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Updated: Oct 1, 2025
Author Spotlight: Advancements in Molecular Biomarker Testing for Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
Performance of an RNA-Based Next-Generation Sequencing Assay for Combined Detection of Clinically Actionable Fusions
Patrice Desmeules1,2, Dominique K Boudreau2, Nathalie Bastien1,2
1Service of Anatomic Pathology and Cytology, Institut Universitaire de Cardiologie et de Pneumologie de Québec-Université Laval, Québec City, Québec, Canada.
Introduction:
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.
Insights
This study presents a new RNA sequencing assay for non-small cell lung cancer (NSCLC) that efficiently detects key mutations and fusions. The assay offers a unified workflow for biomarker identification in NSCLC patients.
Area of Science:
- Molecular diagnostics
- Oncology
- Biomarker discovery
Background:
- Non-small cell lung cancer (NSCLC) management requires comprehensive biomarker assessment.
- Next-generation sequencing (NGS) identifies molecular alterations but presents analytical challenges.
- Parallel DNA and RNA workflows increase complexity, cost, and turnaround time.
Purpose of the Study:
- To evaluate the performance of a 15-gene RNA panel using anchored multiplex PCR.
- To enable simultaneous detection of oncogenic fusion transcripts and hotspot small variants in NSCLC.
- To offer a streamlined workflow for biomarker identification in NSCLC.
Main Methods:
- Validation using formalin-fixed, paraffin-embedded NSCLC samples (N=58), cell lines, and controls.
- An exploratory prospective cohort (N=87) was used for further evaluation.
- Anchored multiplex PCR-based 15-gene RNA panel for combined mutation and fusion detection.
Main Results:
- Raw assay sensitivity reached 83% for hotspot mutations and 93% for fusions.
- Sensitivity improved to 100% after filtering for nucleic acid quality and sequencing metrics.
- Driver alterations were identified in at least 58% of cases in the prospective cohort.
Conclusions:
- The ultrafocused RNA-NGS assay provides a unified workflow for simultaneous detection of mutations and fusions in NSCLC.
- This assay is an advantageous option for identifying actionable gene targets.
- The method simplifies laboratory operations for NSCLC biomarker assessment.
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