Centro Infantil Boldrini, Campinas, Brazil.
Pediatr Blood Cancer. 2010 Dec 15;55(7):1278-86. doi: 10.1002/pbc.22709. Epub 2010 Aug 20.
Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD), which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL). We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method. Here, we report on gene rearrangement frequencies and offer guidelines for the application of the technique.
Two hundred thirty-three children had DNA extracted from bone marrow. Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR. PCR products were submitted to homo/heteroduplex analysis. A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions.
At least one clonal marker could be detected in 98% of the patients, and two markers in approximately 80%. The most commonly rearranged genes in precursor B-cell ALL were IgH (75%), TCRD (59%), IgK (55%), and TCRG (54%). The most commonly rearranged genes for T-ALL were TCRG (100%) and TCRD (24%). The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells.
We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases. In addition, these reactions are suitable for MRD monitoring, especially when aiming the selection of patients with high MRD levels (≥ 10(-2)) at the end of induction therapy. Such an approach would be very useful in centers with limited financial resources.
免疫球蛋白 (Ig) 和 T 细胞受体 (TCR) 基因重排在儿童急性淋巴细胞白血病 (ALL) 中是微小残留病 (MRD) 的最佳预测指标之一，可作为特定标志物。我们最近通过简化的 PCR 方法报告了缓解诱导期间 MRD 的预后价值。在此，我们报告基因重排频率，并提供该技术的应用指南。
从 233 名儿童的骨髓中提取 DNA。使用共识引物和常规 PCR 扩增 Ig 和 TCR 基因重排。将 PCR 产物提交进行同/异源双链分析。设计了一个计算机程序，使用最小的引物和反应组合来定义用于克隆检测的目标组合。
98%的患者至少可以检测到一个克隆标记，约 80%的患者可以检测到两个标记。前体 B 细胞 ALL 中最常重排的基因是 IgH（75%）、TCRD（59%）、IgK（55%）和 TCRG（54%）。T-ALL 中最常重排的基因是 TCRG（100%）和 TCRD（24%）。引物的灵敏度限于在 100 个正常细胞中检测到 1 个白血病细胞。
我们建议对于每种 ALL 亚型，进行 8 次 PCR 反应可以在大多数情况下检测到两个标记。此外，这些反应适用于 MRD 监测，尤其是在诱导治疗结束时旨在选择 MRD 水平较高（≥10(-2)）的患者时。这种方法在资源有限的中心非常有用。
