Somatic Mutations and Deletions of the E-Cadherin Gene Predict Gastric Cancer

胃癌体细胞突变

VOLUME31 NUMBER7 MARCH12013

JOURNALOFCLINICALONCOLOGY

ORIGINALREPORT

SomaticMutationsandDeletionsoftheE-CadherinGenePredictPoorSurvivalofPatientsWithGastricCancer

GiovanniCorso,JoanaCarvalho,DanieleMarrelli,CarlaVindigni,BeatrizCarvalho,RaquelSeruca,FrancoRoviello,andCarlaOliveira

Seeaccompanyingeditorialonpage838;http://wendang.chazidian.com/

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GiovanniCorso,DanieleMarrelli,andFrancoRoviello,UniversityofSienaandInstitutoToscanoTumori;CarlaVindigni,AziendaOspedalieraUniversitariaSenese,Siena,Italy;GiovanniCorso,Joana

Carvalho,RaquelSeruca,andCarlaOliveira,InstituteofMolecularPathologyandImmu-nologyoftheUniversityofPorto(IPATIMUP);GiovanniCorso,Joana

Carvalho,RaquelSeruca,andCarlaOliveira,UniversityofPorto,Portugal;BeatrizCarvalho,VUUniversityMedicalCenter,Amsterdam,http://wendang.chazidian.comonJanuary22,2013.SupportedbythePortugueseFounda-tionforScienceandTechnology(Proj-ectsNo.POCTI/SAU-OBS/58111/2004,PIC/IC/82923/2007,PTDC/SAU-GMG/72168/2006,PTDC/SAU-GMG/110785/2009,andPTDC/SAU-ONC/110294/2009;PhDGrantsNo.SFRH/BD/40090/2007-GCandSFRH/BD/44074/2008-JC);salarysupporttoC.O.fromPOPH–QREN/Type4.2;EuropeanSocialFundandPortugueseMinistryofScienceandTechnology;EuropeanMolecularBiol-ogyOrganizationshort-termfellowship(ASTF338.00-2008-JC);CalousteGulbenkianFoundationshort-termfellowship(nr92352-JC);andIstitutoToscanoTumori(GrantNo.ITT-2007).IPATIMUPisanAssociateLaboratoryofthePortugueseMinistryofScience,Technology,andHigherEducationandispartiallysupportedbythePortugueseFoundationforScienceandTechnology.BothG.C.andJ.C.contributedequallytothiswork.

Authors’disclosuresofpotentialcon ictsofinterestandauthorcontributionsarefoundattheendofthisarticle.

Correspondingauthor:CarlaOliveira,PhD,IPATIMUP–CancerGenetics,RuaDrRobertoFrias,s/n;4200-465Porto,Portugal;e-mail:carlaol@ipatimup.pt.©2013byAmericanSocietyofClinicalOncology

0732-183X/13/3107-868/$20.00DOI:10.1200/JCO.2012.44.4612

ABSTRACT

Purpose

Theprognosisofgastriccancer(GC)ispoor,andthemolecularpathogenesisplayersarevastlyunknown.SurgeryremainstheprimaryoptioninGCtreatment.TheaimofthisstudywastoinvestigatetheimpactofsomaticCDH1alterationsinprognosisandsurvivalofpatientswithGC.

PatientsandMethods

AseriesofpatientswithsporadicandfamilialGC(diffuseandintestinal;n 246)wereanalyzedforsomaticCDH1mutations,promoterhypermethylation,andlossofheterozygosity(LOH)bypolymer-asechainreactionsequencing.E-cadherinproteinexpressionwasdeterminedbyimmunohistochem-istry.Associationsbetweenmolecular,clinicopathologic,andsurvivaldatawereanalyzed.

Results

CDH1somaticalterationswerefoundinapproximately30%ofallpatientswithGC.BothhistologictypesofsporadicGCdisplayedLOHin7.5%,mutationsin1.7%,andhypermethylationin18.4%ofpatients.PrimarytumorsfromhereditarydiffuseGC,lackinggermlineCDH1alterations,showedexclusivelyCDH1promoterhypermethylationin50%ofpatients.FamilialintestinalGC(FIGC)tumorsshowedLOHin9.4%andhypermethylationin17.0%.CDH1alterationsdidnotassociatewithaparticularpatternofE-cadherinexpression.Importantly,theworstpatientsurvivalrateamongallGCsanalyzedwasseeninpatientswithtumorscarryingCDH1structuralalterations,preferentiallythosebelongingtoFIGCfamilies.

Conclusion

CDH1somaticalterationsexistinallclinicalsettingsandhistotypesofGCandassociatewithdifferentsurvivalrates.TheirscreeningatGCdiagnosismaypredictpatientprognosisandislikelytoimprovemanagementofpatientswiththisdisease.

JClinOncol31:868-875.©2013byAmericanSocietyofClinicalOncology

INTRODUCTION

Gastriccancer(GC)isthesecondleadingcauseofcancer-relateddeathsworldwide,affectingclosetoonemillionpeopleperyear.1Themajorityofpa-tientswithGCpresentwithadvanceddiseaseatdi-agnosis(stagesIIIandIV),renderingtheprognosisextremelypoor,witha5-yearoverallsurvivalrateoflessthan25%.2-4MostpatientswithGCareasymp-tomaticduringtheearlystagesofdisease,thusdelay-ingtheinitialdiagnosis.Although,somestudieshavepinpointedimprovementsonpatientsurvivalwithperioperativeandadjuvanttreatmentmodali-ties,surgicalresectionisstilltheprimarycurativetreatmentforlocalizedGC;however,lessthan50%ofpatientsareeligibleforresection.5,6Duringthelastdecade,severalgeneticandepigeneticchanges

underlyinggastriccarcinogenesishavebeeneluci-dated.7,8Nonetheless,theirclinicalusefulnessislimitedbecauseofthelackofsystematicgenetic-clinicalcorrelations.

GCishistologicallyclassi edintotwomajortypes,intestinalanddiffuse.9Theintestinaltypeischaracterizedbywell-differentiatedglandularstruc-tures,whereasthediffusetypeconsistsofindividu-allyin ltratingneoplasticcellsthatmayhavesignetringmorphology,doesnotformglandularstruc-tures,andhasapoorerpatientprognosis.10,11ThemajorityofGCs(90%)appearinasporadicsetting.Theremaining10%ofGCsshowfamilialclustering,andofthese,only1%to3%constitutehereditaryforms.12AmongGCswithfamilialaggregationandwithhistologyinformation,thefollowingspeci csyndromescanbeidenti ed:familialintestinalGC

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StructuralCDH1AlterationsandPoorSurvivalinGastricCancer

(FIGC)andhereditarydiffuseGC(HDGC;OnlineMendelianInher-itanceinManNo.137215).13Sofar,nogermlinedefectshavebeenassociatedwithFIGC,whereasgermlinemutationsanddeletionsofE-cadherin(CDH1)aretheunderlyinggeneticdefectin45%offam-ilieswithclinicaldiagnosisofHDGC.14-16

Furthermore,70%ofCDH1mutation–negativeHDGCpro-bandsdisplaygermlinemonoallelicCDH1RNAdownregulation(al-lelicimbalance)reinforcingtheroleofCDH1locusinthisdisease.17HDGCtumorsappearwhencompletesomaticCDH1inactivationisacquired,leadingtoreducedorabsentE-cadherinexpression.18,19Thisoccursthroughsecondhitmechanisms,pursuingtheKnudson’smodeloftumorsuppressorgeneinactivation.20,21CDH1promoterhypermethylationisthemostfrequentsecondhitinactivationmech-anisminHDGCprimarytumors(50%to70%oftumors),whereasasecondmutationordeletion(lossofheterozygosity[LOH]/intragenicdeletions)waslessfrequentlyidenti ed.22-24

Inthesporadiccontext,thefrequencyofCDH1somaticmuta-tionsiscontroversial,andpercentagesbetween3%andgreaterthan50%havebeenreportedforGCswithadiffusecomponent.25-28LOHattheCDH1locuswasnotedindiffuseandintestinalhistotypes,withratesrangingfrom11%to39%and36%to46%,respectively.29-31EpigeneticsilencingthroughCDH1promoterhypermethylationhasbeenreportedinfrequenciesthatvaryfrom50%to83%indiffusetumorsandfrom6.25%to50%inintestinaltumors.28-32Importantly,thepresenceofconcomitantCDH1inactivationmechanismswasrarelydescribedinthissetting.28,29,33Nevertheless,mostofthesestud-ieshavebeenlimitedbytheseriessize,thetypeofmolecularmecha-nismsstudied,differentmethodologicapproaches,andthelackofextensivecorrelationswithclinicalparameters,thushamperingpo-tentialtranslationofgeneticdataintomedicalpractice.34

ThegoalofthisstudywastoanalyzetheimpactofsomaticCDH1alterationsinprognosisandsurvivalofpatientswithGC.Inthisstudy,weperformedacomprehensiveanalysisofsomaticCDH1mutations,LOH,andpromoterhypermethylationin246patientswithsporadicandfamilialGC.Additionally,weanalyzedtherelationshipbetweensomaticalterationsunderlyingCDH1inactivationandsurvivalrates,clinicopathologiccharacteristicsofpatientswithGC,andE-cadherinexpressionintumors.

materialusingthePuregeneDNAPuri cationKit(GentraSystems,Minne-apolis,MN).

CDH1PromoterHypermethylationAnalysis

CDH1promotermethylationanalysiswascarriedoutin160basepairsupstreamofthetranslationstartsite,encompassing17CpGsites,aspreviouslydescribed23(Appendix,onlineonly).PrimersequencesarelistedinAppendixTableA2(onlineonly).

CDH1SomaticMutationScreening

FiftynanogramsoftumorDNAfrom86diffuseGCsweresubjectedtomutationscreeningattheCDH1hotspotregion(exons7to10),aspreviouslydescribed.23Eachmutationwascon rmedbyanindependentpolymerasechainreactionampli cationfollowedbysequencinganalysis.Primerse-quencesarelistedinAppendixTableA2(onlineonly).

LOHAnalysis

LOHanalysiswasperformedusingthreeintragenicCDH1markers(thepromoter 161C/A[rs16260],theexon132076T/C[rs1801552],andthe3 -untranslatedregionsingle-nucleotidepolymorphisms[rs1801026])andthreeproximalanddistalmicrosatellitemarkers(D16S3025,D16S496,andD16S3067) ankingtheCDH1locus,asdescribed.23Onlyinformativemark-erswereconsideredforLOHanalysis,andpositivesampleswererepeatedtwice(Appendix).PrimersequencesarelistedinAppendixTableA2(onlineonly).

E-CadherinImmunohistochemistry

Formalin- xed,paraf n-embeddedtissuescomprising207GCswerestainedwithmouseE-cadherinmonoclonalantibodyHECD-1(dilution1:200;Zymed,SanFrancisco,CA)followedbyincubationwithavidin-biotincomplexanddiaminobenzidine.E-cadherinimmunoreactivitywasevaluatedontumorandnormaltissuesconsideringthepredominantexpressionpat-tern—normal(completemembranestaining),aberrant(cytoplasmicandhet-erogeneousstaining),orabsent(nostaining).

StatisticalAnalysis

Analyseswereperformedusingcommerciallyavailablestatisticalsoft-ware(SPSS,version14.0;SPSS,Chicago,IL).StatisticalassociationsbetweenclinicopathologiccharacteristicsandCDH1somaticalterationswereassessedusingthe 2testforcategoricalvariablesandthettestoranalysisofvarianceforcontinuousvariables.SurvivalcurveswereestimatedusingtheKaplan-Meiermethodandwerecomparedusingthelog-ranktest.MultivariateanalysiswasperformedusingaCoxproportionalhazardsregressionmodelbyconsideringthefollowingriskfactors:sex,age(oldervthemedianageoryounger),tumorlocation(othervantrum),Laurenhistotype(nonintestinalvintestinal),depthoftumorinvasion(pT2-4vpT1),lymphnodeinvolvement(pN1-3vpN0),presenceofsystemicmetastasis(M1vM0),andRcategory(R1orR2vR0).Postoperativemortalitywasassessed,withdeathsunrelatedtotumorrecur-renceconsideredcensoredobservationsatthetimeofdeath.P .05wasconsideredstatisticallysigni cant.

PATIENTSANDMETHODS

Patients

All246patientswithGCenrolledontothisstudywereadmittedattheDivisionofGeneralSurgeryandSurgicalOncology,UniversityofSiena(Siena,Italy)andwereclassi edashavingsporadic(n 174)orfamilial(n 72)GC.PatientswithfamilialGCwereclassi edashavingHDGC(n 19)orFIGC(n 53)usingclinicalcriteriade nedbytheInternationalGastricCancerLinkageConsortium13(AppendixTableA1,onlineonly).ThepresenceofCDH1germlinemutationswasdiscardedinallpatientswithHDGCinthiscohort.PatientswithGCclassi cationmorethanpT1N http://wendang.chazidian.comrmedconsentwasobtainedfromallpatients,andthestudywasapprovedbythehospital’sethicscommittee(Appendix,onlineonly).

DNAExtraction

TissueareasforDNAextractionwerehistologicallyveri edtocontainaminimumof70%to80%ofneoplasticcells.DNAwasisolatedfromfrozen

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RESULTS

AfterscreeningofsomaticCDH1promoterhypermethylation,LOH,andmutationsinexons7to10,GCswereclusteredinthefollowinggroupsregardingCDH1alterations:patientswithmethylationonly(namedepigenetic);patientswithLOHormutation,with/withoutmethylation(namedstructural);andpatientswithoutCDH1altera-tions(namednegative).

CDH1StructuralandEpigeneticAlterationsinOverallGC

Overall,77(31.3%)of246GCscarriedsomaticCDH1altera-tions.Epigeneticalterationswerefoundin51(20.7%)of246GCs,andstructuralalterationsweredetectedin26(10.6%)of246GCs.Specif-icallywithinstructuralalterations,LOHalonewasdetectedin18

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(7.3%)of246GCs,mutationalonewasfoundinthree(1.2%)of246GCs(c.1109A G[p.Asp370Gly],c.IVS9 5G A,andc.1105_1106insACCAAC),and ve(2%)of246GCspresentedLOHconcomi-tantlywithCDH1promoterhypermethylation(Table1,AppendixFigsA1andA2,onlineonly).

PatientswithtumorswithCDH1structuralalterationsdisplayedpooreroverallsurvival(P .017;Fig1;allcases)thanpatientswithnegativetumorsortumorscarryingCDH1epigeneticalterations.CDH1StructuralandEpigeneticAlterationsin

DifferentClinicalSettingsandHistologicTypesofGC

WeevaluatedthedistributionofsomaticCDH1alterationsamongclinicalsettings(sporadicandfamilial)andhistotypes(intes-tinalanddiffuse)ofGCandcorrelatedthese ndingswithoverallpatientsurvival.TheoverallfrequencyofCDH1alterationswassimi-larinsporadic(51of174tumors;29.3%)andfamilial(26of72tumors;36.1%)tumors(Table1).Speci callywithinthesporadic

Fig1.Kaplan-Meiercurvesshowingtheprobabilityofoverallsurvivalforpatientswithgastriccancer(GC),accordingtoCDH1alterations(epigenetic,structural,ornegative)andstrati edaccordingtoGCclinicalsettings(sporadicorfamilial)andhistologictype(intestinalordiffuse).870

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胃癌体细胞突变

StructuralCDH1AlterationsandPoorSurvivalinGastricCancer

setting,32(18.4%)of174GCsdisplayedepigeneticalterationsand19(10.9%)of174GCspresentedstructuralalterations.Withinthefamil-ialsetting,19(26.4%)of72GCsharboredepigeneticalterations,whereasseven(9.7%)of72GCsdisplayedstructuralalterations(Table1;AppendixTableA3,onlineonly).Nosigni cantcorrelationwasobservedbetweenGCclinicalsettingsandoverall/speci cCDH1al-terations(P .05;AppendixTableA4,onlineonly).However,pa-tientswithtumorswithCDH1structuralalterations,frombothsporadicandfamilialsettings,hadapoorersurvivalratethanpatientswithnegativetumorsorwithCDH1epigeneticalterations(P .046andP .002,respectively;Fig1).

RegardingGChistotypesindependentoftheclinicalsetting,theoverallfrequencyofCDH1alterationswas27.5%(44of160GCs)inintestinalGCsand38.4%(33of86GCs)indiffuseGCs(Table2).Whereaswithinintestinaltumors,15%carriedepigeneticalterationsand12.5%carriedstructuralalterations,indiffusetumors,31.4%carriedepigeneticalterationsand7%carriedstructuralalterations.Althoughdiffusetumorscarriedepigeneticalterationsmoreoftenthanstructuralalterations(P .007;Table2),thesemolecularfea-turesdidnotconferdifferentoverallsurvival(P .230,Fig1).Incontrast,andalthoughintestinaltumorscarriedsimilarfrequenciesofepigeneticandstructuralalterations,Kaplan-Meierplotsshowedthatpatientswithintestinaltumorscarryingstructuralalterationshadalowerprobabilityofsurvivalthanpatientswithintestinaltumorsneg-ativefororcarryingCDH1epigeneticalterations(P .041;Fig1).Infactfromthe26GCswithstructuralalterations,20(76.9%)wereoftheintestinaltype(P .007;Table3).

Weobservedthatwithinsporadicandfamilialsettings,pa-tientswithtumorscarryingstructuralalterationshadworseoverallsurvival.Moreover,wealsoveri edthatpatientswithintestinal-typetumorscarryingstructuralalterations,independentoftheclinicalsetting,hadworseoverallsurvival.Therefore,wenextassessedwhethertheworseoverallsurvivalofpatientswithintes-tinaltumorswithstructuralalterationswasafeatureoffamilialand/orsporadicsettings.

Intestinaltumorsbelongingtosporadicandfamilial(FIGC)set-tingsdidnotdifferinthefrequencyofepigenetic(14%sporadic,17%FIGC)andstructuralalterations(12.1%sporadic,13.2%FIGC;Table1,AppendixTableA3,Fig2).Nonetheless,theKaplan-MeierplotsshowedstatisticaldifferencesintermsofoverallsurvivalonlyforpatientsbelongingtoFIGCfamilies.ThepatientswithFIGCcarryingtumorswithstructuralalterationshadthepoorestsurvivalrate(P .001;Fig2).Althoughitdidnotreachstatisticalsigni cance(P .148),patientswithintestinaltumorsbelongingtothesporadicsettingandcarryingstructuralalterationsalsohadapoorersurvival

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ratecomparedwithpatientscarryingnegative/epigeneticalterations(Table1,AppendixTableA3,Fig2).

Withindiffuse-typetumors,tumorsbelongingtothesporadicsettingdisplayedepigenetic(25.4%)andstructural(8.9%)alterations,whereasHDGCtumors(52.6%)harboredexclusivelyepigenetical-terations(Table1,AppendixTableA3).Kaplan-MeierplotsshowednoassociationbetweenthetypeofCDH1-speci calterationsandoverallsurvivalofpatientswithsporadicdiffuseorHDGCtumors(P .259andP .631,respectively;Fig2).However,despitethelackofstatisticalsigni cance,thefewpatientswithsporadicdiffuse-typetumorscarryingstructuralalterationshadthelowestsurvivalrates(P .259;Fig2).

CDH1StructuralandEpigeneticAlterationsandClinicopathologicFeaturesofGC

Wenexttriedtounderstandwhetherthepresenceofoverallorspeci cCDH1alterationscorrelatedwithclinicopathologicparame-tersoftumorsandpatients(Table3).ThisanalysisrevealedthatpatientswithtumorscarryingoverallCDH1alterationsdisplayedsigni cantlymorefrequentlylymphnodemetastases(P .021)andmoreadvancedtumors(P .029),inparticularinvasivestageIII.Theotherclinicopathologicparameters,includingage,sex,liverorperito-nealmetastases,radicalityofresection,extentofgastrectomy,lymph-adenectomy,depthofinvasion,familialaggregation,andevenLaurenhistotype,werenotsigni cantlyassociatedwiththepresenceofoverallCDH1alterations(Table3).

Whenconsideringthespeci ctypeofCDH1alterationsinthedifferentGCgroups(epigeneticvstructuralvnegative),weobservedthatpatientscarryingtumorsdisplayingepigeneticCDH1alterationsmorefrequentlyhadthediffusehistotype(P .007)and84.3%hadlymphnodemetastases(P .02).WealsoobservedthatpatientscarryingtumorswithstructuralCDH1alterationsmorefrequentlyhadintestinalhistotype(20of26patients,76.9%;P .007)andweremainlymales(23of26patients,88.5%;P .004),and46.1%hadbeensubmittedtoR1/2resections(comparedwith21.6%withepigeneticalterationsand24.8%negativeforalterations;P .09;Table3).CDH1StructuralandEpigeneticAlterationsandE-CadherinImmunoexpressionPattern

WealsoevaluatedE-cadherinexpressionandcorrelateditwithtumorhistologyandCDH1alterations.Of207patientsanalyzed,73.4%showedaberrantexpression,14.5%displayedcompletelossofE-cadherin,and12.1%retainedtheproteinatthecellmembrane(Table4).Inbothsporadicandfamilialsettings,therewasasigni cant

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associationbetweencompletelossofE-cadherinexpressionanddif-fusehistology(sporadic:P .001;familial:P .017;Table4).

Inaddition,therewerenodifferenceswhencomparingE-cadherinimmunohistochemistry(IHC)patterninGCscarryingoverallorspeci cCDH1alterationswiththepatterninthosewithoutalterations(P .479andP .275,respectively;Table3).TheseresultsrevealedthattheCDH1alterationsanalyzeddonotgenerateaspeci cE-cadherinIHCpatternand,moreimportantly,that60%ofGCswithoutE-cadherinexpressionandapproximately70%ofGCswith

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aberrantexpressionarenegativeforCDH1alterations(AppendixTableA5,onlineonly).

DISCUSSION

GCisahighlyheterogeneousdiseasewhereevensimilarclinicalandpathologicfeaturesleadtodistinctoutcomes.7,9,11,36Theseobserva-tionsindicatedthatstagingsystems,basedonclinicalandpathologic

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