张敏跃 分子生物学进展 肿瘤标志物.ppt

,DiscoveryofCancerBiomarkers,Statistics,Morethan11millionpeoplearediagnosedwithcancereveryyear.Itisestimatedthattherewillbe16millionnewcaseseveryyearby2020.Fromatotalof58milliondeathsworldwidein2005,canceraccountsfor7.6million(or13%)oftheglobalmortality.Deathsfromcancerintheworldareprojectedtocontinuerising,withanestimated9millionpeopledyingfromcancerin2015and11.4milliondyingin2030.IntheUSin2006,over1.4millionnewcasesofcancerwerediagnosed.Overhalfamillionpeoplediedfromthisdisease,accountingforapproximately25%ofalldeathsintheUSeachyear,HowtoImprovetheSituation?,PreventionDetectionCancerisadiseaseofgeneticprogressionthatisoftenassociatedwithspecificmolecular,geneticandhistologicalchanges.Theabilitytodevelopbiomarkersthatcandetectthecriticalcomponentsofthesehallmarksofcancertogetherprovidesapowerfulbasisfordiagnosing,monitoringandpredictingoutcomeandresponsetotreatment.,HowtoImprovetheSituation?,Thegoalofcancerbiomarkerfieldistodevelopsimplenon-invasiveteststhatindicatecancerrisk,allowearlycancerdetection,classifytumorssothatthepatientcanreceivethemostappropriatetherapyandmonitordiseaseprogression,regressionandrecurrence.3.Treatment,ConceptofCancerBiomarkers,DefinitionofbiologicalmarkersBiologicalmarkers(biomarkers)havebeendefinedbyHulkaandcolleagues(1990)as“cellular,biochemicalormolecularalterationsthataremeasurableinbiologicalmediasuchashumantissues,cells,orfluids.”HulkaBS.Overviewofbiologicalmarkers.In:Biologicalmarkersinepidemiology(HulkaBS,GriffithJD,WilcoskyTC,eds),pp315.NewYork:OxfordUniversityPress,1990.Morerecently,thedefinitionhasbeenbroadenedtoinclude“biologicalcharacteristicsthatcanbeobjectivelymeasuredandevaluatedasanindicatorofnormalbiologicalprocesses,pathogenicprocesses,orpharmacologicalresponsestoatherapeuticintervention”NaylorS.Biomarkers:currentperspectivesandfutureprospects.ExpertRevMolDiagn3:525529,2003.,ConceptofCancerBiomarkers,2.FormsofcancermarkersHormones,metabolites，aswellasdifferentfunctionalsubgroupsofproteinssuchasenzymes,glycoproteins,oncofetalantigensandreceptors.Furthermore,otherchangesintumors,suchasgeneticmutations,amplificationsortranslocations,andchangesinmicroarray-generatedprofiles(geneticsignatures),arealsoformsoftumormarkers.Themarkersareproducedeitherbythetumoritselforbyothertissues,inresponsetothepresenceofcancerorotherassociatedconditions,suchasinflammation.Cancerbiomarkerscanalsobeprocessessuchasapoptosis,angiogenesisorproliferation.,ConceptofCancerBiomarkers,3.FactorsthatareidealforatumormarkerProducedbythetumorcellsandentersthecirculationPresentatlowlevelsintheserumofhealthyindividualsandthosewithbenigndiseasebutincreasessubstantiallyincancer(preferablyinonecancertypeonly)EasilyquantifiablewithaninexpensiveassayPresentindetectable(orhigherthannormal)quantitiesatearlyorpreclinicalstagesQuantitativelevelsofthetumormarkerreflectthetumorburdenHighdiagnosticsensitivity(fewfalsenegatives)andspecificity(fewfalsepositives),ConceptofCancerBiomarkers,3.Factorsthatareidealforaserologicaltumormarker,ConceptofCancerBiomarkers,4.Typesofcancerbiomarkers4.1.Diagnostic(screening)biomarkerAmarkerthatisusedtodetectandidentifyagiventypeofcancerinanindividual.ThesemarkersareexpectedtohavehighspecificityandsensitivityForexample,thepresenceofBenceJonesproteininurineremainsoneofthestrongestdiagnosticindicatorsofmultiplemyeloma,ConceptofCancerBiomarkers,4.2.PrognosticbiomarkerThistypeofmarkerisusedoncethediseasestatushasbeenestablished.Thesebiomarkersareexpectedtopredicttheprobablecourseofthediseaseincludingitsrecurrence,andtheythereforehaveanimportantinfluenceontheaggressivenessoftherapy.Forexample,intesticularteratoma,humanchorionicgonadotropinandalfa-fetoproteinlevelscandiscriminatetwogroupswithdifferentsurvivalrates.,ConceptofCancerBiomarkers,4.3.Stratification(predictive)biomarkerThistypeofmarkerservestopredicttheresponsetoadrugbeforetreatmentisstarted.Thismarkerclassifiesindividualsaslikelyrespondersornonresponderstoaparticulartreatment.Thesebiomarkersmainlyarisefromarray-typeexperimentsthatmakeitpossibletopredictclinicaloutcomefromthemolecularcharacteristicsofapatientstumor.,Currentapplicationsoftumormarkersandtheirlimitations,Currentapplicationsoftumormarkersandtheirlimitations,Cancerbiomarkersthatarecurrentlyinclinicaluse,Cancerbiomarkersthatarecurrentlyinclinicaluse,Theclinicalphasesoftestinganewcancerdrug,Phase1Determinationsoftoxicity,pharmacokinetics,andoptimaldoselevelsphase2DeterminationsofbiologicefficacyPhase3Definitivecontrolledtrialsofeffectsonclinicalendpoints.Foreachphase,guidelinesexistforsubjectselection,outcomemeasures,relevantcomparisonsforevaluatingstudyresults,andsoforth.,PhasesofbiomarkerdevelopmentMargaretSP;etal.UniversityofWashington(2001),1.PreclinicalexploratorystudiesPrimaryAims1)Toidentifyleadsforpotentiallyusefulbiomarkers.2)Toprioritizeidentifiedleads.Inthisphase,tumorandnon-tumorspecimensarecompared.Strategiessuchasgeneexpressionprofiling,mass-spectrometry-basedmethodsandotherapproachestobiomarkerdiscoverycanbeusedToidentifygenesorclustersofgenes(orproteins)thatappeartobeoverexpressedorunderexpressedintumortissuerelativetocontroltissue.Toidentifycharacteristicsuniquetotumortissuethatmightleadtoideasforclinicaltestsfordetectingcancer.Thedevelopmentofstatisticalalgorithmsforselectingpromisingbiomarkersfromalargepoolofbiomarkersisanactiveareaofresearch.,PhasesofbiomarkerdevelopmentforearlydetectionMargaretSP;etal.UniversityofWashington(2001),1.PreclinicalexploratorystudiesSpecimenSelectionTumortissuefromcasesubjectsshouldbeobtainedatdiagnosisandbeforetreatmentbecausetreatmentmayinterferewiththebehaviorofthebiomarker.Noncancercontrolsubjectsshouldbeselectedsothatfactorspotentiallyinfluencingthebiomarker,otherthanthecanceritself,aretightlymatchedtothoseofthecancercasesubjects.Thesefactorsmightincludeage,sex,race,andpossiblylifestyle-relatedcharacteristics,suchassmokinghabits.,PhasesofbiomarkerdevelopmentforearlydetectionMargaretSP;etal.UniversityofWashington(2001),1.PreclinicalexploratorystudiesSpecimenSelectionFactorsshouldbeconsideredwhenselectingtumorSpecimen1，样品的一致性：取材部位、肿瘤亚型、年龄、性别、种族、生活习惯等。2，样品处理方式的一致性：预处理条件、保存条件（包括时间）、处理条件、操作等。Factorsshouldbeconsideredwhenselectingnontumor(control)Specimen1，对照样品和肿瘤样品的对等性2，对照样品和肿瘤样品的处理方式的对等性,PhasesofbiomarkerdevelopmentforearlydetectionMargaretSP;etal.UniversityofWashington(2001),PreclinicalexploratorystudiesSampleSizesThenumberdependsontheobjectiveofthestudyandtheextentofthevariabilityofthebiomarkerinthestudy.Thefollowingfactorscontributetovariability:thenumberandrelativeprevalenceofthecancersubtypesamongthestudysamplesthecapacitiesofthebiomarkerstodiscriminateamongthedifferentcancersubtypesthenumberofbiomarkersunderstudythenumberofcaseandcontrolsubjectsandthestatisticalalgorithmusedtoselectpromisingbiomarkers.,PhasesofbiomarkerdevelopmentforearlydetectionMargaretSP;etal.UniversityofWashington(2001),2.AssaydevelopmentandvalidationPrimaryAimToestimatetheTPRandFPRorROCcurvefortheclinicalbiomarkerassay,toassessitsabilitytodistinguishsubjectswithcancerfromsubjectswithoutcancer.Aclinicalassaythatusesaspecimenofchoice(usuallysomethingthatcanbeobtainednoninvasively)isdevelopedinthisphase.Thepatientsassessedinthisphasehaveestablisheddisease.Theutilityoftheassayindetectingdiseaseearlyisnotdemonstratedinthisphase.,PhasesofbiomarkerdevelopmentforearlydetectionMargaretSP;etal.UniversityofWashington(2001),3.RetrospectivelongitudinalclinicalrepositorystudiesPrimaryAims1)Toevaluate,asafunctionoftimebeforeclinicaldiagnosis,thecapacityofthebiomarkertodetectpreclinicaldisease.2)Todefinecriteriaforapositivescreeningtestinpreparationforphase4.Repositoriesofclinicalspecimens,collectedandstoredfromacohortofapparentlyhealthysubjectsmonitoredfordevelopmentofcancer,areusedinphase3ofthebiomarkerevaluation.,PhasesofbiomarkerdevelopmentforearlydetetionMargaretSP;etal.UniversityofWashington(2001),4.ProspectivescreeningstudiesPrimaryAimTodeterminetheoperatingcharacteristicsofthebiomarkerbasedscreeningtestinarelevantpopulationbydeterminingthedetectionrateandthefalsereferralrate.Inthisphase,individualsarescreenedwiththeassayanddiagnosticproceduresareappliedtothosewhoscreenedpositive.Thiscanhelptoestablishthetumorstageorthenatureofthediseaseatthetimeofdetection.,PhasesofbiomarkerdevelopmentforearlydetectionMargaretSP;etal.UniversityofWashington(2001),5.RandomizedcontroltrialsPrimaryAimToestimatethereductionsincancermortalityaffordedbythescreeningtest.,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,Genomiclevelcancerbiomarkerdiscovery1.1.GenomicaberrationSequencing:TheCancerGenomeAtlas(TCGA)isapplyinglarge-scalegenomesequencingtechnologytoidentifynovelgenesinvolvedincancerpathogenesis.Comparativegenomichybridization(CGH)array-CGH(aCGH)Spectralkaryotyping(SKY)1.2.SNPSequencingSNParray1.3.Epigeneticalternations,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,2.Transcriptionallevelcancerbiomarkerdiscovery2.1.mRNAexpressionprofilecDNA-micro-array,Oligo-micro-arrayDifferentialdisplay-PCR(DD-PCR)Serialanalysisofgeneexpression(SAGE),cDNALibrarySubtraction,etc.,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,2.Transcriptionallevelcancerbiomarkerdiscovery2.2.miRNAPotentialimportanceofmiRNAsascancerbiomarkersExpressionofmicroRNAs(miRNAs)invarioustissueshasbeenassociatedwithavarietyofdiseases,includingcancers.SerummiRNAscontainfingerprintsforvariousdiseases.RelatedtechniquesSequencingmiRNA-array,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,3.Translationallevelcancerbiomarkerdiscovery3.1.Protein(orsubtypes:enzymes,antibodies,secretedproteins,etc)2-dimensioalelectrophoresis/massspectrometry(2-DE/MS)Surface-enhancedlaserdesorptionionizationtime-of-flightmassspectrometrytechnology(SELDI-TOP-MS):proteomicpatterndiagnosticsMulti-dimensionalproteinidentificationtechnology(MudPIT)/MS,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,PrincipleofSELDI-TOF-MS,Onemicrolitreofraw,unfractionatedserumisappliedtothesurfaceofaprotein-bindingchip.Thechipisrinsedtoremoveunboundproteins,treatedwithaMATRIXCOMPOUND,washedanddried.Alaserirradiatesanddesorbstheadherentproteins.Thetime-of-flight(TOF)oftheionisdetectedbyanelectrode.Aproteomicsignatureoftheserumiscreated.,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,ProteomicpatterndiagnosticsWiththisapproach,theunderlyingidentityoftheindividualcomponentsofthepatternisnotnecessaryforitsuseasapotentialdiagnosticfordisease.,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,3.Translationallevelcancerbiomarkerdiscovery3.1.Protein(orsubtypes:enzymes,antibodies,secretedproteins,etc)2-dimensioalelectrophoresis/massspectrometry(2-DE/MS)Surface-enhancedlaserdesorptionionizationtime-of-flightmassspectrometrytechnology(SELDI-TOP-MS):proteomicpatterndiagnosticsMulti-dimensionalproteinidentificationtechnology(MudPIT)/MS,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,PrincipleofMudPIT,Strategiesandtechniquesfordiscoveryofcancerbiomarkers,4.Post-translationalmodificationsofproteins(cleavageproducts,alteredglycosylation,etc)4.1.Cleavageproductsoftumour-derivedproteinshavebeenproposedaspotentialcancerbiomarkers.4.2.Alteredproteinglycosylationincancerisanothersourceofpotentialcancerbiomarkers.Identificationofglycosylatedproteinsreliesonvariousglyco-capturestrategies,ameansofglycosylated-proteinsub-selectionbyaffinitychromatography.Electron-transferdissociation(电子转移解离)allowslabilemodificationstoremainintactwhileobtainingpeptidesequenceinformation,enablingthestudyofmodificationssuchasglycosylationandphosphorylation.,Currenttumormarkersunderdevelopment,ContributionofoncoproteomicstocancerbiomarkerdiscoveryPublished:2April2007MolecularCancer2007,6:25,PotentialimportanceofmiRNAsascancerbiomarkers,1.HistoryDiscoveredinCaenorhabditiselegansin1993andformallynamedin2001Havebeenidentifiedineveryplantandanimalspeciesexamined2.Features2.1.GeneralfeaturesLength:AclassofnoncodingRNAs,1825nucleotidesSpeciecs:miRNAshavebeenidentified5withupto1,000predicted.Location:miRNAsareencodedbyDNAthatmaybesituatedintheexonsorintronsofgenesorscatteredamongintergenicDNA,PotentialimportanceofmiRNAsascancerbiomarkers,2.2.Transcriptionandmaturation(i)nuclearprocessingintoaprimarymiRNA(pri-miRNA)andthenaprecursor(pre-miRNA);(ii)exportintothecytoplasm;(iii)furtherprocessingintomaturemiRNA;(iv)incorporationintoanRNA-inducedsilencingcomplex(RISC)withanArgonauteproteincatalyst,PotentialimportanceofmiRNAsascancerbiomarkers,2.3.FunctionandtargetsThemiRNA-RISCcomplexhybridizestonucleotidesequencesofvaryingcomplementarityinthe3untranslatedregion(UTR)ofmRNAandinhibitsproteinsynthesisordegradesthetargetmRNAPlayskeyrolesintheregulationoffundamentalcellularprocesses,PotentialimportanceofmiRNAsascancerbiomarkers,DysregulatedexpressionofmicroRNAs(miRNAs)invarioustissueshasbeenassociatedwithavarietyofdiseases,includingcancers.miRNAsexpressedincancermayactlikeoncogenesortumor-suppressorgenesbyregulatingproliferationand/orapoptosis.NormalandmalignanttissueshavespecificmiRNAsignaturesandshowdifferentialexpressionacrosstumortypes.OverexpressionorlackofexpressionofspecificmiRNAsappearstocorrelatewithclinicallyaggressiveormetastaticphenotype.miRNAexpressionhastissuespecificityandhasbeenusedfortumorclassification.CancerbiomarkerprofilingwithmicroRNAsApril2008,NatureBiotechnologyVol26/4.,PotentialimportanceofmiRNAincancer,HerewedemonstratethatmiRNAsarepresentintheserumandplasmaofhumansandotheranimalssuchasmice,rats,bovinefetuses,calves,andhorses.ThelevelsofmiRNAsinserumarestable,reproducible,andconsistentamongindividualsofthesamespecies.EmployingSolexa,wesequencedallserummiRNAsofhealthyChinesesubjectsandfoundover100and91serummiRNAsinmaleandfemalesubjects,respectively.WealsoidentifiedspecificexpressionpatternsofserummiRNAsforlungcancer,colorectalcancer,anddiabetes,providingevidencethatserummiRNAscontainfingerprintsforvariousdiseases.Throughtheseanalyses,weconcludethatserummiRNAscanserveaspotentialbiomarkersforthedetectionofvariouscancersandotherdiseases.CharacterizationofmicroRNAsinserum:anovelclassofbiomarkersfordiagnosisofcancerandotherdiseases.CellResearch,2008,18:997-1006.,Organizations,1.TheInternationalCancerBiomarkersConsortium(ICBC)(/science/international_biomarker)UndertheleadershipofDr.LeeHartwell,PresidentandDirectorofFredHutchinsonCancerResearchCentertheICBCispioneeringanewmodelforbiomarkerdiscoveryanddevelopingtechnologiesandmethodologiestomakeitpossible.,Organizations,1.TheInternationalCancerBiomarkersConsortium(ICBC)(/science/international_biomarker)ThegoaloftheInternationalCancerBiomarkerConsortium(ICBC)istoadvancemedicalresearchandimprovepatientoutcomesbydiscoveringbiomarkers(indicators)formultipletypesofcancer.Throughalarge-scaleeffortsimilartotheHumanGenomeProject,theconsortiumaimstofacilitatehighlycoordinatedresearchandbyleveragingresourcesandexpertisefromaroundtheworldtoovercomethecurrentobstaclesinbiomarkerresearch.,Organizations,1.TheInternationalCancerBiomarkersConsortium(ICBC)(/science/international_biomarker)Atthesametime,theICBCwillprovideastructureforinternationalteamstoworktogetheronglobalissuessuchasadoptionofdatastandardsandthesharingdataaswellasonscientificdetailssuchasthelogisticsoftissuesamplesharingandinvestigationofmousemodelsofcancer.,Organizations,2.NationalInstituteofHealths(NIH)NationalCancerInstitute(NCI)()CancerBiomarkersResearchGroupThisgrouppromotesandsupportsresearchtoidentify,develop,andvalidatebiologicalmarkersforearliercancerdetectionandriskassessment.Thegroupintegratesbasicandclinicalsciencestudiesalongwithcomputational,statisticalandepidemiologicapproachesforacomprehensiveunderstandingofbiomarkers.ItcoordinatestheEarlyDetectionResearchNetwork.,Organizations,3.EarlyDetectionResearchNetwork(EDRN)()AninitiativeoftheNationalCancerInstitute(NCI),bringstogetherdozensofinstitutionstohe