MRD檢測在急性白血病分層治療中的意義

北京大學人民醫(yī)院北京大學血液病研究所微量殘留病檢測在急性白血病分層治療中的意義MRD:TheleukemicpopulationundectablebymorpglogicmethodshasbeendefinedasMRD

MinimalResidualDisease(MRD)MRDisatermusedwhenthereisevidence(immunophenotypic,molecular,orcytogenetic)thatleukemiccellsremainintheBMbutthereareinsufficientcellstobedetectedbyroutineexaminationunderthemicroscope.MRD檢測的方法MRD監(jiān)測的臨床意義

MRD檢測在分層治療中的意義FCM-MRD檢測的結果MRD檢測方法PCR:基因---定性、定量（RQ-PCR）b.多參數FCM：免疫標志發(fā)病時尋找免疫標志和基因標志進行檢測目的基因：融合基因：BCR/ABL,AML1/ETO,PML/RAR

基因重排：IgH，TCR(10-4-10-5)基因突變：FLT3-ITD,NPM1(10-5)基因表達增加：WT1,PRAME((10-3)優(yōu)點：靈敏度高---10-5-10-6/5copes/10-5

特異性強缺點:1.融合基因：應用范圍有限：ALL:10-30%，AML:30-50%2.IgH/TCR:90%,操作復雜，費時,需要基因測序，特異引物/探針(每例患者特異）3.容易污染，出現(xiàn)假陽性。RQ-PCR檢測MRD特點優(yōu)點：靈敏：ALL-10-4,AML-10-3-10-4

（獲取細胞相關）定量單位：細胞%快速：檢測當天即可知結果操作簡便應用范圍廣：90%缺點：表型的變化:假陰性受前體B細胞(Hematogones)的干擾：形態(tài)幼稚，表達CD34,TDT在小兒、化療后、SCT后比例增加>5%需要較高的分析水平和技能應用不夠廣泛，需要建立標準化操作FCMMRD檢測的特點定義：正常骨髓/PB中不表達或表達比例較低的免疫表型白血病相關的免疫表型(LAIP)（LeukemiaAssociatedImmunoPhenotype）FCM-MRD：白血病細胞的特異抗原：NG2(7.1)IM/MRD檢測的抗原CD34,CD33---：正常分化抗原：表達于正常細胞的不同系列、分化階段（非白血病細胞所特異）交叉系列抗原:B、T、髓、NK細胞抗原非同期抗原共表達:CD15/CD117CD34/CD64

抗原表達量異常：表達強度過高、過低或不表達異常的光散射信號：FSC和SSC

LAIP的分類基礎：熟悉正常細胞不同分化階段抗原出現(xiàn)的順序和表達量的規(guī)律Cytometry(communicationsinClinicalcytometry)38:139-152(1999)誘導緩解后MRD水平與累計復發(fā)率Coustan-SmithE.BLOOD,2000;96:2691JohnsHopkinsMostinformationdisplaysNo.ofcases(%)ACD19/CD45/CD20/CD10(N=82)CD45vsCD1048(59)FSCvsCD1013(16)FSCvsCD2011(13)CD10vsCD204(5)76(93)BCD19/CD45/CD9/CD34(N=77)CD45vsCD3427(35)FSCvsCD3417(22)CD34vsCD916(21)72(94)A+B81(99)

Leukemia(1999)13,558-567AChildren’sOncologyGroupStudy（FCM-MRD)

Blood.2008;111:5477誘導緩解后MRD檢測的意義（D29）,N=2134誘導后MRD水平對早期和晚期復發(fā)的影響Relapse-freesurvivalYearsNCISR伴好遺傳學特性患者MRD與EFS關系YearsYears4,108天PBMRD的意義多變量分析鑒別預后非常好的一組患者NCI-SR+DTTEL-AML1+MRD-D8andD2912%成人-ALL-FCMBlood.2003;10:4695CD7/CD5/CD3CD4/CD8/CD3CD7/CD2/CD3CD7/CD34/CD38CD19/CD34/CD45CD10/CD13/CD19CD5/CD33/CD20CD34/CD38/CD19TDT/CD10/CD19CD34/CD22/CD19B-ALLN=73T-ALLN=29Spain<0.05%N=43>0.05%N=44<0.5%N=21>0.5%,N=42Day+33Day+14MRD水平與RFS11.76%(n=12/102),Day14MRD-/<0.03%,RFSOF90%at5yearsUnivariateMultivariateWholeCRVariablePPPAge30/600.0020.05NWBC30000ul0.030.05NPhCh.+/-0.04N0.04TimetomCRD14/D33<0.0001NND35-MRD0.05%0.0010.010.02對RFS的多變量分析Statusofminimalresidualdiseaseafterinductionpredictsoutcomeinbothstandardandhigh-riskPh-negativeadultacutelymphoblasticleukaemia.ThePolishAdultLeukemiaGroupALL4-2002MRDStudyN=116BritishJournalofHaematology,2008:142,227CD7/CD2/CD3CD7/CD5/CD3CD7/CD38/CD34CD7/CD4/CD8TDT/CD7/Ccd3CD7/CD1a/CD3cCD3/CD7/CD3CD10/CD20/CD19CD34/CD22/CD19CD34/CD38/CD19CD45/CD34/CD19TDT/CD10/CD19CD58/CD52/CD19CD33/CD13/CD19CD15/CD117/CD19CD65/CD56/CD19CD7/CD2/CD10B-ALLT-ALL誘導緩解后MRD與復發(fā)關系誘導后MRD狀態(tài)與治療的關系對復發(fā)率和leukaemiafree-survival的多變量分析Riskandresponse-basedclassificationofchildhoodB-ALLChildren’sOncologyGroup(COG)Blood:2007;109:926–935.Retrospective,CCG:1988-1995,POG:1986-1999,N=6238,Age:1-22yCOGriskclassificationschemeRiskfactors:Age:10yWBC:50000/LCytogenetics:TEL/AML1,Trisomies(4,10.17),BCR/ABL,MLL,Day-14marrowresponse:M1<5%.M2:5-25%,M3:>25%blastDay-29MRD-FCMCNS/TDChildhoodB-Precursor-ALLAge:10,WBC:50000/L

:HighRisk<:StandardRiskTEL/Tris,D8/15,29BM,D29MRD,CNSorTD/MLL+,M1,M1<0.1%.-/nLowRisk-,M1,M1<0.1%-/n+-,D15M2/3,>0.1-1.0%.+/+SRHRD8/15BM,D29BM/MRD,CNSorTD/MLL+,M1,M1<0.1%.-/nD15M2/3,>0.1-.0%.+/+HRHRRandomizedAugmentedAugmentedBCR/ABL.MLL+SER.DI<0.81/44Ch.NR,D29-M2/MRD>1.0%+D43-M2,3/MRD>1.0%,VHRRiskandresponse-basedclassificationofchildhoodB-ALLBlood:2007;109:926–935.PediatricOncologyGroup(POG)Children’sCancerGroup(CCG)Riskandresponse-basedclassificationofchildhoodB-ALLPrecursor-B-ALLT-ALLALLN=2854N=422N=3341Notarget54(2%)29(7%)88(3%)≥1target2800(96%)393(93%)3253(97%)≥2targets2662(93%)379(88%)3089(92%)≥3targets2189(77%)281(67%)2510(75%)Nosensitivetarget173(6%)38(9%)217(6%)1sensitivetarget561(20%)95(22%)671(20%)≥2sensitivetargets2066(72%)260(62%)2365(71%)IgHTCR基因的陽性率和敏感性TFlohr，Leukemia(2008)22,771–782AIEOP-BFMALL2000PCRMRD指導的危險度分層AIEOP-BFMALL2000化療流程MRD指導的分層標準HR:PPR;NR,BCR/ABL,MLL/AF4QR-PCR檢測MRD流程Event-freesurvivalcumulativeincidenceofrelapseBlood.2010;115:3206MolecularresponsetotreatmentredefinesallprognosticfactorsinchildrenandadolescentswithB-cellprecursoracuteLymphoblasticleukemia:resultsin3184patientsoftheAIEOP-BFMALL2000studyTEL/AML1+favorableDNAindex(>1.16and<1.6)PCR-MRD對預后好患者的影響PCR-MRD對Ph+

患者的影響SRIRHRPCR-MRD對Ph-患者的影響多變量分析CML歐洲白血病網(ELN)最新推薦伊馬替尼400mg/d初始治療失敗的定義:3個月未達到CHR,6個月未達到任何CyR,12個月未達到PCyR,18個月未達到CCyR,任何時間,丟失之前達到的CCyR或CHR

疾病進展或出現(xiàn)耐藥的Abl激酶突變

推薦采用第二代TKI尼洛替尼治療慢性期加速/急變期NCCN最新版治療指南推薦對于伊馬替尼400mg/d初始治療患者，出現(xiàn)以下事件推薦患者接受尼洛替尼治療:3個月未達到血液學反應或者血液學復發(fā),6個月未達到任何CyR,12個月未達到PCyR或者細胞遺傳學復發(fā),18個月未達到CCyR或者細胞遺傳學復發(fā),

CML患者CCR后BCR-ABLmRNA動態(tài)變化與imatinib應用QinYZ,LiuYR,ZhuHH,etal.IntJLabHematol2008;30:317“○”：CCR“●”：Ph+其中1例患者由CCR進展至急變期，BCR-ABL升高2.5log,但未檢測出Ph染色體Q