急性髓細(xì)胞白血病的整體遺傳特征輪廓預(yù)后相關(guān)性研究

PrognosticRelevanceofIntegratedGeneticProfilinginAcuteMyeloidLeukemia急性髓細(xì)胞白血病的整體遺傳特征輪廓預(yù)后相關(guān)性研究JayP.Patel,MithatG?nen,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.急性髓細(xì)胞白血病是一種有關(guān)表達(dá)和臨床結(jié)果異構(gòu)性疾病。最近發(fā)現(xiàn)的體細(xì)胞突變的預(yù)后價(jià)值尚用于急性髓細(xì)胞白血病治療的臨床三期試驗(yàn)的系統(tǒng)評價(jià)。MethodsWeperformedamutationalanalysisof18genesin398patientsyoungerthan60yearsofagewhohadAMLandwhowererandomlyassignedtoreceiveinductiontherapywithhigh-doseorstandard-dosedaunorubicin.Wevalidatedourprognosticfindingsinanindependentsetof104patients.我們選擇年齡小于60歲的AML398例隨機(jī)分配到高劑量組或標(biāo)準(zhǔn)劑量組，接受柔紅霉素的誘導(dǎo)治療，治療后對18個(gè)基因作突變的分析。并另選擇104例患者進(jìn)行驗(yàn)證了預(yù)后結(jié)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).我們可以確定97.3%的患者至少有一個(gè)細(xì)胞發(fā)生了改變。在FLT3基因中存在串聯(lián)重復(fù)（FLT3-ITD），在MLL基因中存在部分串聯(lián)重復(fù)（MLL-PTD），ASXL1，PHF6突變與降低總體生存率相關(guān)(P=0.001forFLT3-ITD,P=0.009forMLL-PTD,P=0.05forASXL1,andP=0.006forPHF6)；CEBPA和IDH2突變與總生存率改善相關(guān)(P=0.05forCEBPAandP=0.01forIDH2)。NPM1突變的有利作用僅限于NPM1和IDH1或IDH2同時(shí)發(fā)生突變患者。我們確定的改進(jìn)AML患者危險(xiǎn)分層之間遺傳因素預(yù)測因子，該因子獨(dú)立于年齡、白細(xì)胞計(jì)數(shù)、誘導(dǎo)劑量和治療后緩解治療，并通過獨(dú)立的隊(duì)列驗(yàn)證這些預(yù)測因子的重要性。ConclusionsWefoundthatDNMT3AandNPM1mutationsandMLLtranslocationspredictedanimprovedoutcomewithhigh-doseinductionchemotherapyinpatientswithAML.ThesefindingssuggestthatmutationalprofilingcouldpotentiallybeusedforriskstratificationandtoinformprognosticandtherapeuticdecisionsregardingpatientswithAML.(FundedbytheNationalCancerInstituteandothers.)我們發(fā)現(xiàn)DNMT3A和NPM1突變和MLL易位預(yù)測高劑量誘導(dǎo)化療的AML患者的轉(zhuǎn)歸改善結(jié)果。研究結(jié)果表明，突變譜可能被用于AML患者的危險(xiǎn)分層和預(yù)后預(yù)測和治療決策Previousstudieshavehighlightedtheclinicalandbiologicheterogeneityofacutemyeloidleukemia(AML).1-4。However,arelativelysmallnumberofcytogeneticandmolecularlesionshavesufficientrelevancetoinfluenceclinicalpractice.5Theprognosticrelevanceofcytogeneticabnormalitieshasledtothewide-spreadadoptionofriskstratification,withpatientsdividedintothreecytogeneticallydefinedriskgroupswithsignificantdifferencesinover-allsurvival.6Morerecently,FLT3,NPM1,andCEBPAmutationalanalysiswasshowntoimproveriskstratificationforpatientswhodonothavekaryotypicabnormalities.7AlthoughprogresshasbeenmadeindefiningprognosticmarkersforAML,asubstantialpercentageofpatientslackaspecificabnormalityofprognosticsignificance.Inaddition,thereisconsiderableheterogeneityintheoutcomeforindividualpatientsineachriskgroup.以前的研究都強(qiáng)調(diào)，急性髓細(xì)胞白血病的臨床和生物學(xué)異質(zhì)性。然而，相對較少的細(xì)胞遺傳學(xué)和分子病變具有充分的關(guān)聯(lián)性而影響臨床實(shí)踐。細(xì)胞遺傳異常的預(yù)后相關(guān)性促使廣泛采用危險(xiǎn)因素分層，患者根據(jù)細(xì)胞遺傳學(xué)定義的風(fēng)險(xiǎn)分為三組，其所有生存時(shí)間有顯著差異。最近，NPM1，F(xiàn)LT3，和CEBPA突變分析顯示沒有核型畸形患者改善危險(xiǎn)分層。雖然在確定的預(yù)后標(biāo)記的AML取得了進(jìn)展，相當(dāng)比例的患者缺乏特異性預(yù)后差異。此外，各風(fēng)險(xiǎn)組中存在有相當(dāng)大的異質(zhì)性結(jié)果。RecentstudieshaveidentifiednovelrecurrentsomaticmutationsinpatientswithAML.TheseincludemutationsinTET2,8,9ASXL1,10IDH1orIDH2,11-13DNMT3A,4,14andPHF6.15RetrospectiveanalysessuggestthatasubsetofthesemutationsmayhaveprognosticsignificanceinAML,4,14,16althoughthesefindingshavenotbeenvalidatedwithdetailedclinicalandmutationalannotationinlarge,homogeneouslytreatedcohortsofpatientswithAML.Inaddition,thequestionofwhethermutationalprofilingofalargersetofgenes,includingthesenoveldiseasealleles,improvesprognosticationinAMLhasnotbeeninvestigatedinaclinicaltrialcohort.最近的研究發(fā)現(xiàn)AML患者存在新的復(fù)發(fā)性體細(xì)胞突變。這些突變包括TET2，ASXL1，IDH1和IDH2，DNMT3A，4PHF6?；仡櫺苑治霰砻鳎@些突變可能存在預(yù)后差異性，盡管這些研究沒有經(jīng)過詳細(xì)的臨床和基因突變的進(jìn)行驗(yàn)證，也沒治療證實(shí)。此外，是否進(jìn)行大量的基因輪廓分析，包括這些改善AML的預(yù)后疾病新的等位基因，這一問題尚未通過隊(duì)列研究進(jìn)行臨床驗(yàn)證。Arecentphase3clinicaltrial(E1900;ClinicalTnumber,NCT00049517)fromtheEasternCooperativeOncologyGroup(ECOG)showedthatinductiontherapywithcytarabineplus90mgofdaunorubicinpersquaremeterofbody-surfacearea,ascomparedwithcytarabineplus45mgofdaunorubicinpersquaremeter,improvedtheoutcomesinpatientswithnewlydiagnosedAMLwhowere17to60yearsofage17;asimilarstudyinpatientswhowereolderthan60yearsofageshowedthatdose-intensifieddaunorubicinimprovedoverallsurvivalinpatients60to65yearsofage.18Wehypothesizedthatintegratedmutationalanalysisofallknownmolecularalterationsoccurringinmorethan5%ofpatientswithAMLwouldallowustoidentifynovelmolecularmarkersofoutcomeinAMLandtoidentifymolecularlydefinedsubgroupsofpatientswhowouldbenefitfromdose-intensifiedinductionchemotherapy.最近的來自東部腫瘤協(xié)作組（ECOG）的3期臨床試驗(yàn)表明，與阿糖胞苷加45毫克每平方米柔紅霉素相比，阿糖胞苷加90毫克每平方米體表面積的柔紅霉素誘導(dǎo)治療可改善17至60歲初診AML患者的預(yù)后。類似研究表明年齡60歲以上病人加大劑量的柔紅霉素治療可以提高60到65歲患者總體生存。我們假設(shè)，所有已知的分子改變發(fā)生超過5%的AML患者的基因突變分析，允許我們識別新的結(jié)果分子標(biāo)記，識別受益于增強(qiáng)誘導(dǎo)化療劑量AML患者的亞組。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.我們對從腦電圖e1900試驗(yàn)患獲得的診斷樣本進(jìn)行了突變分析。所有患者提供書面知情同意書。試驗(yàn)組（398例）包括在e1900所有患者可用冷凍細(xì)胞允許DNA提取和突變分析。驗(yàn)證組（104例）組成的第二組病人的樣品儲存在Trizol試劑（Invitrogen公司），也是用來提取DNA作突變分析。我們研究的患者的臨床特征，如與完整的e1900試驗(yàn)相比，在附錄中有表S1可瀏覽詳細(xì)信息。納入分析患者的中位隨訪時(shí)間為從隨機(jī)的誘導(dǎo)治療的時(shí)間計(jì)算，為47.4個(gè)月。如最初由斯洛伐克等人所進(jìn)行的研究，由皮層細(xì)胞遺傳學(xué)中心審查委員會審查，通過染色體核型分析，熒光原位雜交，逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)（RT-PCR）進(jìn)行細(xì)胞遺傳學(xué)損傷檢測。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來自樣本中的55.2%例骨髓（277/502）和44.8%例的外周血（225/502）。我們測序整個(gè)編碼區(qū)的TET2，ASXL1，DNMT3A，CEBPA，PHF6，WT1基因，TP53，EZH2，RUNX1，和PTEN和先前描述的突變NPM1，F(xiàn)LT3，HRAS，KRAS，NRAS，KIT，IDH1和IDH2基因?；蚪M坐標(biāo)和所有本研究中所用的引物序列在附錄中表S2詳細(xì)列出。對配對緩解DNA（即，有完整的誘導(dǎo)緩解后化療患者DNA）是從241的398的參與者在測試隊(duì)列和從65的104在驗(yàn)證隊(duì)列獲取。對變異不能由無法緩解的DNA體細(xì)胞突變驗(yàn)證，在已發(fā)表的文獻(xiàn)中的基因突變的報(bào)道認(rèn)為沒有通過對特定基因的突變狀態(tài)方面的數(shù)據(jù)。進(jìn)一步測序方法的細(xì)節(jié)在附錄中詳細(xì)列舉。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()兩對共同雙突變分析由2*2列聯(lián)表和Fisher精確檢驗(yàn)進(jìn)行分析?；蛲蛔兒图?xì)胞遺傳學(xué)風(fēng)險(xiǎn)分類之間的關(guān)聯(lián)性采用卡方檢驗(yàn)進(jìn)行分析。層次聚類采用Lance-Williams法和complete-linkage算法進(jìn)行。生存時(shí)間測定日期的采用隨機(jī)抽取患者死亡日期和最后隨訪的日期之差計(jì)算取得。生存概率采用Kaplan-Meier法進(jìn)行估計(jì)和患者的突變和未突變的等位基因的比較采用log-rank檢驗(yàn)。多變量分析采用Cox模型的前進(jìn)法進(jìn)行。我們Schoenfeld殘差非零時(shí)間函數(shù)回歸規(guī)模對比例風(fēng)險(xiǎn)進(jìn)行假設(shè)檢驗(yàn)(附錄中表S3)。必要時(shí)，如對不同亞組進(jìn)行處理，通過單因素分析選擇有意義的變量。最后多變量模型采用新的風(fēng)險(xiǎn)分類規(guī)則進(jìn)行分析。當(dāng)如此表示，P值進(jìn)行調(diào)整以控制總Ⅰ類錯(cuò)誤率與通過SAS的PROCMULTTEST程序或R數(shù)據(jù)庫的multtest程序的重復(fù)采樣完整近似零假設(shè)分布的。ResultsFrequencyofGeneticAlterations基因修飾頻數(shù)Somaticalterationswereidentifiedin97.3%ofthepatients.Figure1showsthefrequencyofsomaticmutationsintheentirecohortandtheinterrelationshipsamongthevariousmutations,asrepresentedvisuallywiththeuseofaCircosplot.DataforallmolecularsubsetsareprovidedinFiguresS1andS2andTablesS4andS5intheSupplementaryAppendix.Inparticular,mutationalheterogeneitywasgreaterinpatientswithintermediate-riskAMLthaninpatientswithfavorable-riskorunfavorable-riskriskAML(P=0.01)(Fig.S2DintheSupplementaryAppendix).97.3%的病人發(fā)生體細(xì)胞修飾。根據(jù)Circos圖，圖1表明整個(gè)研究隊(duì)列的體細(xì)胞突變頻數(shù)以及各種突變間的相互關(guān)系。所有分子亞組的數(shù)據(jù)由附錄中圖S1、S2和表S4、S5進(jìn)行表述。尤其是，中度風(fēng)險(xiǎn)AML病人的突變異質(zhì)性比低度風(fēng)險(xiǎn)和惡性風(fēng)險(xiǎn)病人高很多(P=0.01)(附錄中圖S2D)。MutationalComplementationGroups突變互補(bǔ)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).整合的基因突變輪廓分析讓我們多次辨別共同發(fā)生的突變和在E1900病人隊(duì)列中發(fā)生的相互排斥的突變（附錄表S6）。另外，為了記錄core-binding–factor修飾t(8;21)和inv(16)/t(16;16)的KIT突變的經(jīng)常發(fā)生的共出現(xiàn)頻數(shù)，我們發(fā)現(xiàn)IDH1andIDH2的突變和NPM1突變具有顯著的共同出現(xiàn)差異，DNMT3A突變與NPM1,FLT3,andIDH1等位基因有顯著的共同出現(xiàn)差異（與所有對照比較P<0.001）（附錄表S7）。最近我們報(bào)到了IDH1和IDH2突變與TET2相互排斥，進(jìn)一步的突變分析證實(shí)IDH1與IDH2突變也與WT1突變相互排斥（P<0.001）（附錄圖S3和表S8）.也同時(shí)發(fā)現(xiàn)DNMT3A與MLL翻譯相互排斥（P<0.001）。MolecularDeterminantsofOverallSurvival總生存的分子決定因子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，單變量分析顯示，F(xiàn)LT3內(nèi)在銜接復(fù)制(FLT3-ITD)突變和MLL內(nèi)在銜接復(fù)制(MLL-PTD)突變與減少總生存相關(guān)(P=0.001forFLT3-ITDandP=0.009forMLL-PTD)(附錄表S9)，而CEBPA突變和core-binding–factor修飾t(8;21)andinv(16)/t(16;16)與改善總生存相關(guān)(P=0.05forCEBPAandP<0.001forthecore-binding–factor修飾)。另外，PHF6和ASXL1突變與減少總生存相關(guān)(P=0.006forPHF6andP=0.05forASXL1)(附錄圖S4)，IDH2突變與整個(gè)檢驗(yàn)隊(duì)列提高總生存率相關(guān)(3年生存率66%;P=0.01)(附錄圖S5)。IDH2突變的有利效應(yīng)僅在有IDH2R140Q突變的病人出現(xiàn)(P=0.009)(附錄圖S5)。除了MLL-PTD,PHF6,andASXL1的突變結(jié)果，單變量分析中所有的結(jié)果再多因素分析中也是有統(tǒng)計(jì)學(xué)意義(P<0.05,調(diào)整年齡、白細(xì)胞計(jì)數(shù)、移植情況[有vs沒有干細(xì)胞移植],、細(xì)胞遺傳特征)（附錄表S9）。在出現(xiàn)core-binding–factor改變的陽性病人中，KIT突變與降低總生存相關(guān)(P=0.006)，而與nv(16)/t(16;16)改變的病人無相關(guān)性(P=0.19)（附錄圖S6）PrognosticValueofMolecularAlterationsinIntermediate-RiskAMLAML中度風(fēng)險(xiǎn)患者的分子修飾的預(yù)后價(jià)值A(chǔ)mongpatientswithintermediate-riskAMLasdefinedbycytogeneticanalysis(TableS10intheSupplementaryAppendix),FLT3-ITDmutationswereassociatedwithreducedoverallsurvival(P=0.008),afindingthatisconsistentwiththeresultsofpreviousstudies.21ASXL1andPHF6mutationswereassociatedwithreducedsurvival,andIDH2R140Qmutationswithimprovedsurvival,amongpatientswithintermediate-riskAML(TableS10intheSupplementaryAppendix),aneffectsimilartothatintheentirecohort.Inaddition,wefoundthatTET2mutationswereassociatedwithreducedoverallsurvivalamongpatientswithintermediate-riskAML(P=0.007)(Fig.S7intheSupplementaryAppendix).通過細(xì)胞遺傳學(xué)分析定義的AML中度風(fēng)險(xiǎn)患者，F(xiàn)LT3-ITD突變與減少總生存相關(guān)(P=0.008)，該發(fā)現(xiàn)與早期研究一致。在具有中度風(fēng)險(xiǎn)的AML患者中，ASXL1和PHF6突變與降低生存相關(guān)，IDH2R140Q與改善生存相關(guān)（附錄表S10），改結(jié)果與整個(gè)隊(duì)列研究結(jié)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).多因素分析證實(shí)FLT3-ITD突變構(gòu)成AML中度風(fēng)險(xiǎn)患者結(jié)局的主要因素(調(diào)整P<0.001)。依據(jù)FLT3-ITD狀況，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的突變分別與不利結(jié)局相關(guān)。與具有NPM1突變和同時(shí)具有IDH1野生型和IDH2野生型的患者比較，同時(shí)具有NPM1與IDH1或IDH2突變的AML中度風(fēng)險(xiǎn)患者具有改善的三年生存期(89%vs.31%,P<0.001)(附錄圖S8)。然后，我們對具有FLT3-ITD野生型AML中度風(fēng)險(xiǎn)患者分成三類，其三年生存率具有顯著差異(調(diào)整P<0.001)：同時(shí)具有IDH1或IDH2突變和NPM1突變患者（總生存率89%）；具有TET2,ASXL1,PHF6,orMLL-PTD突變患者（總生存率6.3%）；具有野生型TET2,ASXL1,PHF6,andMLL-PTD,而非同時(shí)出現(xiàn)IDH和NPM1的患者（總生存率46.2%）。與正常核心患者的分析結(jié)果相似（附錄圖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突變型基因AML中度風(fēng)險(xiǎn)患者，我們研究發(fā)現(xiàn)CEBPA突變與改善的結(jié)局相關(guān)，三體性8的TET2,DNMT3A與MLL-PTD突變與不利結(jié)局相關(guān)。利用這些數(shù)據(jù)，我們把具有FLT3-ITD突變型的AML中度風(fēng)險(xiǎn)患者分為三類。第一類包括三倍性8或TET2,DNMT3A或MLL-PTD突變型，與不利結(jié)局相關(guān)(三年生存率14.5%)；這一生存率明顯低于第二類、第三類患者的生存率，第二類表現(xiàn)為野生型CEBPA,TET2,DNMT3A,andMLL-PTD(總生存率35.2%;P<0.001)，第三類患者表現(xiàn)為CEBPA突變(總生存率42%;P<0.001)(圖2B)。AML中度風(fēng)險(xiǎn)患者中具有突變株FLT3-ITD和野生型CEBPA,TET2,DNMT3A,和MLL-PTD與具有突變型FLT3-ITD和CEBPA的患者明顯沒有差別(P=0.34)，表明。對于具有FLT3-ITD突變型和正常核型AML患者，同樣的三類危險(xiǎn)因素分類有顯著的預(yù)后價(jià)值（附錄圖S9B）。PrognosticSchemawithIntegratedMutationalandCytogeneticProfiling整體突變和細(xì)胞遺傳分析的預(yù)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).這些研究結(jié)果讓我們形成預(yù)后模式，根據(jù)細(xì)胞遺傳數(shù)據(jù)的綜合突變分析的整合結(jié)果，可以分為三類危險(xiǎn)因素組：一是有利風(fēng)險(xiǎn)輪廓組(中位生存期，沒有達(dá)到;三年生存率64%)，二是中位風(fēng)險(xiǎn)輪廓組(中位生存期25.4月;三年生存率42%)，三是不利風(fēng)險(xiǎn)輪廓組(中位生存期10.1月，三年生存率12%)（附錄圖3A,3B，表S11）。多元統(tǒng)計(jì)分析，突變預(yù)測模式預(yù)測結(jié)局的獨(dú)立因素，包括年齡、白細(xì)胞計(jì)數(shù)、誘導(dǎo)計(jì)量、移植狀態(tài)(調(diào)整P<0.001)。不管術(shù)后緩解治療如何，我們的分類是真實(shí)有效的（自體或異體移植或單獨(dú)強(qiáng)化化療）（附錄圖S10）。Giventhenumberofvariablesinourprognosticclassification,wetestedthereproducibilityofthispredictorinanindependentcohortof104patientsfromtheECOGE1900trial.MutationalanalysisofthevalidationcohortconfirmedthereproducibilityofourprognosticschemaforpredictingtheoutcomeinpatientswithAML(adjustedP<0.001)(Fig.3C).Thepredictivevalueofthemutationalprognosticschemawasindependentofriskwithrespecttotreatment-relateddeath(definedasdeathwithin30daysafterinitiationoftreatment)orlackofresponsetoinductionchemotherapy(i.e.,lackofacompleteremission)inthetestcohortandinthecombinedtestandvalidationcohorts(TableS12intheSupplementaryAppendix).根據(jù)預(yù)后變量的數(shù)目，從ECOGE1900試驗(yàn)患者獨(dú)立選擇104名患者的數(shù)據(jù)，我們檢驗(yàn)了這些預(yù)測指標(biāo)的重現(xiàn)性。有效隊(duì)列的突變分析證實(shí)用來預(yù)測AML患者結(jié)局的預(yù)后預(yù)測模式(調(diào)整P<0.001)(圖.3C)。，突變預(yù)后模式的預(yù)測值獨(dú)立于有關(guān)危險(xiǎn)因素，包括治療相關(guān)死亡（誘導(dǎo)治療三十天內(nèi)死亡）或再檢驗(yàn)隊(duì)列中缺少誘導(dǎo)治療響應(yīng)（如，缺少緩解信息）或綜合試驗(yàn)或合法隊(duì)列（附錄表S12）。GeneticPredictorsofResponsetoInductionChemotherapy誘導(dǎo)治療反應(yīng)的基因預(yù)測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，突變型DNMT3A與AML患者不利結(jié)局相關(guān)。但是，我們也發(fā)現(xiàn)DNMT3A的突變與ECOGE1900隊(duì)列中的不利結(jié)局不相關(guān)(圖4A)(P=0.15)。在ECOGE1900隊(duì)列試驗(yàn)中，患者隨機(jī)分配接受兩種誘導(dǎo)治療，一種是阿糖胞苷加45mg柔紅霉素/平方米，一組是阿糖胞苷加90mg柔紅霉素/平方米。因此，我們假設(shè)高劑量柔紅霉素組能夠改善帶有DNMT3A突變患者生存。確實(shí)，我們發(fā)現(xiàn)DNMT3A突變狀態(tài)對劑量敏感性的化療結(jié)局確實(shí)有影響(圖4B)(P=0.02)。我們根據(jù)治療分組結(jié)局，評價(jià)DNMT3A的突變狀態(tài)效果，發(fā)現(xiàn)高劑量柔和霉素和具有DNMT3A突變患者改善的生存率相關(guān)(P=0.04)(附錄圖.S11A)，而和具有DNMT3A野生型患者改善的生存率不相關(guān)(P=0.15)(附錄圖.S11B)。另外，單因素分析顯示，劑量強(qiáng)化誘導(dǎo)治療與發(fā)生混合性白血病淋巴轉(zhuǎn)移AML患者的結(jié)局改善相關(guān)(P=0.01;P=0.06調(diào)整的多重分析)(附錄圖S11C、S11D)，與NPM1突變的AML患者的結(jié)局改善相關(guān)(P=0.01;P=0.10調(diào)整的多重分析)(附錄圖S11E、S11F和表S13)。因?yàn)镹PM1突變和MLL轉(zhuǎn)移的調(diào)整P值接近檢驗(yàn)水準(zhǔn)，必須在將來的后續(xù)試驗(yàn)中做進(jìn)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.我們把進(jìn)入隊(duì)列的患者分成兩組：一組是具有DNMT3A或NPM1突變，或具有MLL轉(zhuǎn)移的患者，一組是又有DNMT3A和NPM1野生型，沒有發(fā)生MLL轉(zhuǎn)移的患者。強(qiáng)化劑量誘導(dǎo)治療與DNMT3A陽性或者NPM1突變或MLL轉(zhuǎn)移患者生存率的顯著改善相關(guān)(P=0.001)(圖.4C)，與有DNMT3A和NPM1野生型，沒有發(fā)生MLL轉(zhuǎn)移的患者沒有相關(guān)(P=0.67)(Fig.4D)。這一發(fā)現(xiàn)獨(dú)立于一些臨床協(xié)變量，如年齡、白細(xì)胞計(jì)數(shù)、移植反應(yīng)狀態(tài)、治療相關(guān)死亡、化學(xué)療法的反應(yīng)（調(diào)整P=0.008、P=0.34分別和突變與野