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small Cell Lung Cancer Receiving Cisplatin-based Therapy

By Julia Ferguson,2014-02-18 21:21
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small Cell Lung Cancer Receiving Cisplatin-based Therapysmall,Cell,Lung,cell,lung,Small

    small Cell Lung Cancer Receiving

    Cisplatin-based Therapy

    29ChinJCancerRes22l4):296302,2010www.springerlink.com

    HighExpressionofERCC1IsaPoorPrognosticFactorinChinese

    PatientswithNonsmallCellLungCancerReceiving

    Cisplatin-basedTherapy

    Qing-zhiGuo,JieWang,HuaBai,TongtongAn,MeinaWu,JunZhao,LuYang

    JianchunDuan,YuyanWang,Zhi-jieWang

    KeylaboratoryofCarcinogenesisandTranslationalResearch(MinistryofEducation),Departmentof

    MedicalOncology,PekingUniversityCancerHospital&Institute,Beijing100142,China

    CLCnumber:R734.2Documentcode:AArticleID:1000-9604(2010)04.0296.07 DoI:1O.1007,s11670.010.0296.0

    @ChineseAntiCancerAssociationandSpringer-VerlagBerlinHeidelberg2010 ABSTRACT

    objective:todeterminetheroleofexcisionrepaircross

    complementinggrouplgene(ERCCI)and

    ribonucleotidereductasesubunitM1(RRM1)expressioninpredictingresponseandsurvivalinChinesepatientswith

    advancedstagenonsmallcelllungcancer(NSCLC)treatedwithplatinum

    basedchemotherapy.

    Methods:Formalinfixed,paraffin

    embeddedbiopsytissueswereretrosDective1vobtainedfrom160advanced NSCLCpatients.mRNAexpressionlevelsOfERCC1andRRM1weredeterminedbyrea1.timePCR.

    Results:AssociationsbetweenmRNAexpressionlevelofERCClandRRM1andclinic.path

ologicparameters

    wereanalyzed.Onehandredandfortytwo(88.75%)specimensweresuccessfullyamplified.mRNAexpressionlevel

    ofERCClandRRMlwasnegativelyassociatedwithtumorresponse.ERCC1expressionlevelsrangedfrom0.01t0

    78.79[median0.63,mean4.25andstandarddeviation(SD)9.23

    andRRM1from0.00to30.9l(median0.63,mean

    0.87andSD3.36).Byadoptingcutoffvaluesaccordingtomedianexpressionlevels.wefoundthatMSTinpatients

    withlowERCCImRNAlevelswassignificantlylongerinoverallpopulationthaninpatientswithhigherlevels(18.63

    versus13.69months,log

    rank5.73,P=0.017),butwedidnotfoundthesurvivalbenefitofthepatientswithlow expressionlevelofRRM1.(19.07versus14.88,log

    rank1.66,P=O.197).Further,inthemultivariableCoxregression

    modelanalysiswefoundthatlowlevelofERCC1expression,thepresenceofweightlossandtargettherapy,were

    significantprognosticfactorsforsurviva1.

    Conclusion:ERCC1expressionisassociatedwithpatients'survivalinChineseadvancedNSCLCpatientstreated

    withplatinum

    basedregimenandmayserveasabiomarkerinpredictingtumorresponseandclinicaloutcomeinthe

    patientpopulation.

    Keywords:ERCC1;RRM1;NSCLC;Platinum-basedchemotherapy;mRNA;RealtimePCR

    INTRoDUCTIoN

    Lungcancerisamostcommonmalignanttumor

    worldwide.AccordingtotheWoridHealth

    Organization(WHO)in2002,thereare1.35million

    newcasesof1ungcancer.and1.18milliondeaths. accountingfor20.0%incidenceoftotalcancer incidence.23.8%ofcancerdeamsintheworld?J.In

    Chinatheincidenceandmortalityoflungcancerare Received20100329:Accepted2010-0902

    Correspondingauthor.

    Email:wanie_

    cc@yahoo.com

    alsoranksfirstincancer,andcontinueupwardtrend. Itispredictedthatby2010.thenumberofnewcases oflungcancerinChinawillreach441.000with 378,000deaths,beingtheleadingcauseofdeaths fromcancer.Foradvancednonsmallcelllung cancer(NSCLC).platinum.basedregimenchemo. therapyisstillthemaintreatment.andplatinum.based regimenarerecommendedasstandardfirst.1ine setting.butthecurrentefficiencyofchemotherapyis only20%40%.withmediansurvivaltimeof810

    months.Mostofpatientshadthesideeffectsof chemotherapy,butnoeffect.Thepossiblereasonof www.springerlink.comChinJCancerRes224):296-302,2010297 thesignificantdifferencesinefficacyinthe chemotherapyofnonsmallcelllungcancerliesinthe diflferenceofpharmacogenomicswhichresultsin differentdrugefficacy.Thus.accordingtothe djfferentconditionsofeachpatient.todevelop specifictreatmentprogramstogivethemost appropriateandeffectiveindividualizedtherapyisthe trendofthelungcancertreatment.

    Cisplatincancausemonoadductsandintrastrand

orinterstrandcrosslinksinDNAJ.thereby

    preventingitsreplicationandtranscription,leadingto DNAdamageandcelldeath.TheDNAlesionscanbe repairedbyDNArepairedmechanismsandactivities ofmoleculesmayimpacttreatmentactivityof platinum.Theexcisionrepaircrosscomplement.

    tation1(ERCCl1.astructurespecificDNArepair

    endonuc1easeresponsibleforthe5primeincision.is

    oneofthekeyenzymesofnucleotideexcisionrepair (NER)pathway.Anumberofstudieshaveshown thatthelowerexpressionlevelsOfERCC1mRNA.the longersurvivalinpatients,suggestingthatitmaybea predictorofeicacy.prognosticandsurvivalin NSCLCpatientswithcisplatin.containingchemo. therapy0.

    RibonucleotidereductaseMlfRRM11isakey enzymeinvolvedinDNAsynthesis,catalyzingthe biosvnthesisofdeoxyribonucleotidesfromthecor

    respondingribonucleotides,thusplaysanimportant roleintheDNAsynthesisandrepair.Dataindicate thathigherlevelsOfRRM1areassociatedwith chemoresistancetogemcitabine.basedtherapies. UpregulationofRRMlhasbeenpreviouslyobserved pre..clinicallyindifferentgemcitabine..resistant humancelllinesL141.Thepatientswithhigh expressionOfRRM1havealongersurvivalthanthat withlowexpressionofRRM[5-.

    Theaimofourworkistovalidate,ina

    retrospectivestudy,theprognosticandpredict relevanceofERCC1andRRM1mRNAexpression

    levelsinChineseadvancedNSCLCpatientstreated withcisplatin.basedtherapies.

    MI'ERIALSANDMETHoDS

    PatientsandSamples

    Atotalof160formalin.fixed.paraffinembedded

    (FFPE)bronchoscopeorfineneedleaspiration(FNA) biopsytumorsampleswereanalyzedtodetermine ERCC1andRRM1mRNAexpressionlevels.They wereobtainedfromtheunresectablelocaladvanced andmetastaticNSCLCpatientswhoweretreatedin BeringCancerHospital(ThoracicOneologyUnit) betweenSeptember2001andOctober2008.

    enrolledpatientshadclinicaland

    pathologicalinformationincludingage,gender,date ofinitialdiagnosisanddateofdeathorlastfollowup.

    Inaddition,thepatientsenrolledinthisstudymetthe followingcriteria:receivedtwocyclesoffirst.1ine cisplatinbasedchemotherapyandhavingmeasureable lesionsandavailableparaffinembeddedtissue

    samples.ThestudywasapprovedbyBeijingcancer hospitalInstientionalReviewBpard.

    Assessmentof0bjectiveResponseandClinical 0utcomes

    Thetumorresponsewereassessedandconfirmed bycomputertomography(CT),brainmagnetic resonanceimaging(MRI)andbonescintigraphy scanatbaselineandaftereverytwotreatmentcycles. Designationsofcompleteresponse(CR),partial response(PR),stabledisease(SD),andprogressive disease(PD),werebasedonthestandardizedresponse

    definitionsestablishedbyresponseevaluationcriteria insolidtumors(RECIST).Theresponserate(RR)was definedastotalnumberofCRandPRpatientsdivided bythenumberofevaluablepatients.Timeto progression(TTP)wascalculatedfromthedateof initiationofchemotherapytothedateofdisease progression.0verallsurvival(OS,wascalculatedas thetimebetweenthebeginningofchemotherapyand deathorcensoredatlastfollow-up.

    RealtimePCRDetection

    Samplepretreatment

    Preliminarily.archiveslidesofthecollected tumorsampleswerereviewedbypathologists.From eachparaffinblockofrepresentativetumorareas. serialsectionswithathicknessof4urnwereprepared. Malignantcellswereselectedundermicroscope magnification(fromx5tox10)anddissectedfromthe slidesimplybyusingascalpelL.

    RNAisolationandcomplementaryDNAsynthesis RNAwasisolatedfromformalin.fixed.paraffin

    embeddedspecimensusinganovel,proprietary procedure(MiRNeasyFFPE.AfterRNAisolation. cDNAwasderivedfromeachsampleaccordingtoa kitdescribedprocedure(usingthecompanykit)and storedin20.C.

    Reversetranscription-polymerasechainreaction RelativecomplementaryDNAquantitationfor ERCC1,RRM1andaninternalreferencegene 298ChinJCancerRes22J296-302,2010www.springerlink,com

(pactin)wasdoneusingSYBRgreenrealtime

    fluorescentquantitativePCRdetectionmethod SteponeSequenceDetectionSystemApplied Biosystems].

    Thesequencesoftheprimersusedwereas follows:

    ERCClforward5'.GGGAATTTGGCGACGTA

    ATTC.3'.

    reverse5'.GCGGAGGCTGAGGAACAG3'.

    RRMlforward5'AAGAGCAGCGTGCCAGA

    GAT3'.

    reverse5'.ACACATCAAAGACCAGTCCT

    GATTAG.3'.

    B.actin(internalreferencegene)forward5'.TGA GCGCGGCTACAGCTT.3'.

    reverse5'.TCCTTAATGTCACGCACGATT3'.

    9.15,22]

    A11primersusedinthisstudywereintron spanningtoavoidgenomicDNAcontamination.The PCRmixtureconsisted2xPOWERSYBRGREEEN PCRMASTERMIX7.5LLl,cDNA3ul,water4.5plto afinalvolumeof15plfallreagentswerefromPE AppliedBiosystems,FosterCity,CA).

    Cyclingconditionswere500Cfor2minand 95.Cfor10minandfollowedby45cyclesat950C for15sand60.Cfor1minfFluorescencesigna1was collected)andthemeltcurvewasdonetoverifythe productsOfPCR.Relativegeneexpressionlevelsare expressedasratios(differencesbetweentheCt values)betweentwoabsolutemeasurements(genesof

    interest/internalreferencegene).HT-29totalRNA (from)wereusedascontrolcalibratorsoneachplate. Datawereanalysisbythemethodof2'CT.

    rF=2.[cT(g.tge"e).CT(I3-actin)CT(positiegene)CT(p'actin)],.

    Byadoptingcutoffvaluesaccordingtomedian

    expressionlevels.wedividedERCC1andRRMlinto highexpressionandlowexpression[.,,]

    StatisticalAnalysis

    Totestsignificantassociationsbetweenthe continuousvariable'geneexpression'and dichotomousvariablesfpatient'sage.sex.tumor stage,etc.).theMannWhitneytestwasused.The

    Kruska1.llistestwasusedtotestsignificant associationswithmultiplevariablesashistology. Spearmancorrelationcoefficientswerecalculatedto estimatethecorrelationbetweenERCC1andRRM1 mRNAle(els.Kaplan-Meiersurvivalcurvesandthe logranktestwereusedtoanalyzeunivadate distributionsforsurviva1.Cox'sproportionalhazards multivariateanalysiswasusedtoevaluatewhichof thesignificantfactorsattheunivariateanalysishada significantinfluenceonsurviva1.Statistical significancewassetatP=0.05.

    RESUIS

    PatientCharacteristics

    Therewere142(88.75%)specimenswere

    successfullyamplified,andtheremaining18samples werenotquantifiablebecauseoftheminimalamounts ofavailabletissueorbecauseoflargeamountsof necrosisinthetumorsample.Thecharacteristicsof

    the142patientsareshowninTable1.A11thepatients receivedcisplatinbasedchemotherapy.Onehundred

    andeightwerestageIVand34stageIIIBdisease.At thetimeofthesurvivalanalysis,89patientshaddied and53werestillalive.Medianfollow.uptimewas 10.97months.0verallmediansurvivaltimefMST)is 13.27months.

    Table1.Patients'characteristics

    :NPVinOre1bine,cisplatin;?:GPGemcitabine/cisplatin

    TPpaclitaxellcisplatin

    wwu:springerlinkcornChinJCancerRes22f4):296-302.2010299

    ERCCI,RRM1ExpressionLevelsinAdvanced NSCLC

    Real.timePCRwasusedtodetectmRNA

    quantificationofERCCl/RRMlandD.actinwasused astheinternalreferencegene.ERCClexpression levelsrangedfrom0.01t078.79fmedian0.63.mean 4.25andstandarddeviationrSD)9.231andRRMl from0.00to30.91(median0.63.mean0.87andSD 3.36,.Byadoptingcutoffvaluesaccordingtomedian

    expressionlevels,nocorrelationwasfoundbetween age(P=0.440),sex(P=0.07),histology(P=0.283), stage(JD=0.668),presenceofweightlOSSfP=0.07,aIl byMannWhitneytest.exceptthatKruskal-WaIlis testwasusedforhistologY1andERCC1expression. SimilarresultswereobservedbetweenRRMlgene expressionandanyoftheclinical-pathological factorsconsidered.Inaddition,nocorrelationwith geneexpressionprofilesandresponseto

    chemotherapywasfoundfP=0.261.Kruskal-Wallis

    test1.WefoundacorrelationbetweenERCCland RRMl(rs=0.380,P<0.0001),bymeansofthe Spearman'srankcorrelationmethod.fTheresultsof MatchingERCClandRRMlexpressionlevelswere obtainedbytherealtimePCRforeverysample). Therewasabetterdiseasecontrolrateinpatients withlowexpressionofERCC1thanthatofhigh expression.Butwedidnotfindrelationsbetween diseasecontrolrateandtheexpressionOf RRM1(Table2,.

    Table2.ResponseandDCR

    'P=0.

    021:"P=0.290MannWhitneytest

    CorrelationofERCC1andRRM1mRNALevels withTTPandOverallSurvival

    WeusedMedianERCClgeneexpressionvalue asacut.offatunivariatemodelandfoundthatMSTin patientswithlowERCC1mRNAlevelswas significantlylongerinoveral1populationthanin patientswithhigherlevelsf18.63versus13.69 months,1ogrank5.73,P=0.0l7,Figure11.

    

    

    

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