Choy L Y Lois, Peng Wenlei, Jiang Peiyong, Cheng Suk Hang, Yu Stephanie C Y, Shang Huimin, Olivia Tse O Y, Wong John, Wong Vincent Wai Sun, Wong Grace L H, Lam W K Jacky, Chan Stephen L, Chiu Rossa W K, Chan K C Allen, Lo Y M Dennis
Centre for Novostics, Hong Kong Science Park, Pak Shek Kok, Hong Kong SAR, China.
State Key Laboratory of Translational Oncology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong SAR, China.
Clin Chem. 2022 Sep 1;68(9):1151-1163. doi: 10.1093/clinchem/hvac086.
Analysis of circulating tumor DNA has become increasingly important as a tool for cancer care. However, the focus of previous studies has been on short fragments of DNA. Also, bisulfite sequencing, a conventional approach for methylation analysis, causes DNA degradation, which is not ideal for the assessment of long DNA properties and methylation patterns. This study attempted to overcome such obstacles by single-molecule sequencing.
Single-molecule real-time (SMRT) sequencing was used to sequence plasma DNA. We performed fragment size and direct methylation analysis for each molecule. A methylation score concerning single-molecule methylation patterns was used for cancer detection.
A substantial proportion of plasma DNA was longer than 1 kb with a median of 16% in hepatocellular carcinoma (HCC) patients, hepatitis B virus carriers, and healthy individuals. The longest plasma DNA molecule in the HCC patients was 39.8 kb. Tumoral cell-free DNA (cfDNA) was generally shorter than nontumoral cfDNA. The longest tumoral cfDNA was 13.6 kb. Tumoral cfDNA had lower methylation levels compared with nontumoral cfDNA (median: 59.3% vs 76.9%). We developed and analyzed a metric reflecting single-molecule methylation patterns associated with cancer, named the HCC methylation score. HCC patients displayed significantly higher HCC methylation scores than those without HCC. Interestingly, compared to using short cfDNA (area under the receiver operating characteristic [ROC] curve, AUC: 0.75), the use of long cfDNA molecules greatly enhanced the discriminatory power (AUC: 0.91).
A previously unidentified long cfDNA population was revealed in cancer patients. The presence and direct methylation analysis of these molecules open new possibilities for cancer liquid biopsy.
循环肿瘤DNA分析作为癌症治疗工具变得越来越重要。然而，以往研究的重点一直是DNA短片段。此外，亚硫酸氢盐测序作为一种传统的甲基化分析方法，会导致DNA降解，这对于评估长DNA特性和甲基化模式并不理想。本研究试图通过单分子测序克服这些障碍。
使用单分子实时（SMRT）测序对血浆DNA进行测序。我们对每个分子进行片段大小和直接甲基化分析。使用关于单分子甲基化模式的甲基化评分进行癌症检测。
相当一部分血浆DNA长度超过1 kb，在肝细胞癌（HCC）患者、乙肝病毒携带者和健康个体中，中位数为16%。HCC患者中最长的血浆DNA分子为39.8 kb。肿瘤游离DNA（cfDNA）通常比非肿瘤cfDNA短。最长的肿瘤cfDNA为13.6 kb。与非肿瘤cfDNA相比，肿瘤cfDNA的甲基化水平较低（中位数：59.3%对76.9%）。我们开发并分析了一种反映与癌症相关的单分子甲基化模式的指标，称为HCC甲基化评分。HCC患者的HCC甲基化评分显著高于无HCC患者。有趣的是，与使用短cfDNA（受试者操作特征曲线下面积，AUC：0.75）相比，使用长cfDNA分子大大增强了鉴别能力（AUC：0.91）。
在癌症患者中发现了一个以前未被识别的长cfDNA群体。这些分子的存在和直接甲基化分析为癌症液体活检开辟了新的可能性。
