Background Genomic analysis of plasma cell-free DNA is transforming lung cancer care; however, available assays are limited by cost, turnaround time, and imperfect accuracy. Here, we study amplicon-based plasma next-generation sequencing (NGS), rather than hybrid-capture-based plasma NGS, hypothesizing this would allow sensitive detection and monitoring of driver and resistance mutations in advanced non-small cell lung cancer (NSCLC). Patients and methods Plasma samples from patients with NSCLC and a known targetable genotype (EGFR, ALK/ROS1, and other rare genotypes) were collected while on therapy and analyzed blinded to tumor genotype. Plasma NGS was carried out using enhanced tagged amplicon sequencing of hotspots and coding regions from 36 genes, as well as intronic coverage for detection of ALK/ROS1 fusions. Diagnostic accuracy was compared with plasma droplet digital PCR (ddPCR) and tumor genotype. Results A total of 168 specimens from 46 patients were studied. Matched plasma NGS and ddPCR across 120 variants from 80 samples revealed high concordance of allelic fraction (R2 = 0.95). Pretreatment, sensitivity of plasma NGS for the detection of EGFR driver mutations was 100% (30/30), compared with 87% for ddPCR (26/30). A full spectrum of rare driver oncogenic mutations could be detected including sensitive detection of ALK/ROS1 fusions (8/9 detected, 89%). Studying 25 patients positive for EGFR T790M that developed resistance to osimertinib, 15 resistance mechanisms could be detected including tertiary EGFR mutations (C797S, Q791P) and mutations or amplifications of non-EGFR genes, some of which could be detected pretreatment or months before progression. Conclusions This blinded analysis demonstrates the ability of amplicon-based plasma NGS to detect a full range of targetable genotypes in NSCLC, including fusion genes, with high accuracy. The ability of plasma NGS to detect a range of preexisting and acquired resistance mechanisms highlights its potential value as an alternative to single mutation digital PCR-based plasma assays for personalizing treatment of TKI resistance in lung cancer.

中文翻译：

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基于扩增子的无浆细胞DNA下一代测序技术，用于检测晚期非小细胞肺癌的驱动基因和耐药基因突变。

背景血浆中无细胞DNA的基因组分析正在改变肺癌的治疗方法。但是，可用的检测方法受到成本，周转时间和不完善的准确性的限制。在这里，我们研究基于扩增子的血浆下一代测序（NGS），而不是基于混合捕获的血浆NGS，假设这将允许对晚期非小细胞肺癌（NSCLC）的驱动程序和耐药突变进行灵敏的检测和监测。患者和方法在治疗期间收集来自NSCLC和已知可靶向基因型（EGFR，ALK / ROS1和其他罕见基因型）患者的血浆样品，并进行盲法分析以了解肿瘤基因型。使用热点的增强标记扩增子测序和来自36个基因的编码区进行血浆NGS的检测，并采用内含子检测ALK / ROS1融合蛋白。将诊断准确性与血浆液滴数字PCR（ddPCR）和肿瘤基因型进行比较。结果共研究了46例患者的168例标本。来自80个样品的120个变体的匹配血浆NGS和ddPCR显示等位基因分数的高度一致性（R2 = 0.95）。预处理前，血浆NGS对EGFR驱动程序突变的检测敏感性为100％（30/30），而ddPCR的敏感性为87％（26/30）。可以检测到各种罕见的驱动程序致癌突变，包括灵敏地检测到ALK / ROS1融合蛋白（检测到8/9，占89％）。研究了25例对奥西替尼产生耐药性的EGFR T790M阳性患者，可以检测到15种耐药机制，包括三级EGFR突变（C797S，Q791P）和非EGFR基因的突变或扩增，其中一些可以在治疗前或病情发展前几个月检测到。结论该盲法分析证明了基于扩增子的血浆NGS能够以高精度检测NSCLC中所有目标基因型的能力，包括融合基因。血浆NGS检测多种既有和获得性耐药机制的能力凸显了其潜在价值，可替代基于单突变数字PCR的血浆分析用于肺癌TKI耐药的个性化治疗。