Error-corrected sequencing strategies specific for leukemias

Background: Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. Methods: Primary bone marrow samples from pediatric leukemia (n=28) and adult leukemia subjects (n=6), cell line MV4-11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. Results: Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was =0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of =0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. Conclusions: Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking.

背景：儿童白血病具有多样化的基因组特征，与复杂的结构变异相关，包括基因融合、插入缺失以及单核苷酸变异。常规核型分析与荧光原位杂交（Fluorescence in situ hybridization, FISH）技术对小型基因组变异的检测灵敏度不足。下一代测序（Next-generation sequencing, NGS）检测正越来越多地被用于评估各类此类病变。然而，标准NGS因固有错误率较高，无法满足最小残留病（Minimal residual disease, MRD）监测的定量灵敏度要求。方法：本研究纳入28例儿童白血病、6例成人白血病患者的原发性骨髓样本，MV4-11细胞系样本以及1份脐带血样本。基于锚定多重PCR（anchored multiplexed PCR, AMP®）技术、基于扩增子的错误校正测序（error-corrected sequencing, ECS）或人类癌症转录组检测方法，通过分子条形码结合靶向DNA与RNA文库富集技术对样本进行测序。通过计算分析定量评估各类DNA与RNA病变的检测限（limit of detection, LOD），该分析可系统应用于MRD检测。结果：对匹配的白血病患者样本在三个时间点进行分析，分别为确诊时、诱导治疗结束（end of induction, EOI）时以及复发时。与急性淋巴细胞白血病（Acute Lymphoblastic Leukemia, ALL）MRD检测常用的流式细胞术类似，本研究中这类测序策略对单核苷酸变异的检测限为0.001。针对DNA结构变异，FLT3内部串联重复（FLT3 internal tandem duplication, ITD）阳性的细胞系与患者样本的检测限同样为0.001，同时还检测到白血病相关基因中此前未被报道的拷贝数缺失事件。基于RNA的ECS检测发现了多种新型基因融合事件，包括1例急性淋巴细胞白血病患者体内的SPANT-ABL基因融合，该融合可指导临床治疗方案的调整。总体而言，RNA-ECS检测可定量展现RNA分子的复杂特征：其中12%的分子为基因融合转录本、12%为外显子重复转录本、8%为外显子缺失转录本，剩余68%为内含子保留转录本。通过液滴数字PCR（Droplet digital PCR）对RNA-ECS结果进行验证，证实其检测灵敏度可达单mRNA分子水平。结论：综上，本研究中的检测方法可实现对各类克隆性白血病突变的高灵敏度、全面且同步分析，能够以极高灵敏度追踪疾病不同阶段（确诊、诱导治疗结束及复发）的突变变化。本研究提出的检测策略与结果证实，可利用NGS开展MRD动态监测。