应用多重巢式RT-PCR初筛急性髓系白血病融合基因及其临床意义

Application of multiplex nested RT-PCR assay for screening the fusion genes in acute myeloid leukemia and its clinical significance

目的探讨应用多重巢式逆转录聚合酶链反应（RT-nPcR）初筛急性髓系白血病（AML）融合基因的临床价值。方法根据WH02008AML分型指南建立检测16种融合基因（AML1-EVI1、AMLl-ETO、AML1-MDSl、AML1-MTG16、MLL-AF9、MLL—AF6、MLL—AF10、MLL—ENL、MLL—MLL、PML—RARα、PLZF．RARα、NPM1-RARα、CBFB-MYH11、DEK—CAN、SET-CAN、TLS—ERG）的RT-nPCR体系，对356例AML患者同时采用RT-nPCR和核型分析方法检测染色体交互易位情况，针对阳性患者进一步采用分离PCR（split—outPCR）和荧光原位杂交（FISH）方法进行验证。结果在356例AML患者中相应融合基因阳性者172例，阳性率为48-31％，高于核型分析相应染色体易位检出率（31．46％）（t=70.314，P〈0．01）。在少见和难以识别的染色体易位中，RT-nPCR比核型分析、FISH更具有优势（X2=96．074，P〈0．01）。结论RT-nPCR可便捷、有效、准确地检测AML患者融合基因异常，作为AML初筛的检测手段为疾病诊断及疗效判定提供重要依据，同时也为微小残留病监测及预后评估提供重要信息。

Objective To investigate the clinical value of multiplex nested reverse transcription PCR （RT-nPCR） in screening acute myeloid leukemia （AML） fusion genes. Methods A novel multiplex RT-nPCR assay was developed to detect 16 AML-related fusion genes （AML 1-EVI 1, AML 1-ETO, AML 1- MDS1, AML1-MTG16, MLL-AF9, MLL-AF6, MLL-AF10, MLL-ENL, MLL-MLL, PML-RARct, PLZF- RARer, NPM1-RARct, CBFB-MYHll, DEK-CAN, SET-CAN and TLS-ERG） according to 2008 WHO classification of AML. The chromosome reciprocal translocations of 356 AML cases were detected by multiplex RT-nPCR and karyotyping. The positive samples were further confirmed by split-out PCR and FISH. Results The fusion genes were detected in 172 patients with the positive detection rate of 48.31% （172/356）, which was higher than that of karyotyping （31.46%） （^（2=70.314, P〈0.01 ）. Multiplex RT-nPCR is superior to karyotyping and FISH in identifying the rare, cryptic chromosome translocation （）（=96.074, P〈0.01）. Conclusion The multiplex RT-nPCR used in this study can quickly, effectively and accurately screen the fusion genes in AML patients, which can provide important evidence for assessing diagnosis and treatment, and also provide necessary information for minimal residual disease （MRD） and prognosis.