极限稀释定量PCR法动态监测儿童急性淋巴细胞性白血病MRD

Dynamic Monitoring of MRD in Children with Acute Lymphoblastic Leukemia by Quantitative PCR Method of Limited Dilution

为探讨一种简便易行检测儿童急性淋巴细胞性白血病 (AL L)微小残留白血病 (MRD)的方法 ,应用 Chelex10 0为介质抽提 4 1例 AL L 患儿不同病期未染色骨髓涂片 DNA,以 TCR Vδ2 Dδ3基因重排为标志 ,采用极限稀释定量PCR法扩增 ,动态监测 MRD的消长与临床病情变化的关系。结果发现 :2 4例患儿在初诊时检出 TCR Vδ2 Dδ3基因重排(5 9% ) ,灵敏度均为 4个拷贝。经诱导缓解后 ,12例 TCR Vδ2 Dδ3基因重排仍为阳性 ,MRD值为 2× 10 - 6 ～ 1× 10 - 3,其中 7例 MRD值于 3～ 6个月内迅速下降转阴 ,骨髓检测未见复发 ,另 5例 MRD持续阳性或 MRD持续高值 ,早期复发。骨髓检测复发患儿缓解期时 MRD平均值为 1.9× 10 — 3明显高于同期骨髓检测未复发患儿的 3× 10 - 6 (P<0 .0 1)。余 12例诱导缓解后 TCR Vδ2 Dδ3基因重排转阴 ,MRD值均 <1× 10 - 6 ,其中 3例 MRD由阴性转阳后加强化疗力度 ,未复发。结果提示 :以 Chelex 10 0为介质抽提 DNA进行 PCR扩增 ,操作简便 ,敏感度高 ;动态监测 MRD的消长对指导临床治疗 。

To explore a simple and easy method to detect the minimal residual disease (MRD) in children with acute lymphoblastic leukemia (ALL), DNA was extracted from stored bone marrow smears with chelex 100 at different periods in 41 children with ALL. Using TCR Vδ 2 Dδ 3 rearrangement fragments as gene markers, and PCR amplification of quantitative method of limited dilution, the relationship between MRD levels and clinical progress was dynamically monitored. It was found that Vδ 2 Dδ 3 gene rearrangement was detected in 24 of all 41 cases at initial diagnosis(59 %). The sensitivity was 4 copies. After complete remission (CR), 12 cases of Vδ 2 Dδ 3 gene rearrangement were also positive with the MRD value being 2×10 -6 -1×10 -3 . MRD levels were decreased rapidly to turn to negative in 7 cases within 3-6 months. Bone marrow examination revealted no relapse, while in the remaining 5 cases, MRD was persistently positive or MRD levels was persistently higher. Relapse occurred at the early stage. Bone marrow examination showed the mean MRD value was 1.9×10 -3 at CR period,significantly higher than that (3×10 -6 ) of unrelapse cases at the same time ( P <0.01). In another 12 cases out of 24 cases, Vδ 2 Dδ 3 gene rearrangement turned into negative after CR with MRD value bing <1×10 -6 . Among them, 3 cases with MRD being converted from negative were not relapse after intensive chemotherapy. The results suggested that the method of PCR after extraction of DNA with chelex 100 was simple and sensitive; Dynamic monitoring of MRD levels might play an important role in guiding clinical treatment, evaluating the chemotherapy efficiency and predicting relapse.