急性髓细胞白血病的整体遗传特征轮廓预后相关性研究

PrognosticRelevanceofIntegratedGeneticProfilinginAcuteMyeloidLeukemia急性髓细胞白血病的整体遗传特征轮廓预后相关性研究JayP.Patel,MithatGönen,Ph.D.,MariaE.Figueroa,M.D.,HugoFernandez,M.D.,ZhuoxinSun,Ph.D.,JanisRacevskis,Ph.D.,PieterVanVlierberghe,Ph.D.,IgorDolgalev,B.S.,SabrenaThomas,B.S.,OlgaAminova,B.S.,KetyHuberman,B.S.,JaniceCheng,B.S.,AgnesViale,Ph.D.,NicholasD.Socci,Ph.D.,AdrianaHeguy,Ph.D.,AthenaCherry,Ph.D.,GailVance,M.D.,RodneyR.Higgins,Ph.D.,RhettP.Ketterling,M.D.,RobertE.Gallagher,M.D.,MarkLitzow,M.D.,MarcelR.M.vandenBrink,M.D.,Ph.D.,HillardM.Lazarus,M.D.,JacobM.Rowe,M.D.,SelinaLuger,M.D.,AdolfoFerrando,M.D.,Ph.D.,ElisabethPaietta,Ph.D.,MartinS.Tallman,M.D.,AriMelnick,M.D.,OmarAbdel-Wahab,M.D.,andRossL.Levine,M.D.AbstractBackgroundAcutemyeloidleukemia(AML)isaheterogeneousdiseasewithrespecttopresentationandclinicaloutcome.Theprognosticvalueofrecentlyidentifiedsomaticmutationshasnotbeensystematicallyevaluatedinaphase3trialoftreatmentforAML.急性髓细胞白血病是一种有关表达和临床结果异构性疾病。最近发现的体细胞突变的预后价值尚用于急性髓细胞白血病治疗的临床三期试验的系统评价。MethodsWeperformedamutationalanalysisof18genesin398patientsyoungerthan60yearsofagewhohadAMLandwhowererandomlyassignedtoreceiveinductiontherapywithhigh-doseorstandard-dosedaunorubicin.Wevalidatedourprognosticfindingsinanindependentsetof104patients.我们选择年龄小于60岁的AML398例随机分配到高剂量组或标准剂量组，接受柔红霉素的诱导治疗，治疗后对18个基因作突变的分析。并另选择104例患者进行验证了预后结果ResultsWeidentifiedatleastonesomaticalterationin97.3%ofthepatients.WefoundthatinternaltandemduplicationinFLT3(FLT3-ITD),partialtandemduplicationinMLL(MLL-PTD),andmutationsinASXL1andPHF6wereassociatedwithreducedoverallsurvival(P=0.001forFLT3-ITD,P=0.009forMLL-PTD,P=0.05forASXL1,andP=0.006forPHF6);CEBPAandIDH2mutationswereassociatedwithimprovedover-allsurvival(P=0.05forCEBPAandP=0.01forIDH2).ThefavorableeffectofNPM1mutationswasrestrictedtopatientswithco-occurringNPM1andIDH1orIDH2mutations.WeidentifiedgeneticpredictorsofoutcomethatimprovedriskstratificationamongpatientswithAML,independentlyofage,white-cellcount,inductiondose,andpost-remissiontherapy,andvalidatedthesignificanceofthesepredictorsinanindependentcohort.High-dosedaunorubicin,ascomparedwithstandard-dosedaunorubicin,improvedtherateofsurvivalamongpatientswithDNMT3AorNPM1mutationsorMLLtranslocations(P=0.001)butnotamongpatientswithwild-typeDNMT3A,NPM1,andMLL(P=0.67).我们可以确定97.3%的患者至少有一个细胞发生了改变。在FLT3基因中存在串联重复（FLT3-ITD），在MLL基因中存在部分串联重复（MLL-PTD），ASXL1，PHF6突变与降低总体生存率相关(P=0.001forFLT3-ITD,P=0.009forMLL-PTD,P=0.05forASXL1,andP=0.006forPHF6)；CEBPA和IDH2突变与总生存率改善相关(P=0.05forCEBPAandP=0.01forIDH2)。NPM1突变的有利作用仅限于NPM1和IDH1或IDH2同时发生突变患者。我们确定的改进AML患者危险分层之间遗传因素预测因子，该因子独立于年龄、白细胞计数、诱导剂量和治疗后缓解治疗，并通过独立的队列验证这些预测因子的重要性。ConclusionsWefoundthatDNMT3AandNPM1mutationsandMLLtranslocationspredictedanimprovedoutcomewithhigh-doseinductionchemotherapyinpatientswithAML.ThesefindingssuggestthatmutationalprofilingcouldpotentiallybeusedforriskstratificationandtoinformprognosticandtherapeuticdecisionsregardingpatientswithAML.(FundedbytheNationalCancerInstituteandothers.)我们发现DNMT3A和NPM1突变和MLL易位预测高剂量诱导化疗的AML患者的转归改善结果。研究结果表明，突变谱可能被用于AML患者的危险分层和预后预测和治疗决策Previousstudieshavehighlightedtheclinicalandbiologicheterogeneityofacutemyeloidleukemia(AML).1-4。However,arelativelysmallnumberofcytogeneticandmolecularlesionshavesufficientrelevancetoinfluenceclinicalpractice.5Theprognosticrelevanceofcytogeneticabnormalitieshasledtothewide-spreadadoptionofriskstratification,withpatientsdividedintothreecytogeneticallydefinedriskgroupswithsignificantdifferencesinover-allsurvival.6Morerecently,FLT3,NPM1,andCEBPAmutationalanalysiswasshowntoimproveriskstratificationforpatientswhodonothavekaryotypicabnormalities.7AlthoughprogresshasbeenmadeindefiningprognosticmarkersforAML,asubstantialpercentageofpatientslackaspecificabnormalityofprognosticsignificance.Inaddition,thereisconsiderableheterogeneityintheoutcomeforindividualpatientsineachriskgroup.以前的研究都强调，急性髓细胞白血病的临床和生物学异质性。然而，相对较少的细胞遗传学和分子病变具有充分的关联性而影响临床实践。细胞遗传异常的预后相关性促使广泛采用危险因素分层，患者根据细胞遗传学定义的风险分为三组，其所有生存时间有显著差异。最近，NPM1，FLT3，和CEBPA突变分析显示没有核型畸形患者改善危险分层。虽然在确定的预后标记的AML取得了进展，相当比例的患者缺乏特异性预后差异。此外，各风险组中存在有相当大的异质性结果。RecentstudieshaveidentifiednovelrecurrentsomaticmutationsinpatientswithAML.TheseincludemutationsinTET2,8,9ASXL1,10IDH1orIDH2,11-13DNMT3A,4,14andPHF6.15RetrospectiveanalysessuggestthatasubsetofthesemutationsmayhaveprognosticsignificanceinAML,4,14,16althoughthesefindingshavenotbeenvalidatedwithdetailedclinicalandmutationalannotationinlarge,homogeneouslytreatedcohortsofpatientswithAML.Inaddition,thequestionofwhethermutationalprofilingofalargersetofgenes,includingthesenoveldiseasealleles,improvesprognosticationinAMLhasnotbeeninvestigatedinaclinicaltrialcohort.最近的研究发现AML患者存在新的复发性体细胞突变。这些突变包括TET2，ASXL1，IDH1和IDH2，DNMT3A，4PHF6。回顾性分析表明，这些突变可能存在预后差异性，尽管这些研究没有经过详细的临床和基因突变的进行验证，也没治疗证实。此外，是否进行大量的基因轮廓分析，包括这些改善AML的预后疾病新的等位基因，这一问题尚未通过队列研究进行临床验证。Arecentphase3clinicaltrial(E1900;ClinicalTnumber,NCT00049517)fromtheEasternCooperativeOncologyGroup(ECOG)showedthatinductiontherapywithcytarabineplus90mgofdaunorubicinpersquaremeterofbody-surfacearea,ascomparedwithcytarabineplus45mgofdaunorubicinpersquaremeter,improvedtheoutcomesinpatientswithnewlydiagnosedAMLwhowere17to60yearsofage17;asimilarstudyinpatientswhowereolderthan60yearsofageshowedthatdose-intensifieddaunorubicinimprovedoverallsurvivalinpatients60to65yearsofage.18Wehypothesizedthatintegratedmutationalanalysisofallknownmolecularalterationsoccurringinmorethan5%ofpatientswithAMLwouldallowustoidentifynovelmolecularmarkersofoutcomeinAMLandtoidentifymolecularlydefinedsubgroupsofpatientswhowouldbenefitfromdose-intensifiedinductionchemotherapy.最近的来自东部肿瘤协作组（ECOG）的3期临床试验表明，与阿糖胞苷加45毫克每平方米柔红霉素相比，阿糖胞苷加90毫克每平方米体表面积的柔红霉素诱导治疗可改善17至60岁初诊AML患者的预后。类似研究表明年龄60岁以上病人加大剂量的柔红霉素治疗可以提高60到65岁患者总体生存。我们假设，所有已知的分子改变发生超过5%的AML患者的基因突变分析，允许我们识别新的结果分子标记，识别受益于增强诱导化疗剂量AML患者的亚组。MethodsPatientsWeperformedmutationalanalysisondiagnosticsamplesobtainedfrompatientsintheECOGE1900trial.Allpatientsprovidedwritteninformedconsent.Thetestcohort(398patients)comprisedallpatientsintheE1900trialforwhomviablyfrozencellswereavailableforDNAextractionandmutationalprofiling.Thevalidationcohort(104patients)comprisedasecondsetofpatientsforwhomsampleswerebankedinTrizolreagent(Invitrogen),whichwasusedtoextractDNAformutationalstudies.Theclinicalcharacteristicsofthepatientswestudied,ascomparedwiththecompleteE1900trialcohort,areprovidedinTableS1intheSupplementaryAppendix,availablewiththefulltextofthisarticleatNEJM.org.Themedianfollow-uptimeforthepatientsincludedintheanalysis,calculatedfromthetimeofrandomizationforinductiontherapy,was47.4months.Cytogeneticanalysis,fluorescenceinsituhybridization,andreverse-transcriptase–polymerase-chain-reaction(RT-PCR)assaysforrecurrentcytogeneticlesionswereperformedasdescribedinitiallybySlovaketal.6andasusedpreviously,17withcentralreviewbytheECOGCytogeneticSubcommittee.我们对从脑电图e1900试验患获得的诊断样本进行了突变分析。所有患者提供书面知情同意书。试验组（398例）包括在e1900所有患者可用冷冻细胞允许DNA提取和突变分析。验证组（104例）组成的第二组病人的样品储存在Trizol试剂（Invitrogen公司），也是用来提取DNA作突变分析。我们研究的患者的临床特征，如与完整的e1900试验相比，在附录中有表S1可浏览详细信息。纳入分析患者的中位随访时间为从随机的诱导治疗的时间计算，为47.4个月。如最初由斯洛伐克等人所进行的研究，由皮层细胞遗传学中心审查委员会审查，通过染色体核型分析，荧光原位杂交，逆转录-聚合酶链反应（RT-PCR）进行细胞遗传学损伤检测。MutationalAnalysisThesourceoftheDNAwasbonemarrowinthecaseof55.2%ofthesamples(277of502)andperipheralbloodinthecaseof44.8%(225of502).WesequencedtheentirecodingregionsofTET2,ASXL1,DNMT3A,CEBPA,PHF6,WT1,TP53,EZH2,RUNX1,andPTENandtheregionsofpreviouslydescribedmutationsforFLT3,NPM1,HRAS,KRAS,NRAS,KIT,IDH1,andIDH2.ThegenomiccoordinatesandsequencesofalltheprimersusedinthisstudyareprovidedinTableS2intheSupplementaryAppendix.PairedremissionDNA(i.e.,DNAfrompatientswhohadacompletere-missionafterinductionchemotherapy)wasavail-ablefrom241ofthe398participantsinthetestcohortandfrom65ofthe104inthevalidationcohort.DataonvariantsthatcouldnotbevalidatedasbonafidesomaticmutationsowingtounavailableremissionDNAandtheabsenceofreportsofthemutationsinthepublishedliteratureofsomaticmutationswerecensoredwithrespecttomutationalstatusforthatspecificgene.FurtherdetailsofthesequencingmethodsareavailableintheSupplementaryAppendix.DNA来自样本中的55.2%例骨髓（277/502）和44.8%例的外周血（225/502）。我们测序整个编码区的TET2，ASXL1，DNMT3A，CEBPA，PHF6，WT1基因，TP53，EZH2，RUNX1，和PTEN和先前描述的突变NPM1，FLT3，HRAS，KRAS，NRAS，KIT，IDH1和IDH2基因。基因组坐标和所有本研究中所用的引物序列在附录中表S2详细列出。对配对缓解DNA（即，有完整的诱导缓解后化疗患者DNA）是从241的398的参与者在测试队列和从65的104在验证队列获取。对变异不能由无法缓解的DNA体细胞突变验证，在已发表的文献中的基因突变的报道认为没有通过对特定基因的突变状态方面的数据。进一步测序方法的细节在附录中详细列举。StatisticalAnalysisThemutualexclusivityofpairsofmutationswasevaluatedwiththeuseoftwo-by-twocontingencytablesandFisher’sexacttest.Theassociationbetweenmutationsandcytogeneticriskclassificationwastestedwiththeuseofthechi-squaretest.HierarchicalclusteringwasperformedwiththeuseoftheLance–Williamsdissimilarityformulaandthecomplete-linkagealgorithm.Survivaltimewasmeasuredfromthedateofrandomizationtothedateofdeathforpatientswhodiedandtothedateofthelastfollow-upforthosewhowerealiveatthetimeoftheanalysis.SurvivalprobabilitieswereestimatedwiththeuseoftheKaplan–Meiermethodandwerecom-paredbetweenpatientswithamutationandthosewithoutmutantallelesbymeansofthelog-ranktest.MultivariateanalyseswereconductedwiththeuseoftheCoxmodelwithforwardse-lection.Wecheckedtheproportional-hazardsassumptionbytestingforanonzeroslopeinaregressionofthescaledSchoenfeldresidualsonfunctionsoftime(TableS3intheSupplementaryAppendix).Whennecessary,suchasintheanalysesperformedinvarioussubsets,theresultsoftheunivariateanalyseswereusedtoselectthevariablestobeincludedintheforwardvariablesearch.Finalmultivariatemodelsinformedthedevelopmentofnovelrisk-classificationrules.Whensoindicated,Pvalueswereadjustedtocontrolthefamily-wiseerrorratewiththeuseofthecompletenulldistributionapproximatedbyresamplingobtainedthroughthePROCMULTTESTprograminSASorthemulttestlibraryinR.19Theonlyexceptionwastheadjustmentintestsoftheeffectofmutationsontheresponsetotheinductiondose,forwhichastep-downHolmprocedurewasusedtocorrectformultipletesting.AllanalyseswereperformedwiththeuseofSASsoftware,version9.2(),andtheRstatisticalpackage,version2.12()两对共同双突变分析由2*2列联表和Fisher精确检验进行分析。基因突变和细胞遗传学风险分类之间的关联性采用卡方检验进行分析。层次聚类采用Lance-Williams法和complete-linkage算法进行。生存时间测定日期的采用随机抽取患者死亡日期和最后随访的日期之差计算取得。生存概率采用Kaplan-Meier法进行估计和患者的突变和未突变的等位基因的比较采用log-rank检验。多变量分析采用Cox模型的前进法进行。我们Schoenfeld残差非零时间函数回归规模对比例风险进行假设检验(附录中表S3)。必要时，如对不同亚组进行处理，通过单因素分析选择有意义的变量。最后多变量模型采用新的风险分类规则进行分析。当如此表示，P值进行调整以控制总Ⅰ类错误率与通过SAS的PROCMULTTEST程序或R数据库的multtest程序的重复采样完整近似零假设分布的。ResultsFrequencyofGeneticAlterations基因修饰频数Somaticalterationswereidentifiedin97.3%ofthepatients.Figure1showsthefrequencyofsomaticmutationsintheentirecohortandtheinterrelationshipsamongthevariousmutations,asrepresentedvisuallywiththeuseofaCircosplot.DataforallmolecularsubsetsareprovidedinFiguresS1andS2andTablesS4andS5intheSupplementaryAppendix.Inparticular,mutationalheterogeneitywasgreaterinpatientswithintermediate-riskAMLthaninpatientswithfavorable-riskorunfavorable-riskriskAML(P=0.01)(Fig.S2DintheSupplementaryAppendix).97.3%的病人发生体细胞修饰。根据Circos图，图1表明整个研究队列的体细胞突变频数以及各种突变间的相互关系。所有分子亚组的数据由附录中图S1、S2和表S4、S5进行表述。尤其是，中度风险AML病人的突变异质性比低度风险和恶性风险病人高很多(P=0.01)(附录中图S2D)。MutationalComplementationGroups突变互补基因群Integratedmutationalanalysisallowedustoidentifyfrequentlyco-occurringmutationsandmutationsthatweremutuallyexclusiveintheE1900patientcohort(TableS6intheSupplementaryAppendix).Inadditiontonotingfrequentco-occurrenceofKITmutationswithcore-binding–factoralterationst(8;21)andinv(16)/t(16;16),wefoundsignificantco-occurrenceofIDH1andIDH2mutationswithNPM1mutationsandofDNMT3AmutationswithNPM1,FLT3,andIDH1alleles(P<0.001forallcomparisons)(TableS7intheSupplementaryAppendix).WerecentlyreportedthatIDH1andIDH2mutationsweremutuallyexclusivewithTET2mutations20;detailedmutationalanalysisrevealedthatIDH1andIDH2mutationswerealsomutuallyexclusivewithWT1mutations(P<0.001)(Fig.S3andTableS8intheSupplementaryAppendix).WealsoobservedthatDNMT3AmutationsandMLLtranslocationsweremutuallyexclusive(P<0.01).整合的基因突变轮廓分析让我们多次辨别共同发生的突变和在E1900病人队列中发生的相互排斥的突变（附录表S6）。另外，为了记录core-binding–factor修饰t(8;21)和inv(16)/t(16;16)的KIT突变的经常发生的共出现频数，我们发现IDH1andIDH2的突变和NPM1突变具有显著的共同出现差异，DNMT3A突变与NPM1,FLT3,andIDH1等位基因有显著的共同出现差异（与所有对照比较P<0.001）（附录表S7）。最近我们报到了IDH1和IDH2突变与TET2相互排斥，进一步的突变分析证实IDH1与IDH2突变也与WT1突变相互排斥（P<0.001）（附录图S3和表S8）.也同时发现DNMT3A与MLL翻译相互排斥（P<0.001）。MolecularDeterminantsofOverallSurvival总生存的分子决定因子Univariateanalysisrevealed,aspreviouslydescribed,21,22thatFLT3internaltandemduplication(FLT3-ITD)mutationsandMLLpartialtandemduplication(MLL-PTD)mutationswereassociatedwithreducedoverallsurvival(P=0.001forFLT3-ITDandP=0.009forMLL-PTD)(TableS9intheSupplementaryAppendix),whereasCEBPAmutationsandcore-binding–factoralterationst(8;21)andinv(16)/t(16;16)wereassociatedwithimprovedoverallsurvival(P=0.05forCEBPAandP<0.001forthecore-binding–factoralterations).2,23Inaddition,PHF6andASXL1mutationswereassociatedwithreducedoverallsurvival(P=0.006forPHF6andP=0.05forASXL1)(Fig.S4intheSupplementaryAppendix).IDH2mutationswereassociatedwithanimprovedrateofoverallsurvivalintheentiretestcohort(3-yearrate,66%;P=0.01)(Fig.S5intheSupplementaryAppendix).ThefavorableeffectofIDH2mutationswasfoundexclusivelyinpatientswithIDH2R140Qmutations(P=0.009)(Fig.S5intheSupplementaryAppendix).Allthefindingsintheunivariateanalysiswerealsosignificantinthemultivariateanalysis(P<0.05,withadjustmentforage,white-cellcount,transplantationstatus[didvs.didnotundergostem-celltransplantation],andcytogeneticcharacteristics)(TableS9intheSupplementaryAppendix),withtheexceptionofthefindingsforMLL-PTD,PHF6,andASXL1mutations.KITmutationswereassociatedwithreducedoverallsurvivalamongpatientswhowerepositiveforthet(8;21)core-binding–factoralteration(P=0.006)butnotamongpatientswiththeinv(16)/t(16;16)alteration(P=0.19)(Fig.S6intheSupplementaryAppendix).正如先前描述，单变量分析显示，FLT3内在衔接复制(FLT3-ITD)突变和MLL内在衔接复制(MLL-PTD)突变与减少总生存相关(P=0.001forFLT3-ITDandP=0.009forMLL-PTD)(附录表S9)，而CEBPA突变和core-binding–factor修饰t(8;21)andinv(16)/t(16;16)与改善总生存相关(P=0.05forCEBPAandP<0.001forthecore-binding–factor修饰)。另外，PHF6和ASXL1突变与减少总生存相关(P=0.006forPHF6andP=0.05forASXL1)(附录图S4)，IDH2突变与整个检验队列提高总生存率相关(3年生存率66%;P=0.01)(附录图S5)。IDH2突变的有利效应仅在有IDH2R140Q突变的病人出现(P=0.009)(附录图S5)。除了MLL-PTD,PHF6,andASXL1的突变结果，单变量分析中所有的结果再多因素分析中也是有统计学意义(P<0.05,调整年龄、白细胞计数、移植情况[有vs没有干细胞移植],、细胞遗传特征)（附录表S9）。在出现core-binding–factor改变的阳性病人中，KIT突变与降低总生存相关(P=0.006)，而与nv(16)/t(16;16)改变的病人无相关性(P=0.19)（附录图S6）PrognosticValueofMolecularAlterationsinIntermediate-RiskAMLAML中度风险患者的分子修饰的预后价值Amongpatientswithintermediate-riskAMLasdefinedbycytogeneticanalysis(TableS10intheSupplementaryAppendix),FLT3-ITDmutationswereassociatedwithreducedoverallsurvival(P=0.008),afindingthatisconsistentwiththeresultsofpreviousstudies.21ASXL1andPHF6mutationswereassociatedwithreducedsurvival,andIDH2R140Qmutationswithimprovedsurvival,amongpatientswithintermediate-riskAML(TableS10intheSupplementaryAppendix),aneffectsimilartothatintheentirecohort.Inaddition,wefoundthatTET2mutationswereassociatedwithreducedoverallsurvivalamongpatientswithintermediate-riskAML(P=0.007)(Fig.S7intheSupplementaryAppendix).通过细胞遗传学分析定义的AML中度风险患者，FLT3-ITD突变与减少总生存相关(P=0.008)，该发现与早期研究一致。在具有中度风险的AML患者中，ASXL1和PHF6突变与降低生存相关，IDH2R140Q与改善生存相关（附录表S10），改结果与整个队列研究结果相似。MultivariateanalysisrevealedthatFLT3-ITDmutationsconstitutedtheprimarypredictorofoutcomeinpatientswithintermediate-riskAML(adjustedP<0.001).AsubsequentmultivariateanalysisaccordingtoFLT3-ITDstatusshowedthatinpatientswithwild-typeFLT3-ITD,mutationsinTET2,ASXL1,PHF6,andMLL-PTDwereindependentlyassociatedwithanadverseout-come.Patientswithintermediate-riskAMLwhohadbothNPM1andIDH1orIDH2mutationshadanimproved3-yearrateofoverallsurvival,ascomparedwithpatientswhohadmutantNPM1andbothwild-typeIDH1andwild-typeIDH2(89%vs.31%,P<0.001)(Fig.S8intheSupplementaryAppendix).Wethenclassifiedpatientswithintermediate-riskAMLwhohadwild-typeFLT3-ITDintothreecategories,withmarkeddifferencesinthe3-yearrateofoverallsurvival(adjustedP<0.001):patientswithIDH1orIDH2mutationsandNPM1mutations(overallsurvival,89%);patientswithTET2,ASXL1,PHF6,orMLL-PTDmutations(overallsurvival,6.3%);andpatientswithwild-typeTET2,ASXL1,PHF6,andMLL-PTD,withoutco-occurringIDHorNPM1mutations(overallsurvival,46.2%)(Fig.2A).Similarresultswereobtainedwhentheanalysiswasrestrictedtopatientswithanormalkaryotype(Fig.S9AintheSupplementaryAppendix).多因素分析证实FLT3-ITD突变构成AML中度风险患者结局的主要因素(调整P<0.001)。依据FLT3-ITD状况，一些列多因素分析表明，在FLT3-ITD野生型的患者中，TET2,ASXL1,PHF6,和MLL-PTD的突变分别与不利结局相关。与具有NPM1突变和同时具有IDH1野生型和IDH2野生型的患者比较，同时具有NPM1与IDH1或IDH2突变的AML中度风险患者具有改善的三年生存期(89%vs.31%,P<0.001)(附录图S8)。然后，我们对具有FLT3-ITD野生型AML中度风险患者分成三类，其三年生存率具有显著差异(调整P<0.001)：同时具有IDH1或IDH2突变和NPM1突变患者（总生存率89%）；具有TET2,ASXL1,PHF6,orMLL-PTD突变患者（总生存率6.3%）；具有野生型TET2,ASXL1,PHF6,andMLL-PTD,而非同时出现IDH和NPM1的患者（总生存率46.2%）。与正常核心患者的分析结果相似（附录图S9A）。Inpatientswithintermediate-riskAMLwhohadmutantFLT3-ITD,wefoundthatCEBPAmutationswereassociatedwithanimprovedout-comeandthattrisomy8andTET2,DNMT3A,andMLL-PTDmutationswereassociatedwithanadverseoutcome.Weusedthesedatatoclassifypatientswithintermediate-riskAMLwhohadmutantFLT3-ITDintothreecategories.Thefirstcategoryincludedpatientswithtrisomy8orTET2,DNMT3A,orMLL-PTDmutations,whichwereassociatedwithanadverseoutcome(3-yearrateofoverallsurvival,14.5%);thisrateofsurvivalwassignificantlylowerthantheratesamongpatientsinthesecondcategory,thosewithwild-typeCEBPA,TET2,DNMT3A,andMLL-PTD(overallsurvival,35.2%;P<0.001),andpatientsinthethirdcategory,thosewithCEBPAmutations(overallsurvival,42%;P<0.001)(Fig.2B).Therateofsurvivalamongpatientswithintermediate-riskAMLwhohadmutantFLT3-ITDandwild-typeCEBPA,TET2,DNMT3A,andMLL-PTDdidnotdiffersignificantlyfromtherateamongpatientswithmutantFLT3-ITDandmutantCEBPA(P=0.34),suggestingthatthepresenceofmutationsassociatedwithanunfavorable-riskprofilemorepreciselyidentifiespatientswithmutantFLT3-ITDwhowillhaveadverseoutcomesofAMLthandoestheabsenceofCEBPAmutationsalone.ThesesamethreeriskcategoriesalsohadsignificantprognosticvalueinpatientswithAMLwhohadmutantFLT3-ITDandanormalkaryotype(Fig.S9BintheSupplementaryAppendix).具有FLT3-ITD突变型基因AML中度风险患者，我们研究发现CEBPA突变与改善的结局相关，三体性8的TET2,DNMT3A与MLL-PTD突变与不利结局相关。利用这些数据，我们把具有FLT3-ITD突变型的AML中度风险患者分为三类。第一类包括三倍性8或TET2,DNMT3A或MLL-PTD突变型，与不利结局相关(三年生存率14.5%)；这一生存率明显低于第二类、第三类患者的生存率，第二类表现为野生型CEBPA,TET2,DNMT3A,andMLL-PTD(总生存率35.2%;P<0.001)，第三类患者表现为CEBPA突变(总生存率42%;P<0.001)(图2B)。AML中度风险患者中具有突变株FLT3-ITD和野生型CEBPA,TET2,DNMT3A,和MLL-PTD与具有突变型FLT3-ITD和CEBPA的患者明显没有差别(P=0.34)，表明。对于具有FLT3-ITD突变型和正常核型AML患者，同样的三类危险因素分类有显著的预后价值（附录图S9B）。PrognosticSchemawithIntegratedMutationalandCytogeneticProfiling整体突变和细胞遗传分析的预后模式Theseresultsallowedustodevelopaprognosticschemathatintegratedourfindingsfromthecomprehensivemutationalanalysiswithcytogeneticdatatoidentifythreeriskgroups:agroupwithafavorable-riskprofile(mediansurvival,notreached;3-yearrateofoverallsurvival,64%),agroupwithanintermediate-riskprofile(mediansurvival,25.4months;3-yearrateofoverallsurvival,42%),andagroupwithanadverse-riskprofile(mediansurvival,10.1months;3-yearrateofoverallsurvival,12%)(Fig.3Aand3B,andTableS11intheSupplementaryAppendix).Inmultivariateanalysis,themutationalprognosticschemapredictedtheoutcomeindependentlyofage,white-cellcount,inductiondose,andtransplantationstatus(adjustedP<0.001).Ourclassificationheldtrueregardlessofthetypeofpost-remissiontherapy(autologousorallogeneictransplantationorconsolidationchemotherapyalone)(Fig.S10intheSupplementaryAppendix).这些研究结果让我们形成预后模式，根据细胞遗传数据的综合突变分析的整合结果，可以分为三类危险因素组：一是有利风险轮廓组(中位生存期，没有达到;三年生存率64%)，二是中位风险轮廓组(中位生存期25.4月;三年生存率42%)，三是不利风险轮廓组(中位生存期10.1月，三年生存率12%)（附录图3A,3B，表S11）。多元统计分析，突变预测模式预测结局的独立因素，包括年龄、白细胞计数、诱导计量、移植状态(调整P<0.001)。不管术后缓解治疗如何，我们的分类是真实有效的（自体或异体移植或单独强化化疗）（附录图S10）。Giventhenumberofvariablesinourprognosticclassification,wetestedthereproducibilityofthispredictorinanindependentcohortof104patientsfromtheECOGE1900trial.MutationalanalysisofthevalidationcohortconfirmedthereproducibilityofourprognosticschemaforpredictingtheoutcomeinpatientswithAML(adjustedP<0.001)(Fig.3C).Thepredictivevalueofthemutationalprognosticschemawasindependentofriskwithrespecttotreatment-relateddeath(definedasdeathwithin30daysafterinitiationoftreatment)orlackofresponsetoinductionchemotherapy(i.e.,lackofacompleteremission)inthetestcohortandinthecombinedtestandvalidationcohorts(TableS12intheSupplementaryAppendix).根据预后变量的数目，从ECOGE1900试验患者独立选择104名患者的数据，我们检验了这些预测指标的重现性。有效队列的突变分析证实用来预测AML患者结局的预后预测模式(调整P<0.001)(图.3C)。，突变预后模式的预测值独立于有关危险因素，包括治疗相关死亡（诱导治疗三十天内死亡）或再检验队列中缺少诱导治疗响应（如，缺少缓解信息）或综合试验或合法队列（附录表S12）。GeneticPredictorsofResponsetoInductionChemotherapy诱导治疗反应的基因预测RecentstudieshaveshownthatmutantDNMT3AwasassociatedwithadverseoutcomesinpatientswithAML.4,14However,wefoundthatDNMT3AmutationswerenotassociatedwithadverseoutcomesintheECOGE1900cohort(Fig.4A)(P=0.15).IntheECOGE1900trial,patientswererandomlyassignedtoinductiontherapywithcytarabinepluseither45mgofdaunorubicinpersquaremeteror90mgofdaunorubicinpersquaremeter.17Wethereforehypothesizedthathigh-dosedaunorubicinimprovedtheoutcomesinpatientswithdaunorubicinwhohadDNMT3Amutations.Indeed,wefoundthatDNMT3Amutationalstatushadasignificanteffectontheout-comewithdose-intensivechemotherapy(Fig.4B)(P=0.02).WethenassessedtheeffectsofDNMT3Amutationalstatusontheoutcomeaccordingtotreatmentgroupandfoundthathigh-dosedaunorubicinwasassociatedwithanimprovedrateofsurvivalamongpatientswithmutantDNMT3A(P=0.04)(Fig.S11AintheSupplementaryAppendix)butnotamongpatientswithwild-typeDNMT3A(P=0.15)(Fig.S11BintheSupplementaryAppendix).Inaddition,univariateanalysisrevealedthatdose-intensifiedinductiontherapywasassociatedwithanimprovedoutcomeinpatientswithAMLwhohadMLLtranslocations(P=0.01;P=0.06withadjustmentformultipletesting)(Fig.S11CandS11DintheSupplementaryAppendix)andinthosewhohadNPM1mutations(P=0.01;P=0.10withadjustmentformultipletesting)(Fig.S11EandS11FandTableS13intheSupplementaryAppendix).BecausetheadjustedPvaluesforNPM1mutationsandMLLtranslocations(P≤0.10)areclosetostatisticalsignificance,theyshouldbestudiedfurtherinprospectivetrials.最近的研究表明，突变型DNMT3A与AML患者不利结局相关。但是，我们也发现DNMT3A的突变与ECOGE1900队列中的不利结局不相关(图4A)(P=0.15)。在ECOGE1900队列试验中，患者随机分配接受两种诱导治疗，一种是阿糖胞苷加45mg柔红霉素/平方米，一组是阿糖胞苷加90mg柔红霉素/平方米。因此，我们假设高剂量柔红霉素组能够改善带有DNMT3A突变患者生存。确实，我们发现DNMT3A突变状态对剂量敏感性的化疗结局确实有影响(图4B)(P=0.02)。我们根据治疗分组结局，评价DNMT3A的突变状态效果，发现高剂量柔和霉素和具有DNMT3A突变患者改善的生存率相关(P=0.04)(附录图.S11A)，而和具有DNMT3A野生型患者改善的生存率不相关(P=0.15)(附录图.S11B)。另外，单因素分析显示，剂量强化诱导治疗与发生混合性白血病淋巴转移AML患者的结局改善相关(P=0.01;P=0.06调整的多重分析)(附录图S11C、S11D)，与NPM1突变的AML患者的结局改善相关(P=0.01;P=0.10调整的多重分析)(附录图S11E、S11F和表S13)。因为NPM1突变和MLL转移的调整P值接近检验水准，必须在将来的后续试验中做进一步研究。Wethenseparatedthepatientsinourcohortintotwogroups:patientswithmutationsinDNMT3AorNPM1orwithMLLtranslocationsandpatientswithwild-typeDNMT3AandNPM1andnoMLLtranslocations.Dose-intensiveinductiontherapywasassociatedwithamarkedimprovementintherateofsurvivalamongpatientswhowerepositiveforDNMT3AorNPM1mutationsorMLLtranslocations(P=0.001)(Fig.4C)butnotamongpatientswithwild-typeDNMT3AandNPM1andnoMLLtranslocations(P=0.67)(Fig.4D).Thisfindingwasindependentoftheclinicalcovariatesofage,white-cellcount,andstatuswithrespecttotransplantation,treatment-relateddeath,andresponsetochemotherapy(adjustedP=0.008andP=0.34forpatientswithmutantandwild-typegenes,respectively),suggestingthathigh-doseanthracyclinechemotherapyprovidesabenefitingeneticallydefinedsubgroupsofpatientswithAML.我们把进入队列的患者分成两组：一组是具有DNMT3A或NPM1突变，或具有MLL转移的患者，一组是又有DNMT3A和NPM1野生型，没有发生MLL转移的患者。强化剂量诱导治疗与DNMT3A阳性或者NPM1突变或MLL转移患者生存率的显著改善相关(P=0.001)(图.4C)，与有DNMT3A和NPM1野生型，没有发生MLL转移的患者没有相关(P=0.67)(Fig.4D)。这一发现独立于一些临床协变量，如年龄、白细胞计数、移植反应状态、治疗相关死亡、化学疗法的反应（调整P=0.008、P=0.34分别和突变与野