Translational Research Laborator, Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, USA; Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, USA.
Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, USA.
Ann Oncol. 2018 Apr 1;29(4):1049-1055. doi: 10.1093/annonc/mdy005.
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).
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.
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.
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.
游离血浆 DNA 的基因组分析正在改变肺癌治疗；然而，现有的检测方法受到成本、周转时间和不完美准确性的限制。在这里，我们研究基于扩增子的血浆下一代测序（NGS），而不是基于杂交捕获的血浆 NGS，假设这将允许在晚期非小细胞肺癌（NSCLC）中敏感地检测和监测驱动基因和耐药突变。
在接受治疗的同时收集了患有 NSCLC 且具有已知可靶向基因型（EGFR、ALK/ROS1 和其他罕见基因型）的患者的血浆样本，并对肿瘤基因型进行盲法分析。采用增强标记扩增子测序对 36 个基因的热点和编码区以及 ALK/ROS1 融合的内含子进行检测，进行了血浆 NGS。将诊断准确性与血浆液滴数字 PCR（ddPCR）和肿瘤基因型进行了比较。
共研究了 46 例患者的 168 份标本。在 80 个样本的 120 个变体中，匹配的血浆 NGS 和 ddPCR 的等位基因分数高度一致（R2=0.95）。在治疗前，血浆 NGS 检测 EGFR 驱动突变的灵敏度为 100%（30/30），而 ddPCR 为 87%（26/30）。可以检测到全谱罕见的驱动致癌突变，包括敏感检测 ALK/ROS1 融合（8/9 例，89%）。在研究了 25 例对奥希替尼产生耐药性且 EGFR T790M 阳性的患者后，可检测到 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，其中一些可在治疗前或进展前数月检测到。
这项盲法分析表明，基于扩增子的血浆 NGS 能够以高精度检测 NSCLC 中包括融合基因在内的多种靶向基因型。血浆 NGS 检测多种预先存在和获得的耐药机制的能力突出了其作为替代基于单突变数字 PCR 的血浆检测用于个性化治疗肺癌 TKI 耐药的潜在价值。
