一种新的液-液cfDNA提取方法，用于大肠癌癌症患者样本的靶向测序：一项中试研究

摘要(中文译文)

血液中由肿瘤释放的游离DNA（cell-free DNA，cfDNA），也称为循环肿瘤DNA（circulating tumor DNA，ctDNA），已成为一种有前景的癌症检测和监测生物标志物。从血浆液体活检中提取ctDNA是一种新兴的、微创的方法，用于检测与结直肠癌相关的基因突变。ctDNA突变分析的一个障碍是获得足量且质量良好的DNA，以兼容下游诊断检测，尤其是在检测血浆体积有限的样本时。我们开发并评估了一种新型液-液提取方法，该方法经过优化可浓缩和纯化血浆样本中的ctDNA。使用参考物质评估了分析性能，并在靶向panel下一代测序条件下与市售金标准提取试剂盒进行了比较。对16例结直肠癌患者的血浆样本进行了初步研究。此外，还纳入了一项独立的、盲法外部验证研究，以确认该工作流程的分析有效性。在使用参考物质的分析测试中，与市售金标准提取试剂盒相比，该新型提取试剂盒在靶向panel下一代测序条件下实现了更高的DNA产量和更高的肿瘤突变检出率。在使用该液-液提取方法工作流程对16例结直肠癌患者样本进行的初步研究中，在低体积血浆中平均可检出73.4%（262/357）的肿瘤突变，高于既往一项荟萃分析中血浆cfDNA 50%的合并肿瘤突变检出率。血浆和肿瘤样本的平行检测可增强对潜在可干预基因的检出。同时，该工作流程在所有样本中实现了完全一致性，为其诊断精确性和灵敏度提供了客观的第三方验证。这些结果表明，将液-液血浆提取与靶向panel下一代测序相结合，在识别结直肠癌（colorectal cancer，CRC）患者癌症相关突变方面具有良好的性能表现。

Abstract

Cell-free DNA (cfDNA) in the bloodstream released from tumor, also known as circulating tumor DNA (ctDNA), has emerged as a promising biomarker for cancer detection and monitoring. Extraction of ctDNA from plasma liquid biopsies is an emerging approach with minimal invasiveness to detect gene mutations associated with colorectal cancer. One hurdle to ctDNA mutational analyses is obtaining sufficient quantity and good quality of DNA which is compatible with downstream diagnostic testing, especially when testing samples with limited volumes of plasma. We developed and evaluated a novel liquid-liquid extraction method optimized to concentrate and purify ctDNA from plasma samples. Analytical performance was assessed using reference materials and compared to a gold standard commercially available extraction kit under targeted panel next-generation sequencing. A pilot study was conducted on plasma samples from 16 colorectal cancer patients. Additionally, an independent, blinded external validation study was included to confirm the analytical validity of this workflow. In analytical testing using reference materials, the novel extraction kit achieved higher DNA yield and higher tumor mutation detection rate compared to a gold standard commercially available extraction kit under targeted panel next-generation sequencing. In the pilot study of 16 colorectal cancer patient samples using this workflow with the liquid-liquid extraction method, an average of 73.4% (262/357) of the tumor mutations could be detected in low volumes of plasma, which is higher than the 50% pooled tumor mutation detection rate of plasma cfDNA in a previous meta-analysis. Parallel testing of plasma and tumor samples can enhance the detection of potentially actionable genes. Meanwhile, the workflow achieved full concordance across all samples, providing objective, third-party verification of its diagnostic precision and sensitivity. These results demonstrate the promising performance of integrating liquid-liquid plasma extraction with targeted panel next-generation sequencing to identify cancer-associated mutations in colorectal cancer (CRC) patients.

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