Desmeules Patrice, Boudreau Dominique K, Bastien Nathalie, Boulanger Marie-Chloé, Bossé Yohan, Joubert Philippe, Couture Christian
Service of Anatomic Pathology and Cytology, Institut Universitaire de Cardiologie et de Pneumologie de Québec-Université Laval, Québec City, Québec, Canada.
Research Center, Institut Universitaire de Cardiologie et de Pneumologie de Québec-Université Laval, Québec City, Québec, Canada.
JTO Clin Res Rep. 2022 Jan 10;3(2):100276. doi: 10.1016/j.jtocrr.2022.100276. eCollection 2022 Feb.
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.
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).
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%).
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.
随着非小细胞肺癌（NSCLC）获批的治疗适应症不断增加且新适应症不断涌现，它成为了典型的肿瘤类型，需要对多种生物标志物进行前期评估以指导临床管理。新一代测序技术能够识别不同类型的分子改变，每种改变都面临特定的分析挑战。使用并行DNA和RNA工作流程进行文库制备可以克服其中大部分挑战，但这会增加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检测板的性能特征，用于联合检测临床相关的致癌融合转录本和热点小变异。
使用福尔马林固定、石蜡包埋的NSCLC临床样本（N = 58）以及细胞系和商业对照来验证该检测方法的分析性能，随后进行了一项探索性前瞻性队列研究（N = 87）。
该检测方法对热点突变和融合的原始灵敏度分别为83%和93%，在根据关键检测指标进行筛选后达到100%。这些指标包括核酸输入的数量和质量以及该检测方法中包含的管家基因引物的测序指标。在前瞻性队列中，大多数病例（≥58%）都检测到了驱动改变。
这种高度聚焦的RNA新一代测序检测方法提供了一种有利的选择，采用单一统一的工作流程同时检测NSCLC中临床相关的热点突变和融合，重点关注可操作的基因靶点。
