Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, 360 Longwood Avenue, Boston, MA, 02215, USA.
Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA, 02215, USA.
Lung Cancer. 2019 Aug;134:96-99. doi: 10.1016/j.lungcan.2019.06.004. Epub 2019 Jun 5.
Plasma genotyping represents an opportunity for convenient detection of clinically actionable mutations in advanced cancer patients, such has been well-documented in non-small cell lung cancer (NSCLC). Oncogenic gene fusions are complex variants that may be more challenging to detect by next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA). Rigorous evaluation of plasma NGS assays in the detection of fusions is needed to maximize clinical utility.
Additional plasma was collected from patients with advanced NSCLC and ALK, ROS1, or RET gene fusions in tissue who had undergone clinical plasma NGS using Guardant360(G360, Guardant Health). We then sequenced extracted cfDNA with a plasma NGS kit focused on known driver mutations in NSCLC (ctDx-Lung, Resolution Bioscience) with cloud-based bioinformatic analysis and blinded variant calling.
Of 16 patients assayed known to harbor anALK, ROS1, or RET in tumor, G360 detected fusions in 7 cases, ctDx-Lung detected fusions in 13 cases, and 3 cases were detected by neither. Of the 7 fusions detected by both assays, G360 reported lower mutant allelic fractions (AF). In cases missed by G360, tumor derived TP53 mutations were often detected confirming presence of tumor DNA. Raw sequencing data showed that inverted or out-of-frame variants were overrepresented in cases detected using ctDx-Lung but not by G360.
Focusing on complex, clinically actionable mutations using tumor as a reference standard allows for evaluation of technical differences in plasma NGS assays that may impact clinical performance. Noting the heterogeneity of fusion sequences observed in NSCLC, we hypothesize that differences in hybrid capture techniques and bioinformatic calling may be sources of variations in sensitivity among these assays.
血浆基因分型为检测晚期癌症患者的临床可操作突变提供了便利，这在非小细胞肺癌（NSCLC）中已有充分的记录。致癌基因融合是复杂的变体，通过检测血浆无细胞游离 DNA（cfDNA）的下一代测序（NGS）可能更具挑战性。需要对血浆 NGS 检测融合的方法进行严格评估，以最大程度地提高临床实用性。
从组织中具有 ALK、ROS1 或 RET 基因融合的晚期 NSCLC 患者中采集额外的血浆，这些患者已经接受了 Guardant360（G360，Guardant Health）的临床血浆 NGS 检测。然后，我们使用专注于 NSCLC 已知驱动突变的血浆 NGS 试剂盒（ctDx-Lung，Resolution Bioscience）对提取的 cfDNA 进行测序，该试剂盒具有基于云的生物信息学分析和盲变异调用。
在已知肿瘤中存在 ALK、ROS1 或 RET 的 16 例患者中，G360 在 7 例中检测到融合，ctDx-Lung 在 13 例中检测到融合，而 3 例均未检测到。在两种检测方法均检测到的 7 种融合中，G360 报告的突变等位基因分数（AF）较低。在 G360 漏检的病例中，经常检测到肿瘤衍生的 TP53 突变，证实存在肿瘤 DNA。原始测序数据显示，在使用 ctDx-Lung 检测到的病例中，倒置或失框变体过度表达，但 G360 未检测到。
以肿瘤为参考标准，专注于复杂的临床可操作突变，可评估可能影响临床性能的血浆 NGS 检测方法的技术差异。鉴于 NSCLC 中观察到的融合序列的异质性，我们假设杂交捕获技术和生物信息学调用的差异可能是这些检测方法之间灵敏度变化的来源。
