实时定量荧光反转录-聚合酶链反应检测急性白血病AML1／ETO融合基因

Real-time RT-PCR for detection and quantification of AML1/ETO leukemia fusion gene

目的建立实时定量荧光反转录-聚合酶链反应（RQRT—PCR）的方法并用来检测急性髓系白血病（AML）-M2患者中AML1／ETO融合基因的拷贝数，观察患者体内融合基因阳性率、该融合基因转录水平的变化情况及AML1／ETO融合基因阳性患者对治疗的反应。方法利用含AML1／ETO融合基因的Kasumi-1细胞株构建质粒标准品并制作标准曲线，检测25例AML—M2患者的骨髓及外周血标本45份，个别患者连续监测融合基因转录表达水平。25例患者均同时行流式细胞术免疫分型检测及骨髓细胞染色体检查，确诊后给予MA方案进行诱导缓解。结果7例（28％）AML—M2初发确诊患者检测到AML1／ETO阳性（AML1／ETO：ABL为0．01～19．2），其中5例（20％）有t（8；21）（q22；q22）。连续监测患者融合基因转录表达水平与临床缓解和复发的变化情况相吻合。7例AML1／ETO融合基因阳性患者均在MA治疗1个疗程后达完全缓解，AML1／ETO融合基因下降3个数量级。其余18例完全缓解11例。连续监测7例AML1／ETO融合基因阳性患者6个月均处于完全缓解状态。结论RQRT—PCR技术成熟、操作简便，检测白血病融合基因结果准确稳定，对于临床明确诊断、具体分型、动态观测肿瘤负荷、选择治疗方案、评估治疗效果和预后都有较大价值。

Objective To set up real-time quantitative RT-PCR technique and measure leukemia fusion gene transcripts in patients with AML of FAB-M2 subtype, and also to investigate the positive rate in patients and the relationship between the AML1/ETO mRNA levels and the response rate after chemical therapy. Methods The plasmid containing the AML1/ETO fusion gene sequences were constructed from myeloid cell lines Kasumi-1 （expressing AML1/ETO） to establish the standard curves. A TaqMan based realtime quantitative RT-PCR was performed to measure aberrant fusion gene transcripts in 45 samples of peripheral blood （PB） or bone marrow （BM） from 25 newly diagnosed patients with AML-M2. All these 25 patients were diagnosed by the FCM （flow cytometry） and bone marrow molecular cytogenetics, and received the induce remission therapy with MA （Mitoxantrone ＋ Ara-C）. Results As a result, the AML1/ETO fusion gene transcripts were detected in 7 （28 %） out of 25 AML-M2 patients （the ratios of AML1/ETO/ABL vary from 0.01 to 19.2）, in which 5 patients were found t（8;21）（q22;q22）. The transcript level of AML1/ETO fusion gene varied from the clinical situation of patients. These 7 patients with AML1/ETO fusion gene got complete remission （CR） after the first MA therapy, and the fusion gene reduced by 3 log in AML1/ETO/ABL. Only 11 patients got CR in 18 patients without AML1/ETO fusion gene. By following up these 7 patients with AML1/ ETO fusion gene kept persistent CR for 6 months. Conclusion It was concluded that real-time quantitative PCR is a reliable, innovative and promising technology with high sensitivity and speciality. It has potential clinical value for diagnosis, tumor typing, treatment selection, measuring the tumor load, monitoring fusion gene expression level and evaluating therapeutic strategy. It is worthy to apply in the clinical practice.