微量殘留病檢測在急性白血病

微量殘留病檢測在急性白血病分層治療中的意義1整理pptMRD:TheleukemicpopulationundectablebymorpglogicmethodshasbeendefinedasMRD

MinimalResidualDisease(MRD)MRDisatermusedwhenthereisevidence(immunophenotypic,molecular,orcytogenetic)thatleukemiccellsremainintheBMbutthereareinsufficientcellstobedetectedbyroutineexaminationunderthemicroscope.2整理pptMRD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(jié)果3整理pptMRD檢測方法PCR:基因---定性、定量〔RQ-PCR〕b.多參數(shù)FCM：免疫標志發(fā)病時尋找免疫標志和基因標志進行檢測4整理ppt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5整理ppt優(yōu)點：靈敏：ALL-10-4,AML-10-3-10-4〔獲取細胞相關(guān)〕定量單位：細胞%快速：檢測當天即可知結(jié)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6整理ppt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(guān)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〕7整理ppt

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的分類根底：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8整理ppt9整理pptCytometry(communicationsinClinicalcytometry)38:139-152(1999)10整理pptTheeffectofend-inductionMRDonCIRCoustan-SmithE.BLOOD,2000;96:269111整理pptJohnsHopkinsMostinformationdisplaysNo.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)13,558-56712整理ppt第8天形態(tài)結(jié)果與29天MRD關(guān)系〔N=1016〕兩組抗體=90%FractionofpatientsMRD+atday29

Day8morphologyDay8bloodnegativeDay8bloodpositiveM1(<5%)4/258(1.65)25/255(9.8%)M2/M3(5-25%/>25%)2/51(3.9%)70/260(27%)Totals6/309(1.9%)96/515(18.6%)Leukemia(2003)17,1566-157213整理pptAChildren’sOncologyGroupStudy〔FCM-MRD)Blood.2021;111:5477誘導緩解后MRD檢測的意義〔D29〕,N=213414整理pptTheeffectofend-inductionMRDonearlyandlaterelapseRelapse-freesurvivalYears15整理pptEFSofNCISRpatientswithfavorablegeneticfeatures.YearsYears16整理pptPrognosticsignificanceofday-8PBMRD17整理pptCoxmultivariateanalysis18整理pptIdentificationofasubgroupofpatientswithexcellentoutcome12%Best-prognosispatients:NCI-SR+DTorTEL-AML1+MRD-atbothD8andD2919整理ppt成人-ALL-FCM20整理pptBlood.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=29Spain21整理ppt<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%at5years22整理pptUnivariateMultivariateWholeCRVariablePPPAge30/600.0020.05NWBC30000ul0.030.05NPhCh.+/-0.04N0.04TimetomCRD14/D33<0.0001NND35-MRD0.05%0.0010.010.02MultivariateAnalysisofthePrognosticImpactonRFS23整理pptStatusofminimalresidualdiseaseafterinductionpredictsoutcomeinbothstandardandhigh-riskPh-negativeadultacutelymphoblasticleukaemia.ThePolishAdultLeukemiaGroupALL4-2002MRDStudyN=116BritishJournalofHaematology,2021:142,227CD7/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-ALL24整理pptKaplan–MeierestimatesofrelapseaccordingtoMRDevaluationafterinduction25整理pptImpactofMRDstatusafterinductiononoutcomeaccordingtothetreatmentactuallyreceived.26整理pptUnivariateandmultivariateanalysisofprognosticfactorsassociatedwithincreasedriskofrelapseanddecreasedleukaemiafree-survival.27整理pptRiskandresponse-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/TD28整理pptChildhoodB-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%,VHR29整理pptRiskandresponse-basedclassificationofchildhoodB-ALLBlood:2007;109:926–935.PediatricOncologyGroup(POG)Children’sCancerGroup(CCG)Riskandresponse-basedclassificationofchildhoodB-ALL30整理pptPrecursor-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(2021)22,771–782AIEOP-BFMALL2000PCRMRD-directedriskgroupstratification31整理pptAIEOP-BFMALL2000化療流程32整理pptMRD-directedriskgroupstratificationHR:PPR;NR,BCR/ABL,MLL/AF433整理pptQR-PCR檢測MRD流程34整理pptEvent-freesurvivalcumulativeincidenceofrelapseBlood.2021;115:3206MolecularresponsetotreatmentredefinesallprognosticfactorsinchildrenandadolescentswithB-cellprecursoracuteLymphoblasticleukemia:resultsin3184patientsoftheAIEOP-BFMALL2000study35整理pptTEL/AML1+favorableDNAindex(>1.16and<1.6;B)PrognosticimpactofPCR-MRD36整理pptPh+

patientsPrognosticimpactofPCR-MRD37整理pptPrognosticimpactofPCR-MRDinPH-pB-ALLpatientswithinsubgroupsaccordingtoALL-BFM95criteria(N=3184)SRIRHR38整理pptResultsoftheunivariateandmultivariateanalyses39整理ppt正常B細胞的分化規(guī)律北京大學人民醫(yī)院血液病研究所

40整理pptCD10CD34CD19與CD45關(guān)系41整理pptCD22CD20CD19與CD45關(guān)系42整理pptCD38CD58CD19與CD45關(guān)系43整理pptI:CD34+CD10+cIg-sIg-(Com-B-ALL)II:CD34-CD10+cIg+sIg-(pre-B-ALL)III:CD34-CD10-cIg+sIg+(matureB-ALL)Note:noCD19+CD10-CD34+(Pro-B-ALL)44整理ppt成人與兒童B-ALL各亞型中LAIP發(fā)生率363/403(90.1)236/262(90.1)127/141(90.1)總8/15(53.3)e8/12(69.2)c0/3(0)a成熟-/-59/75(78.7)d35/46(76.1)b24/29(82.8)Pre-/+238/255(93.3)148/159(93.1)90/96(93.8)Com+/+58/58(100)45/45(100)13/13(100)Pro+/-總成人兒童CD34/CD10B-ALL亞型中國實驗血液學雜志2006；14：85345整理pptCampana.Cytometry38:139-152(1999)46整理pptMRD+0.1%CD45-CD10st+CD34s+分析全部CD19+的細胞，以Hematogones作為內(nèi)對照觀察每個雙參數(shù)的圖形，無僵硬設門優(yōu)點：防止表型改變所致假陰性不依賴于發(fā)病時的IM缺點：不易掌握47整理ppt4色FCM檢測B-ALLMRD的臨床意義98例：兒童78，成人20，671份標本，6.8

次/人，10.8月

/人，3.1月/次中國血液學雜志2006；27：302有無總體初次IM72例26例98BM復發(fā)12719復發(fā)前MRD+9615復發(fā)前MRD-101復發(fā)前MRD+%90%100%93.70%復發(fā)前<4MNT213

兩組四色抗體：通用：CD34/CD10/CD45/CD19

個體化：CD38,CD123,CD13,CD33,CD58,CD20/CD45/CD19

無IM:CD20/CD22/CD45/CD19orCD20/CD38/CD45/CD19連續(xù)監(jiān)測，間隔<3M。如MRD+？,需要證實時，在<2W。48整理ppt發(fā)病1-3個月MRD+和MRD-患者累計復發(fā)率P=0.00149整理ppt12歲女Pro-B-ALL連續(xù)檢測結(jié)果08.6.1108.8.1909.3.2609.6.30LAIP+%38.05（-）（-）0.03WT14.20.020.090.11IgH（+）（-）（-）（-）09.7.909.8.2009.9.2209.11.3LAIP+%0.090.116.5934.16WT10.090.069.917.2IgHN（-）N（+）50整理pptMRD+=0.45%7色-FCM-MRD-B-ALLCD10/CD34/CD123/CD38/CD58/CD45CD19CD19CD10CD10CD34CD38CD10CD58CD34CD45CD45CD123CD123CD10CD45CD45SSCSSC51整理pptAMLMRD檢測52整理pptNBM-CD34+細胞亞群分析CD7CD56CD3CD33CD38CD15CD1bCD13CD19HLA-DRCD117HLA-DRCD3453整理ppt

CD34+/SSClowCD117+/SSClow

相對比例（%）實際比例（%）相對比例（%）實際比例（%）CD11b+2.71±2.300.012±0.0113.36±1.840.028±0.011CD19+6.66±3.970.030±0.0211.23±0.720.009±0.005CD56+8.18±4.910.031±0.0215.89±4.670.025±0.020CD7+12.24±4.400.048±0.0266.83±2.180.051±0.021CD15+20.53±5.120.091±0.02829.59±4.720.256±0.085CD9+33.45±12.130.112±0.06214.17±3.370.076±0.028CD33+74.23±26.730.248±0.13043.05±10.710.319±0.130CD13+87.12±7.270.340±0.15445.49±14.560.397±0.199CD117+/CD34+89.14±5.920.316±0.14251.95±9.850.388±0.130HLA-DR+93.96±4.300.333±0.14687.97±6.770.660±0.293CD38+93.31±3.410.365±0.13191.96±3.770.694±0.304正常骨髓CD34+/SSClowCD117+/SSClow細胞中的抗原表達〔x±SD〕中華血液學雜志2021；29：12154整理pptLAIP陽性患者中LAIP+細胞比例（%）NBM中LAIP+細胞比例（%）對數(shù)差*CD34+CD7+44.09(9.44-93.46)0.042(0.016-0.109)3.02(2.35-3.35)CD34+CD56+41.65(10.18-94.07)0.027(0.005-0.087)3.19(2.58-3.54)CD34+CD11b+26.10(4.17-83.68)0.007(0.000-0.039)3.57(2.78-4.08)CD34+CD15+45.05(12.92-83.43)0.090(0.038-0.143)2.70(2.16-2.97)CD117+CD34+CD9+40.16(16.00-56.83)0.093(0.040-0.138)2.64(2.24-2.79)CD117+CD34-CD11b+41.65(20.48-93.59)0.049(0.004-0.145)2.93(2.62-3.28)CD117+CD34-CD15+35.92(27.99-45.96)0.097(0.038-0.263)2.57(2.46-2.68)CD117+CD34-CD9+36.65(21.08-97.22)0.036(0.019-0.351)3.01(2.77-3.43)CD117+CD15-11b+60.06(21.96-85.86)0.025(0.003-0.044)3.38(2.94-3.54)CD34+CD33-HLA-DR+63.53(21.60-90.31)0.042(0.004-0.182)3.18(2.71-3.33)610例AML和正常骨髓中LAIP細胞的表達差異中華血液學雜志2007；28：73119種抗體，四色組合，LAIP+=86%55整理ppt診斷時AMLM2患者特點：SSC小CD117+CD33+CD13-HLA-DR-LAIP:CD117+CD34-CD33+HLA-DR-和CD117+CD34-CD33+CD13-56整理ppt診斷后23個月〔CR〕MRD〔+〕=0.25%診斷后27個月〔CR〕MRD〔+〕=0.80%57整理pptAML治療后MRD(+)和MRD(-)患者RFS3-4M1-2M5-6M中國實驗血液學雜志2021;17:551N=95AML601份BMLAIP+58整理pptMRD+=0.33%7色-FCM-MRD-AMLCD56/CD19/CD33/CD34/CD117/CD45CD34CD38CD56CD117CD34CD56CD19CD19CD33CD33CD33CD33CD56CD38CD33CD45CD11759整