基于超深度测序技术的儿童白血病微量残留病研究

Compelling evidences from many studies support the roles of minimal residual disease (MRD) monitoring on stratified diagnosis, dynamic therapeutic intervention, precisely assessing early treatment response and predicting early recurrence of childhood acute leukemia. However, the MRD marker of immunophenotypic combinations by flow cytometry, fusion gene or TCR/Ig rearrangement by real-time PCR cannot cover all patients and the leukemia-associated immunophenotype transfer and leukemia specific TCR/Ig rearrangement changes will happened during the treatment course. To determine the MRD of childhood leukemia, we performed the whole genome sequence and bioinformatics analysis on 113 trios new diagnostic, complete remission and relapsed samples. After removing the common germline cell mutations, we found the genomic variants (i.g., single nucleotide variant, SNVs and insertion/deletion variants, In/Dels) can be used as leukemia markers on MRD monitorning. By comparing the genetic differences of new diagnostic and relapsed samples with complete remission bone marrow samples, we identified 971 geneswhich carry recurrent SNVs and/or In/Dels, we named these variants as tumor-specific variants (TSVs). Based on these information and research progress in recent years, we develop the target capture probes to screen the TSVs on each leukemic sample. To measurement MRD by the next-generation sequencing technology accurately, we also develop the new next-generation sequencing library named Duo-rolling amplification (Duo-R) method. This assay can identify one read with mutated nucleotide on 1,000,000 wild type reads with the sensitivity above 10-6. In this study, we will screen 4 or more genomic TSVs and perform the MRD monitoring by ultra-depth sequencing on each sample from 100 patients’ sequential samples, including the new diagnostic samples, complete remission samples, relapse samples as wells as the samples after stopping treatment if disease not recurrence. Then we will compare these MRD values with MRD determined by flow cytometry. Through the above study, we will establish the theoretical and experimental basis of childhood leukemia MRD detection and monitoring technological system by the next-generation sequencing.

微量残留病（Minimal Residual Disease, MRD）对于儿童急性白血病的诊断分层、疗效评估、动态干预和预后预测至关重要，传统的MRD标志聚焦于Ig和TCR重排、融合基因和白血病细胞相关免疫表型，均存在标志容易丢失和不能覆盖所有患者的缺点，相比较而言基因组水平的单碱基变异和插入缺失变异具有无可比拟的优势。本课题通过对343份儿童白血病的初发、缓解和复发成对样本的全基因组测序和生物信息学分析，锁定了971个儿童白血病肿瘤特异性变异基因，用于定制儿童白血病MRD靶向捕获探针，在基因组水平筛选新发样本的MRD标志；通过采用滚环扩增和链内矫正原理建立基于Duo-R建库和超高深度测序的MRD检测方案，用于白血病治疗不同阶段骨髓连续样本的MRD监测，并与基于流式细胞技术的MRD检测结果进行对比。通过上述研究将为建立基于下一代测序技术的儿童白血病MRD检测技术体系提供实验和理论依据。

微量残留病（Minimal Residual Disease, MRD）对于儿童白血病的诊断分层、疗效评估、动态干预和预后预测至关重要，传统的MRD标志聚焦于Ig和TCR重排、融合基因和白血病细胞相关免疫表型，均存在不能覆盖所有患者和标志容易丢失的缺点，相比较而言基因组水平的单碱基变异和插入缺失变异具有无可比拟的优势。本课题通过对343份儿童白血病的初发、缓解和复发成对样本的全基因组测序和生物信息学分析，锁定了971个儿童白血病肿瘤特异性变异基因，用于定制儿童白血病MRD靶向捕获探针，在基因组水平筛选新发样本的MRD标志。为探讨急性淋巴细胞白血病（ALL）的复发机制，通过对103个诊断为复发的种系进行了全基因组测序，并对16例208个系列标本进行了超深测序。与药物应答有关的12个基因（NR3C1、NR3C2、46 TP53、NT5C2、FPGS、CREBBP、MSH2、MSH6、PMS2、WHSC1、PRPS1和PRPS2）富含复发特异性体细胞改变。极早期复发组（诊断后<9个月）患病率为17%，早期复发组（9-36个月）患病率为65%，晚期复发组（36个月以上）患病率为32%。在6例复发病例中观察到多个亚克隆携带同一耐药基因突变的趋同进化，1例通过单细胞测序证实。数学模型和突变特征分析表明，早期复发耐药的获得通常是两步过程，即一个持久的克隆在最初的治疗中存活，然后在治疗过程中获得真正的耐药突变。相反，很早的复发是由已经存在的耐药克隆引起的。在早期和晚期复发中发现了两个新的复发特异性突变特征，其中一个是由硫嘌呤治疗引起的，但在2540例泛癌诊断样本和129例非ALL复发中没有发现。在27%的复发ALL中检测到新的特征，并导致NT5C2、PRPS1、NR3C1和TP53中46%的获得性耐药突变。这些结果表明化疗诱导的耐药突变促进了一部分儿童ALL复发。