Abstract
IntroductionWith 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. MethodsFormalin-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). ResultsThe 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%). ConclusionsThis 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.
Highlights
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
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
The alterations with the highest clinical interest are found in several genes (EGFR, ALK, ROS1, BRAF, RET, ERBB2, MET, KRAS, and NTRKs) and consist of various single nucleotide variants (SNVs), insertions and deletions, copy number variations, and oncogenic fusions and isoforms.[2,3]
Summary
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. Hybrid-capture panels offer the possibility to deal with a single analyte, but even large comprehensive panels are subject to miss fusions and alternative splicing events, as intron length and repetitive regions are factors complicating the capture of relevant genomic regions.[4,5] sequential DNA and RNA-NGS are being proposed as the optimal strategy to capture all possible alterations,[6] this entails a risk for tissue exhaustion inherent to small specimens and increased overall turnaround time, laboratory workload, and costs All these factors must be considered along with analytical performance when selecting the proper NGS strategy for first-line molecular diagnosis, such as for NSCLC, a tumor type with high volume and a crucial need for robust oncogenic fusion detection
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