基于DNA的高通量测序已广泛应用于选择非小细胞肺癌（NSCLC）患者的靶向治疗。基于RNA的高通量测序已被证明在检测融合和外显子跳跃突变方面是有价值的，并被美国国家综合癌症网络指南推荐用于这些突变类型。作者开发了一个面向实体瘤的基于RNA的杂交面板，用于靶向肿瘤驱动基因。实验和生物信息学流程进行了优化，以检测融合，单核苷酸变异（SNV）和插入/缺失（indel）。总共，1253个非小细胞肺癌患者的正规化石蜡埋藏样本通过DNA和RNA面板测序进行分析，以评估RNA面板在检测多种类型的突变中的表现。在分析验证中，RNA面板实现了1.45-3.15个拷贝/纳克对于SNV和0.21-6.48个拷贝/纳克对于融合的检测限制。在1253个正规化的石蜡埋藏的非小细胞肺癌样本中，RNA面板识别了共124个融合事件和26个MET外显子14跳跃事件，其中由DNA面板测序漏检了14个融合和6个MET外显子14跳跃突变。通过将DNA面板用作参考，RNA面板的阳性协议百分比和阳性预测值分别为98.08％和98.62％，用于检测可靶向SNV，分别为98.15％和99.38％，用于检测可靶向indel。并行DNA和RNA测序分析表明RNA测序面板检测多种类型临床可操作的突变的准确性和鲁棒性。简化的实验工作流程和低样本消耗将使RNA面板测序成为临床测试的一个潜在有效的方法。©2023年美国癌症协会。

DNA-based next-generation sequencing has been widely used in the selection of target therapies for patients with nonsmall cell lung cancer (NSCLC). RNA-based next-generation sequencing has been proven to be valuable in detecting fusion and exon-skipping mutations and is recommended by National Comprehensive Cancer Network guidelines for these mutation types.The authors developed an RNA-based hybridization panel targeting actionable driver oncogenes in solid tumors. Experimental and bioinformatics pipelines were optimized for the detection of fusions, single-nucleotide variants (SNVs), and insertion/deletion (indels). In total, 1253 formalin-fixed, paraffin-embedded samples from patients with NSCLC were analyzed by DNA and RNA panel sequencing in parallel to assess the performance of the RNA panel in detecting multiple types of mutations.In analytical validation, the RNA panel achieved a limit of detection of 1.45-3.15 copies per nanogram for SNVs and 0.21-6.48 copies per nanogram for fusions. In 1253 formalin-fixed, paraffin-embedded NSCLC samples, the RNA panel identified a total of 124 fusion events and 26 MET exon 14-skipping events, in which 14 fusions and six MET exon 14-skipping mutations were missed by DNA panel sequencing. By using the DNA panel as the reference, the positive percent agreement and the positive predictive value of the RNA panel were 98.08% and 98.62%, respectively, for detecting targetable SNVs and 98.15% and 99.38%, respectively, for detecting targetable indels.Parallel DNA and RNA sequencing analyses demonstrated the accuracy and robustness of the RNA sequencing panel in detecting multiple types of clinically actionable mutations. The simplified experimental workflow and low sample consumption will make RNA panel sequencing a potentially effective method in clinical testing.© 2023 American Cancer Society.