实时定量聚合酶链反应检测微小残留白血病的临床研究  

Real-time quantitative study of minimal residual disease in childhood B cell acute lymphoblastic leukemia

作　　者：贾月萍[1] 刘桂兰[1] 张乐萍[1] 

机构地区：[1]北京大学人民医院儿科,100044

出　　处：《中华儿科杂志》2004年第8期600-604,共5页Chinese Journal of Pediatrics

摘　　要：目的 探讨并研究实时定量聚合酶链反应(RQ—PCR)技术定量检测小儿急性淋巴细胞白血病(ALL)微小残留病(MRD)的临床适用性和临床价值。方法 以免疫球蛋白重链(IgH)基因重排作为ALL的肿瘤标志,应用RQ—PCR、胚系探针策略,定量检测了34例B细胞ALL(B—ALL)患儿的MRD,并对其中的16例患儿进行了缓解期MRD的定量动态追踪观察。结果在34例B—ALL患儿的初治标本中IgH基因单克隆重排16例,对16例单克隆IgH重排靶基因进行序列分析发现,V片段使用最频繁的是V3家族,J片段使用最频繁的是J4和J6。在16个靶基因中,RQ—PCR的检测敏感度有9例为10-4,6例为10-5,1例为10-3,非特异性扩增见于6例患儿。16例初治患儿的标准曲线相关系数均为0.99以上,斜率均值为-3.34±0.37,截距均值为24.30±2.95。对16例患儿随访期样本的MRD动态追踪研究发现,复发患儿的MRD水平较高且复发前有动态增加。诱导化疗结束时的MRD水平与ALL高危因素无明显相关性(P>0.05)。结论 研究表明,RQ—PCR技术胚系探针策略检测ALL患儿的MRD具有临床适用性,随访期标本MRD的定量检测及动态追踪监测具有临床价值。Objective The study was aimed to investigate the feasibility and clinical significance of quantitative detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) by real-time quantitative polymerase chain reaction (RQ-PCR). Methods Clonal IgH gene rearrangements of samples at diagnosis were identified by standard PCR assay with consensus primers. Monoclonal IgH gene rearrangements were analyzed using DNAPLOT software. Upstream primers were designed with the Primer Express software and allele specific oligonucleotide developed complementary to the V-D or D-J junction. Samples at diagnosis were serially diluted to generate the patient specific standard curves. RQ-PCR method was used to quantify the MRD of the follow up samples collected at five time points during chemotherapy. To check the quantity and quality of DNA, the investigators used RQ-PCR analysis for the albumin gene. Results Totally 16 monoclonal IgH gene rearrangements were identified from 34 patients with B-ALL The analysis of the 16 monoclonal rearrangements showed that the most frequently used V segment was from V3 family and J segment from J4 and J6. The RQ-PCR sensitivity of 10 -4 to 10 -5 was mostly reached. Nonspecific amplification was seen in 6 patients. The number of inserted and deleted nucleotides did not appear to be related to the sensitivity ( P > 0. 05 ) . The correlation coefficients of all 16 standard curves were excellent ( ≥0. 99). The mean slope of the standard curves was - 3. 4 ± 0. 37 and the mean intercept was 24. 3 ±2. 95. MRD analysis of follow up samples from the 16 patients showed an association between high degree of MRD and relapse. There was no apparent relationship between MRD degree at the end of induction chemotherapy and other high risk factors of AT.I.( P > 0. 05 ). Conclusion The study showed that the above approach with RQ-PCR was applicable to clinical detection of MRD in childhood ALL. Quantitative and dynamic study of MRD was of prognostic importance.

关 键 词：实时定量聚合酶链反应 检测 微小残留 急性淋巴细胞白血病 小儿 

分 类 号：R733.7[医药卫生—肿瘤]