Introduction: ROS1 gene fusions are a well‐characterized class of oncogenic driver found in approximately 1% to 2% of NSCLC patients. ROS1‐directed therapy in these patients is more efficacious and is associated with fewer side effects compared to chemotherapy and is thus now considered standard‐of‐care for patients with advanced disease. Consequently, accurate detection of ROS1 rearrangements/fusions in clinical tumor samples is vital. In this study, we compared the performance of three common molecular testing approaches on a cohort of ROS1 rearrangement/fusion‐positive patient samples. Methods: Twenty‐three ROS1 rearrangement/fusion‐positive clinical samples were assessed by at least two of the following molecular testing methodologies: break‐apart fluorescence in situ hybridization, DNA‐based hybrid capture library preparation followed by next‐generation sequencing (NGS), and RNA‐based anchored multiplex polymerase chain reaction library preparation followed by NGS. Results: None of the testing methodologies demonstrated 100% sensitivity in detection of ROS1 rearrangements/fusions. Fluorescence in situ hybridization results were negative in 2 of 20 tested samples, the DNA‐based NGS assay was negative in 4 of 18 tested samples, and the RNA‐based NGS assay was negative in 3 of 19 tested samples. For all three testing approaches, we identified assay characteristics that likely contributed to false‐negative results. Additionally, we report that genomic breakpoints are an unreliable predictor of breakpoints at the transcript level, likely due to alternative splicing. Conclusions: ROS1 rearrangement/fusion detection in the clinical setting is complex and all methodologies have inherent limitations of which users must be aware to correctly interpret results.

中文翻译：

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在一组阳性患者样本中检测 ROS1 重排的分子检测方式的比较

简介：ROS1 基因融合是一类特征明确的致癌驱动因子，见于约 1% 至 2% 的 NSCLC 患者。与化疗相比，针对这些患者的 ROS1 导向疗法更有效，副作用更少，因此现在被认为是晚期疾病患者的标准治疗。因此，准确检测临床肿瘤样本中的 ROS1 重排/融合至关重要。在这项研究中，我们比较了三种常见分子检测方法对一组 ROS1 重排/融合阳性患者样本的性能。方法：通过以下至少两种分子检测方法评估 23 个 ROS1 重排/融合阳性临床样本：分离荧光原位杂交、基于 DNA 的混合捕获文库制备，然后是下一代测序 (NGS)，以及基于 RNA 的锚定多重聚合酶链反应文库制备，然后是 NGS。结果：没有一种测试方法在检测 ROS1 重排/融合方面表现出 100% 的灵敏度。20 个测试样品中有 2 个的荧光原位杂交结果为阴性，18 个测试样品中有 4 个基于 DNA 的 NGS 检测为阴性，基于 RNA 的 NGS 检测在 19 个测试样品中的 3 个为阴性。对于所有三种测试方法，我们确定了可能导致假阴性结果的分析特征。此外，我们报告说基因组断点是转录水平断点的不可靠预测因子，可能是由于选择性剪接。结论：