Table_7_Whole-genome informed circulating tumor DNA analysis by multiplex digital PCR for disease monitoring in B-cell lymphomas: a proof-of-concept study.xlsx

IntroductionAnalyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA).MethodsIn 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up.ResultsA total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value

本研究所涉及的液体活检旨在分析肿瘤特异性异常，以促进在治疗期间及随访过程中对可测量残留疾病（MRD）的检测。本研究评估了在诊断时采用全基因组测序（WGS）对淋巴瘤进行测序以识别患者特异性结构变异（SVs）和单核苷酸变异（SNVs），从而实现纵向、多靶点微滴数字PCR分析（ddPCR）的无细胞DNA（cfDNA）的临床潜力。方法：在9例B细胞淋巴瘤（弥漫性大B细胞淋巴瘤和滤泡性淋巴瘤）患者中，通过30倍全基因组测序对配对的肿瘤和正常组织标本进行了全面的基因组分析。针对患者特异性，设计了多重微滴数字PCR（m-ddPCR）检测多个SNVs、插入缺失（indels）和/或SVs的实验，SVs检测的灵敏度达到0.0025%，SNVs/indel检测的灵敏度达到0.02%。M-ddPCR被应用于分析在原发性及/或复发治疗期间及随访过程中，按临床关键时间点收集的血浆中分离出的cfDNA。结果：全基因组测序共鉴定出164个SNVs/indels，包括30个已知与淋巴瘤发病机制功能相关的变异。最常见的突变基因包括KMT2D、PIM1、SOCS1和BCL2。WGS分析进一步确定了复发的SVs，包括t(14;18)(q32;q21)（IGH::BCL2）和t(6;14)(p25;q32)（IGH::IRF4）。诊断时的血浆分析显示，88%的患者存在阳性循环肿瘤DNA（ctDNA）水平，且ctDNA负荷与基线临床参数（LDH和沉降率）相关（p值