyan Zheng and Junluan Mo contributed equally to this work1Department of Clinical Pharmacology Hunan Key Laboratory ofPharmacogenetics and National Clinical Research Center for GeriatricDisorders Xiangya Hospital Central South University Changsha People™s Republic of China2Institute of Clinical Pharmacology Engineering Research Center for appliedTechnology of Pharmacogenomics of Ministry of Education Central SouthUniversity Changsha People™s Republic of ChinaFull list of author information is available at the end of the adaptive immunity [] Abnormal RNA or DNA RNADNA hybridization and cyclic dinucleotides derived frommicrobes are usually considered pathogenassociated molecular patterns PAMPs [ ] Cells associated with innate immunity recognize different microbial PAMPsthrough specific PRRs thereby playing key roles in hostresistance to microbial infection [] The pathways governing RNA recognition such as retinoid acid induciblegene I RIGIlike receptors have been reviewed elsewhere and will not be covered herein In the case of DNArecognition one of the best known PRRs is Tolllike receptor TLR9 which senses extracellular CpG hypomethylated DNA that has entered the cytosol through thephagosomelysosome system [] In addition the AIM2like receptor AIM2 inflammasome can be triggered afterthe entry of doublestranded DNA dsDNA into 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 0cZheng Molecular Cancer Page of cytosolic compartment which induces the proteolyticmaturation of proinflammatory cytokines such as IL1and IL18 and the activation of gasdermin D leading topyroptosis [“] Nevertheless the most notable PRR iscGAS a direct cytosolic dsDNA sensor which was identified by Dr Chen™s group in [] Once cGAS bindsto dsDNA the cGASSTING pathway is activated to further induce the expression of type I IFNs and other inflammatory cytokinesthus triggering innate immuneresponses [] Mounting evidence suggests that cGASSTING signaling not only plays pivotal roles in the hostdefense against microbialinfection but also modulatestumorigenesis Hence in this review we summarize themechanism of cGASSTING activation and elaboratefindings regarding its dual effects on tumor developmentCurrent advances in the use of STING agonists as a novelstrategy for antitumor therapy are also reviewedInsights into the cGASSTING signal transductioncascadecGAS is an innate immune sensor that identifies variouscytosolic dsDNAincluding DNA with viral bacterialmitochondrial micronuclei and retroelement originswhich can be mainly divided into pathogenderivedDNA and selfDNA Table In the cytoplasm cGAS isactivated by interacting with dsDNA in a sequence[“]independent butStructural and biochemical analyses have revealed thatthe Cterminal lobe of cGAS contains a conserved zinclengthdependent mannerionbinding module that mediates DNA binding andcGAS dimerization [ ] DNA ligands promotecGAS activation primarily by inducing conformationalchanges around the catalytic site and in the DNAbinding structures of cGASthe GScontaining loopundergoes conformational change to maintain stabilitywhich is a major mechanism of cGAS activation byDNA [] In addition to the primary DNAbinding sitementioned above the secondary site located beside theprimary site is a helix formed between strands 78and several surfaceexposed loops [] The proximity ofthe two DNAbinding sites in cGAS leads to a cGASDNA complex assembly in which two cGAS moleculesembrace two molecules of dsDNA [ ] The cGASdimers are anized in œheadtohead alignment nextto the DNA [] and thus form stable œladderlike networks between one long curved dsDNA helix or two independent dsDNA strands [ ] In this way eachindividual cGASdsDNA complex can be cooperativelystabilized and can lead to stronger enzymatic activitywhich may provide a possible explanation for longerdsDNA as more likely to activate cGAS [] In additionlong DNA is more efficient than short DNA in drivingthe liquidliquid phase separation of cGAS and the formation ofcriticallydependent on the concentration of cGAS and DNA inthe cytoplasm [] cGAS and dsDNA are spatially concentratedcGASdimerization and activation [“] Once cGAS andcGAS liquidlike dropletsin liquiddropletsistofacilitateTable Classification of the cytosolic dsDNA that activates the cGASSTING signaling axisClassificationSelfDNASource of dsDNAMicronucleiPossible mechanismsRupture of the micronuclei membrane leads to exposureof chromatin DNA that is recognized by cGAS whichactivates the cGASSTING pathwayReferences[]MitochondrionNuclear RNAPathogenderived DNADNA virusHSV1 HSV2 KSHV adenovirus vacciniavirus cytomegalovirus papillomavirusmurine gammaherpesvirus RetrovirusHIV SIV murine leukemia virusRNA virusWest Nile virus dengue virus VSVSARSCOV2BacteriaListeria monocytogenes Mycobacteriumtuberculosis Listeria Shigella FrancisellaChlamydia and NeisseriaMitochondrial stress induces mtDNA leakage into thecytosol thus activating the STING pathway and inducingproduction of cytokinesFacilitated by endogenous retroelements nuclear RNAcan be reversely transcribed into DNA that activatescGASSTING signaling[][]DNA viruses invade host cells and release pathogenderivedDNA to induce STING activation[“]DNA intermediates generated from reverse transcription maybe recognized by cGAS to stimulate downstream STINGsignaling[]Infection with RNA viruses might cause cellular damage andcell death which results in the release of cellular DNA andfurther activation of the cGASSTING axis SARSCoV2 bindingto ACE2 can lead to excessive angiotensin II signaling thatactivates the STING pathway in mice[“]Bacteria produce CDNs such as cyclic diGMP and cyclicdiAMP which can directly bind to and activate STING[ “]HSV1 herpes simplex virus HSV2 herpes simplex virus KSHV Kaposi sarcoma“associated herpesvirus HIV human immunodeficiency virus SIV simianimmunodeficiency virus VSV vesicular stomatitis virus CDNs cyclic dinucleotides and SARSCOV2 severe acute respiratory syndrome coronavirus 0cZheng Molecular Cancer Page of dsDNA interacts structural switches rearrange the catalytic pocket to enable cGAS to catalyze the synthesis of²²cyclic GMPAMP ²²cGAMP with ATP andGTP as substrates The first step in this process is theformation of a linear dinucleotide ²pppG ²²pAwith ATP serving as the donor and ²OH on GTP serving as the acceptor Then the intermediate product flipsover in the catalytic pocket placing GTP at the donorposition and AMP at the acceptor position to form asecond ²² phosphodiester bond [ ] Notablyalthough dsRNA or singlestrand DNA ssDNA is ableto bind to cGAS neither can rearrange the catalyticpocket which may explain the exclusive activation ofcGAS by dsDNA Ultimately cGAMP acts as a secondmessenger to bind to and activate STING a small endoplasmic reticulum ERlocated protein KD withfour putative transmembrane domains [ ] Normally in a resting state STING is retained in the ER byinteracting with the Ca2 sensor stromalinteractionmolecule STIM1 [] The cytosolic ligandbindingdomain LBD of STING exists as the most functionalunit capable of integrating with ²² cGAMP or CDNscyclic dinucleotides such as cdiAMP cdiGMP or ²²cGAMP from bacteria Upon interaction the obviousclosure of the ligand binding pocket in the LBD is observed which is related to the activation of STING []Next STING transforms into a tetramer through a highorder oligomerization reaction and is translocated fromthe ER to the perinuclear area facilitated by cytoplasmiccoat protein complex II COPII and ADPribosylationfactor ARF GTPases [ ] In the Golgi STING ispalmitoylated atCys88 andCys91 a posttranslational modification necessary forSTING activation [] Modified STING recruits thekinase TANKbinding kinase TBK1 in turn the Cterminal domains of STING are phosphorylated byTBK1 and then phosphorylated STING recruits interferon regulatory factor IRF3 which is also phosphorylated by TBK1 and dimerizes ultimately dimerizedIRF3 enters the nucleus and exerts its function in thetranscription of type I IFNs and interferonstimulatedgenes ISGs [] In parallel STING can also bind toand stimulate IκB kinase IKK to mediate the production of nuclear factorκB NFκBdriven inflammatorygenes Upon signal transduction termination STING istransferred to endolysosomes for degradation [] Considering that cGAMP can be transferred through gapjunctions or delivered in viralexosome packages cGASSTING signaling may be activated in the cytoplasmwithout dsDNA [ ] Moreover newly produced typeI IFNs activate heterodimer interferon receptors IFNAR1 and IFNAR2 through paracrine signaling and thusinduce the transcription of ISGs [ ] In summaryonce virusderived DNA and selfDNA are located intwo cysteine residuesthe cytoplasm they can be sensed by cGAS and a cGASdsDNA complex is formed to catalyze the synthesis of ²²cGAMP with ATP and GTP Then ²²cGAMP and bacteriaderived CDNs induce STING activation and mediate the release of downstream type IIFNs TNFα and IL6 which are prerequisites for antimicrobial defense and antitumor effects The wholeprocess shows that the dsDNAcGASSTING axis canlead to the activation of both innate and adaptive immunity Fig The antitumor functions of the cGASSTINGsignaling pathwayRecent evidence has revealed the close association of thecGASSTING pathway with cancer development Thissignaling pathway is generally regarded as a potent regulator of cancer immunity A STINGmediated immunesupportive microenvironment can hamper malignancyoccurrence []stressbyTumor cell cytosolic dsDNA induces STING activationUnder normal circumstances DNA is strictly unaffiliatedwith the cytoplasm in eukaryotic cells to avoid autoimmunity [] However DNA leaks aberrantly in tumorcells [ ] Cancer cells share common features including genome instability tumor suppressor gene mutation or deletion oxidativeand vigorousmetabolism [] Under these intense states nuclear andmitochondrial DNA is fragile and easily damaged whichleads to eventual DNA leakage in the forms of micronuclei chromatin fragments andor free telomeric DNA[ ] Chromosomal instability CIN is the primary source of cytoplasmic DNA in malignant cells andis generally associated with tumor progression distantmetastasis and therapeutic tolerance [] Excessive proliferation of cancer cells results in unstable genomes [] usuallychromosomal missegregation during mitosis Due to defects in segregation lagging chromosomes generate micronuclei in a cellcycledependent manner [] The vulnerable membraneof micronuclei easily exposes the inner DNA to the cytoplasm and activates the cGASSTING signaling axis [] Exogenous stimuli such as chemotherapy and irradiation can also cause DNA damage In addition to leakednuclear DNA oxidative stressinduced mitochondrialDNA leakage is another crucial initiator of STING pathway activation Several anticancer treatments that precisely attack mitochondrial membranes result in effluxand cell death Therefore the permeabilization of mitochondria membranes provides a reasonable explanationfor mitochondrial DNA escape [ ] Other sourcessuch as apoptotic cellderived DNA exosomal DNAExoDNA and transposable elements have also beencharacterized 0cZheng Molecular Cancer Page of Fig The cGASSTING DNA sensing signaling pathway Various DNA derived from virus dying tumor cells or nucleus and mitochondria binds toand activates the cytosolic DNA sensor cGAS cGAS catalyzes the synthesis of ²²cGAMP in the presence of ATP and GTP then ²²cGAMP bindsto the ER adaptor STING which also can be activated by CDNs derived from bacteria Upon activation STING translocates from ER to Golgicompartments where it activates TBK1 and IKK which phosphorylate IRF3 and IκBα respectively Then IRF3 and IκBα dimerize and enter nucleusinitiating the transcription of Type I IFN TNF and IL6 The primary roles of these cytokines are reflected in host defense inflammation andantitumor immunitydemonstrated to evoke cGAS“STING activation intumor cells [ ]Type I IFNs mediators of STING and adaptive antitumoreffectscGASSTING signaling exerts antitumor functions incancer cells both in an autonomous and nonautonomousmanner On the one hand DNA damage can provokeacute STING signal transduction and induce cellularsenescence an irreversible cell cycle arrest state whichthwarts the aberrant proliferation of tumor cells throughacquisition ofsecretoryphenotype SASP which is associated with the releaseof abundantinflammatory mediators proteases andgrowth factors [ ] In contrast to undergoingsenescence tumor cells also directly propel apoptosisprocesses by upregulating proapoptosis protein BCL2associated X BAX and downregulating the BCL2 apoptosis[] On the other hand STINGsenescenceassociatedtheregulatoractivation in tumor cells not only facilitates the transcription of downstream type I IFNs to induce dendriticcell maturation but also recruits supportive immunecells for direct nonspontaneous tumor elimination []STING activation in nonmalignant cells causes tumorsuppressive effects as well STING signaling protectsagainst colitisassociated carcinomas CACs induced byazoxymethane AOM and dextran sulfate sodiumDSS which induce DNA damage in intestinal epithelialcells and further trigger STING activation Downstreamcytokines of STING signaling such as IL1 and IL18prevent neoplastic transformation by facilitating woundrepair More importantly STING signaling can also provoke cytotoxic T cell responses to control tumorigenesis[] Necrotic cancer cells are commonly engulfed byantigenpresenting cells especially the basic leucine zippertranscription factor ATFlike BATF3drivenlineage of dendritic cells DCs [] BATF3 DCs take intumorassociated antigens and migrate towardsthe 0cZheng Molecular Cancer Page of tumordraining lymph node via the lymphatic systemwhere they crossprime tumorspecific CD8 T cellsThen CD8 T cells undergo activation and clonal expansion in the lymph nodes and are trafficked throughblood vessels to kill tumor cells In turn damaged cancercells release more antigens that are further captured byDCs the whole process forms a positive feedback loopcalled the cancerimmunity cycle [] Tumor eradication can be achieved by multiple processes in thecancerimmunity cycle including tumor antigen captureand presentation and T cell priming and activation withtumor antigenspecific T cell priming and activationrelying on DCs and type I IFN release [] The involvement of type I IFNs in innate immune sensing and adaptive immunity provides a reasonable hypothesis forexploring candidate PRR pathways as potential immunomodulators Mice lacking TLR9 myeloid differentiationprimary response gene MyD88 cytosolic RNA sensor MAVS or the purinergic receptor P2X7R maintainintact antitumor immunity responses whereas mice deficient in STING or IRF3 present with impaired CD8 Tcell priming and activation [ ] In fact dying tumorcells can release multiple damageassociated molecularpatterns DAMPs to trigger innate immune responsesin DCs among these released stimuli tumor cellderivedDNA is a pivotal inducer In general the phagocytosis ofapoptotic cells causesimmune silence because ofDNasebased degradation [] Nevertheless tumor cellreleased DNA can be preserved in the DC endolysosomal compartment through an unknown mechanism [] cGAS recognizes DNA invading the cytoplasm andinduces the activation of STING cascades excretion oftype I IFNs and expression of ISGs Additionally undersome physiological conditions such as hypoxia andacidic environments nuclear or mitochondrial DNAmight be packaged in exosomes Exosomal DNAExoDNA animates STING signaling once it is absorbedby tumorinfiltrating DCs [] Finallytumor cellderived cGAMP can also be transferred to host DCs bythe folate transporter SLC19A1 and then directly bindsto STING activating it in DCs [] A recent study moredirectly demonstrated that cellautonomous STING promoted the maintenance of stem celllike CD8 T cellsand augmented antitumor T cell responses and mechanistically cGASSTINGmediated type I interferon signaling reinforced the stem cell“like CD8 T celldifferentiation program mainly by restraining Akt activity []Immune cellderived type I IFNs have crucial functions in antitumor immunity control On the one handtype I IFNs boost cross presentation by various mechanisms first they stimulate the maturation of DCs secondthey slow the endosomelysosome acidificationprocess to prevent engulfed tumor antigen clearance andelevate the expression of MHC I molecules on the cellsurface [ ] finally they accelerate DC migrationtowardslymph nodes where they can crossprimetumorspecific CD8 T cells [] On the other handtype I IFNs drive the expression of multiple chemokinessuch as CXCL9 and CXCL10 both of which are necessary for cytotoxic T lymphocyte CTL transfer and infiltration [] Similarly type I IFNs restrain the defaultimmune suppressive action of regulatory T Treg cellsby downregulating phosphodiesterase PDE4 and upregulating cyclic AMP cAMP [] Consequently typeI IFNs serve as bridges linking the cGASSTING pathway with CD8 T cellmediated antitumor immunityThe antitumor mechanisms of the cGASSTING signaling axis are illustrated in Fig Indeed previous studies revealed that STING activation can stimulate antitumor immune responses inleukemia melanoma glioma and hepatocellular carcinoma [“] Additionally STING expression is downregulated in a wide variety of tumor tissues and celllines according to a pancancer analysis with a smallproportion of tumors approximately bearing silent STING expression [] Lower STING expressionwas found in hepatic carcinoma and gastric cancer compared with its level in corresponding normal tissues andthis lower expression level was correlated with highertumor stage and poorer prognosis [ ] Consistentlycompared with that in the MCFG10A mammary epithelial cell line lower STING expression was detected inmalignant breast cancer cellincluding MCF7HBL100 and T47D cells as well as human melanomacell lines and colorectal adenocarcinoma lines [ ] Collectivelythat cGASSTING signaling might act as a tumor suppressor in certain types of cancersthese findings suggestlinesSTING pathway agonists as cancer therapeuticsThe immunostimulatory potential of the cGASSTINGpathway makes it an attractive pharmacological targetsince its activation in the tumor microenvironmentTME can induce efficient crosspriming oftumorspecific antigens and facilitate the infiltration of effectorT cells Recent drug research has focused on the development of STING agonists because of their potential inanticancer therapy [ ] To date various kinds ofSTING agonists have been discovered and they aremainly divided into the following categories cyclic dinucleotides and their derivates DMXAA and its analogsand small molecular agonists In addition some conventional antitumor therapeutics can also indirectly activateSTING such as chemotherapy radiotherapy RT andtargeted therapy [] In addition STING agonists areable to enhance the efficacy of other anticancer therapeutic agents when used in combination STING 0cZheng Molecular Cancer Page of Fig The antitumor immunity effect of the cGASSTING pathway DNA damage leads to the formation of dsDNA in tumor cells upon itsstimulation STING signaling is activated and promotes the release of Type I IFN which is crucial for DC maturation STING signaling activation inDCs is the core step of the whole cancerimmunity cycle which can be initiated through engulfment of dyingdamaged tumor cells exosometransfer and cGAMP gap junctions Then DCs migrate towards the tumordraining lymph node and crossprime tumor specific CD8 T cells withthe help of Type I IFNs Finally T cells undergo clonal expansion and traffic through the blood vessel to conduct tumor killingagonists and their synergistic use with other remedies isfurther explored in detail belowCyclic dinucleotides CDNsCDNs constitute a main type of STING agonist whichmainly originate from bacteria The known naturalCDNs consist of exogenous cyclic diGMP cdiGMPcdiAMP ²²cGAMP and endogenous ²²cGAMPAmong these cdiGMP cdiAMP and ²²cGAMPare synthesized by bacteria and identified as secondarymessengers that mediate STING signal transduction inprokaryotic cells while ²²cGAMP functions as theinitiator of STING in mammalian cells [] The antitumor potential of these natural dinucleotides was firstproven by the finding that cdiGMP could inhibit theproliferation of human colon cancer cells in vitro andbasal cell proliferation of human cecal adenocarcinomaH508 cells was inhibited with μM cdiGMP []Intraperitoneal ip injection of highdose cdiGMPdirectly activated caspase3 and triggered T1 tumoripcell apoptosis in vitro nmol of cdiGMP reduced thegrowth of T1 tumor cells in vitro by and nmreduced it by while lowdose cdiGMP nmol accelerated the adaptive T cell response by converting a subgroup of myeloidderived suppressor cellsMDSCs into immune stimulatory cells producing IL12injection of ²²cGAMP [] Consistentlymgkg expedited dramatic leukemic elimination in ElTCL1 transgenic mice bearing chronic lymphocyticleukemia CLL and promoted tumor shrinkage of multiple myeloma in vivo [] From the perspective of endogenous CDNs ²²cGAMP mgkg was alsoshown to restrain tumorigenesis and improve the survival rate of mice bearing CT26 colon adenocarcinomain a dosagedependent manner relying on DC activationand T cell crosspriming [] More recently OhkuriT further demonstrated that intratumoral it injection of ²²cGAMP μg25 μLdose on and days after the injection of tumor cells significantly mitigated tumor growth and prolonged the survival of breast 0cZheng Molecular Cancer Page of cancer T1luc squamous cell carcinoma mSCC1colon cancer CT26 and melanoma B16F10 mousemodels [] Notably the it injection of ²²cGAMPinhibited not only tumor growth but also lung metastases in mice bearing B16F10 cellderived tumors suggesting that cGAMPinduced CD8 Tcell priming can drivesystemic antitumor immunity to control local and distant tumor growth []termedvaccineSTINGVAXConsidering the superior properties of STING signaling in activating adaptive immunityit is rational toutilize STING agonists such as CDNs as cancer vaccineadjuvants to increase tumor immunogenicity [] Fu investigated the in vivo therapeutic efficacy of acancercomprisinggranulocytemacrophage colonystimulating factor GMCSF and bacteriaderived or synthetic CDNs Theyobserved that after it injection of STINGVAX with μg of CDNs per vaccine dose the volume of B16melanoma tumors was dramatically reduced in a dosedependent manner Compared to mice receiving GMCSF cancer vaccine alone STINGVAXtreated mice hadmore infiltrating CD8 IFNγ T cells in the tumormicroenvironment The in vivo antitumor effect of STINGVAX was also verified in models of colon carcinomaCT26 pancreatic carcinoma Panc02 and upper aerodigestive squamous cell carcinoma SCCFVII []Although natural CDNs are able to produce robust antitumor immunity their chemical features might hindertheir future application in the clinical setting First native CDNs are easily degraded by enzymes inside the cellor in the bloodstream Second their negatively chargedproperty hydrophilicity and phosphate moieties severelyimpede CDNs from penetrating cell membranes to activate cytosolic STING leading to low bioavailability andpoor retention of the CDNs in specific cells and tissuesThird unintentional toxicities and narrow therapeuticwindows are also unavoidable Thus new strategies toimprove therapeutic efficacy and reduce adverse effectsare urgently needed including drug delivery carrier engineering original structural modification and nonnucleotide agonist screening [] Regarding agonistdelivery Smith reported that biopolymer implantscodelivering cdiGMP μg and chimeric antigen receptor T CART cells resulted in significant tumor regression in mice bearing pancreatic tumors[]Moreoveriv administration of cdiGMPYSK05Lip equivalent to μg of cdiGMP aYSK05liposome delivery system encapsulating cdiGMP led to a tremendous decrease in metastatic lesionsin a B16F10 mouse melanoma model with nearly ofthe injected mice showing resistance against tumor relapsethe adaptive immune responsememory was successfully induced [] Chen alsofound thatliposomalindicating thatinjection ofintravenousintravenousivnanopdelivered cGAMP cGAMPNP could activate the STING axis more effectively than solublecGAMP and converted the immunosuppressive TME toa tumoricidal state in a transplanted B16F10 cell melanoma model and in a genetically engineered triplenegative breast cancer model [] Moreover a recentstudy creatively suggested that modified bacteria mightbe exploited as a selective carrier of STING agonistsIntroduction of a dinucleotide cyclasecoding gene intothe Escherichia coli Nissle strain was an attempt at realizing this effect however advancements to the systemare needed []tobysnakeApartdigestionresistancecompoundatoms The modifiedfrom improving delivery methods CDNswith superior properties are currently being synthesized and tested For instance to prevent enzymatichydrolysis of cGAMP the nonbridging oxygen atomsin cGAMP phosphodiester linkages were replaced by²²sulfurcGsAsMP showed resistance against degradation byENPP1 a major ²²cGAMP hydrolasetherebyleading to a longer halflife and sustained high affinity for human STING hSTING[] Syntheticdithio mixedlinkage CDNs with both Rp Rp R Rand Rp Sp R S dithio diastereomers possessed notonlyvenomphosphodiesterase but also enhanced affinityforSTING A novel superior modified product ML RRS2 CDA also termed ADUS100 had the potencyto activate all hSTING variants and mouse STINGmSTING ADUS100 had higher efficiency in activating STING signaling than endogenous or exogenous CDNs mainly because of its enhanced stabilityand lipophilicity Its powerful tumor elimination effect was extensively demonstrated in multiple murinemodelsincluding B16 melanoma T1 breast cancer and CT26 colon cancer with all treated animalsshowing significant and durable tumorregressionafter itinjection of ADUS100 three mg doseswhen tumor volumes reached mm3 [] TheremarkableforhSTING laid the foundation for its clinical use Related clinicaltrials of ADUS100 are outlined inTable In addition to ADUS100 some other novelSTING agonists have been well designed IACS8779and IACS8803 are two highly potent ²²thiophosphate CDN analogs that induced striking systemicantitumorin a B16 melanoma murineinjection μg on and daysmodel after itposttumor implantation compared with ADUS100or cGAMP [] The characteristics and preclinicalapplications of all these mentioned CNDs are summarized in Table Because of the structural modification and optimization of delivery strategiestheapplication range and efficacy of CDNs have beenand high affinityresponsescurativeeffect 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonistsClassificationCharacteristicsApplicationmodelsNatural CDNagonistscdiGMPPoor membrane permeabilitysuitable for various codeliverytechnologiesColon cancer H508cells T1 metastaticbreast cancerTreatmentinformation μM nmol ip nmol ip nmol ip²²cGAMP²²cGAMPHigher binding affinity formSTING than for hSTINGHigher affinity for hSTING thanits lineage isomers binds tovarious STING nucleotidepolymorphisms observed inhumans easily degraded byphosphodiesteraseimpermeable to the cellmembraneChronic lymphocyticleukemia mgkg ipmultiple myeloma mgkg ipCT26 colonadenocarcinoma mgkgbreast cancer T1lucsquamous cellcarcinomasmSCC1 μg25 μLdose it μg25 μLdose itcolon cancer CT26 μg25 μLdose itmelanoma B16F10 μg25 μLdose itTherapeutic effects References[ ][][ ]Inhibitsproliferation tumorregression tumorregressionAccelerates TcellresponseLeukemiaeliminationSuppressesgrowthRestrainstumorigenesisImproves survivalrateDelays tumrowthDelays tumrowthDelays tumrowthDelays tumrowthSTINGVAXSyntheticCDNagonistsPotent in vivo antitumor efficacyin multiple therapeutic modelsof established cancercGAMPNPsBiopolymer scaffolds cdiGMP and CAR T cellscdiGMPYSK05Lip²²cGsAsMPADUS100IACS8779IACS8803NonCDNagonistsFAALiposomal nanops NPsdeliver cGAMP intracellularlymore effectively than realizedwith soluble cGAMPEradicates tumors moreeffectively than systemicdeliveryYSK05 is a lipid that can efficientlydeliver cdiGMP to the cytosolpossesses high fusogenic activitywhich enhances endosomalescapeMore resistant to degradation byENPP1 tenfold more potent atinducing IFN secretion potentialuse as a cancer vaccine adjuvantImproves stability and lipophilicityhigher affinity for hSTING thannatural CDN agonists capable toactivate all hSTING variants andmSTINGStimulates a superior systemicantitumor response thanADUS100 and cGAMPCauses hemorrhagic necrosisfailed in a phase I clinical trialdue to species specificity μg CDNs itReduces tumorvolume[]B16 melanomacolon carcinomaCT26pancreaticcarcinoma Panc02B16F10 melanomaivTNBCCreates atumoricidal state[]Pancreatic cancer μg cdiGMPTumor regression[]B16F10 mousemelanoma μg cdiGMP ivDecreasesmetastasisTHP1 monocytesB16 melanomathree mg doses it T1 breast cancerthree mg doses itMC26 colon cancerthree mg doses itDurable tumorregressionDurable tumorregressionDurable tumorregression[][][]B16 melanoma μg on day and posttumor implantationAntitumorresponse[]Murine colontumorsExtensive tumorrejection[ ]DMXAAFirst discovered as a vascularRat mammary mgkg ipHigh anticancer[ 0cZheng Molecular Cancer Page of Table Characteristics and preclinical applications of different STING agonists ContinuedClassificationCharacteristicsApplicationmodelsTreatmentinformationInduces proinflammatory cytokinesin a STINGdependent mannerHuman fibroblastsAntiviral activity[]Selectively induces STINGdependentsynthesis and secretion of bioactiveIFNs no evidence of binding directlyto STINGActivates STING
2
collagen triple helix repeat containing1 cthrc1 anextracellular matrix ecm protein was identified in thescreening of diï¬erentially expressed sequences between balloon injury and normal arteries the evolution of cthrc1can be traced back to at least million years ago and theconserved genes were not found in invertebrates cthrc1has complicated interactions with various intracellular andextracellular matrices in diï¬erent ways of secretion [ ]cthrc1 increases the activity of collagen promoter throughbinding to ligands and could contribute to vascular remodeling by limiting collagen matrix deposition and promoting cellmigration cthrc1 promotes the recruitment of m2macrophages and regulates tgf and notch pathways toaccelerate wound healing in a mouse model of acute woundhealing as a coupling factor cthrc1 can be secretedby osteoclasts and ‚uence bone formation and remodelingby acting on osteoblasts and osteocytes [ ] cthrc1 maypromote il1induced apoptosis of chondrocytes by activating the jnk12 pathway the anti‚ammatoryeï¬ect of cthrc1 expressed on activated synovial cellswas also found in a collagen antibodyinduced arthritismodel besides cthrc1 can regulate physiologicalfunctions such as fat and glycogen synthesis and promoteautonomous activity [ ]therefore as a secreted protein cthrc1 is involved inmultiple pathophysiologies a remarkable eï¬ect is that thehigh expression of cthrc1 promotes tumorigenesis anddevelopment through positive regulation of tumor spreadinvasion migration adhesion and metastasis cthrc1exerts its eï¬ects through several signaling pathways such as 0cmediators of ‚ammationgpa gvp grd gsp gan gip gtp gip grd gfk gek gechydrophobic regiongxy repeatcysteinesnglycosylationnh2signal peptidecollagendomainc c c cc cc ccoohfigure the structure of the cthrc1 protein the construct of cthrc1 contains an nh2terminal peptide for extracellular secretion ashort collagen triple helix repeat of amino acids and a coohterminal globular domain the prolinerich hydrophobic domain liesbetween the 1st and 30th amino acids and serves as a signal peptide for transport to the endoplasmic reticulum cthrc1 comprises acollagen domain between amino acids and and the protein contains cysteine residues corresponding to about cysteine inthe final protein what is more its only amino acid posttranslational modification is the glycosylation of asparagine at position integrin faktgf wntsrcfak mekerkpi3kakterk hif1α and pkcδerk signaling pathways in this we focus on the advances in the signalingpathways mediated by cthrc1 in tumors the structural characteristics andexpression of cthrc1 the structural features of cthrc1 the cthrc1 geneis located at chromosome 8q223 and it contains five exonsin humans and four exons in mice it covers kb onthe direct strand and can be transcribed into kb mrnathe amino acid sequence identity between human and ratcthrc1 proteins was and no homologs were foundin lower species [ ]protein nglycosylationsecreted cthrc1 exists primarily as a dimer kdaand a trimer kda as well as multimers of the trimericcthrc1 kda and kda the construct of cthrc1contains an nh2terminal peptide for extracellular secretiona short collagen triple helix repeat of amino acids and acoohterminal globular domain [ ] similar molecularweight and structural characteristics to adiponectin alsoexplain why cthrc1 can form high molecular weightcomplexes the biological activity of cthrc1 isrestricted to the highly conserved amino acids at thecterminal region and the cterminal region of cthrc1contains a putative nglycosylation site that stabilizescthrc1promotescthrc1 to tether to the cell membrane which promotesactin polymerization and cell polarity a short collagen motif with glyxy repeats presents in c1qtumornecrosis factorαrelated proteins ctrps which appearsto be responsible for the trimerization of protein and renders molecule susceptible to cleavage by collagenase seefigure however dimeric cthrc1 would not be susceptible to cleavage by collagenase [ ] the molecularweight of secreted cthrc1 kda appears to be largerthan that of cellular cthrc1 kda cthrc1 has fourdiï¬erentabout kda to kda the fulllength of cthrc1 accountsfor both secreted and cellular cthrc1 glycosylatedprotein cthrc1 with a signal sequence is related to ecmisoforms with molecular weights ofalsoand contains a variable short collagenlike motif intriguingly cthrc1 plays a role in inhibiting structural proteins unlike other members of the collagen family moreover leclair found that cthrc1 cleaved atthe nterminus by plasmin shows better inhibition of collagen synthesis compared to fulllength cthrc1 in thepac1 cell line these studies suggested that cthrc1might obtain biologicalthrough proteolyticprocessingactivity the expression of cthrc1 cthrc1 is transientlyexpressed by fibroblasts in remodeling adventitia and bysmooth muscle cells in the neointima of injured tissuehowever cthrc1 is not detected in normal arteries ininjured arteries and skin the expression of cthrc1 isassociated with myofibroblasts and locates in the sites ofcollagen matrix deposition in micethe first exon ofcthrc1 was targeted to be replaced with a galactosidase expression construct which demonstrated the expression of cthrc1 in inner ear hair cells there iscthrc1 expression in many mesenchymalderived cellsduring body growth and tissue repair in mouseembryos cthrc1 is expressed in visceral endodermnotochord neuraltube developing kidney and heartabundant expression of cthrc1 is observed in developincluding cartilage primordia growth plateing skeletoncartilagein adultscthrc1 is expressed only in bone matrix and periosteum cthrc1 is also found in the matrix of calcifyingatherosclerotic plaques and mineralized bone of skeletaltissues in humans in other tissues the sites of cthrc1expression overlap considerably with interstitial collagensand transforming growth factor tgf family membersparticularly bone morphogenetic proteins bmps the sitesof cthrc1 expression are characterized by the presence ofactive tgf and abundant collagen synthesis cthrc1mrna expression levels are increased in response to bmp4bmp2 and tgf furthermore tgf signaling could leadto a significant increase in neointimal lesion formation the expression of cthrc1 is also positively correlatedwith tumor lymph node metastasis tumornodemetastasistnm stage and disease prognosis however its potentialand periosteumbone matrix 0cmediators of ‚ammationregulatory mechanisms in the tumor environment have notyet been elucidated the molecules that regulate theexpression of cthrc1cthrc1 is abnormally expressed in several solid tumorsespecially in gastric cancer pancreatic cancer hepatocellularcarcinoma keloid breast cancer colorectal cancer crcepithelial ovarian cancer esophageal squamous cell carcinoma escc cervical cancer nonsmallcell lung carcinoma nsclc melanoma and so on [ “] andmolecules that regulate the expression of cthrc1 includemirnas lncrnas waif1 and dpagt1 mirnas microribonucleic acids mirnas which canregulate gene expression are a class of noncoding singlestranded small rnas of about nucleotides that can inhibitthe mrna translation process by exclusively promoting thedegradation of several mrnas in many tumors mirnas such as mir30c mir9 mir520d5p mir1555pmir98 let7b mir155 mir101 and mir217 can regulate the expression of cthrc1mir30c could regulate cthrc1 at a posttranscriptionallevelin breast cancer it downregulates the cthrc1mediated gsk3catenin signal and inhibits tumor cellproliferation invasion and migration in addition mir30ccan also upregulate baxcaspase9caspase3 a downstreamsignal of cthrc1 inhibiting apoptosis in hepatocellular carcinoma cthrc1 downregulates mir1555p throughthe activation of gsk3involved wntcatenin signalingto promote tumor formation and mir98 dramaticallydownregulates cthrc1 by directly targeting the ²utr ofcthrc1 suppressing hepatocellular carcinoma formation mir9 could inhibit the migration of schwann cell bytargeting cthrc1 following sciatic nerve injury therebyinactivating downstream rac1 gtpase mir520d5pis significantly downexpressed and suppresses cell proliferation migration and invasion by targeting cthrc1 in crc as the second mirna following lin4 in caenorhabditiselegans let7b may directly target cthrc1 and function asa tumor suppressor gene in gastric cancer [ ] in esccmir101 and mir217 could inhibitthe expression ofcthrc1 mir30 could downregulate the expressionof cthrc1 and downstream signal molecules such as mmp and mmp2 to inhibit the invasion and migration ofnsclc cells a recent study found that mir155 downregulation and cthrc1 upregulation were observed incrc moreover overexpression of mir155 can silencedownstream cthrc1 thereby inhibiting cell proliferationand inducing apoptosis of cells to prevent tumor progressionand metastasis in conclusion the negative regulation ofcthrc1 by mirna has the potential to become a noveldirection for cancer treatment in the futurelncrnas metastasisassociated lung adenocarcinomatranscript i malat1 is a large infrequently spliced longnoncoding rna lncrna which could genetically increasecthrc1 activity to regulate lung cancer cell migration the silence of malat1 could also inhibit the expression ofcthrc1 which is a positive regulator of escc furtheranother lncrna named nonmmug014387 could also regulate cthrc1 and activate the wntpcp pathway to promote schwann cell proliferation at the site of injury waif1 wntactivated inhibitory factor waif1 issilenced by promoter hypermethylation in various cancers[ ] lcmsms analysis using liquid chromatographyand mass spectrometry analysis of samples of cthrc1binding membrane proteins indicates that the largest partof cthrc1 binds the waif1 receptor recent researchsuggests that waifi expression is activated by suppressingmethylation of its promoter activated waif1 downregulates the expression of wntcatenin target genes to inhibitthe development of endometrial adenocarcinoma thebinding of cthrc1 to waif1 could promote osteoblast differentiation therefore cthrc1waif1 interactioncan be a potential therapeutic target in the futurepromoter hypomethylation dpagt1 nglycosylation is essential for the migrationpattern of immune cells and its dysregulation is related tovarious diseases including cancer in human escc the overexpression of cthrc1 is associated with hyperglycosylationandincreased nglycosylation is associated with preferential localization ofcthrc1 in wound cells and nglycosylation facilitatesthe promigratory function of cthrc1 dolichylphosphatenacetylglucosaminephosphotransferase dpagt1 thegene that encodes the first enzyme and ratelimiting enzymein the assembly of lipidlinked oligosaccharide precursors inthe endoplasmic reticulum is related to the formation ofmature intercellular adhesion complexes as anupstream regulator of nglycosylation status of ecadherindpagt1 could upregulate cthrc1 by increasing proteinturnover indicating that nglycosylation can also stabilizecthrc1 besides tgf and fak could also regulate the expression of cthrc1 in diï¬erent signaling pathways it should behighlighted that cthrc1 not only is the result of tumor progression but also plays a predominant regulatory role in theprogression and metastasis of many solid tumors [ “]in summary many molecules can regulate the expressionand activity of cthrc1 and together with cthrc1 as novelantitumor molecular targets for the treatment of cancer inthe future signaling pathways mediated by cthrc1involved in the progression andmetastasis of tumorthe ‚uence of cthrc1 on various events in tumor progression is based on its regulation of various signaling pathways such as tgf wntintegrin fak srcfakmekerk pi3kakterk hif1α and pkcδerk signaling pathways see figure these properties™ pathwaysaï¬ected by cthrc1 play an essential role not only in tissueremodeling after injury regulation of ossification and other 0cmediators of ‚ammationcthrc1tgf𝛽t𝛽r2wnt3alrp56cthrc1wnt5aror12cthrc1cthrc1𝛽𝛼𝛼𝛽integrincxrc4t𝛽r1p ppsmad23smad4cthrc1dvlpdvlaxinapc𝛽cateninck1𝛼gsk3dpagt1stabilization𝛽cateninpkc𝛿daamrac1rhoajnkrockpsrcfakpaxgrb2rasrafmekerkpfakpi3kaktmtorfra1crebmmpmekerkhif𝛼vegfecminvasionpsmad23smad4𝛽catenintcflefcjunap1snailcyclin d1g1mg2smetastasisangiogenesisproliferationfigure signaling pathways mediated by cthrc1 involved in the progression and metastasis of tumor tgf signaling pathway isquite complex especially in terms of its eï¬ects which are often contradictory depending on location and time there exists a criticalnegative feedback regulatory loop between tgfsmad23 signaling pathway and cthrc1 wnt signaling includes wntcatenincanonical pathway and cateninindependent noncanonical pathway in the canonical wnt signaling fzd receptor and lrp5lrp6coreceptor are transduced to catenin signaling cascade for the maintenance of stem and progenitor cells in the noncanonical wntsignaling fzd receptor and ror2ptk7ryk coreceptor are transduced to rhoa jnk signaling cascades for the control of tissuepolarity cell adhesion or cell movement the downstream molecules of the wntpcp pathway mainly include the small gtpase familysuch as rac1 rhoa and jnk which play essential roles in cancer cell migration and invasion cthrc1 signal via waif1 canactivate pkcδ which is an essential component of the wntpcp pathway furthermore pkcδ is responsible for the activation of thecthrc1induced erk signaling pathway in cthrc1integrin signaling pathway the upregulation of cthrc1 is related to theprogression and metastasis of several cancers through the activation of several key signaling molecules including src fak paxillin mekerk and rac1 fak promotes cancer cell migration by regulating focal adhesion formation and turnover which involve activation of srcand paxillin fra1 is activated by cthrc1 through the mapkmekerk signaling which leads to the upregulation of cyclin d1 andthat promotes cell proliferation fra1 also induces snail1mediated mmp14 expression to facilitate escc cell invasion migration andmetastasis pi3kakt signaling pathway induces emt change and mmp2mmp9 expression hif1α and vegf are activated bycthrc1 through activating the pi3kaktmtor signaling pathway which promotes tumor angiogenesis cthrc1 also participates intumor cell migration and invasion through hif1αcxcr4 signalsphysiological processes but also in the development of cancerand metastasis negative feedback regulation of cthrc1 and cell typespecific tgf signaling pathway as the most potentgrowth factor involved in wound healing tgf is releasedby platelets at the site of injury ‚uencing ‚ammatoryresponse angiogenesis reepithelialization ecm and remodeling tgf superfamily members include tgf activin and bmps smad158 mediates bmp signaling whilesmad23 mediates tgf and activin signalingcthrc1 has been reported to have a relationship withthe tgf family since its discovery as their expression sitesoverlap significantly tgf1 and bmp4 can induce thetranscription and expression of cthrc1 in nih3t3 cells cthrc1 can activate tgf signaling via an elevationin smad2smad3 phosphorylation activated smad23 formsa complex with smad4 and accumulates in the nucleus causing an increase in collagen type i deposition during vascularremodeling [“] there exists a critical negative feedbackregulatory loop between tgf1 and cthrc1 the conserved region of amino acids in cthrc1 proteincan bind to phosphosmad3 cthrc1 is induced by tgf1 via phosphosmad3 binding to the promoter with subsequenttranscription activation and in turn cthrc1inhibits tgf1 signaling by accelerating proteasomal degradation of phosphosmad3 which inhibits collagen deposition tgf can enhance the migration and invasioncharacteristics of endothelial cells by regulating the secretionand expression of mmp2 and mmp9 therefore inhibiting cthrc1mediated tgf signaling pathways mayeï¬ectively suppress the invasion and angiogenesis of cancercells [ ]however the mechanism of tgf involved in tumorprogression is very complex even in the same tumor typetgf has many diï¬erent roles in tumor progression forexample the activation of nuclear factor of activated tcellsnfats can drive the switch of the tumorsuppressive function of tgf towards tumor progression [ ] tgfincreases the level of cthrc1 in crc cells highly 0cmediators of ‚ammationexpressed cthrc1 promotes epithelialmesenchymal transition emt and tumor metastasis through the smad2smad3 activation of tgf pathway cthrc1 can alsoinhibit the tgfsmad pathway and yap nuclear translocation thereby inhibiting type i collagen synthesis metabolites such as bile acid may induce cthrc1 to activatethe tgfsmad2smad3 pathway to mediate liver fibrosisand may progress towards hepatocellular carcinoma in the polyvinyl alcohol sponge model cthrc1 activates tgf and notch pathways to promote the recruitment of m2 macrophages however cthrc1 maydownregulate tgf expression during the late remodelingphase of wound healing tgf regulates the expressionof cthrc1 in a concentrationdependent manner inkeloids and excess cthrc1 reverses collagen synthesis therefore these results of the regulation betweencthrc1 and tgf are not contradictory other than thatcthrc1 has no inhibitory eï¬ect on tgf signaling inendothelial cells these results indicate that the regulation of tgf by cthrc1 may play a role in other interstitial cells of the tumor microenvironment and that thisregulation is cell typespecific the further exploration ofdetailed molecular mechanism by which cthrc1 activatesthe tgf pathway may resolve these disputes mutual regulation between cthrc1 and wnt pathwaysto promote tumor progression and metastasis wnt familyare secreted glycoproteins include wnt1 wnt1 wnt3awnt4 wnt5a wnt5b wnt6 wnt7a and wnt7b andparticipate in the process of numerous oncogenic and development progress [“] wnt5a is a member of the wntprotein family and plays an essential role in the pathologicalprocess of neuropathy and malignant tumors [“] wntproteins activate the wntcatenin canonical pathway andcateninindependent noncanonicalamongwhich the planar cell polarity pcp pathway and wntcalcium ca2 pathway are the most widely studied [“]current reports indicate that cthrc1 is mainly involvedin tumor progression through the canonical wntcateninand noncanonical wntpcp pathwayspathway wntcatenin canonical signaling pathway in thewntcatenin canonical pathway wnt proteins bind tofrizzled fzd receptor and lipoprotein receptorrelatedprotein lrp56 coreceptor in the absence of wntsignaling the cytoplasmic catenin form the œdestructioncomplex composed with the casein kinase 1α ck1αglycogen synthase kinase 3 gsk3 adenomatous polyposis coli apc and axin which activates the emt topromotethroughcthrc1wntcatenin [ ] the level of cateninis maintained as low by the series of eventsincludingpriming phosphorylation by ck1α at ser45 and subsequently at thr41 ser37 and ser33 by gsk3 [ ]when secreted wnt ligands are accumulated wnt combines with fzd receptor and lrp56 coreceptors lead toactivation of dishevelled dvl protein the activateddvl is phosphorylated and translocated to the fzd recepthe catenintorcausing the dissociation ofand metastases cancerinvasionœdestruction complex and the cytosolic accumulation ofcatenin as the cytosolic catenin accumulates rasproteins are accumulated due to the absence of gsk3mediated phosphorylation the stabilized ras proteins atthe plasma membrane activate rafmitogenactivatedprotein kinase mekextracellular signalregulated kinaseerk cascade besides cytosolic catenin subsequently translocates into the nucleus and forms a complexwith the tcell factor tcf or lymphoid enhancer factorlef the complex activates the expression oftargetgenes involving proliferation and transformation such ascmyc cjun ccnd1 gene encoding cyclin d1 epidermal growth factor receptor egfr cd44 cd133 andleucinerich repeatcontaining g proteincoupled receptor lgr5 [“]the wntcatenin signaling pathway plays an indispensable role in the occurrence and development of manytypes of cancer cthrc1induced nuclear translocation ofcatenin was observed in nclh23 cells and luciferaseassay showed that catenintcf transcriptional activitywas enhanced in contrast the knockdown of cthrc1reduced the catenintcf transcriptional activity whichshows that cthrc1 regulates the invasiveness of nsclccells through the wntcatenin pathway similarlycthrc1 activates snail1 through the wntcatenin signaling pathway to promote emt in epithelial ovarian cancer during the development and metastasis of crccthrc1 may promote the activation of the wnt signalingpathway through anos1 it can also participate in thewntcatenin pathway to regulate the malignant behaviorof hepatocellular carcinoma with gsk3 many cancers usually metastasize to bone in advanced stages cthrc1secreted by osteoclasts promotes basic fibroblast growth factor bfgf expression in osteoblasts by stimulating wntcatenin signaling which may induce the development of cancerous bone lesions but not mediate vascular production the constitutive activation of the wntcatenin pathway leads to carcinogenesis in tumors cthrc1 promotes catenin nucleartranslocation and inducestranscription of downstream target genes such as cmycand cyclin d1 in the nucleus reduces cell adhesion and promotes cell proliferation subsequently tumor cell invasion and metastasis occurredinterestingly another reported that catenincould act on the cthrc1 promoter region and promotetranscription nglycosylation stabilizes cthrc1 in oralsquamous cell carcinoma oscc specimens by reducingprotein turnover rate and cthrc1 is positively feedbackregulated by the dpagt1canonical wnt pathway therebyactivating noncanonical wnt pathways to drive tumor cellmigration and invasion in contrast the overexpressionof cthrc1 in hek293t cell and gastrointestinal stromaltumor gist cell significantly inhibited the canonical wntpathway but activated the noncanonical wntpcp pathway[ ] based on the evidence reviewed above it can be indicated that crosstalk between the canonical wntcateninpathway and noncanonical wntpcp pathway and themutual regulation of wntcatenin and cthrc1 acceleratethe process of tumor progression 0cmediators of ‚ammation wntpcp noncanonical signaling pathway earlyreports suggest that cthrc1 activates the pcp pathwayduring inner ear development cthrc1 can interactwith multiple extracellular components of wnt signalingfzd proteins and wntpcp coreceptor ror2 the components form a cthrc1wntfzdror2 complex to activatethe wntpcp pathway selectively and transmit signals fromthe cellsurface complex to the nucleus by dvlrhoarac1jnkatf2cjun cascade promoting cancer cell protrusionsproliferation migration and invasion [ “]cthrc1 is capable of coordinating three small rho gtpasesrac1 rhoa and cdc42 which are the leading performers ofwntpcp to promote cell migration in cervical cancercthrc1 cooperates with e6e7 human papillomavirushpv to activate the noncanonical wntpcp pathway whichaggravates tumor malignancy in pancreatic cancer andhuman urothelial carcinoma wnt5aror2 signaling is associated with emt and promotes tumor cellinvasion andmetastasis [ ] in gist cthrc1 appears to activatethe wntpcp pathway in a dosedependent manner andwnt5apcprho axis determinesinvasionpromoting activity of cthrc1 a recent study demonstrated that cthrc1 could promote erk and jnkphosphorylation by activating pcp signaling pathways inhuman umbilical vein endothelial cells huvecs and promote tumor angiogenesis whatit wasobserved that the paracrine cthrc1 controls the expression of ang2 via noncanonical wnt pathway activationof erkdependent ap1 in huvecs hence overand above that associated with the canonical wntcateninpathway noncanonical wnt signaling pathways interactwith other signaling pathwaysis moretumorthe cthrc1 binds integrin and triggers a series ofsignaling cascades to promote tumor progressionand metastasis integrin faksrc signaling pathway integrins aretransmembrane receptors that promote cellecm adhesion with two subtypes of α and it can participate ina variety of physiological activities such as tumor progression and migration cthrc1 promotes hepatocellular carcinoma cell invasion by activating the rhoarhoassociated kinase rock pathway and facilitates adhesionof hepatocellular carcinoma cells to ecm through induction of integrin 1 expression and activation of focal adhesion kinase fak another study of hepatocellularcarcinoma suggests that cthrc1 inhibits anoikis andincreases tumor cell survival by activating integrin expression cell adhesion to fibronectin is mediatedby integrin 1 previous researches have demonstrated that targeting the integrin 3fak signaling couldenhance the antitumor activity and attenuate cancermetastasiscancernsclc and escc [“] guo found that phosphorylated fak was significantly reduced in mice witheoc xenograft tumors and inhibition of fak did notinterfere with integrin 3 expression in vivo howeverthe overexpression of cthrc1 leads to the upregulationincluding melanomaendometrialof integrin 3 in model mice proving that cthrc1 interacts with integrin 3 and promotes fak phosphorylationat tyr397 thereby promoting ovarian cancer cell adhesionmigration and invasion the high level of cthrc1 is connected with the progression and metastasis of pancreatic cancers through the activation of several key signaling molecules including the steroidreceptor coactivator src fak paxillin mek erk andrasrelated c3 botulinum toxin substrate rac1 srcfak signaling cascade takes a regulative role in regulating the formation of protein complexes at focal adhesionsin the migration and metastasis of cancer cells srccan correspond to integrinecm interaction and is recruitedto form the srcfak complex which permits fak to beactive [ ] then fak activates src and paxillin by regulating focal adhesion formation and turnover paxillin a focaladhesion adaptor molecule serves as a scaï¬oldfor the organization and the activation of raf mek anderk [ ] furthermore paxillin can stimulate rac1which is a ras superfamily member of small guanosine triphosphatase gtpase and a critical factor in cytoskeleton reorganization regulation of gene expression and cell proliferationand cellular transformation [“]erk2mediated paxillin phosphorylation promotes fakadhesion to focal adhesions additionally the inhibitionof fakpaxillin interaction results in decreased phosphorylation of fak and its targets which in turn changes cell adhesion and migration thisinspired thedevelopment of anticancer drugs targeting fak faksrc complex plays essential functions in tgfinduced hepatocyte emt models such as upregulating mmp9 and fibronectin and downregulating ecadherin evidence has mekerk and pi3kakterk signaling pathwaycthrc1 interacts with integrin to trigger a series of signalcascades because src can phosphorylate other fak sites itcan recruit proteins containing src homology sh2domains such as grb2 subsequentlythe downstreamrasmapk pathway and the phosphatidylinositol kinase pi3k akt cascades are activated to participatein cellular response cthrc1 activates fosrelatedantigen1 fra1 through mapkmekerk signalingwhich leads to the upregulation of cyclin d1 and promotescell proliferation fra1 a fos family transcription factoralso induces snail1mediated mmp14 expression to facilitate escc cellinvasion migration and metastasis snail1 transcriptionaltriggeringemt and inducing tumor cell invasion knockingdown cthrc1 will change the phosphorylation level oferk12 and thus regulate the pathological process of endometriosisfrequent upregulation ofcthrc1 observed in human colon cancer cells may bedue to a cpg demethylation event in the exon regionof the gene kim tested the luciferase reporter geneusing the erkresponsive elk1 promoter proposing thatcthrc1 upregulates mmp9 through erk activation further treatment with mek12 inhibitors can reduce tumorcell invasion and erk activation and aggressiveness arereduced by knocking down cthrc1 theis essentialfactorforem 0cmediators of ‚ammationcthrc1 promotes invasiveness and metastasis of hepatocellular carcinoma through the activation of pi3kproteinkinase b pkbakterkcamp responseelementbinding protein creb signaling pathway which inducesemt change and mmp2mmp9 expression cthrc1is highly expressed in hepatitis b virus hbv associatedhepatocellular carcinoma hbv activates nuclear factorkappa b nfκb and creb through the erkcjun nterminal kinase cjnk pathway to stimulate cthrc1expression in addition hypoxiainducible factor 1α hif1α and vascular endothelial growth factor vegf are activated by cthrc1 through activating the pi3kaktmammalian target of rapamycin mtor signaling pathwaywhich promotesis morecthrc1 enhances colony formation migration and invasion of hepatocellular carcinoma cells by downregulatingtumor suppressor p53 and stimulating invasionassociatedfactor mmp9 tumor angiogenesis whatstudies on myocardial infarction mi have also foundthat activation of infarct repair cardiac fibroblasts ircfinvolves cthrc1 expression and pi3kakt signaling pathway in ovarian cancer cells gene silencing cthrc1 doesnot alter mmp9 expression or phosphorylate mek theinvasionpromoting eï¬ect of cthrc1 on eoc cells dependson downstream pi3kakt and erk12 signaling dominatedby egfr besides the invasion and metastasis of endometrial cancer are closely related to the upregulation ofcthrc1mediated cx3cr1 in macrophages this processregulates the integrin 3pi3kakt pathway which alsopromotes the recruitment of m2like macrophages cthrc1 activates hifα pathway and contributes totumor angiogenesis as mentioned above cthrc1 in hepatocellular carcinoma can induce hif1α to promote tumorangiogenesis and regulate downstream mmps and tumorsuppressor gene p53 by activating the pi3kakt signalingpathway in human squamous cell carcinoma hif1αoverexpression stimulates vegfc upregulation and induceslymphangiogenesis and tumor cell invasion ding pinpointed that cthrc1 and hif1α were upregulated inthe nucleus of cthrc1 overexpressed gc cells hif1αinhibitors reduced cthrc1induced cxcr4 expressionfurthermore it was found that inhibition of hif1α expression and inhibition of cxcl12cxcr4 signals all alleviatetumor cell migration and invasion therefore cthrc1 canparticipate in tumor cell migration and invasion throughhif1αcxcr4 signals in gc in short cthrc1 canaï¬ect the expression of hif1α which is not only related tolymphangiogenesis but also closely related to tumor progression and invasion a novel signaling pathway the potential role of pkcδerk in tumors protein kinase c δ pkcδ has beenimplicated in various epithelial tumors such as prostatebreast and crc [ ] activated pkcδ causes angiogenesis and tumor growth of prostate tumors by increasingnadph oxidase activity and hif1α expression levels pkcδ can also inhibit the wntcatenin pathwayin colon cancer cells however a recent study illustrated that mek and pkcδ inhibitors could blockcthrc1induced erk phosphorylation and that pkcδphosphorylation was not inhibited by mek inhibition surprisingly inhibition of plc a membraneassociated enzymethat activates pkcδ to promote bone formation in noncanonical wnt signals did not inhibit cthrc1induced alkaline phosphatase alp activity therefore waif1 bound bycthrc1 activates pkcδ and erk to stimulate osteoblastdiï¬erentiation which is a novel signaling pathway unrelatedto the noncanonical wnt pathway therefore pkcδsignal may explain the role of cthrc1 in tumor progressions such as angiogenesis and bone metastasisto put it briefly cthrc1 may be involved in manyother signaling pathways including mirna and lncrnawhich interact with or crosstalk with the tgf wnt andintegrin erk pathways and jointly participate in tumordevelopment and metastasis see table conclusiontumor development and metastasis a complex processinvolving cell adhesion and proteolytic degradation of theecm depends not only on the cancer cells but also on theinteraction between the cancer cells and their microenvironment complementary dna microarray analysis also demonstrated that the cthrc1 gene is expressed in mosthuman solid cancers as we all know cthrc1 is a secretedecm protein which can inhibit collagen deposition and participate in tumor invasion and metastasis even thoughcthrc1 was first discovered more than a decad
0
" micrornas mirnas have been reported to have important regulatory roles in the progression of several types of cancer including cervical cancer cc however the biological roles and regulatory mechanisms of mirnas in cc remain to be fully elucidated the aim of the present study was to examine the functions of mirnas in cc and the possible mechanisms using a microarray it was identified that mirna15a5p mir15a5p was one of the most downregulated mirnas in cc tissues compared with adjacent noncancerous tissues the low expression of mir15a5p was observed in cc tumor tissues with distant metastasis and in cc cell lines in addition the effects of mir15a5p upregulation on cell viability apoptosis invasion and migration of cc cells were investigated using cck‘ flow cytometry transwell and wound healing assays respectively it was demonstrated that upregulation of mir‘15a‘5p significantly suppressed the viability migration and invasion and promoted the apoptosis of siha and c33a cells furthermore yesassociated protein yap1 a well‘known oncogene was confirmed to be directly targeted by mir15a5p and was found to be negatively regulated by mir15a5p further correlation analysis indicated that mir15a5p expression was negatively correlated with yap1 expression in cc tissues notably overexpression of yap1 abrogated the tumor suppressive effects of mir15a5p in cc cells taken together these present findings indicated that the mir15a5pyap1 axis may provide a novel strategy for the clinical treatment of cccorrespondence to professor xu chen department of obstetrics and gynaecology huashan hospital north fudan university jingpohu road baoshan shanghai pr chinaemail xuchenccx163comcontributed equallykey words cervical cancer microrna15a5p cell viability migration invasion yesassociated protein introductioncervical cancer cc is a type of malignant tumor commonly presenting in women in cc cases are diagnosed each year and it accounts for of all female cancerassociated mortalities each year worldwide despite advances in the therapeutic strategies for cc including targeted therapies and immunotherapy the prognosis of cc remains poor due to the abnormal growth of epithelial cells thus it is imperative to clarify the molecular interactions occurring during the initiation and progression of ccmicrornas mirnas are a family of short noncoding rnas with an average length of nucleotides which negatively regulate target gene expression through either translation repression or rna degradation accumulating evidence has indicated that mirnas may function as oncogenes or tumor suppressors depending on their target mrna in various types of cancer including cc for example yang reported that mir214 inhibits the growth of cc cells by the regulation of its target enhancer of zeste homolog dong demonstrated a suppressive role of mir217 in the development of cc cells via targeting rhoassociated protein kinase chen reported that mir499a promotes the proliferation cell cycle progression colony formation migration and invasion of cc cells by targeting srybox transcription factor in addition several mirnas serve as diagnostic biomarkers in patients with cc such as mir152 and mir365 despite the aforementioned findings the roles of mirnas in the development of cc require further investigationin the present study a mirna microarray was performed to investigate the expression profiles of mirnas in cc tissues and the most downregulated mirna identified mir‘15a‘5p was selected for further analysis the potential role and underlying mechanism of mir15a5p in cc cells were also investigated the present results suggest that mir15a5p may serve as a therapeutic target for ccmaterials and methodspatients and samples in total paired cervical samples tumor tissues and adjacent noncancerous tissues were 0cchen mir15a inhibits cervical cancer cell growthobtained from female patients with cc who underwent cervical surgical resection without preoperative systemic therapy at the department of obstetrics and gynecology huashan hospital north of fudan university shanghai china between may and december the median age of the patients was years range years among all patients there were patients with metastatic cc and with nonmetastatic cc the matched nontumor adjacent tissue was obtained cm beyond the boundary of cc tissue all tissue samples were immediately snapfrozen in liquid nitrogen and stored at ‘Ëšc until use the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university written informed consent for participation in the study was obtained from all patientsmirna expression profiling total rna from cc tissues three randomly selected paired tumor tissues and adjacent noncancerous tissues was extracted using mirneasy mini kit qiagen gmbh the samples were assessed using the mircury lna„¢ array v180 agilent technologies inc the procedure and imaging processes were performed as described previously cell culture human cc cell lines hela c33a caski and siha 293t cells and normal cervical epithelial cells ect1e6e7 were obtained from the american type culture collection all cells were cultured in dmem sigmaaldrich merck kgaa supplemented with vv fbs sigmaaldrich merck kgaa plus uml penicillinstreptomycin at ˚c with co2reverse transcription‘quantitative pcr rt‘qpcr total rna was extracted from tissues or cell lines using trizol reagent invitrogen thermo fisher scientific inc for mirna rt cdna was generated from ng total rna samples using taqman„¢ microrna reverse transcription kit applied biosystems thermo fisher scientific inc at ˚c for min for mrna rt cdna was synthesized using primescript rt reagent kit takara bio inc at ˚c for min qpcr for mirna and mrna was performed using the sybrgreen i realtime pcr kit applied biosystems thermo fisher scientific inc on an abi system applied biosystems thermo fisher scientific inc the reaction was performed under the following conditions ˚c for min followed by cycles at ˚c for sec and ˚c for sec and a final extension at ˚c for sec the primers for qpcr analysis were as follows mir15a5p forward 'aat gtt gcc cgt aat gcc3' and reverse 'ccc aag cgg aga aag gaa3' u6 forward 'gct tcg gca gca cat ata cta aaa t3' and reverse 'cgc ttc acg aat ttg cgt gtc at3' yesassociated protein yap1 forward 'cgg tcc act tca gtc tcc3' and reverse 'gag tgt ggt gga cag gta ctg3' and gapdh forward 'gtg gtg aag acg cca gtg ga3' and reverse 'cga gcc aca tcg ctc aga ca3' the expression levels of mir15a5p and yap1 were normalized to the expression of u6 and gapdh respectively the relative expression of each gene was calculated using the ‘ˆ†ˆ†cq method cell transfection the mir15a5p mimic mimic negative control nc mir15a5p inhibitor inhibitor nc yap1 overexpression plasmid pcdnayap1 and pcdnavector were all provided by guangzhou ribobio co ltd when c33a and siha cells 5x105 cellswell in 6well plates grew to confluence mir‘15a‘5p mimic nm mimic nc nm mir15a5p inhibitor nm inhibitor nc nm pcdnayap1 µg or pcdnavector µg were transfected into cells at ˚c for h using lipofectamine® invitrogen thermo fisher scientific inc the sequences were as follows mir15a5p mimic 'uag cag cac aua aug guu ugu g3' mimic nc 'uuc ucc gaa cgu guc acg utt3' mir15a5p inhibitor 'cac aaa cca uua ugu gcu gcu a3' and inhibitor nc 'cag uac uuu ugu gua gua caa3'in addition small interfering rna targeting yap1 si‘yap1 and the negative control targeting a non‘specific sequence siscramble were provided by thermo fisher scientific inc siha and c‘33a cells were transfected with the sirnas nmoll using lipofectamine invitrogen thermo fisher scientific inc the sequences of si‘yap1 and siscramble were as follows siyap1 'ctc agg atg gag aaa ttt a3' and siscramble 'ttc tcc gaa cgt gtc acg t3' at h posttransfection the cells were harvested for further analysis and the inhibition efficiency was determined by western blottingcell viability the c33a and siha cells were seeded in 96well plates at a density of 5x103well overnight following transfection the cell viability was measured using a cck8 assay briefly µl cck‘ solution was added to each well and cultured for h at ˚c the absorbance of the samples at nm was detected using a microplate reader biorad laboratories inccaspase‘ activity following transfection c33a and siha cells were harvested and the caspase3 activity was measured using a caspase3 activity assay kit beyotime institute of biotechnology according to the manufacturer's protocolcell apoptosis the apoptosis of c33a and siha cells was examined using flow cytometry following transfection c33a and siha cells were collected and the apoptotic cells were identified using an annexin v‘fitc apoptosis detection kit abcam according to the manufacturer's protocol after washing with cold pbs the cells were resuspended in binding buffer followed by staining with annexin v and propidium iodide for min in the dark at room temperature the fluorescence was measured using a facscan flow cytometer beckman coulter inc and then analyzed by flowjo v871 software flowjo llcimmunofluorescence assay following transfection c33a and siha cells were fixed in absolute ethyl alcohol for min at room temperature after washing twice with pbs the fixed cells were stained with primary antibody targeting cleavedcaspase3 cat no c ell signaling technology inc for h at room temperature subsequently an antirabbit conjugated antibody with fitc cat no f0382 sigmaaldrich merck kgaa was added for h in the 0cinternational journal of molecular medicine dark fluorescence images were obtained using an inverted fluorescence microscope magnification x200cell invasion assays transwell chambers 8µm pore bd biosciences coated with matrigel bd biosciences were used for the invasion assay briefly c‘33a and siha cells 8x104 were seeded in the top chamber with serumfree medium while the lower chamber contained culture medium with fbs following incubation for h the cells were fixed in paraformaldehyde solution beyotime institute of biotechnology for min and stained with crystal violet beyotime institute of biotechnology for min at room temperature images were captured with an inverted microscope olympus corporation magnification x100wound healing assay for the wound healing assay c33a and siha cells were seeded onto 12well plates 2x105 cellswell and h after transfection a scratch was made using a 10µl pipette tip in the confluent cell monolayer then cells were washed twice with pbs and incubated in dmem without fbs the wound healing images were captured at and h after scratching using an inverted light microscope olympus corporation magnification x100 the wound healing rate was calculated using imagej software v146 national institutes of healthdual‘luciferase reporter assay mirna target prediction tools including miranda httpmirandaorguk and targetscan httptargetscanorg were used to search for the putative targets of mir15a5p pgl3yap1 widetype or pgl3yap1 mutant type pgl3yap1mut promega corporation were cotransfected with mir15a5p mimics into 293t cells in 24well plates 2x105well using lipofectamine invitrogen thermo fisher scientific inc at h post‘transfection the luciferase activities were analyzed using the dualluciferase reporter assay system promega corporation with renilla luciferase activity as an internal control western blot analysis western blotting was performed as previously described briefly cells were lysed using radio immunoprecipitation assay buffer beyotime institute of biotechnology and the protein concentration was determined using the bicinchoninic acid assay total protein µglane was separated by sdspage and electrophoretically transferred onto a polyvinylidene difluoride membrane emd millipore subsequently membranes were blocked with skim milk for h at ˚c overnight each membrane was probed with primary antibodies against yap1 cat no and β‘actin cat no at ˚c overnight all primary antibodies were obtained from cell signaling technology inc subsequently the membrane was incubated with horseradish peroxidaseconjugated goat antirabbit igg cat no abcam at room temperature for h βactin served as the loading control and for normalization of protein expression the protein bands were developed using ecl kit ge healthcare and expression levels were quantified using imagej v146 national institutes of healthstatistical analysis all data are presented as mean ± standard deviation the correlation between mir15a5p and yap1 levels was evaluated using spearman's correlation analysis pairwise comparisons were performed by student's ttest and comparisons among groups were analyzed by oneway anova followed by tukey's posthoc test p005 was considered to indicate a statistically significant differenceresultsmir‘15a‘5p is downregulated in cc to examine the potential involvement of mirnas in the development of cc microarray analysis was performed to evaluate the mirna expression profiles between cc tissues and adjacent noncancerous tissues of differently expressed mirnas identified in the tumor group mirnas exhibited decreased expression and mirnas demonstrated increased expression compared with that in adjacent noncancerous tissues fig 1a among the aberrant mirnas the present study focused on mir15a5p for subsequent experiments due to its suppressive role in a variety of other cancer types such as endometrial cancer and chronic myeloid leukemia subsequently rtqpcr was performed to detect the expression of mir15a5p in pairs of tumor tissues and adjacent noncancerous tissues the results revealed that the level of mir15a5p was significantly lower in tumor tissues compared with that in adjacent noncancerous tissues fig 1b it was also observed that mir15a5p was expressed at a significantly lower level in tumor tissues with distant metastasis compared with in tumors tissues without distant metastasis fig 1c indicating that mir15a5p downregulation is associated with cc metastasis in addition rtqpcr was used to examine the mir15a5p level in four cc cell lines hela c33a caski and siha and the normal cervical epithelial cell line ect1e6e7 which was used as a control as expected mir‘15a‘5p was significantly lower in the four cc cell lines compared with ect1e6e7 cells fig 1d siha and c33a cells were selected for further experiments as they demonstrated the lowest expression of mir15a5p among all cell lines examinedupregulation of mir‘15a‘5p inhibits cell viability and promotes cell apoptosis in an attempt to understand the biological function of mir15a5p mir15a5p expression was upregulated or downregulated in the cultured siha and c33a cells by transfection with mir15a5p mimic or inhibitor respectively mir‘15a‘5p expression was significantly increased after mir15a5p mimic transfection whereas it was significantly decreased following mir‘15a‘5p inhibitor transfection in both siha and c33a cells fig 2a the present study then investigated the effect of mir15a5p expression on cell viability and the results demonstrated that the viability of siha and c‘33a cells was significantly inhibited by overexpression of mir15a5p whereas it was significantly enhanced by knockdown of mir‘15a‘5p compared with the negative control group fig 2b and c to assess the effects of mir15a5p upregulation on the apoptosis of siha and c33a cells caspase3 expression level and activity were analyzed by immunofluorescence and caspase‘ activity assays respectively as presented in fig 2d and e the expression of cleaved caspase3 and caspase3 activity was increased in siha and c33a cells transfected with 0cchen mir15a inhibits cervical cancer cell growthfigure mir‘15a‘5p is downregulated in cc tissues and cell lines a heat map presents significant differentially expressed mirnas in cc tissues and matched adjacent noncancerous tissues n3 green indicates downregulation and red indicates upregulation b mir15a5p expression was measured by rtqpcr in pairs of cc tissues and matched adjacent noncancerous tissues c mir15a5p expression was measured in tumor tissues with distant metastasis and tumors tissues without distant metastasis by rtqpcr d mir15a5p expression was detected in four cervical cancer cell lines hela c33a caski and siha and the normal cervical epithelial cells ect1e6e7 data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs ect1e6e7 cells mir microrna cc cervical cancer rtqpcr reverse transcriptionquantitative pcr mir15a5p mimic compared with the mimic nc groups furthermore the results of flow cytometry demonstrated that the extent of apoptosis was significantly increased after mir15a5p mimic transfection compared with the mimic nc groups fig 2f taken together these results indicate that overexpression of mir15a5p inhibits cell viability by inducing cell apoptosisupregulation of mir‘15a‘5p inhibits the invasion and migra‘tion of cc cells the present study further investigated whether overexpression of mir15a5p could reduce the invasiveness and migratory potential of cc cells using a transwell assay it was identified that the invasive capacities of siha and c‘33a cells were significantly inhibited by mir‘15a‘5p mimic whereas they were increased by mir15a5p inhibitor compared with the nc groups furthermore the wound healing assay results also demonstrated a significant reduction of cell migration in siha and c33a cells following mir15a5p overexpression however the migration of siha and c‘33a cells was significantly enhanced by mir15a5p inhibition fig 3c and d collectively the present data suggest that overexpression of mir15a5p suppresses the invasive and migratory abilities of cc cellsyap1 is a direct target of mir‘15a‘5p using the targetscan and miranda algorithms yap1 was found to have a putative target site of mir15a5p in its 'utr fig 4a to validate the possibility that yap1 is a direct target gene of mir15a5p a luciferase reporter assay was then performed the data revealed that mir‘15a‘5p mimic significantly inhibited the luciferase activity in the constructs containing the wildtype 0cinternational journal of molecular medicine figure overexpression of mir15a5p suppresses cell viability and promotes cell apoptosis siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis a transfection efficiency was assessed by reverse transcription‘quantitative pcr cell viability was measured by cck8 assay at indicated times for b siha and c c33a cells d the expression of cleaved caspase3 was determined by immunofluorescence assay magnification x200 e the caspase‘ activity was detected by a commercial caspase‘ activity kit f cell apoptosis was measured by flow cytometry data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs mimic nc p005 p001 vs inhibitor nc mir microrna nc negative control od optical density pi propidium iodidebinding site of yap13'utr while it had no evident effects on the activity of yap13'utrmut by contrast mir15a5p inhibitor significantly increased luciferase activity without any evident effects on yap13'utrmut activity fig 4b subsequently to further detect the potential regulation of yap1 by mir15a5p the expression of yap1 protein was measured in cc cells by western blotting as presented in fig 4c the expression of yap1 was significantly decreased upon ectopic expression of mir15a5p suggesting that high expression of yap1 was partly due to the downregulation of mir15a5p in cc cells in addition it was identified that the mrna level of yap1 was significantly increased in cervical cancer compared with the control and inversely correlated with mir15a5p expression levels in cancer tissues fig 4d and e these results indicated that yap1 is a downstream gene of mir15a5p in cc 0cchen mir15a inhibits cervical cancer cell growthfigure overexpression of mir15a5p suppresses cell invasion and migration siha and c33a cells were transfected with the mir15a5p mimic or inhibitor for h and then cells were used for analysis invasion of a siha and b c‘33a cells was measured by a transwell assay magnification x200 the migration of c siha and d c33a cells was assessed by a wound healing assay the images were taken at and h after gaps were generated wound healing was quantified by the distance of the wounded region with an absence of cells data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs mimics nc p001 vs inhibitor nc mir microrna nc negative controlyap1 inhibition suppresses cell viability promotes cell apop‘tosis and inhibits invasion and migration previous evidence has shown that yap1 exerts an oncogenic function in several types of human cancer such as breast and lung cancer as the findings of the present study revealed that yap1 is upregulated in cc it was hypothesized that yap1 may act as an oncogenic gene in cc to confirm this hypothesis siha and c33a cells were transfected with siyap1 or siscramble western blot assay revealed that yap1 was notably downregulated following transfection with siyap1 fig 5a functionally yap1‘knockdown significantly suppressed the cell viability and induced cell apoptosis compared with the siscramble group fig 5b and c furthermore knockdown of yap1 significantly suppressed the invasive and migratory abilities of siha and c33a cells fig 5d and e suggesting that yap1 may play an oncogene role in the development of ccoverexpression of yap1 moderates the negative functions of mir‘15a‘5p on cell viability migration and invasion to ascertain whether yap1 is involved in the inhibitory effects of mir15a5p on cc cells the present study cotransfected pcdnayap1 andor mir15a5p mimic as well as their controls into siha and c33a cells the overexpression efficiency was verified by western blotting as shown in fig 6a yap1 was notably increased in siha and c33a cells after pcdna‘yap1 transfection subsequently the cell viability apoptosis invasion and migration were evaluated overexpression of yap1 significantly abolished the inhibitory effects of mir15a5p upregulation on the viability of siha and c33a cells fig 6b the increased apoptosis induced by mir15a5p overexpression was also reversed by overexpression of yap1 fig 6c furthermore overexpression of yap1 significantly reversed the inhibitory effects of mir‘15a‘5p on cell invasion and migration fig 6d and e in addition it was identified that overexpression of yap1 alone significantly promoted cc cell viability inhibited cell apoptosis and enhanced the invasion and migration compared with blank control group suggesting the oncogenic role of yap1 in cc cells these results indicate that mir15a5p exerts its tumor suppressive role in cc at least partially through yap1 0cinternational journal of molecular medicine figure yap1 is a direct target of mir15a5p a schematic of the yap1 'utr containing the mir15a5p binding sites b luciferase assay of 293t cells co‘transfected with firefly luciferase constructs containing the yap1 wt or mut '‘utrs and mir‘15a‘5p mimics mimics nc mir‘15a‘5p inhibitor or inhibitor nc as indicated n3 p001 c siha and c33a cells were transfected with the mir15a5p mimic and mimic nc for h and the expression levels of yap1 protein were determined by western blotting p001 vs mimic nc d yap1 expression was measured by reverse transcriptionquantitative pcr in cc tissues and matched adjacent noncancerous tissues n40 p001 e spearman's analysis was used to analyze the correlation between the expression of yap1 and the expression of mir15a5p expression in cervical cancer tissues r p001 data are expressed at the mean ± standard deviation n3 of one representative experiment yap1 yesassociated protein mir microrna 'utr 'untranslated region wt wildtype mut mutant nc negative controldiscussionin the present study mir15a5p was shown to be decreased in cc tissues and cell lines and associated with cc metastasis furthermore overexpression of mir15a5p inhibited the cc cell viability invasion and migration and promoted cell apoptosis while inhibition of mir15a5p demonstrated the opposite effects additionally yap1 was confirmed as a functional target of mir15a5p ectopic expression of which significantly reversed suppression of mir‘15a‘5p the present data indicated that mir15a5p may function as a tumor suppressor in cc progression by inhibiting yap1 expressiona number of studies have shown that mirnas participate in the development of cc for example xia reported that mir374b overexpression suppresses cell proliferative and invasive abilities via affecting forkhead box m1 expression yao also demonstrated that mir641 upregulation restricts cc cell growth in vitro and in vivo xu reported that mir2185p suppresses the progression of cc via the lynnfκb signaling pathway yuan demonstrated that overexpression of mir138 suppresses cc cell growth in vivo these findings suggest that targeting mirnas may be an effective therapeutic strategy for cc in the present study based on microarray expression data it was identified that mir15a5p is one of the most markedly downregulated mirnas in cc tissues notably previous studies have reported that mir15a5p functions as a tumor suppressor in several human cancer types although mir15a5p has been found to be downregulated in cc to the best of our knowledge the tumorigenic role and mechanism remain unknown therefore the present study focused on mir15a5p in cc for molecular analyses in the 0cchen mir15a inhibits cervical cancer cell growthfigure yap1 inhibition suppresses cell viability promotes cell apoptosis and inhibits invasion and migration siha and c33a cells were transfected with siyap1 or siscramble and then cells were harvested for further study a the expression of yap1 was measured by western blotting b cell viability was measured by cck‘ assay c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration assessed by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p001 vs siscramble yap1 yesassociated protein mir microrna si small interfering rnafigure mir15a5p inhibits cell viability and induces cell apoptosis by targeting yap1 a siha and c33a cells were transfected with the pcdnayap1 plasmid for h and then the protein expression of yap1 was measured by western blotting subsequently siha and c33a cells were cotransfected with the pcdnayap1 plasmid and mir15a5p mimic for h and then cells were used for analysis b viability of siha and c33a cells was measured by cck8 assay at indicated times c the cell apoptosis was assessed by flow cytometry d cell invasion was measured by transwell assay e cell migration was measured by a wound healing assay data are expressed at the mean ± standard deviation n3 of one representative experiment p005 p001 vs blank group p001 mir microrna yap1 yesassociated protein microarray expression data the expression levels of numerous mirnas exhibited significant changes such as mir137 which demonstrated the most significant upregulation in cc tissues miao reported that mir137 upregulation inhibits cc cell invasion migration and epithelialmesenchymal transition by suppressing the tgfβsmad pathway 0cinternational journal of molecular medicine notably mir15a3p has also reported to exhibit differential expression and induce apoptosis in human cc cells although the present study did not detect the expression change of mir15a3p in the microarray expression data the expression of mir15a3p in four cc cell lines was examined and the results demonstrated that mir15a3p was also downregulated in cc cells compared with ect1e6e7 cells data not shown however the role and regulatory mechanisms of mir15a3p on invasion and migration remain unclear the function of more mirnas in cc will be investigated in the futureprevious studies have reported that mir15a5p has the potential to suppress cell growth and inhibit the progression of human cancers by regulating its downstream target genes for example luo demonstrated that overexpression of mir15a5p causes cellular growth inhibition and suppression of migration by targeting cyclin e1 in breast cancer wu and guo found that mir15a overexpression suppressed the cell proliferation and invasion by suppression of bmi1 translation in gastric cancer gc as well as pancreatic cancer pc of note several studies have reported aberrant expression of mir15a5p in cc tissues or cells however the role and mechanism of mir15a5p in cc remain largely unknown the present results demonstrated that overexpression of mir15a5p inhibited cell viability cell migration and invasion and induced cell apoptosis in siha and c33a cells while inhibition of mir15a5p demonstrated the opposite effects indicating that mir15a5p may serve as tumor suppressive role in cc yap1 a transcriptional coactivator and oncogene has been found to play an important role in different types of carcinoma for example liu reported that yap1 overexpression promotes the invasion migration and growth of colon cancer cells yu demonstrated that knockdown of yap1 causes a significant inhibition of the growth and migration of renal cell carcinoma cells in vitro and in vivo notably yap1 has been verified to target mir‘15a‘5p to suppress cell growth and metastasis in gastric adenocarcinoma and colon cancer however whether yap1 is a target of mir15a5p in cc remains unclear in the present study yap1 was confirmed to be a target of mir‘15a‘5p and its protein expression levels were negatively regulated by mir15a5p further investigation indicated that yap1 was significantly increased in cc tissues and inversely correlated with mir15a5p in cc tissues furthermore yap1 was confirmed to act as an oncogene gene in cc cells and its overexpression partly abrogated the inhibitory effect induced by enhanced expression of mir15a5p in cc cells taken together the present study demonstrates that mir15a5p exerts its tumor suppressive role in cc cells by targeting yap1due to the limitation in experimental conditions and funds further research in the future is required to investigate whether mir15a5p serves its role via other downstream targets in addition the present study investigated the cellular function of mir15a5p and its underlying mechanism in cc however in vivo studies and clinical trial data are required to validate the preliminary in vitro results obtained therefore the function of mir15a5p in cc needs to be further investigated in vivoin conclusion the present results demonstrated that mir15a5p suppresses the viability migration and invasion of cc cells by directly targeting yap1 based on these findings it is proposed that the mir15a5pyap1 axis may serve as a novel biomarker for new targets in cc therapyacknowledgementsnot applicablefundingfunding was received from the scientific research project of shanghai science and technology commission grant nos and availability of data and materialsthe datasets used andor analysed during the current study are available from the corresponding author on reasonable request authors' contributionsrc hl tz xy and sx performed the experiments contributed to data analysis and wrote the paper rc hl tz xy and sx analysed the data xc conceptualized the study design and contributed to data analysis and experimental materials all authors read and approved the final manuscriptethics approval and consent to participateall individuals provided written informed consent for the use of human specimens for clinical research the experimental protocols were approved by the ethics committee of huashan hospital north of fudan university patient consent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interests references alldredge jk and tewari ks clinical trials of antiangiogenesis therapy in recurrentpersistent and metastatic cervical cancer oncologist tsikouras p zervoudis s manav b tomara e iatrakis g romanidis c bothou a and galazios g cervical cancer screening diagnosis and staging j buon fang j zhang h and jin s epigenetics and cervical cancer from pathogenesis to therapy tumour biol wang j liu y wang x li j we j wang y song w and zhang z mir1266 promotes cell proliferation migration and invasion in cervical cancer by targeting dab2ip biochim biophys acta mol basis dis zhu l zhu l s
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" phytolaccaceae species in china are not only ornamental plants but also perennial herbs that areclosely related to human health however both largescale fulllength cdna sequencing and reference genevalidation of phytolaccaceae members are still lacking therefore singlemolecule realtime sequencing technologywas employed to generate fulllength transcriptome in invasive phytolacca americana and noninvasive exotic picosandra based on the transcriptome data rtqpcr was employed to evaluate the gene expression stability in thetwo plant species and another indigenous congener p acinosaresults total of gb and gb clean reads of p americana and p icosandra were generated including and full length nonchimeric flnc reads respectively transcript clustering analysis of flnc readsidentified and consensus isoforms including and highquality ones after removingredundant reads and transcripts were obtained based on structure analysis total and alternative splicing variants and simple sequence repeats ssr as well as and completecoding sequences were detected separately furthermore and lncrna were predicted and and transcripts were annotated respectively subsequently seven reference genes in the two plant species and anative species p acinosa were selected and evaluated by rtqpcr for gene expression analysis when tested indifferent tissues leaves stems roots and flowers 18s rrna showed the highest stability in p americana whetherinfested by spodoptera litura or not ef2 had the most stable expression in p icosandra while ef1α was the mostappropriate one when attacked by s litura ef1α showed the highest stability in pacinosa whereas gapdh wasrecommended when infested by s litura moreover ef1α was the most stable one among the three plant specieswhenever germinating seeds or flowers only were consideredcontinued on next page correspondence yiwangynueducn1yunnan key laboratory of plant reproductive adaption and evolutionaryecology yunnan university kunming china2laboratory of ecology and evolutionary biology state key laboratory forconservation and utilization of bioresources in yunnan yunnan universitykunming chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cliu bmc plant biology page of continued from previous page fulllength transcriptome of p americana and p icosandra were produced individually based on thetranscriptome data the expression stability of seven candidate reference genes under different experimentalconditions was evaluated these results would facilitate further exploration of functional and comparative genomicstudies in phytolaccaceae and provide insights into invasion success of p americanakeywords phytolaccaceae smrt sequencing fulllength transcriptome analysis reference gene evaluation rtqpcr phytolacca americana is a member of the phytolaccaceae family and is native to northeast america becauseof its ornamental and medicinal applications it was introduced into china in unfortunately it hasevolved into an invasive species and spread to mostareas of the country especially in central and southernchina compared to noninvasive exotic congener p icosandra and native congener p acinosa p americana isof interest because it exhibits multiple biological activities such as plant pesticides antimicrobial propertyheavy metal accumulation capacity [“]in order to investigate the mechanisms of various bioactivities of p americana further transcriptomewidestudy is necessary to facilitate reports have showed thatjasmonic acidinduced and cadmiumtreated transcriptome data of p americana have been obtained by illumina hiseq and illumina hiseq platformrespectively [ ] howeverthese data were bothachieved by second generation sequencing sgs whichcould not produce fulllength transcripts genomic dataof p americana was available at the sra under projectprjna544344 but it™s 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 it™s 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 chen™s 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 what™s 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 what™s 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 [ ] what™s 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 ˆ’ °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 ˆ’ °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 manufacturer™s instructionsthe rna quality and purity were measured by using ananophotometer n60 implen germany and the agilent bioanalyzer system agilent technologies causa samples only with a ratio of to a ratio between and and a rin value morethan were chosen for the sequencing library construction an equal amount of total rnas from four different tissues of the same plant species were mixed asone sample for fulllength transcriptome sequencingtotal rnas from the samples collected for referencegene evaluation was also extracted individually as described above for each sample cdna was prepared byusing μg of total rna following the recommendedinstructions of fastquant rt kit with gdnase tiangenbiotech co ltd beijing chinapacbio cdna library preparation and smrt sequencingfulllength cdna was synthesized by using the smarter„¢ pcr cdna synthesis kit clontech ca usathe generated cdna was then reamplified using pcrafter end repairing smrt adaptor with a hairpin loopstructure was ligated to the cdna via exonucleasedigesting the cdna library was constructed after quality measurement of the cdna library smrt sequencingwas performed using the pacific bioscience sequel platform following the provided protocolillumina cdna library construction and secondgenarationsequencingthe extracted mrna was purified using oligo dtattached magnetic beads fragmentation was conducted inthe nebnext first strand synthesis reaction bufferfirststrand cdna was acquired based on the randomhexamers and then the secondstrand cdna was synthesized with dntps rnase h and primestar gxldna polymerase the synthesized cdna was purifiedwith ampure xp beads after end repairing adding polya and adaptor ligation ampure xp beads were used forsize selection the generated cdna was then amplifiedfor building cdna libraries the qualified libraries werepair end sequenced on illumina nova platformquality filtering and error correction of long readsraw smrt sequencing reads were filtered by removingpolymerase reads less than bp and sequence accuracyless than after removing adaptor subreads were obtained clean data was produced with subreads morethan bp ccss were produced from clean data withparameters of full passes and accuracy over after examining the coexistence of ² and ² adaptorsand poly a tail fulllength transcripts were selectedduring the processes of library preparation the chimericsequences formed by the direct linkage of two cdnatemplate strands due to the low concentrations ofadaptor or smrtbell are called artificial chimeric sequences the nonchimeric sequences in the fulllength 0cliu bmc plant biology page of transcripts are the fulllength nonchimeric flncsequencesas smrt sequencing generates a high error rate it isnecessary to perform error correction iterative clustering was used first to obtain consensus isoforms and thefulllength consensus sequences from iterative clusteringfor error correction were refined using quiver [ ]moreover the raw illumina sgs reads were filtered toremove adaptor sequences and low quality reads anderror correction of lowquality isoforms was conductedusing the sgs reads with the software proovread inbriefly the short reads of illumina rnaseq data weremapped to the low quality isoforms and then the basein the low quality isoform was replaced by the particularbase that had the maximum number
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tetrastigma hemsleyanum diels et gilg t hemsleyanum mostly known as œsan ye qing is a kind of folk plant because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource it mainly grows in the eastern central southern and south western provinces of china such as zhejiang jiangsu guangxi fujian and yunnan provinces peng and wang t hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models xu as an edible plant the leaves of t hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body sun while the aerial parts of t hemsleyanum developed as potential new traditional chinese medicine tcm preparations guo corresponding author ningbo research institute of zhejiang university ningbo zhejiang people™s republic of china email address px4142163com x peng 101016jjep2020113247 received may received in revised form july accepted august ofethnopharmacology2642021113247availableonline12august2020037887412020elsevierbvallrightsreserved 0ct ji abbreviations t hemsleyanum tetrastigma hemsleyanum diels et gilg tcm uplcesiqtofmsms ultra high performance liquid traditional chinese medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκb 5hydroxytryptamine norepinephrine dopamine prostaglandin e2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin mic gsh mda nfκb 5ht ne da pge2 mapk mitogenactivated protein kinase lps celegans caenorhabditis elegans tnfα il1 il6 il12p40 interleukin subunit p40 stnfr1 soluble tnf receptors il10 il1 il4 inos tlr4 md2 myd88 myeloid differentiation protein jnk gpt got alp sod interleukin interleukin interleukin inducible no synthase tolllike receptor myeloid differentiation factor2 cjun nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory the root tubers of t hemsleyanum are extensively used either alone or in combination with other herbal medicines in tcm clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain sun chen and guo therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal in february t hemsleyanum was awarded as the new œeight famous kinds of tcm in zhejiang province meant that it has become a key object of industrialization development of zhejiang™s dominant large varieties of medicinal materials in covid19 broke out and has caused more than deaths in china and infection cases have been reported in more than countries hua shi xuan fei mixture approval number of zhejiang medicine z20200026000 which composed of t hemsleyanum has been approved by zhejiang provincial drug administration for clinical treatment of covid19 futhermore the modern pharmacological studies had shown that t hemsleyanum also had effects of antiinflammatory ji antioxidant hossain antivirus ding antitumor lin antipyretic yang and wang antihepatic injury ma et al immunomodulatory xu antibacterial chen hypoglycemic ru 2018ab etc numerous reports have demonstrated that the biological activities of t hemsleyanum are attributed to its many chemical components fu wang has reported isolated alkaloids from the aerial parts of t hemsleyanum wang ru extracted a novel polysaccharide tdgp3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin a secretory immunoglobulin a epithelialmesenchymal transition alt ast ha ln tbili tp ifnÎ iga siga emt mmps matrix metalloproteinase timps matrixmetallo proteinase cytc cat gshpx glutathione peroxidase tregs tgf cox2 foxp3 pdl taoc ccl4 cef hvj vsv a f s1 s2 pef cff eaf baf cytochrome c catalase regulatory t cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast hemagglutinating virus of japan vesicular stomatitis virus alkalicontaining extract of t hemsleyanum ketonecontaining extract of t hemsleyanum crude extract of t hemsleyanum crude extract of t hemsleyanum petroleum ether extractions of t hemsleyanum ethanol extract chloroform extractions of t hemsleyanum ethanol extract ethyl acetate extractions of t hemsleyanum ethanol extract nbutanol extractions of t hemsleyanum ethanol extract t hemsleyanum with a molecular weight of — da by enzymolysisultrasonic assisted extraction method ru 2019ab large amounts of flavonoids were found in leaves aerial parts and root tubers of t hemsleyanum xu 2014ab deng yu in addition t hemsleyanum also contains a variety of functional components such as anic acids hu phenolic acids liu minerals fan amino acids fu etc in recent years wild resources of t hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments in it was listed in the preferentially protected crop germplasm resources of zhejiang province based on our team™s preliminary research peng peng 2016ab li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of t hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization materials and methods the available information about the traditional uses phytochemicals and pharmacological properties of t hemsleyanum was searched via web of science google scholar pubmed science direct china national knowledge infrastructure cnki and springer search using chinese or english as the retrieval languages the keywords used include t hemsleyanum root tubers of t hemsleyanum radix tetrastigma ofethnopharmacology26420211132472 0ct ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words all references were from experimental studies and published prior to april were reviewed all chemical structures were drawn using chemdraw pro software heatclearing were botanical characteristics t hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose it is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus the optimum ph is between and the root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter fig the epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section the stem of t hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate the leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base the flowers of t hemsleyanum are small yellow green and ovate the flowering stage of t hemsleyanum ranges from april to june and the fruit phase is normally from august to november when the flower withered it will form a small green round fruit with the size of millet when it is mature the fruit will turn from green to red the berries are spherical and soft spherical traditional uses t hemsleyanum belonging to the family vitaceae was firstly recorded in ben cao gang mu ming dynasty ad the aliases of sanyeqing include shi hou zi shi bao zi shi lao shu lan shan hu lei dan zi po shi zhu tu jing wan sou jia feng san ye dui golden wire hanging gourd golden bell golden wire hanging potato etc the root tubers or whole grass of t hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of tcm such as zhi wu ming shi tu kao qing dynasty wu jiangxi herbal medicine common folk herbal medicine in zhejiang all of these ancient works described the effects of toxicityremoving t dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women national compilation team of chinese herbal medicine in the tcm culture the properties of t hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional chinese medicine and zhong hua ben cao shanghai science and technology press the channel tropism was lung heart liver and kidney meridians decocting with water or mashing for external application are the traditional possess methods of t hemsleyanum considering its extensive traditional effects many prescriptions containing t hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies excitingly it has reported that jinlian disinfection drink containing san ye qing combined with interferon can treat covid19 he jinqi tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was wei moreover zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage iii primary liver cancer jiang and gong in addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites ji chemical compounds of themsleyanum the chemical constituents of t hemsleyanum have been widely investigated sun sun zeng xu 2014ab fu fan chen ding 2015a fig the aerial part a root tuber b and raw herb c of t hemsleyanum ofethnopharmacology26420211132473 0ct ji b ding a total of one hundred and fortytwo compounds have been isolated and identified from t hemsleyanum until now the information about compound name molecular weight compound formula detection method analysis sample is summarized in table flavonoids and their glycosides modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from t hemsleyanum lin zhang table to date fiftyone flavonoids and their glycosides have been extracted and identified from t hemsleyanum in this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on c3² and c4™ on the b ring of flavonoid aglycone at present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry uplcesiqtofms has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution our team used uplcesiqtofms to identify chemical constituents from the aerial part of t hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc sun according to the report liu total flavonoids of t hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase mapk and nuclear factorκb nfκb in lung tissue moreover the flavonoids of t hemsleyanum had the activity of antilung cancer wei luteolin a flavonoid found in t hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers muhammad it is certain that t hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs polysaccharide saccharide is another important active ingredient extracted from t hemsleyanum shao polysaccharide has great potential in clinical application because of its unique pharmacological activity however due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures guo table the prescriptions and traditional uses of t hemsleyanum in china prescriptions name qingteng fengshi qufengshi yaojiu main composition jiu traditional use t hemsleyanum parabarium chunianum tsiang zanthoxylum nitidum roxb dc t hemsleyanum deeringia amaranthoides lam merr blumea aromatica wall dc t hemsleyanum deeringia amaranthoides lam merr zanthoxylum nitidum roxb dc panax notoginseng burk fh chen t hemsleyanum gypsum lonicera japonica thunb houttuynia cordata thunb ophiopogon japonicus linn f kergawl t hemsleyanum t hemsleyanum lysimachia christinae hance imperata cylindrica citrus reticulata blanco t hemsleyanum ginsenoside astragalus propinquus schischkin t hemsleyanum nepeta cataria l lonicera japonica thunb saposhnikovia divaricata trucz schischk huatuo fengtongbao capsule sanyeqing gypsum decoction sanyeqing power zhonggan mixture jinqi tablet hua shi xuan fei mixture extracted the polysaccharides from roots of t hemsleyanum rtp1 rtp2 and rtp3 were successively found by protein precipitation and purification moreover further study indicated rtp31 was high purity polysaccharide with a molecular weight of kda and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively ru 2018ab extracted a polysaccharide thp from t hemsleyanum with the average molecular weight estimated as kda the results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of in ru 2019ab successfully extracted polysaccharide thdp3 from t hemsleyanum with molecular weight of kda which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of moreover tdgp3 mainly consists of †’4αdgalap1†’ †’4dgalp1†’ and †’4αdglcp1†’ residues as backbones and dmanp1†’ †’36dmanp1†’ and αdaraf1†’residues as branches phenolic acids phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring as a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects twenty three phenolic acids no52“ table have been reported in the aerial parts of t hemsleyanum such as caffeic acid chlorogenic acid 1ogalloyldglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid there were twentyone phenolic acids in the root tuber of t hemsleyanum some of which were the same as aerial parts alkaloids alkaloids are a group of basic anic compounds containing nitrogen that exist in nature alkaloids are stored in small quantities in t hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare wang fu extracted the aerial parts of t hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and treatment of joint pain wind cold dampness arthralgia treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture treatment of infantile hyperpyretic convulsion treatment of blood avalanche leucorrhea treatment of liver cancer treatment of malignant tumor treatment of covid19 usage oral administration “ ml once times a day oral administration ml once times a day oral administration capsules once times a day references ministerial standard ministerial standard ministerial standard one dose a day decoct twice in water and take it “ times after mixing oral administration oral administration ml once times a day oral administration capsules once times a day oral administration ml once times a day xu gao jiang and gong wei zhejiang provincial drug administration ofethnopharmacology26420211132474 0ct ji detection mode analysis parts of sample reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber sun sun zeng sun sun sun zeng zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun zeng sun zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun xu 2014b sun zeng sun zeng sun zeng zeng sun sun sun sun xu 2014b sun xu 2014b sun zeng sun xu 2014b sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun fu sun sun xu 2014b fan xu 2014b fan sun continued on next page uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms table chemical constituents isolated from the different parts of t hemsleyanum name flavonoids and their glycosides quercetin quercitrin quercetin3oglucoside quercetin3orutinoside quercetin3galactoside quercetin3oxylosylglucoside quercetin3oxylosylglucose7orhamnoside orientin orientin2²²orhamnoside isoorientin isoorientin2²²orhamnoside isoorientin cid0 ²²oxyloside vitexin vitexin2²²orhamnoside vitexin2²²oglucoside vitexin2²²oarabinoside isovitexin isovitexin2²²orhamnoside isovitexin2²²oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8cxylosyl6cglucoside apigenin6cαlarabinose8cdglucose eriodictyol eriodictyolohexoside i eriodictyolohexoside ii luteolin luteolin6 8dichexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3oneohesperidin kaempferol3orhamnoside kaempferol7orhamnose3oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3ocarfuran7orhamnosyl glucoside daidzein biochanin a procyanidin dimmer procyanidin b1 procyanidin b2 procyanidin trimer phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr13cnmr ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms ofethnopharmacology26420211132475 0ct ji table continued name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1ogalloyldglucose protocatechol glucoside epigallocatechin vanillic acid1ofuran celery glucosyl ester protocatechuic acid1ofuran celery glucosyl ester methoxyphenol1ofuran glycosyloglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid scid0 trolline fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid dimeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid terpenoids and steroids sitosterol daucosterol campesterol stigmasterol 6obenzoyl daucosterol ergosterol taraxerone taraxerol αamyrine pteroside z ganoderic acid h 3epipapyriferic acid oleanic acid saponins ginsenoside rh1 detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr lcms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms gcms tcl hnmr cnmr ms gcms gcms ir hnmr eims ir hnmr ms ir hnmr ms ir hnmr ms ir eims uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms hnmr cnmr ms analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber reference sun sun sun sun sun sun zeng sun xu 2014b zeng zeng zeng zeng xu 2014b xu 2014b chen fu fu fu fu fu fu fu fu fu fu sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun chen ding sun sun guo ru ru ru ru sun sun sun ding uplcesiqtofmsms root tuber sun continued on next page ofethnopharmacology26420211132476 0ct ji table continued name ginsenoside rh2 vinaginsenoside r1 amino acid and derivatives phenylalanine pyroglutamic acid glutimic acid hexose tryptophan lglutamic acid detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part reference sun sun sun sun sun sun sun respectively a carboline by comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and scid0 trolline the chemical structures were shown in fig identified as indole anic acids and derivatives the biologically essential anic acids have been isolated and characterized from t hemsleyanum as well ten anic acids and seventeen fatty acids were identified from the aerial parts and root tuber of t hemsleyanum most of which were found in the aerial parts except stearic acid propanoic acid and dihydroxy octadecenoic acid all the anic acids and fatty acids are listed in no112“ and no95“ of table respectively terpenoids and steroids terpenoids and steroids are other kinds of secondary metabolites of t hemsleyanum thirteen of these compounds have been isolated and identified no122“ table liu yang liu isolated and identified αamyrine sitosterol ergosterol taraxerone taraxerol from the aerial part of t hemsleyanum in addition daucosterol campesterol stigmasterol 6obenzoyldaucosterol pteroside z ganoderic acid h 3epipapyriferic acid and oleanic acid were successively separated tuber roots of t hemsleyanum liu and yang from the inanic elements the mineral elements of tcm are indispensable supplements to the bioactive components which are closely related to the efficacy toxicity and side effects of tcm wu wu et al demonstrated that t hemsleyanum contained twentyseven different mineral elements namely li be na mg al k ca v cr mn fe co ni cu zn ga as se rb ag cd cs ba hg ti pb u moreover ca cu ni ba al k have higher loading values which are the characteristic elements of t hemsleyanum wang wang has indicated that the contents of fe mn zn and cu in three populations of t hemsleyanum cultivated in different environments were “ “ “ “ mg kgcid0 respectively pharmacology the ethnomedical uses of t hemsleyanum have stimulated various pharmacological studies on it the extracts and isolated compounds from t hemsleyanum showed a variety of bioactivities such as antiviral antibacterial antioxidant antipyretic analgesic hepatoprotective immunoregulatory and antitumor activity the detailed pharmacological activities of t hemsleyanum were presented in table and summarized as follows antiviral activity according to yang™s literatures yang the nitrogenous alkalicontaining extract a ketonecontaining extract f crude extract s1 and crude extract s2 of t hemsleyanum had different antiviral effect on mice and chicken embryo fibroblast cef infected with hemagglutinating virus of japan hvj influenza virus pr6 vesicular stomatitis virus vsv specifically s2 strongly inhibited the proliferation of influenza virus pr6 with at the concentration of mgml and mgml s1 has obvious antiviral effect on hvj at the concentrations of mgml and mgml both f and s1 displayed a strong suppressive effect on the plaque formation of vsv in vivo a f s1 s2 have different degrees of antiviral activity when the concentration of a was gkg the protective rate was up to and that of s1 gkg was however the author did not give the sample preparation method ding had demonstrated compounds quercetin3orutinoside kaempferol kaempferol3glucoside quercitrin quercetin kaempferol3orutinoside procyanidin dimmer and epicatechin which were isolated from t hemsleyanum were positively related to the activity of t hemsleyanum against h1n1 influenza virus the ethyl acetate extracts of t hemsleyanum have been shown to obviously restrain the secretion of hbsag and hbeag released by hbv with the ic50 values of “ mgl however the specific mechanism of action needs to be further confirmed yang and wu wang had proved that the nbutanol and ethyl acetate extraction of t hemsleyanum had antiviral activity against rsv and were superior to ribavirin with the ec50 values of mgl wang moreover the t hemsleyanum extracts had different degrees of inhibition to different hiv1 strains the ec50 values were between μgml and μgml and the
0
" Innovation Primary liver cancer PLC is a fatal disease that affects millions of livesworldwide PLC is the leading cause of cancerrelated deaths and theincidence rate is predicted to rise in the coming decades PLC can becategorized into three major histological subtypes hepatocellular carcinoma HCCintrahepatic cholangiocarcinoma ICC and combinedHCCICC These subtypes are distinct with respect to epidemiology clinicopathological features genetic alterations and clinical managementswhich are thoroughly summarized in this review The state of treatmentstrategies for each subtype including the currently approved drugs andthe potential novel therapies are also discussedKEYWORDS PRIMARY LIVER CANCER HEPATOCELLULAR CARCINOMA INTRAHEPATIC CHOLANGIOCARCINOMA COMBINED HCCICC PLC THERAPYIntroductionPrimary liver cancer PLC is a deadly malignancy with significant histological and biological heterogeneity and ranks as the fourth leading cause ofcancerrelated death worldwide12 Therefore it has become a major publichealthy challenge Over the past decades the morbidity and mortality associated with PLC have steadily risen According to Globocan's latest GlobalCancer Statistics Report cases of liver cancer were reported worldwide in accounting for of the total cancer cases in the sameperiod while deaths totaled accounting for of total cancerdeaths3 On the basis of annual projections the World Health anization estimates that patients will die from liver cancer in Incidenceand mortality of PLC differ widely between regions The highest incidenceof PLC was observed in East Asia and in subSaharan Africa4 In particularChina experiences the highest number of cases of PLC with a high incidencerate cases100000 inhabitants5PLC manifests as three subtypes hepatocellular carcinoma HCC intrahepatic cholangiocarcinoma ICC and combined HCCICC cHCCICCwhich differ notably in epidemiology clinicopathological morphology geneticalteration and appropriate therapeutic responses HCCs are primarily relatedto viral infection alcohol abuse and metabolic syndrome6 whereas ICCs aremainly associated with chronic liver ‚ammation and biliary tract diseases78 Risk factors for development of cHCCICC include overweightobsess nonalcoholic steatohepatitis and liver cirrhosis910 HCCs show asolid and trabecular pattern with local invasion restricted to the liver11“whereas ICCs are ductular papillary or solid tumor structures with highmetastasis to distal ans14“ cHCCICCs are the combination of theHCC and ICC phenotypes present in liver tissue and are classified into separate combined and mixed cHCCICC subclasses which are more aggressiveand have a poorer prognosis217“The three PLC subtypes have distinct genetic alterations and molecularpatterns HCCs are associated with genetic alterations in specific chromosomal regions and genes including TERT promoter mutation TP53 deletionand WNT signaling CTNNB1 and AXIN1 activation22“ ICCs show aunique mutational landscape with recurrent mutations with the genetic alterations in TP53 KRAS isocitrate dehydrogenase IDH and fibroblastgrowth factor receptor FGFR gene fusions30“ Combined cHCCICCsshow strong ICClike features whereas mixed cHCCICCs show HCClikefeatures3637 Understanding the molecular alterations that initiate variousPLC subtypes is of great importance for us to decipher the mechanisms oftumorigenesis Genetic alterations can be transformed into biomarkersthat may represent new therapeutic targets affectthe treatmentdecisions and ultimately improve the treatment of liver cancer patientsHCCs mainly respond to targeted therapy immunotherapy and antiviralagents while ICC patients benefit from classical chemotherapy targetedtherapy and immunotherapy Based on the pathological classification andthe molecular features of cHCCICCs combined cHCCICCs should betreated with similar therapies to ICCs whereas mixed cHCCICCs are treatedmore like HCCs In this review we systematically summarize the epidemiology pathogenesis genetic alteration and treatment for each subtype andcomprehensively describe current therapy drugs and the potential novel therapies for PLCEpidemiology and Risk Factors HCC HCC represents the major histologic subtype accounting for approximately of all cases of PLC The riskfactors for HCC includes hepatitis B virus HBVhepatitis C virus HCV infection aflatoxin B1 alcoholic abuse and nonalcoholic metabolic symptomssuch as diabetes and obesity6 According to the Global Burden of Diseasefrom to HBV and HCV accounted for liver cancer deaths alcohol for and other causes for deathsIn particular of all HCC cases worldwide are reported from China38 dueto the locally high prevalence of HBV infectionICC As the second most common liver carcinoma following HCC ICCaccounts for around of PLC cases with a high incidence of per population worldwide annually39 The most common risk factorsfor ICC are biliary tract diseasesincluding choledochal cysts cholelithiasis choledocholithiasisliver flukes viral hepatitis metabolic syndromeand other risk factors including tobacco and alcohol use and cirrhosis7Recently the incidence of ICC has been increasing more rapidly owing torisk factors8 including increasing chronic liver disease and environmentaltoxins and is found more often due to improved diagnostic tools andimagingcHCCICC cHCCICC presents as a heterogeneous tumor showing both hepatocyte and cholangiocyte differentiation and has a poor prognosis40cHCCICC is a rather rare tumor with an incidence rate less than Thepoor prognosis associated with cHCCICC is due to the limited treatment options and difficulty of diagnosis To date the largest cohort analysis whichincluded patients diagnosed with cHCCICC between and across registries41 reported that the incidence of cHCCICC in men andwomen was and per per year respectively with the averageage of years at diagnosis One and 5year causespecific survival rates forcHCCICC were and respectively with the median survival of months Among racial groups cHCCICCs are most common in Asianraces and Pacific Islanders Obesity nonalcoholic steatohepatitis and livercirrhosis were observed in some cHCCICC cohorts910 and are potentialrisk factors for cHCCICCClinicopathological Features HCC HCC shows a solid trabecular andpseudoglandular pattern with a high density of tumor cells It has three subtypes welldifferentiated HCC moderately differentiated HCC and poorlyllThe Innovation August 0cnoitavonnIehTReviewdifferentiated HCC11“ Welldifferentiated HCCs are often small less than cm in diameter and are composed of cells with a higher nuclear to cytoplasmic ratio arranged in a thin trabecular pattern with rare pseudoglandularstructures Moderately differentiated HCCs are usually larger tumors largerthan cm showing polygonal tumor cells in a thick trabecular arrangementwith a frequent pseudoglandular pattern Poorly differentiated HCCs arecomposed of pleomorphic tumor cells in a solid or compact growth patternICC ICC can be divided into two subtypes a small duct type that originatesfrom small intrahepatic ductules with no or minimal mucin production and alarge bile duct type that arises from large intrahepatic ducts proximal to thebifurcation of the right and left hepatic ducts with high mucin production ability14“ Further ICC shows three different growth patterns mass formingMF periductal ltrating PI and intraductal growth IG42 MF ICC is afirm multilobulated unencapsulated whitegray tumor owing to its extensivedesmoplastic stroma The PI subtype shows extensive ltration along theintrahepatic hilum structure and the IG subtype is usually restricted to tubeswith papillary structures MF ICC is the most common type associated with apoor prognosis while IG type is rare but has a favorable prognosis17cHCCICC Though the phenomenon of HCC and ICC being present in thesame liver was first described in cHCCICC was not systematicallydescribed until when it was classified into three subtypes dependingon the location of HCC and ICC type A separate type has separate nodulesof hepatocellular and bile duct carcinoma type B combined type showscontiguity with intermingling but with clearly defined areas type C mixedtype presents as intimate association without clear boundaries18 In another classification system with three subtypes was established type Icollision tumors is the simultaneous occurrence of both HCC and ICC inthe same patient type II transitional tumors is an identifiable intermediatetransition between HCC and ICC type III fibrolamellar tumors resemblesthe fibrolamellar variant of HCC but also contains mucinproducing pseudoglands19 Presently the World Health anization WHO classificationis commonly used in which cHCCICC is classified into two main types theclassic type and the stem cell SC type subtype with SC features with theSC type subdivided into three subtypes including the typical subtype intermediate subtype INT and cholangiocellular type43The lack of a unified classification system greatly adds to the difficulty forcHCCICC research and the clinicopathological characteristics of cHCCICCremain illdefined cHCCICC can exhibit stemprogenitor cell phenotypesconsisting of small cells with scant cytoplasm hyperchromatic nucleiembedded within a thick desmoplastic stroma a high nuclearcytoplasmicratio and increased mitotic activity1 In addition the immunohistochemistryhas identified stemnessrelated markers KRT19 CD56 EpCAM CD117CD113 OV6120 cHCCICC clinicopathologic characteristics include morefrequent multifocallesions more microvascular emboli and portal veinand lymph node invasion all of which indicate a poor prognosis21Genetic Alterations HCC Widescale genomic studies have revealedthat hundreds of somatic DNA alterations accrue in HCC including chromosome aberrations and mutations Highlevel DNA amplifications are enrichedin chromosome locations 6p21 and 11q13 in HCC44 which occur in “of cases Recently some oncogenic genes were identified in the regions offrequent DNA gain For example LINC01138 is an oncogenic long intergenicnoncoding RNA located in this region which has been identified as a driver ofHCC45 VEGFA and CCND1FGF19 have also identified in these regions andare potential therapeutic targets46 Loss of heterozygosity on chromosome8p is a frequent event in HCC47 These DNA alterations are often associatedwith cancer progression due to the deletion of tumor suppressor genesIntriguingly in these regions a variety of vulnerability genes have recentlybeen identified For example TSLNC8 was characterized as a tumor suppressor gene on chromosome 8p12 the region that shows allelic loss in HCC andwas shown to inhibit the proliferation and metastasis of HCC48 The geneticmutations of HCC have been well studied Mutations in the TERT promoteroccur in approximately of cases and cause recurrent viral insertion ofHBV49 Deletion mutations in TP53 are the most frequent genetic alterationsaccounting for about of cases22“ and are thought to be the initiatingevent driving the formation of precursor lesions Mutated genes in WNTsignaling CTNNB1 and AXIN1 and chromatin remodeling ARID1A accountfor approximately “ of cases22“ Accumulation of activating mutations in oncogenes including activation of AKT or mTOR and of the oxidativestress pathway activation occurs throughout tumor progression and couldbe potentially targeted with molecular therapies in the futureICC ICC shows a unique mutational landscape with recurrent mutationscompared with HCC It harbors the genetic alterations in TP53 KRASARID1A BAP1 IDH1 IDH2 PIK3CA SMARCB1 EPHA2 SMAD4 GNAS andPBRM1 as well as FGFR gene fusions30“ Gain of function of IDH1 andIDH2 mutation on R132 and R172 two hotpot codons was observed in“ of ICC cases32 Fusions amplifications translocations and rearrangements of FGFR genes are found in ICC and are closely related to theinitiation and progression of ICC50 The activating mutation of KRAS “ is another frequent genomic alteration in ICC315152 The KRAS mutationoften exists concurrently with FGFR2 fusions and IDH mutations suggestinga possible cooperative role in ICC pathogenesis5354 In addition recentstudies have shown that BRAF and Notch are considerably more prevalentin ICC and function in ICC pathogenesis55cHCCICC cHCCICCs are genetically complex tumors The combined subtype of cHCCICC shows strong ICClike features with the high expression ofEPCAM KRT19 PRDM5 and KRAS The mixed subtype of cHCCICC showsHCClike features with the high expression levels of AFP GPC3 APOE SALL4and AFP8136The most frequent mutation observed in cHCCICCs is TP53 with a strikingly high mutation frequency much higher than that in HCC “ and ICC “ Interestingly several studies have foundthat the disruption of Trp53 alone in livers of mice can induce the formationof cHCCICC3757 which further implies that TP53 may be the driver gene incHCCICC It is notable that Nestin a type VI intermediate filament IF proteinthat is commonly used as a neuroectodermal SC marker is highly expressedin cHCCICC and is strongly associated with poorer prognosis36 Hence Nestin may be a promising biomarker for cHCCICCChallenges and Limitations of Current Treatment Strategies ResectionTransplantation Local and Regional Therapies HCC The commonlyused staging system for HCC is the Barcelona Clinic Liver Cancer staging system Figure HCCs in the very early stage or intermediate stage can betreated with local regional therapies which include radiofrequency ablationRFA resection da Vinci surgery laparoscopic surgery or traditional surgery transplantation orthotopic liver transplantation piggyback transplantation split liver transplantation auxiliary liver transplantation percutaneousethanoltranscatheter arterial chemoembolizationTACE58injections PEI orICC Surgery is currently the only curative treatment for ICCs but only aminority of patients in early stages are considered candidates for resectionIn surgery ICC is usually treated with hepatic resection to achieve negativeresection margins59 For patients with locally unresectable ICC tumor ablation such as RFA or hepatic arterybased therapies like yttrium90 radioembolization appear promising59“cHCCICC An accurate diagnosis is of paramount importance for thetreatment of cHCCICC Currently major hepatectomy is the optimal management for cHCCICC65 The rarity of this cancer as well as the lack of biomarkers have made this cancer difficult to diagnosis and manage Surgicalresection remains the only curative option for patients with cHCCICCThe treatment options for cHCCICC are similar to those for HCC and ICCand include surgery radiation yttrium90 radioembolization chemotherapycombined radiation and chemotherapy combined surgery and chemotherapy and triple therapy surgery radiation and chemotherapy4166“ Arecently retrospective analysis from to of PLC patientsincluding cHCCICC HCC and ICC patients who underwentresection found that although cHCCICC is more poorly differentiated thanHCC and ICC it had a similar 5year survival rate and respectively and 3year recurrence rate respectively70Systemic Chemotherapy HCC Systemic chemotherapy has limited efficacy on HCC several clinicaltrials of chemotherapy have shownlow response rates and worse toxicity without a significant improvement inThe Innovation August wwwcellcomtheinnovation\x0cReviewFigure Barcelona Clinic Liver Cancer Staging Systemand Corresponding Treatment Options The schematic diagram illustrates therapeutic choice by which a treatmenttheoretically recommended for a different stage is the besttreatment option 1L firstline 2L secondline ECOGEastern Cooperative Oncology Group M metastasis stageN nodal stage PEI percutaneous ethanolinjection PSperformance status T tumor stage TACE transarterialchemoembolization TARE transarterialradioembolizationY90 Y90 radioembolizationTheInnovation[5FU]including gemcitabine and doxorubicinbasedthe overall survival OStreatment FOLFOX 5fluorouracilleucovorin oxaliplatin andPIAF cisplatininterferon alpha2bdoxorubicin5FU71“ This suggestsa limited role for traditional chemotherapy in the treatment of advanced HCCICC Current firstline standard of treatment for ICC is the combination ofgemcitabine and platinumderived chemotherapy Figure 2B With the poorprognosis the median survival of advanced ICC patients is less than one yearVery limited effective treatments are available for patients who progress onfirstline chemotherapy so there is a high medical demandFirstLine Treatment Effective molecular targeted therapy and immunotherapy is lacking so chemotherapy with gemcitabine platinum compoundsand fluoropyrimidines is still the mainstream of standard treatment for unresectable ICCThe primary chemotherapy for ICC is gemcitabine which was establishedas the firstline therapy for advanced biliary tract cancer BTC in In the randomized controlled ABC02 phase III clinical trial comparedthe benefit of gemcitabine plus cisplatin CisGem chemotherapy with thesingle agent gemcitabine75 This study showed an advantage for CisGemin OS months versus months hazard ratio [HR] confidence intervalPFS months versus months p This effectiveness wasconfirmed in a Japanese randomized phase II study BT22 median OS months versus months HR Based on these promising results CisGem is currently regarded as the standard of care in the firstlinetreatment for advanced cholangiocarcinoma[CI] “ and progressionfree survivalOther than cisplatin gemcitabine plus other agents such as oxaliplatin S1capecitabine bevacizumab and Nabpaclitaxel have also been considered asthe firstline choices for advanced cholangiocarcinoma based on the promising outcomes from several phase II or III trials77“A recent multicenter randomized phase III clinical trial NCT01470443showed that XELOX has the comparable efficacious effect to GEMOX interms of tumor response survival rate OS and PFS and safety Also XELOXhas an advantage of low hospital visits compared with GEMOX Thus XELOXcould be an alternative for cholangiocarcinoma therapiesSecondLine Treatment There is no established standard secondlinechemotherapy for advanced cholangiocarcinoma and all regimens haveshown limited efficacy with a median PFS of around months and medianOS of about months92FOLFOX Lfolinic acid 5FU and oxaliplatin is an optional secondlinetreatment option based on the randomized phase III multicenter labelABC06 study NCT01926236 FOLFOX showed increased benefit for median OS months and months and OS rate months and compared with months and for the control groupactive symptom control [ASC] arm92cHCCICC In contrastCurrently several phase II and III chemotherapy clinical trials are under wayTable Combined therapy with chemotherapy shows promise in the treatment of cholangiocarcinoma selective internal radiotherapy SIRT pluschemotherapy or hepatic arterialinfusion plus systemic chemotherapyboth had antitumor activity and are promising for the treatment of ICC9394to surgerybased treatments for resectablecHCCICC systemic therapy is the nonstandard option for advanced and unresectable cHCCICC based on the standard treatment strategy for the unresectable HCC or ICC Chemotherapy for advanced or unresectable cHCCICCis largely understudied with only a few case reports and some retrospectivestudies having been published91095“ Recently a multicenter retrospectiveanalysis has been conducted by Kobayashi and colleagues10According to dividedgroup treatment with gemcitabine plus cisplatinn 5FU plus cisplatin n sorafenib monotherapy n others n they found that patients with platinumcontaining treatment had longer OS time than those treated by sorafenib monotherapyshowing OS of months CI “ months CI “ months CI “ and months CI “respectivelyA similar conclusion was drawn in another retrospective study of cHCCICC patients with receiving gemcitabinebased therapygemcitabine platinum or gemcitabine 5FU or targeted agents sorafenib9 Median PFS favored gemcitabineplatinum and gemcitabine5FU and months respectively over sorafenib monotherapy monthsllThe Innovation August 0cnoitavonnIehTReviewABFigure Treatment Strategy for Advanced HCC and ICC The schematic illustration represents FDAapproved drugs for treatment of advanced HCC and ICC Firstlinedrugs for HCC include sorafenib lenvatinib atezolizumab plus bevacizumab tremelimumab plus durvalumab and donafenib whereas for ICC the combination ofgemcitabine and cisplatin is currently proposed as first line The bottom row represents corresponding secondline therapies that come in when patients are not suitable fortheir firstline therapyMolecular Targeted Therapy HCC FirstLine Drugs Sorafenib Sorafenib was the first US Food and Drug Administration FDAapproved firstline systemic targeted drug for advanced HCC It is an oral smallmoleculemultikinase inhibitor targeting VEGFR1 VEGFR2 VEGFR3 PDGFRb andRaf Two large international multicenter clinical trials SHARP and AsianPacific have proved that sorafenib can suppress tumor progression and prolong OS in patients with advanced HCC102103 These trials showed that sorafenib can increase PFS and OS by months in patients with advancedHCC in Western countries As the first generation of targeted drugs forHCC sorafenib has been used for over a decade During this time many patients have benefitedthough others quickly developed resistance tosorafenib104Lenvatinib Lenvatinib is becoming available for HCC patients whodevelop sorafenib resistance Lenvatinib is an oral tyrosine kinase inhibitorinhibiting VEGFR1“ FGFR1“ PDGFR RET and KIT In August theFDA approved lenvatinib for firstline treatment of patients with unresectableHCC after lenvatinib was proved to be noninferior to sorafenib in the phase REFLECT trial105Median OS in the lenvatinib arm and sorafenib arm was months and months HR CI respectively The adverse effectswere hypertension diarrhea and decreased appetite withlenvatinib and palmarplantar erythrodysesthesia diarrhea decreased weight hypertension and decreased appetite with sorafenibDonafenib Similar to sorafenib donafenib is a novel multikinase inhibitortargeting RAF kinase and various receptor tyrosine kinases RTKs includingVEGF receptor VEGFR and BRAF106 According to the report from International Conference of the American Society of Clinical Oncology CSCOdonafenib significantly improves OS over sorafenib versus monthswith fewer side effects and higher patient tolerance for advanced HCC patients in its phase IIIII label trial107 The grade and above adverse reaction rates for donafenib and sorafenib were and respectivelyThus donafenib was recommended as the firstline therapy in the CSCOguidelines for HCCSecondLine Drugs Regorafenib Regorafenib an oral multikinase inhibitor inhibits the activity of protein kinases involved in multiple biological processes such as tumorigenesis tumor angiogenesis distant metastasisand tumor immune escape These kinases include VEGFR “ TIE2RAF1 KIT RET RAF BRAF PDGFR FGFR and CSF1R The randomized doubleblind multicenter phase III clinical trial RESORCE showed that regorafenib significantly improves the OS of patients as compared with the placebofrom to months HR p Grade “ adverse eventswere reported in of patients receiving regorafenib and of patientsreceiving the placebo In regorafenib received FDA approval as the secondline drug for the treatment of patients with advanced HCC who fail torespond to the sorafenib treatmentCabozantinib Cabozantinib is an oral inhibitor and targets multiple kinasesincluding VEGFR2 cMET RET ROS1 TYRO3 MER KIT TRKBFLT3 TIE2 as well as the GAS6 receptor AXL109110 It was originallyapproved for medullary thyroid cancer in and advanced renal carcinoma in According to the randomized doubleblind multicenter phase clinical trial conducted across centers in countries median OS was months for patients receiving cabozantinib and months for patientstreated with placebo HR p Median PFS was monthsand months respectively Grade or adverse events occurred in of patients in the cabozantinib arm and in the placebo arm Theobserved hepatotoxicity can be mostly controlled through dose modifications Based on the encouraging results of prolonged OS and PFS cabozantinib received its FDA approval for HCC in Ramucirumab Ramucirumab is a completely human monoclonalantibody that can specifically inhibit VEGFR2112 For patients with alphafetoprotein R400 ngmL and who have been previously treated with sorafenib ramucirumab was approved as a monotherapy by the FDA on May The Innovation August wwwcellcomtheinnovation\x0cTable Systemic Therapies Currently or Promising Approved for Advanced HCC and ICCReviewTargetTherapy LineApproved YearTrialDrugsHCCSorafenib NexavarLenvatinib LenvimaRegorafenib StivargaNivolumab OpdivoVEGFR2 VEGFR3 PDGFRb RAF kinasesFGFR VEGFR PDGFRa RET KITTie2 VEGFR PDGFR FGFRPD1Cabozantinib CabometyxcMet VEGFR2 AXL RETPembrolizumab KeytrudaRamucirumab CYRAMZAPD1VEGFR2Nivolumab ipilimumab Opdivo YervoyPD1 CTLA4Atezolizumab bevacizumabTremelimumab durvalumabDonafenibApatinibICCGemcitabine cisplatinPemigatinib PemazyreIvosidenibPDL1VEGFPD1 CTLA4VEGFR BRAFVEGFR2chemotherapyFGFR1“IDH12TheInnovationpromisingpromisingpromisingpromisingSHARP AsianPacificREFLECTRESORCECHECKMATE040CELESTIALKEYNOTE224REACH2Cohort of CHECKMATE040IMbravel50NCT02519348NCT02645981NCT02329860ABC02FIGHT202promisingClarlDHyApproval was based on REACH NCT02435433 a randomized doubleblind multicenter phase III study of patients with AFP R400 ngmL whohad disease progression after sorafenib or were intolerant to sorafenib113More recently a study further confirmed the efficacy of ramucirumab inelderly patients with HCC and elevated AFP after sorafenib in REACH andREACH2 with a survival benefit observed across all age subgroups and atolerable safety profile supporting its value irrespective of age including forpatients R75 years114Apatinib Apatinib a tyrosine kinase inhibitor targeting VEGFR2 significantly prolonged OS and PFS in Chinese patients with advanced HCC whohad previously been treated with sorafenib andor chemotherapy accordingto the results of a randomized placebocontrolled phase III trial conducted in sites in China115 Median OS was almost months longer for patients whoreceived apatinib compared with patients receiving the placebo monthsversus months and median PFS was more than months longer months versus months115 The most common grade or worseadverse events occurred at a rate of in the apatinib arm and inthe placebo arm With the significantly prolonged OS and PFS and a manageable safety profile apatinib has potential to become a new secondline therapy for liver cancerNovel Therapeutic Targets Even with all these available treatments Table the median PFS for HCC patients remains less than a year Thus noveltreatment is still a critical unmet need for treatment of HCC Based on thegenomic profile and biomarkers reported in HCC several clinical trials targeting various pathways are currently ongoing Table Recently a firstinhuman phase I study NCT02508467 of fisogatinib BLU554 an orally bioavailable inhibitor of human FGFR4 demonstrated its antitumor activity in HCCand further validated that the aberrant FGF19FGFR4 signaling pathwaymay be a driver event116 In addition the TGFb1 receptor type I inhibitor galunisertib also showed an acceptable safety and prolonged OS outcome in combination with sorafenib in a phase II trial NCT01246986117118 Other potential candidatesincluding the cyclindependent kinase CDK inhibitorsregulating the cell cycle pathways ribociclib palbociclib119120 abemacicliband milciclib as well as the cMET inhibitors tepotinib121 and tivantinib122are being evaluated in HCC clinical trialsICC Moleculartargeted therapy controls tumor cell proliferationapoptosis adhesion and movement by inhibiting the surface molecules oftumor cell membranes and thereby inhibiting intracellular signaling pathways ICC genetic alterations primarily include FGFR IDH epidermal growthfactor EGFR and breast cancer type susceptible protein associated protein1 BAP1123“ Genetic alterations of these genes all have implicationsfor therapy At present a variety of molecular targeted drugs are in the clinicalresearch stage Table some of which have made progress in the treatment of ICC Table FGFR Inhibitors The most promising target therapy for cholangiocarcinoma identified in recent years is the inhibitor of the fibroblast growth factorFGF signaling pathway which consists of members labeled FGF1“FGF15 FGF19 called FGF1519 and four interacting transmembrane receptors FGFR1“ FGF signals regulate cell proliferation in which FGFR2fusions occurred in “ of ICC patients and are considered as a promising therapeutic target3351127128 Currently several FGFR inhibitors are being evaluated in clinical trials for cholangiocarcinomas with FGFR geneticaberrationsPemigatinib INCB054828 Pemigatinib is the first and only targeted therapy so far approved in by the FDA for the treatment of this rare cancerIt is a selective potent oral inhibitor of FGFR and Approval wasbased on findings from the phase II FIGHT202 trial NCT02924376 whichenrolled patients with locally advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements cohort A other FGFFGFR genetic alterations cohort B or no FGFFGFR genetic alterations cohort CFor those in cohort A treatment with pemigatinib resulted in a median OSof months and median PFS of months The FIGHT202 study suggests that locally advanced or metastatic cholangiocarcinoma patientswith FGFR2 fusions or rearrangements may benefit from potent oralFGFR1 and inhibitor treatment Median PFS was months for patientswith FGFR2 alterations months for patients with other FGFFGFR alterations and months for those with no alterations in these genes MedianOS was months months and months for the respective cohorts130 With the promising results of phase II the phase III clinical trial ofpemigatinib is currently underway NCT03656536llThe Innovation August 0cnoitavonnIehTDrugTargeted TherapyCabozantinibLenvatinibDonafenibMilciclibPalbociclibRibociclibGalunisertib versus LY2157299 sorafenib versus placebo sorafenibImmunotherapyVEGFRVEGFRVEGFRCDK2CDK46CDK46TGFbToripalimab versus placeboNivolumab versus placeboNivolumab versus sorafenibPD1PD1PD1Hospices Civils de Lyonrecruitingphase Eisai Pharmaceuticals IndiaPvt Ltdnot yetrecruitingphase NCT03963206NCT04297254completedphase phase NCT02645981Suzhou ZelgenBiopharmaceuticalsTiziana LifeSciencesPfizeractive notrecruitingactive notrecruitingphase phase Texas Universityrecruitingphase Eli Lillyactive notrecruitingphase NCT03109886NCT01356628NCT02524119NCT02178358NCT03412773NCT03859128NCT03383458ReviewTable Selected Ongoing Systemic Therapy Clinical Trials for Advanced HCCTargetSponsorStatusPhaseEnrollmentTrial IdentifierTislelizumab versus sorafenibPD1BeiGeneactive notrecruitingphase Shanghai Junshi Biosciencerecruitingphase phase BristolMyers Squibbrecruitingphase BristolMyers Squibbactive notrecruitingphase NCT02576509Pembrolizumab versus placeboPD1Merck Sharp Dohmerecruitingphase AvelumabPDL1Seoul National UniversityHospitalactive notrecruitingphase Combined TherapyLenvatinib pembrolizumabversus lenvatinib placeboCS1003 lenvatinib versusplacebo lenvatinibVGFR PD1Merck Sharp Dohmeactive notrecruitingphase VGFR PD1CStone Pharmaceuticalsrecruitingphase Tislelizumab regorafenibversus placebo regorafenibVEGF PD1National Taiwan UniversityHospi
2
incidence and death rate of nonsmall cell lung cancer NSCLC in China ranks the first among the malignant tumors Circular RNA circRNA was reported to be involved in the progression of NSCLC Our study aimed to investigate the underlying mechanism of circ_0020123 in NSCLC progressionMethods Quantitative realtime polymerase chain reaction qRTPCR was used to detect the expression of circ_0020123 miR5905p and Thrombospondin THBS2 in NSCLC tissues and cells Cell proliferation and migration were examined by Cell Counting Kit8 CCK8 assay and Transwell assay respectively Flow cytometry assay was used to detect the apoptosis of NSCLC cells The protein levels of Ki67 matrix metalloprotein9 MMP9 Cleavedcaspase9 Cleavedcasp9 and THBS2 were detected by Western blot The targets of circ_0020123 and miR5905p were predicted by starBase and TargetScan and then confirmed by dualluciferase reporter assay and RNA immunoprecipitation RIP assay The animal experiment showed the effect of circ_0020123 on tumor growth in vivoResults The expression of circ_0020123 was upregulated in NSCLC tissues and cells Functionally circ_0020123 downregulation inhibited the proliferation and migration and promoted the apoptosis of NSCLC cells Interestingly circ_0020123 directly targeted miR5905p and inhibition of miR5905p reversed the knockdown effects of circ_0020123 on NSCLC cells More importantly THBS2 was a target of miR5905p and THBS2 overexpression reversed the effects of circ_0020123 knockdown on cell proliferation migration and apoptosis in NSCLC cells Finally suppression of circ_0020123 inhibited tumor growth in vivo through miR5905pTHBS2 axisConclusion Circular RNA circ_0020123 regulated THBS2 by sponging miR5905p to promote cell proliferation and migration and inhibit cell apoptosis in NSCLC cellsKeywords NSCLC Circ_0020123 miR5905p THBS2Highlights Circ_0020123 was upregulated and downregulation of circ_0020123 inhibited cell proliferation migration and promoted cell apoptosis in NSCLC cellsCorrespondence bskrju163comDepartment of Thoracic Surgery Lianyungang Second People™s Hospital No Hailian East Road Haizhou District Lianyungang Jiangsu China Circ_0020123 directly targeted miR5905p and miR5905p downregulation reversed the knockdown effects of circ_0020123 on NSCLC progression THBS2 acted as a target of miR5905p and overthe effects of expression of THBS2 reversed circ_0020123 knockdown on NSCLC progression Downregulation of circ_0020123 suppressed tumor growth in vivo through miR5905pTHBS2 axis The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cWang a0et a0al Cancer Cell Int Page of BackgroundLung cancer has the highest incidence of total cases and is the most common cause of cancer death of total cancer deaths in worldwide [] Lung cancer can be divided into several histological subtypes according to the location and the tendency of metastasis Small cell lung cancer SCLC accounts for about of all lung cancer cases [] However nonsmall cell lung cancer NSCLC accounts for of lung cancer and the a0years overall survival rate OS is only about [] Therefore it is important to find the effective treatment and potential molecular targets of NSCLC progressionCircular RNA circRNA is a single stranded RNA molecule with a closed circular structure Recently amounts of circular DNA have been discovered and most of which were thought to be the byproducts of typical splicing [ ] Previous reports indicated that the expression of circRNA was tissuespecific and the change of its expression intensity was associated with some diseases [“] Furthermore circRNA was involved in the occurrence and development of the disease and might be used as a potential biomarker in clinical diagnosis prognosis and treatment of diseases [ ] For example circ_0039569 facilitated cell proliferation and migration of renal cell carcinoma by sponging miR34a5p to regulate CC Chemokine ligand CCL22 [] Meanwhile hsa_circ_0043256 participated in the progression of NSCLC cells by mediating the cinnamaldehyde treatment [] A previous report suggested that circ_0020123 acted as an oncogene in NSCLC and circ_0020123 regulated zincfingerenhancer binding protein ZEB1 and enhancer of zeste homolog EZH2 by competitively binding with miR144 to induce cell progression and migration [] These reports suggested that circ_0020123 was a vital factor in the pathogenesis of NSCLC and its function and molecular mechanism need to be further studiedAs a small endogenous RNA microRNA miRNA is essential in regulating gene expression and plays a potential role in the exploitation of biomarkers [] Recently some aggregated miRNAs have been found in prostate cancer such as miR221222 miR143145 miR23b27b241 and miR1133a which were downregulated and had tumor inhibiting functions [] A previous study found that circulating miR5905p could be used as routine diagnostic tools for lung cancer and as a potential prognostic marker for liquid biopsy Besides overexpression of miR5905p reduced the development of NSCLC cells and regulated the expression of epithelialmesenchymal transformation EMTrelated proteins by targeting the signal transducers and activators of transcription STAT3 [] However the precise mechanism by which miR5905p affects NSCLC needs further investigationThrombospondin THBS2 as a secreted protein was confirmed to be highly expressed in different cancers including cervical cancer [] colorectal cancer [] and NSCLC [] A previous report suggested that THBS2 was involved in the proliferation apoptosis and antiautophagy regulation of cervical cancer cells by miR20a [] Tian et a0al found the expression and clinicopathological features of THBS2 in colorectal cancer were significantly correlated with the prognosis of cancer and might be used as a biomarker of prognosis [] However the molecular function of THBS2 in NSCLC remains poorly definedIn this study the targeting relationship between circ_0020123 and miR5905p was firstly detected The effects of circ_0020123 on cell proliferation migration apoptosis and tumor growth were performed by gain and lossoffunction experiments and molecular biology techniquesMaterials and a0methodsPatients and a0specimensNSCLC tissues and the adjacent healthy lung tissues were taken from NSCLC patients in the Lianyungang Second People™s Hospital All volunteers signed written informed consents NSCLC tissues and the adjacent tissues were immediately frozen in liquid nitrogen and kept at ˆ’ a0 °C for further experiments This research was approved by the Ethics Committee of Lianyungang Second People™s HospitalCell culture and a0cell transfectionTwo NSCLC cell lines A549 and H1299 and one normal lung cell line IMR90 were obtained from the Beijing Concorde Cell Library Beijing China A549 H1299 and IMR90 cells were cultivated in Dulbecco™s modified eagle medium DMEM HyClone Logan UT USA supplementing with fetal bovine serum FBS HyClone and cultured in an incubator at a0„ƒ with CO2Small interfering RNA siRNA targeting circ_00201231 sicirc_00201231 and sicirc_00201232 short hairpin RNA shRNA targeting circ_0020123 shcirc_0020123 miR5905pinhibitors siRNA negative control siNC shNC and NCinhibitors were all obtained from Biomics Biotech Jiangsu China Full length of THBS2 cDNA Sangon Biotech Shanghai China was subcloned into pcDNA31 plasmid EKBioscience Shanghai China Then cell transfection was performed by Lipofectamine Thermo Fisher Scientific Waltham MA USA 0cWang a0et a0al Cancer Cell Int Page of RNA isolation and a0quantitative real‘time polymerase chain reaction qRT‘PCRThe TRIzol reagent Invitrogen Carlsbad CA USA was used for extracting the total RNAs Next the reversed transcription was carried out by RTPCR kit Invitrogen The qRTPCR was performed using the ABI SYBR Green Master Mix Invitrogen The primers in our study were as follows F5²TTC GGA CGA CCG TCA AAC AT3² and R5²AGG ATC CCT GCA CCA CAA TG3² for circ_0020123 F5²TGA AAG ACG TGA TGG CAC AC3² and R5²CTT CCA TTT TGG for miR5905p F5²AGA AGG GGT TTT TGG3 ² CTG GGG CTC ATT TG3² R5²AGG GGC CAT CCA CAG TCT TC3² for glyceraldehyde3phosphate dehydrogenase GAPDH [] F5²GCG GCT GGG TCT ATT TGT C3² and R5²GCA GGA GGT GAA GAA CCA TC3² for THBS2 [] F5²ATT GGA ACG ATA CAG AGA AGATT3² and R5²GGA ACG CTT CAC GAA TTT G3² for U6 [] GAPDH and U6 were the internal parametersCell Counting Kit‘ CCK‘ assayThe proliferation viability of A549 and H1299 cells were detected by the CellCounting Kit8 MSK Wuhan China A549 and H1299 cells were cultivated into a 96well plate with a density of × a0cellswell and incubated in a0°C for or a0h Then a0μL fresh medium and CCK8 solution was added After incubation at a0°C for a0h the OD values were detected by the Multiskan Ascent microplate reader Abcam Cambridge MA USATranswell assayTranswell chamber Corning Life Sciences Corning NY USA was used to detect cell migration Firstly the serumfree DMEM Thermo Fisher Scientific was fixed with cell suspension cells and seeded into the upper chamber and the DMEM containing serum was put into the lower chamber After incubation for a0h the cells in lower surface of the upper chamber were treated with formaldehyde solution for a0 h and then thoroughly washed Finally the migrated cells were stained with crystal violet Corning Life Sciences and observed by using a microscopeFlow cytometryFirstly A549 and H1299 cells were cultured and PBS was used for washing cells Then the binding buffer was used to resuspend cells and the Annexin Vfluorescein isothiocyanate VFITCpropidium iodide PI Apoptosis Detection Kit Thermo Fisher Scientific was used to stain cells Finally cell apoptosis was detected by flow cytometry Thermo Fisher ScientificWestern blot analysisThe total proteins of NSCLC tumors or cells were collected by RIPA lysis buffer Sangon Biotech Then the proteins were separated by Sodium dodecyl sulphate polyacrylamide gel electrophoresis SDSPAGE and transferred to polyvinylidene fluoride PVDF membranes Thermo Fisher Scientific The skimmed milk was added and incubated with primary antiGAPDH antibody Invitrogen Carlsbad CA USA antiβactin antibody Invitrogen antiKi67 antibody Invitrogen antimatrix metalloprotein9 MMP9 antibody Invitrogen antiCleavedcaspase9 Cleavedcasp9 antibody Invitrogen or antiTHBS2 antibody Invitrogen at a0°C overnight Finally the membranes were incubated with the secondary antibody for a0 h at room temperature The results were viewed using Kodak film developer Fujifilm JapanDual‘luciferase reporter assaysThe wild type circ_0020123 sequences circ_0020123WT mutant circ_0020123 sequences circ_0020123MUT wild type THBS2 ²UTR sequences THBS2WT mutant THBS2 ²UTR sequences THBS2MUT were cloned into pGL3 luciferase reporter plasmid Promega Madison WI USA Then the plasmid and miR5905p or miRNC were cotransfected into A549 and H1299 cells by Lipofectamine Thermo Fisher Scientific After transfection for a0h the DualLuciferase Reporter Assay System Promega was performed to detect the luciferase activityRNA immunoprecipitation RIPFirstly the Magna RIP RNAbinding Protein Immunoprecipitation Kit gzscbio Guangzhou China was performed to verify the relationship between circ_0020123 and miR5905p In brief the magnetic beads and antiAgo2 antibody Abcam were added into cells and incubated for a0h Then the proteinase K and the phenol“chloroformisoamyl alcohol reagent were added for purifying RNAs Finally qRTPCR was used to measure circ_0020123 enrichmentAnimal experimentsThe 4weekold BALBc male nude mice Vitalriver Beijing China were raised in a sterile environment for 0cWang a0et a0al Cancer Cell Int Page of experiments Then PBS was used to suspend A549 cells × transfected with shcirc_0020123 or shNC Next the nude mice were divided into two groups n A549 cells transfected with shcirc_0020123 or shNC were shcirc_0020123 or shNC inoculated into the nude mice The tumor volume was detected every a0 days After a0days the nude mice were euthanatized and the tumor weight was detected Besides the tumor tissues from each group were collected to detect the expression of circ_0020123 miR5905p and THBS2 The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second People™s HospitalStatistical analysisThe software GraphPad Prism was performed for statistical analysis The data was displayed as mean ± standard deviation SD The significant difference was calculated by Student™s t test and oneway analysis of variance ANOVA P was considered as statistically significantResultsCirc_0020123 was a0upregulated in a0NSCLC tissues and a0cellsTo begin with qRTPCR was used to detect the expression of circ_0020123 the result showed that circ_0020123 was significantly upregulated in NSCLC tissues compared with the adjacent healthy tissues Fig a0 1a Similarly the expression of circ_0020123 in NSCLC cells A549 and H1299 was markedly higher than that in normal cells IMR90 Fig a01b From these data it is speculated that circ_0020123 might be acted as an oncogene in NSCLCFig Circ_0020123 was upregulated in NSCLC tissues and cells a QRTPCR was used to detect the expression of circ_0020123 in adjacent healthy tissues n and tumor tissues n b The expression of circ_0020123 in normal cell line IMR90 and NSCLC cell lines A549 and H1299 was detected by qRTPCR P Downregulation of a0circ_0020123 inhibited the a0proliferation migration and a0induced apoptosis of a0NSCLC cellsTo investigate the functional effects of circ_0020123 on NSCLC cells sicirc_00201231 and sicirc_00201232 were obtained and transfected into A549 and H1299 cells Firstly the transfection efficiency was detected by qRTPCR Fig a02a Next CCK8 was used to detect the proliferation and the results showed that the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was reduced Fig a0 2b Moreover the migration of A549 and H1299 cells was significantly downregulated by circ_0020123 knockdown Fig a02c In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was obviously higher than transfected with siNC Fig a02d Finally the protein levels of cell proliferationrelated protein Ki67 and cell migrationrelated protein MMP9 were inhibited while cell apoptosisrelated protein Cleavedcasp9 was upregulated in NSCLC cells transfected with sicirc_00201231 or sicirc_00201232 Fig a02e These data suggested that inhibition of circ_0020123 suppressed cell proliferation migration and promoted apoptosis in NSCLC cellsCirc_0020123 directly targeted miR‘‘5pBy searching in the online software starBase the potential binding sites between circ_0020123 and miR5905p were detected Fig a0 3a To confirm that the dualluciferase reporter assay was performed the results showed that the luciferase activity of circ_0020123WT reporter plasmid was reduced by miR5905p mimic while the circ_0020123MUT reporter plasmid activity was not changed in A549 and H1299 cells Fig a03b Furthermore the expression of miR5905p was lower in A549 and H1299 cells compared with that in IMR90 cells Fig a0 3c In contrast miR5905p expression was elevated in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a0 3d Finally the RIP assay was also used to confirm the targeting relationship between circ_0020123 and miR5905p and the results showed that circ_0020123 and miR5905p were enriched in antiAgo2 group Fig a03eMiR‘‘5p downregulation reversed the a0inhibition effects of a0circ_0020123 on a0NSCLC cellsTo further explore the functional effects between circ_0020123 and miR5905p miR5905pinhibitor was established QRTPCR was used to detect the transfection efficiency Fig a0 4a Interestingly miR5905p was upregulated in A549 and H1299 cells transfected with sicirc_00201231 while the expression of miR5905p was recovered in cells transfected with 0cWang a0et a0al Cancer Cell Int Page of Fig Downregulation of circ_0020123 inhibited the proliferation and migration and induced the apoptosis of NSCLC cells a The transfection efficiency of sicirc_00201231 and sicirc_00201232 in A549 and H1299 cells was detected by qRTPCR b CCK8 assay was used to detect the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 c The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was measured by Transwell assay d Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 e The protein levels of cell proliferation related protein Ki67 cell migration related protein MMP9 and cell apoptosis related protein Cleavedcasp9 were detected by Western blot P Fig a0sicirc_00201231 miR5905pinhibitors 4b Moreover circ_00201231 knockdown inhibited cell proliferation and migration while the miR5905p inhibitor reversed these effects Fig a0 4c d In addition the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 was increased which was abolished by miR5905pinhibitor Fig a0 4e Similarly miR5905p inhibitors reversed the effects on the protein levels of Ki67 MMP9 and Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 Fig a0 4f These results that miR5905p downregulation reversed the effects of circ_0020123 downregulation on the proliferation migration and apoptosis of NSCLC cellsindicated MiR‘‘5p targeted THBS2 in a0NSCLC cellsThe THBS2 ²UTR was predicted to contain the binding sites of miR5905p through the online software TargetScan Fig a05a Then the dualluciferase reporter assay was used to confirm the targeting relationship The results showed that cotransfection of miR5905p and THBS2WT significantly limited the luciferase activity in both A549 and H1299 cells the luciferase activity was not altered in cells cotransfected with miR5905p and THBS2MUT Fig a05b Importantly the mRNA and protein level of THBS2 was enahnced in NSCLC cells Fig a05c d We further explored whether circ_0020123 affected the functions of THBS2 in NSCLC cells The mRNA and protein expression of THBS2 were repressed in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 Fig a05e f 0cWang a0et a0al Cancer Cell Int Page of Fig Circ_0020123 directly targeted miR5905p a The binding site between circ_0020123 and miR5905p was detected by the online software starBase b The luciferase activity of circ_0020123WT or circ_0020123MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p was detected by dualluciferase reporter assay c QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells d The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR e RIP assay was used to confirm the relationship between circ_0020123 and miR5905p P THBS2 overexpression reversed the a0effects of a0circ_0020123 knockdown on a0the a0proliferation migration and a0apoptosis of a0NSCLC cellsBased on the work ahead of us the pcDNA31THBS2 was constructed Then the qRTPCR and Western blot were used to detect the transfection efficiency and the THBS2 expression was increased in A549 and H1299 cells transfected with pcDNA31THBS2 Fig a0 6a b In addition the proliferation and migration rates of A549 and H1299 cells transfected with sicirc_00201231 pcDNA31THBS2 were higher than that transfected with sicirc_00201231 Fig a0 6c d Meanwhile a similarly phenomenon was also observed in cell apoptosis the pcDNA31THBS2 significantly reversed the promotion effect of circ_0020123 deletion on cell apoptosis Fig a0 6e Furthermore the effects of circ_0020123 deletion on Ki67 MMP9 and Cleavedcasp9 protein levels were also reversed by THBS2 overexpression Fig a0 6f These data suggested that overexpression of THBS2 could reverse the effects of circ_0020123 downregulation on cell proliferation migration and apoptosisReduction of a0circ_0020123 suppressed tumor growth in a0vivo through a0circ_0020123miR‘‘5pTHBS2 axisTo further explore the function of circ_0020123 in NSCLC cells the shcirc_0020123 was constructed and the xenograft tumor was established Then A549 cells transfected with shcirc_0020123 or shNC were injected into the nude mice The xenograft tumor volume was measured every a0 days after injection and the results showed that tumor volume was smaller shcirc_0020123 group than that in shNC group Fig a07a Moreover tumor weight was inhibited by circ_0020123 knockdown Fig a0 7b Furthermore the expression circ_0020123 and THBS2 was decreased while the miR5905p was increased in xenograft tumor transfected with shcirc_0020123 Fig a0 7c Western blot assay also revealed that the protein level of THBS2 was repressed by circ_0020123 knockdown Fig a07d Finally the digital tomosynthesis DTS was used to detect the number of lung metastatic nodules in xenograft tumor and it was reduced in shcirc_0020123 group Fig a07e The results suggested that downregulation of circ_0020123 inhibited tumor growth in a0vivoDiscussionClinically only a few NSCLC patients were diagnosed at an early stage and treated by surgical resection More than of NSCLC patients were diagnosed with the advanced stage or metastatic tumors [] Thus finding novel biomarkers and therapeutic targets were necessary for the effective diagnosis and treatment of NSCLCRecently circRNA was no longer considered as a random product in the RNA shearing process and its biological significance and function in malignant tumors 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p downregulation reversed circ_0020123 knockdown effects in NSCLC cells a QRTPCR was used to detect the expression of miR5905p in A549 and H1299 cells transfected with miR5905pinhibitors b The expression of miR5905p in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was detected by qRTPCR c The proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors was tested by CCK8 assay d Transwell assay was used to measure the migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors e Flow cytolysis assay was used to detect the apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 miR5905pinhibitors were detected by Western blot P had received more and more attention Previous reports revealed that circ_0020123 was involved in the development of NSCLC [] Moreover the level of circ_0020123 was elevated in NSCLC cells [] Consistently we found that the expression of circ_0020123 was markedly higher in NSCLC tissues and cells Moreover this research indicated that inhibition of circ_0020123 suppressed the proliferation migration and induced apoptosis of NSCLC cells in a0 vitro Besides circ_0020123 promoted tumor growth in a0vivoEndogenous circRNAs could act as microRNA sponges to inhibit their function and some studies linked miRNA sponges to human diseases including cancer [] A previous study indicated that circRNA ctransferrin receptor cTFRC regulated TFRC by sponging miR107 to facilitate bladder carcinoma development [] MiR5905p was studied in different cells such as airway smooth muscle cells [] colon epithelial cells [] and NSCLC cells [] However the potential relationship between miR5905p and circRNA has not been researched In this study circ_0020123 directly targeted miR5905p and miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC progression These data provided a clue to the therapeutic strategy for NSCLC 0cWang a0et a0al Cancer Cell Int Page of Fig MiR5905p targeted THBS2 in NSCLC cells a The potential binding site between THBS2 ²UTR and miR5905p was predicted by the online software TargetScan b Dualluciferase reporter assay was used to measure the luciferase activity of THBS2WT or THBS2MUT reporter plasmid in A549 and H1299 cells transfected with miRNC or miR5905p c QRTPCR was used to detect the mRNA expression of THBS2 in NSCLC cells d The protein level of THBS2 in NSCLC cells was tested by Western blot e The mRNA expression of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 was detected by qRTPCR f Western blot was used to measure the protein level of THBS2 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201232 P Our study also confirmed that miR5905p could target THBS2 directly in NSCLC cells THBS2 is a calciumbinding protein that binds to and inactivates matrix metalloproteinase MMP genes involved in tissue formation and repair [ ] A previous document suggested that THBS2 acted as a target of miR2213p and participated in lymph node metastasis in cervical cancer [] The data in this research showed that the expression of THBS2 in NSCLC cells was markedly higher than normal healthy cells Furthermore overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells suggesting that circ_0020123 promoted the progression of NSCLC cells through miR5905pTHBS2 axisConclusionIn conclusion our research showed that the expression of circ_0020123 was higher in NSCLC tissues and cells than control and downregulation of circ_0020123 inhibited the proliferation migration and promoted apoptosis of NSCLC cells and also suppressed tumor growth in a0 vivo Moreover circ_0020123 directly targeted miR5905p while miR5905p inhibition reversed the effects of circ_0020123 knockdown on NSCLC cells More importantly circ_0020123 regulated the expression of THBS2 by sponging miR5905p and upregulation of THBS2 reversed the effects of circ_0020123 knockdown on NSCLC cells Therefore our research demonstrated that circ_0020123 enhanced proliferation migration and inhibited 0cWang a0et a0al Cancer Cell Int Page of Fig Overexpression of THBS2 reversed the effects of circ_0020123 knockdown on proliferation migration and apoptosis of NSCLC cells a b The mRNA and protein expression of THBS2 in A549 and H1299 cells transfected with pcDNA31THBS2 was detected by qRTPCR and Western blot c CCK8 assay indicated the proliferation of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 d The migration of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was measured by Transwell assay e The apoptosis of A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 was detected by Flow cytolysis assay f The protein levels of Ki67 MMP9 Cleavedcasp9 in A549 and H1299 cells transfected with sicirc_00201231 or sicirc_00201231 pcDNA31THBS2 were detected by Western blot P apoptosis of NSCLC cells by sponging miR5905p to regulate THBS2results and develop the manuscript All authors read and approved the final manuscriptAbbreviationsNSCLC Nonsmall cell lung cancer circRNA Circular RNA qRTPCR Quantitative realtime polymerase chain reaction CCK8 Cell Counting Kit8 MMP9 Matrix metalloprotein9 Cleavedcasp9 Cleavedcaspase9 Cleavedcasp9 Cleavedcaspase9 RIP RNA immunoprecipitation ZEB1 Zincfingerenhancer binding protein EZH2 Zeste homolog STAT3 Signal transducers and activators of transcription THBS2 Thrombospondin AcknowledgementsNot applicableAuthors™ contributionsLW collaborated to design the study LZ were responsible for experiments analyzed the data YW wrote the paper All authors collaborated to interpret FundingNoneAvailability of data and materialsPlease contact corresponding author for data requestsEthics approval and consent to participateThis research was approved by the Ethics Committee of Lianyungang Second People™s Hospital The animal experiment was approved by the Animal Experimentation Ethics Committee of Lianyungang Second People™s HospitalConsent for publicationAll listed authors have actively participated in the study and have read and approved the submitted manuscript 0cWang a0et a0al Cancer Cell Int Page of Fig Reduction of circ_0020123 suppressed the tumor growth in vivo through circ_0020123miR5905pTHBS2 axis a A total of × A549 cells transfected with shcirc_0020123 or shNC were injected into nude mice to establish the xenograft tumor Tumor volume was measured every d after injection b Tumor weight was measured on d c The expression of circ_0020123 miR5905p and THBS2 in xenograft tumor was measured by qRTPCR d The protein level of THBS2 in xenograft tumor was evaluated by Western blot e The number of lung metastatic nodules in xenograft tumor was detected by digital tomosynthesis DTS P Competing interestsThe authors declare that they have no competing interestsReceived April Accepted July References Bray F Ferlay J Soerjomataram I Siegel RL Torre LA Jemal A Global cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA Cancer J Clin “ Abe H Takase Y Sadashima E Fukumitsu C Murata K Ito T Kawahara A Naito Y Akiba J Insulinomaassociated protein is a novel diagnostic marker of small cell lung cancer in bronchial brushing and cell block cytology from pleural effusions validity and reliability with cutoff value Cancer Cytopathol “Li C Zhang L Meng G Wang Q Lv X Zhang J Li J Circular RNAs pivotal molecular regulators and novel diagnostic and prognostic biomarkers in nonsmall cell lung cancer J Cancer Res Clin Oncol “ Belousova EA Filipenko ML Kushlinskii NE Circular RNA new regulatory molecules Bull Exp Biol Med “ Zhang Z Xie Q He D Ling Y Li Y Li J Zhang H Circular RNA new star new hope in cancer BMC Cancer Li L Chen Y Nie L Ding X Zhang X Zhao W Xu X Kyei B Dai D Zhan S Guo J Zhong T Wang L Zhang H MyoDinduced circular RNA CDR1as promotes myogenic differentiation of skeletal muscle satellite cells Biochim Biophys Acta Gene Regul Mech “ Greco S Cardinali B Falcone G Martelli F Circular RNAs in muscle function and disease Int J Mol Sc
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recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm
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