背景:随着批准的和新兴的治疗适应症清单的扩大,NSCLC是需要预先评估几种生物标志物以指导临床管理的示例性肿瘤类型。下一代测序允许识别不同类型的分子改变,每个人都有具体的分析挑战。使用平行的DNA和RNA工作流程进行文库制备可以克服其中的大多数,但它增加了实验室操作的复杂性,周转时间,和成本。我们描述了基于锚定多重聚合酶链反应的15基因RNA面板的性能特征,用于联合检测临床相关的致癌融合转录本和热点小变体。
方法:福尔马林固定,石蜡包埋的非小细胞肺癌临床样品(N=58)与细胞系和商业对照一起使用,以验证分析的分析性能,其次是探索性前瞻性队列(N=87)。
结果:热点突变和融合的原始检测灵敏度分别为83%和93%,分别,在过滤关键测定指标后达到100%。这些包括核酸输入的数量和质量以及来自测定中包括的管家基因的引物的测序度量。在前瞻性队列中,在大多数情况下(≥58%)发现了驾驶员变更.
结论:这种超聚焦RNA-下一代测序分析提供了一种有利的选择,具有单一统一的工作流程,可同时检测NSCLC中临床相关的热点突变和融合。专注于可操作的基因靶标。
BACKGROUND: With its expanding list of approved and emerging therapeutic indications, NSCLC is the exemplar tumor type requiring upfront assessment of several biomarkers to guide clinical management. Next-generation sequencing allows identification of different types of molecular alterations, each with specific analytical challenges. Library preparation using parallel DNA and RNA workflows can overcome most of them, but it increases complexity of laboratory operations, turnaround time, and costs. We describe the performance characteristics of a 15-gene RNA panel on the basis of anchored multiplex polymerase chain reaction for combined detection of clinically relevant oncogenic fusion transcripts and hotspot small variants.
METHODS: Formalin-fixed, paraffin-embedded NSCLC clinical samples (N = 58) were used along cell lines and commercial controls to validate the assay\'s analytical performance, followed by an exploratory prospective cohort (N = 87).
RESULTS: The raw assay sensitivity for hotspot mutations and fusions was 83% and 93%, respectively, reaching 100% after filtering for key assay metrics. Those include quantity and quality of input of nucleic acid and sequencing metric from primers on housekeeping genes included in the assay. In the prospective cohort, driver alterations were identified in most cases (≥58%).
CONCLUSIONS: This ultrafocused RNA-next-generation sequencing assay offers an advantageous option with single unified workflow for simultaneous detection of clinically relevant hotspot mutations and fusions in NSCLC, focusing on actionable gene targets.