流式细胞术联合实时荧光定量聚合酶链反应动态检测急性髓系白血病微小残留病和WT1基因表达水平

Dynamic monitoring of the expression level of minimal residual disease and WT1 gene by flow cytometry and real time quantitative PCR in acute myeloid leukemia

目的 探讨流式细胞术（FCM）联合实时荧光定量聚合酶链反应（RQ-PCR）动态检测急性髓系白血病（AML）患者微小残留病（MRD）和WT1基因表达水平的临床意义.方法 采用FCM联合RQ-PCR检测42例AML住院患者（除外M3）化疗前后349份骨髓标本的MRD值及WT1基因表达量.结果 化疗前AML患者的WT1基因表达水平与疗效无关（x2=0.166 3,P＞0.5）,首次诱导化疗后形态学完全缓解（CR）患者34例,其中MRD高水平11例,复发率达63.6 ％（7/11）,低水平23例,复发率21.7％（5/23）,两组之间复发率差异有统计学意义（x2=5.729,P＜ 0.025）.WT1基因高水平表达8例,复发率达87.5 ％（7/8）,低水平26例,复发率23.1％（6/26）,两组之间复发率差异有统计学意义（x2=10.749,P＜0.005）.WT1基因的表达水平在CR后下降（t=4.669,P＜0.001）.CR后WT1与MRD均为低水平表达者的复发率[15.0％（3/20）]明显低于其中有一项为高水平表达者的复发率[64.3％（9/14）],两组之间差异有统计学意义（P＜0.05）.且CR后WT1基因及MRD表达量呈正相关（r=0.835,P＜ 0.001）.结论 FCM联合RQ-PCR动态检测AML患者的MRD及WT1基因的表达水平可以作为AML患者MRD的监测指标,同时提高了MRD检测的阳性率,为临床开展个体化治疗及分层治疗提供一定的实验室依据.

Objective To explore the clinical significance of minimal residual disease （MRD） and WT1 in patients with acute myeloid leukemia （AML）.Methods 349 bone marrow specimens of 42 patients with AML （except M3） before and after chemotherapy were collected.The expression level of MRD was determined by flow cytometry （FCM） and WT1 gene was detected by real time quantitative PCR （RQ-PCR）.Results No correlation was observed between WT1 gene expression level of AML patients before chemotherapy with treatment response （x2 =0.166 3,P 〉 0.5）.34 AML patients achieved morphological complete remission （CR） after the first course of inductive chemotherapy.The recurrence rate was significantly higher in patients with high MRD level than that in those with low MRD level （63.6 % vs 21.7 %） （x2 =5.729,P 〈 0.025）,and significantly higher in patients with high WT1 expression than that in those with low WT1 expression （87.5 % vs 23.1%） （x2 =10.749,P 〈 0.005）.WT1 expression level of patients with CR was significantly lower than that of patients without CR （t =4.669,P 〈 0.001）.The recurrence rate of patients with both low level of MRD and WT1 was significantly lower than those of either high level of MRD and WT1 （15.0 % vs 64.3 %,P 〈 0.05）.The level of WT1 in patients with CR had positive correlation with the level of MRD （r =0.835,P 〈 0.001）.Conclusions MRD and WT1 have great importance in evaluating the therapeutic effects among patients with AML and increasing the positive rate of MRD.They also provide experimental basis for the individual and gradation treatment in the clinic.