PO.BCS01.05 · 生物信息与计算
利用分子技术和新型信息学平台灵敏检测罕见cfDNA变异：一种基因组与蛋白质组联合的MRD应用
Sensitive detection of rare cfDNA variants utilizing molecular technologies and a novel informatics platform: A combined genomic and proteomic MRD application
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摘要 Abstract
中文摘要
下一代测序（NGS）的进步正在以快速下降的成本产生海量数据。在保持灵敏度和特异性的同时有效处理数百万条高深度测序读段，是新型分子检测设计中日益增长的挑战。一个关键应用是检测和后续监测血浆中用于分子残留病（MRD）的罕见变异。我们的MRD检测策略利用初步的蛋白质组学复发风险质谱检测，将高风险基线样本分层为可能需要进一步监测的样本，随后设计定制的ddPCR检测，以在高风险人群的随访血浆中标记复发。为了验证用于基因组监测的检测，我们开发了一种新型信息学流程，利用约2400万条读段和100万个结构变异检出，对14例转移性去势抵抗性前列腺癌患者（mCRPCa）队列进行分析。系统性分析组织来源的NGS数据，以鉴定用于监测治疗后血浆标本中分子复发的MRD指纹。我们在整个队列中实现了64—367×的测序深度。数据过滤策略包括质量分值（≥20）、相同同源性（≤5）、结构变异大小，以及多个金标准生物信息学工具（Delly和Manta）的交集。从原始NGS数据到设计引物的分析周转时间为16.5—72小时。所有被评估患者均成功设计出定制引物或有现成引物可用。初步研究鉴定了源自四例具有匹配基线血浆的mCRPCa患者的组织指导变异。在匹配患者的血浆标本中检出了6/6所测变异。值得注意的是，纵向ctDNA分析在一例患者中鉴定出AR p.L702H从循环中清除，该患者在多线AR信号抑制剂（ARSI）进展后，对多西他赛显示出影像学和生化缓解的证据。AR p.L702H是对ARSI的一种获得性耐药机制，携带该改变的亚克隆细胞群的消除可能提示对这些药物的敏感性恢复。此外，ctDNA的下降与血清PSA的下降同步。本报告展示了一种新型MRD检测的设计，它将用于早期且有影响力的复发风险分层的蛋白质组学，与在高复发风险患者随访血浆中经济高效且快速的基因组PCR监测相结合，可用于真实世界临床诊断实验室。
查看英文原文 English abstract
Advances in next-generation sequencing (NGS) are producing massive amounts of data at rapidly declining costs. Effective processing of millions of high-depth sequencing reads while maintaining sensitivity and specificity is a growing challenge in the design of new molecular tests. One critical application is detection and subsequent monitoring of rare variants in plasma for molecular residual disease (MRD). Our MRD testing strategy utilizes a preliminary proteomic risk of recurrence mass spectrometry assay to stratify high-risk baseline samples as likely to need further monitoring, followed by design of bespoke ddPCR assays to mark recurrence in the follow-up plasma of the high-risk population. To qualify assays for genomic monitoring, we developed a novel informatics pipeline utilizing approximately 24 million reads and 1 million structural variant calls to profile a cohort of 14 metastatic castration resistant prostate cancer patients (mCRPCa). Tissue-derived NGS data were systematically analyzed to identify a MRD fingerprint for monitoring molecular recurrence in post-treatment plasma specimens. We achieved sequencing depths of 64-367x across the cohort. Data filtering strategies included quality score (≥20), identical homology (≤5), structural variant size, and intersection of multiple gold-standard bioinformatics tools, Delly and Manta. Analytical turnaround time was 16.5 - 72 hours from raw NGS data to designed primers. Bespoke primers were successfully designed or available off-the-shelf for all evaluated patients. Initial studies identified tissue-informed variants derived from four mCRPCa patients with matched baseline plasma. 6/6 of the tested variants were found in the matched patient plasma specimens. Significantly, longitudinal ctDNA analyses identified clearance of AR p.L702H from circulation in a patient with evidence of radiographic and biochemical response to docetaxel after progression on multiple lines of AR signaling inhibitors (ARSI). AR p.L702H is an acquired resistance mechanism to ARSIs, and the elimination of sub-clonal cell populations harboring the alteration could indicate restored sensitivity to these agents. Additionally, declines in ctDNA coincided with declines in serum PSA. This report showcases the design of a novel MRD test combining proteomics for early and impactful risk of recurrence stratification with cost-effective and rapid genomic PCR monitoring in the follow-up plasma of patients at a high-risk of recurrence, for utility in real-world clinical diagnostics laboratories
利益披露 Disclosure
E. K. Longshore,
Biodesix Employment.
E. Barnett,
Biodesix Employment.
I. T. Erazo,
Biodesix Employment.
L. Jackson,
Biodesix Employment.
H. Halpin,
Biodesix Employment.
R. Pestano,
Biodesix Employment.
P. Schnepp,
Biodesix Employment.
A. Corner,
Biodesix Employment.
H. I. Scher,
Biodesix Employment.
G. A. Pestano,
Biodesix Employment.