实时荧光定量反转录多聚酶链反应技术检测TEL-AML1融合基因阳性急性淋巴细胞白血病

Detection of Translocation ETS Leukemia-Acute Myeloid Leukemia1 Fusion Gene in Acute Lymphoblastic Leukemia Children by Real-Time Fluorescent Quantitative Polymerase Chain Reaction and Its Clinical Signi-ficance

目的建立实时荧光定量反转录(qRT)-PCR方法检测小儿急性淋巴细胞白血病TEL-AML1融合基因的表达水平,探讨其临床意义。方法采用TaqMan探针qRT-PCR技术,2-△△ct相对定量法动态检测20例TEL-AML1融合基因阳性急性淋巴细胞白血病患儿初诊期(20例)、缓解期(20例)、复发期(2例),及15例骨髓形态学正常的非肿瘤非血液病儿童作为对照组的TEL-AML1融合基因表达水平。结果初诊期、缓解期、复发期及对照组的TEL-AML1中位数表达水平分别为0.62、0.31、0.76、0.23,初诊期和复发期基因表达水平均显著高于缓解期及对照组(Pa<0.05),初诊期和复发期的表达水平比较差异无统计学意义(P>0.05)。诱导缓解第33天TEL-AML1水平高表达(>对照组的P75)患儿的复发率、无病生存率期间显著高于和低于低表达患儿(Pa<0.05)。20例患儿首次诱导治疗第33天骨髓均达完全缓解,高表达8例均在强化治疗、维持治疗后TEL-AML1水平下降,但其中1例患儿在维持治疗期间TEL-AML1水平又异常升高后复发,另1例患儿在停药后2个月TEL-AML1水平再上升后复发。诱导缓解第33天qRT-PCR检测8例微小残留病阳性,骨髓形态学、染色体核型分析均阴性,实时反转录-PCR检测5例阳性。结论 qRT-PCR是一种快速、灵敏度高、特异性好的方法,诱导缓解第33天TEL-AML1融合基因水平可用于早期判断预后,TEL-AML1水平的变化可用于监测微小残留病、预测复发,指导个体化治疗。

Objective To establish a real - time fluorescent quantitative reverse transcription - polymerase chain reaction（ qRT - PCR） for detection of TEL- AML1 fusion gene mRNA in acute lymphoblastic leukemia （ALL） children and explore its clinical significance. Methods A qRT-PCR with a TaqMan fluorescence probe and 2 aac, relative quantitative method was used to dynamically detect the ex- pression levels of TEL - AML1 fusion gene in 20 acute lymphoblastic leukemia children with TEL - AML1 fusion gene positive at the time of newly diagnosed （ n = 20 ）, complete remission （ n = 20 ）, recurrence period （ n = 2 ）, and 15 children with normal bone marrow morphology and with non tumor and no blood disease as a control group. Results Median of the expression level of TEL - AML1 fusion gene at newly diagnosed, complete remission, relapse period were 0. 62 ,0. 31,0. 76 ,0. 23 , respectively, and there were significant differences （Po 〈 0.05 ）. The expression levels of TEL - AML1 gene in newly diagnosed and relapsed children were significantly higher than those of the children with remission and the control group（ Pa〈 0.05 ）. The expression levels of TEL - AML1 gene showed no significant difference between newly diag- nosed and relapsed patients（ P 〉 0.05 ）. The recurrence rate and event- free survival rate of patients with high level of TEL- AML1 expression （ 〉 P75 of the control group） on day 33 were significantly higher and lower than those of low expression of children, respectively （P 〈 0. 05 ）. Twenty patients on day 33 at the end of the first induction therapy achieved completely remission by bone marrow morphological exami- nation. After intense and maintenance therapy ,the high levels of the 8 children were declined. But the level of 1 case out of 8 cases abnormally increased and then the patient relapsed during maintenance therapy. After 2 months of ending chemotherapy, another 1 patient also relapsed with high TEL - AML1 level again. The levels of minimal residual disease were positive in 8 cases by qRT - PCR test, negative in 20 cases by bone marrow morphology and karyotype analysis tests, positive in 5 cases by real - time reaverse transcription - PCR test. Conclusions qRT - PCR is a rapid, efficient, sensitive and specific method. The expression level of TEL - AML1 gene on day 33 during induction of remis- sion can be used for early prognosis. The changes of TEL - AML1 levels can be used to monitor minimal residual disease, prediction of relapse and guide individual treatment.