微量殘留病檢測(cè)在急性白血病

1、微量殘留病檢測(cè)在急性白血病微量殘留病檢測(cè)在急性白血病分層治療中的意義分層治療中的意義 MRD:The leukemic population undectable by morpglogic methodshas been defined as MRD Minimal Residual Disease (MRD)MRD is a term used when there is evidence( immunophenotypic, molecular, or cytogenetic) that leukemic cells remain in the BM but there are insu

2、fficient cells to be detected by routine examination under the microscope.MRD檢測(cè)的方法檢測(cè)的方法MRD監(jiān)測(cè)的臨床意義監(jiān)測(cè)的臨床意義 MRD 檢測(cè)在分層治療中的意義檢測(cè)在分層治療中的意義FCM-MRD檢測(cè)的結(jié)果檢測(cè)的結(jié)果MRD檢測(cè)方法檢測(cè)方法a. PCR: 基因基因-定性、定性、 定量（定量（RQ-PCR）b.多參數(shù)多參數(shù)FCM：免疫標(biāo)志免疫標(biāo)志發(fā)病時(shí)尋找免疫標(biāo)志和基因標(biāo)志進(jìn)行檢測(cè)發(fā)病時(shí)尋找免疫標(biāo)志和基因標(biāo)志進(jìn)行檢測(cè)目的基因：目的基因：融合基因：融合基因：BCR/ABL,AML1/ETO,PML/RAR 基因重排：基因

3、重排： IgH，TCR(10-4-10-5) 基因突變：基因突變：FLT3-ITD,NPM1(10-5) 基因表達(dá)增加：基因表達(dá)增加：WT1,PRAME(10-3)優(yōu)點(diǎn)：優(yōu)點(diǎn)： 靈敏度高靈敏度高-10-5-10-6/5 copes /10-5 特異性強(qiáng)特異性強(qiáng)缺點(diǎn)缺點(diǎn): 1. 融合基因：應(yīng)用范圍有限：融合基因：應(yīng)用范圍有限： ALL:10-30%，AML:30-50% 2. IgH/TCR : 90%, 操作復(fù)雜，費(fèi)時(shí)操作復(fù)雜，費(fèi)時(shí),需要基因測(cè)序，特異引需要基因測(cè)序，特異引 物物/探針探針(每例患者特異）每例患者特異） 3.容易污染，出現(xiàn)假陽(yáng)性。容易污染，出現(xiàn)假陽(yáng)性。RQ-PCR檢測(cè)檢測(cè)MRD

4、特點(diǎn)特點(diǎn)優(yōu)點(diǎn)：優(yōu)點(diǎn)： 靈敏：靈敏：ALL-10-4 , AML-10-3-10-4 （獲取細(xì)胞相關(guān)）（獲取細(xì)胞相關(guān)） 定量單位：細(xì)胞定量單位：細(xì)胞% 快速：檢測(cè)當(dāng)天即可知結(jié)果快速：檢測(cè)當(dāng)天即可知結(jié)果 操作簡(jiǎn)便操作簡(jiǎn)便 應(yīng)用范圍廣：應(yīng)用范圍廣：90%缺點(diǎn)缺點(diǎn)： 表型的變化表型的變化:假陰性假陰性 受前體受前體B細(xì)胞細(xì)胞(Hematogones)的干擾：的干擾： 形態(tài)幼稚，表達(dá)形態(tài)幼稚，表達(dá)CD34,TDT 在小兒、化療后、在小兒、化療后、SCT后比例增加后比例增加5% 需要較高的分析水平和技能需要較高的分析水平和技能 應(yīng)用不夠廣泛，需要建立標(biāo)準(zhǔn)化操作應(yīng)用不夠廣泛，需要建立標(biāo)準(zhǔn)化操作FCM MRD

5、檢測(cè)的特點(diǎn)檢測(cè)的特點(diǎn)定義：正常骨髓定義：正常骨髓/PB中不表達(dá)或表達(dá)比例較低的中不表達(dá)或表達(dá)比例較低的 免疫表型免疫表型 白血病相關(guān)的免疫表型白血病相關(guān)的免疫表型(LAIP)（Leukemia Associated ImmunoPhenotype）FCM-MRD：白血病細(xì)胞的特異抗原：白血病細(xì)胞的特異抗原：NG2(7.1)IM/MRD檢測(cè)的抗原檢測(cè)的抗原CD34,CD33-：正常分化抗原：表達(dá)于：正常分化抗原：表達(dá)于正常細(xì)胞的不同系列、分化階段正常細(xì)胞的不同系列、分化階段（非非白血病細(xì)胞所特異）白血病細(xì)胞所特異） 交叉系列抗原交叉系列抗原: B、T、髓、髓、NK細(xì)胞抗原細(xì)胞抗原 非同期抗原共表

6、達(dá)非同期抗原共表達(dá): CD15/CD117 CD34/CD64 抗原表達(dá)量異常：表達(dá)強(qiáng)度過(guò)高、過(guò)低或不表達(dá)抗原表達(dá)量異常：表達(dá)強(qiáng)度過(guò)高、過(guò)低或不表達(dá) 異常的光散射信號(hào)：異常的光散射信號(hào)：FSC和和SSC LAIP的分類的分類基礎(chǔ)：熟悉正常細(xì)胞不同分化階段抗原出現(xiàn)的基礎(chǔ)：熟悉正常細(xì)胞不同分化階段抗原出現(xiàn)的順序順序 和表達(dá)和表達(dá)量量的規(guī)律的規(guī)律Cytometry (communications in Clinical cytometry)38:139-152(1999)The effect of end-induction MRD on CIRCoustan-Smith E.BLOOD,2000;

7、96:2691Johns HopkinsMost information displays No. of cases(%) A CD19/CD45/CD20/CD10 (N=82)CD45 vs CD10 48(59)FSC vs CD10 13(16)FSC vs CD20 11(13)CD10 vs CD20 4(5) 76(93)B CD19/CD45/CD9/CD34 (N=77) CD45 vs CD34 27(35) FSC vs CD34 17(22)CD34 vs CD9 16(21) 72(94)A+B 81(99) Leukemia(1999)13,558-567第第8天形

8、態(tài)結(jié)果與天形態(tài)結(jié)果與29天天MRD關(guān)系關(guān)系 （N=1016）兩組抗體兩組抗體=90% Fraction of patients MRD + at day 29 Day 8 morphology Day 8 blood negative Day 8 blood positive M1 (25%) 2/51(3.9%) 70/260(27%)Totals 6/309(1.9%) 96/515(18.6%)Leukemia(2003)17,1566-1572 A Childrens Oncology Group Study（FCM-MRD) Blood.2008;111:5477 誘導(dǎo)緩解后誘導(dǎo)緩解

9、后MRD檢測(cè)的意義（檢測(cè)的意義（D29）, N=2134The effect of end-induction MRD on early and late relapseRelapse-free survivalYearsEFS of NCI SR patients with favorable genetic features.YearsYearsPrognostic significance of day-8 PB MRDCox multivariate analysisIdentification of a subgroup of patients with excellent outc

10、ome12% Best-prognosis patients:NCI-SR+DT or TEL-AML1+MRD- at both D8 and D29成人成人-ALL-FCMBlood.2003;10:4695 CD7/CD5/CD3CD4/CD8/CD3CD7/CD2/CD3CD7/CD34/CD38CD19/CD34/CD45CD10/CD13/CD19CD5/CD33/CD20CD34/CD38/CD19TDT/CD10/CD19CD34/CD22/CD19B-ALL N=73T-ALL N=29Spain0.05%N=440.5%,N=42Day +33Day +14MRD水平與水平

11、與RFS11.76%(n=12/102), Day 14 MRD-/0.03%, RFS OF 90% at 5 yearsUnivariateUnivariateMultivariateMultivariateWholeWholeCRCRVariableVariableP PP PP PAgeAge30/6030/600.0020.0020.050.05N NWBCWBC30000ul30000ul0.030.030.050.05N NPh Ch.Ph Ch.+/-+/-0.040.04N N0.040.04Time to mCRTime to mCRD14/D33D14/D330.0001

12、0.0001N NN ND35-MRDD35-MRD0.05%0.05%0.0010.0010.010.010.020.02Multivariate Analysis of the Prognostic Impact on RFSStatus of minimal residual disease after induction predictsoutcome in both standard and high-risk Ph-negative adult acutelymphoblastic leukaemia. The Polish Adult Leukemia GroupALL 4-20

13、02 MRD Study N=116British Journal of Haematology, 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-ALLKaplanM

14、eier estimates of relapse according to MRD evaluation after inductionImpact of MRD status after induction on outcome accordingto the treatment actually received.Univariate and multivariate analysis of prognostic factorsassociated with increased risk of relapse and decreasedleukaemia free-survival.Ri

15、sk and response-based classification of childhood B-ALLChildrens Oncology Group (COG)Blood: 2007; 109: 926935. Retrospective,CCG:1988-1995,POG:1986-1999,N=6238,Age:1-22yCOG risk classification schemeRisk factors: Age:10 y WBC:50000/ L Cytogenetics:TEL/AML1,Trisomies(4,10.17),BCR/ABL,MLL, Day-14 marr

16、ow response:M125% blast Day-29 MRD-FCM CNS/TD Childhood B-Precursor-ALL Age:10,WBC:50000/ L :High Risk :Standard RiskTEL/Tris,D8/15,29 BM,D29MRD, CNSorTD/MLL+, M1,M10.1%.-/nLow Risk-, M1,M10.1-1.0%.+/+SRHRD8/15 BM,D29BM/MRD,CNS orTD/MLL+,M1,M10.1-.0%.+/+HRHRRandomizedAugmentedAugmentedBCR/ABL.MLL+SE

17、R.DI1.0%+D43-M2,3/MRD1.0%,VHRRisk and response-based classification of childhood B-ALLBlood: 2007; 109: 926935. Pediatric Oncology Group (POG)Childrens Cancer Group (CCG)Risk and response-based classification of childhood B-ALLPrecursor-B-ALLPrecursor-B-ALLT-ALLT-ALLALLALLN=2854N=2854N=422N=422N=334

18、1N=3341No target54(2%)54(2%)29(7%)29(7%)88(3%)88(3%) 1 target2800(96%)2800(96%)393(93%)393(93%)3253(97%)3253(97%) 2 targets2662(93%)2662(93%)379(88%)379(88%)3089(92%)3089(92%) 3 targets2189(77%)2189(77%)281(67%)281(67%)2510(75%)2510(75%)No sensitive target173(6%)173(6%)38(9%)38(9%)217(6%)217(6%)1 se

19、nsitive target561(20%)561(20%)95(22%)95(22%)671(20%)671(20%) 2 sensitive targets2066(72%)2066(72%)260(62%)260(62%)2365(71%)2365(71%)IgH TCR基因的陽(yáng)性率和敏感性基因的陽(yáng)性率和敏感性T Flohr，Leukemia (2008) 22, 771782AIEOP-BFM ALL 2000PCR MRD-directed risk group stratificationAIEOP-BFM ALL 2000化療流程化療流程MRD-directed risk gro

20、up stratificationHR:PPR;NR,BCR/ABL,MLL/AF4QR-PCR 檢測(cè)檢測(cè)MRD流程流程Event-free survivalcumulative incidence of relapseBlood.2010;115:3206Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute Lymphoblastic leukemia:results in 3184 patients of

21、 the AIEOP-BFM ALL 2000 studyTEL/AML1+favorable DNA index ( 1.16 and 1.6; B)Prognostic impact of PCR-MRDPh+ patientsPrognostic impact of PCR-MRDPrognostic impact of PCR-MRD in PH-pB-ALL patients within subgroups according to ALL-BFM 95 criteria(N=3184)SRIRHRResults of the univariate and multivariate

22、 analyses正常正常B細(xì)胞的分化規(guī)律細(xì)胞的分化規(guī)律北京大學(xué)人民醫(yī)院血液病研究所北京大學(xué)人民醫(yī)院血液病研究所 CD10 CD34 CD19CD10 CD34 CD19與與 CD45 CD45 關(guān)系關(guān)系CD22 CD20 CD19CD22 CD20 CD19與與 CD45CD45關(guān)系關(guān)系CD38 CD58 CD19CD38 CD58 CD19與與 CD45CD45關(guān)系關(guān)系I: CD34+CD10+ cIg- sIg- (Com-B-ALL)II:CD34-CD10+ cIg+ sIg- (pre-B-ALL)III: CD34-CD10- cIg+ sIg+(mature B-ALL)Not

23、e : no CD19+CD10-CD34+(Pro-B-ALL)成人與兒童成人與兒童B-ALLB-ALL各亞型中各亞型中LAIP LAIP 發(fā)生率發(fā)生率363/403(90.1)236/262(90.1)127/141(90.1)總總8/15(53.3)e8/12(69.2)c0/3(0)a成熟成熟 -/-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 +/-總總成人成人兒童兒童CD34/CD10 B-

24、ALL亞型亞型中國(guó)實(shí)驗(yàn)血液學(xué)雜志中國(guó)實(shí)驗(yàn)血液學(xué)雜志 2006；14：853Campana. Cytometry 38:139-152(1999)MRD+0.1%CD45-CD10st+CD34s+分析全部分析全部CD19+的細(xì)胞，的細(xì)胞，以以Hematogones作為內(nèi)作為內(nèi)對(duì)照對(duì)照觀察每個(gè)雙參數(shù)的圖形，觀察每個(gè)雙參數(shù)的圖形，無(wú)僵硬設(shè)門(mén)無(wú)僵硬設(shè)門(mén)優(yōu)點(diǎn)：避免表型改變所優(yōu)點(diǎn)：避免表型改變所致假陰性致假陰性不依賴于發(fā)病時(shí)的不依賴于發(fā)病時(shí)的IM缺點(diǎn)：不易掌握缺點(diǎn)：不易掌握4 4色色FCMFCM檢測(cè)檢測(cè)B-ALL MRDB-ALL MRD的臨床意義的臨床意義9898例：兒童例：兒童7878，成人，成人2

25、0 20 ，671671份標(biāo)本，份標(biāo)本，6.8 次/人，10.8月月 /人， 3.1月/次中國(guó)血液學(xué)雜志中國(guó)血液學(xué)雜志 2006；27：302有有無(wú)無(wú)總體總體初次初次IMIM7272例例2626例例9898BMBM復(fù)發(fā)復(fù)發(fā)12127 71919復(fù)發(fā)前復(fù)發(fā)前MRD+MRD+9 96 61515復(fù)發(fā)前復(fù)發(fā)前MRD-MRD-1 10 01 1復(fù)發(fā)前復(fù)發(fā)前MRD+%MRD+%90%90%100%100%93.70%93.70%復(fù)發(fā)前復(fù)發(fā)前4M NT4M NT2 21 13 3 兩組四色抗體：通用：兩組四色抗體：通用：CD34/CD10/CD45/CD19 個(gè)體化：個(gè)體化：CD38,CD123,CD13

26、,CD33,CD58,CD20/CD45/CD19 無(wú)無(wú)IM: CD20/CD22/CD45/CD19 or CD20/CD38/CD45/CD19連續(xù)監(jiān)測(cè)，間隔連續(xù)監(jiān)測(cè)，間隔3M。如。如MRD+？,需要證實(shí)時(shí)，在需要證實(shí)時(shí)，在2W。0102030405060010203040506070M RD+;50%,n=12M RD-:7.5%,n=40隨 訪時(shí)間 （月 ）累 積復(fù) 發(fā) 率發(fā)病發(fā)病1-31-3個(gè)月個(gè)月MRDMRD+ +和和MRDMRD-患者累計(jì)復(fù)發(fā)率患者累計(jì)復(fù)發(fā)率P=0.00112歲歲 女女 Pro-B-ALL 連續(xù)檢測(cè)結(jié)果連續(xù)檢測(cè)結(jié)果08.6.1108.6.1108.8.1908.8

27、.1909.3.2609.3.2609.6.3009.6.30LAIP+%LAIP+%38.0538.05（- -）（- -）0.030.03WT1WT14.24.20.020.020.090.090.110.11IgHIgH（+ +）（- -）（- -）（- -）09.7.909.7.909.8.2009.8.2009.9.2209.9.2209.11.309.11.3LAIP+%LAIP+%0.090.090.110.116.596.5934.1634.16WT1WT10.090.090.060.069.99.917.217.2IgHIgHN N（- -）N N（+ +）MRD+=0.45

28、%7色色-FCM-MRD-B-ALLCD10/CD34/CD123/CD38/CD58/CD45CD19CD19CD10CD10CD34CD38CD10CD58CD34CD45CD45CD123CD123CD10CD45CD45SSCSSCAML MRD檢測(cè)檢測(cè)NBM-CD34+細(xì)胞亞群分析細(xì)胞亞群分析CD7CD56CD3CD33CD38CD15CD1bCD13CD19HLA-DRCD117HLA-DRCD34CD34+/SSClowCD117+/SSClow相對(duì)比例（相對(duì)比例（%）實(shí)際比例實(shí)際比例（%）相對(duì)比例相對(duì)比例（%）實(shí)際比例（實(shí)際比例（%）CD11b+2.712.300.0120.0

29、113.361.840.0280.011CD19+6.663.970.0300.0211.230.720.0090.005CD56+8.184.910.0310.0215.894.670.0250.020CD7+12.244.400.0480.0266.832.180.0510.021CD15+20.535.120.0910.02829.594.720.2560.085CD9+33.4512.130.1120.06214.173.370.0760.028CD33+74.2326.730.2480.13043.0510.710.3190.130CD13+87.127.270.3400.15445

30、.4914.560.3970.199CD117+/CD34+89.145.920.3160.14251.959.850.3880.130HLA-DR+93.964.300.3330.14687.976.770.6600.293CD38+93.313.410.3650.13191.963.770.6940.304 正常骨髓正常骨髓CD34+/SSClow CD117+/SSClow細(xì)胞中的抗原表達(dá)（細(xì)胞中的抗原表達(dá)（xSD） 中華血液學(xué)雜志中華血液學(xué)雜志2008；29：121LAIP陽(yáng)性患者中陽(yáng)性患者中LAIP+細(xì)細(xì)胞比例（胞比例（%）NBM中中LAIP+細(xì)胞細(xì)胞比例（比例（%）對(duì)數(shù)差對(duì)數(shù)差*C

31、D34+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 (0.040-0.13

32、8)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例例AML和正常骨髓中和