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Author Spotlight: Advancements in Molecular Biomarker Testing for Non-Squamous Non-Small Cell Lung Cancer
Published on: September 8, 2023
A mass spectrometry assay to simultaneously analyze ROS1 and RET fusion gene expression in non-small-cell lung cancer
Priyanga Wijesinghe1, Gerold Bepler, Aliccia Bollig-Fischer
1Barbara Ann Karmanos Cancer Institute and Department of Oncology, Wayne State University School of Medicine, Detroit, MI.
Introduction:
ROS1 and RET gene fusions were recently discovered in non-small-cell lung cancer (NSCLC) as potential therapeutic targets with small-molecule kinase inhibitors. The conventional screening methods of these fusions are time-consuming and require samples of high quality and quantity. Here, we describe a novel and efficient method by coupling the power of multiplexing polymerase chain reaction and the sensitivity of mass spectrometry.
Methods:
The multiplex mass spectrometry platform simultaneously tests samples for the expression of nine ROS1 and six RET fusion genes. The assay incorporates detection of wild-type exon junctions immediately upstream and downstream of the fusion junction to exclude false-negative results. To flag false-positives, the system also comprises two independent assays for each fusion gene junction.
Results:
The characteristic mass spectrometric peaks of the gene fusions were obtained using engineered plasmid constructs. Specific assays targeting the wild-type gene exon junctions were validated using complimentary DNA from lung tissue of healthy individuals. The system was further validated using complimentary DNA derived from NSCLC cell lines that express endogenous fusion genes. The expressed ROS1-SLC34A2 and CCDC6-RET gene fusions from the NSCLC cell lines HCC78 and LC-2/ad, respectively, were accurately detected by the novel assay. The assay is extremely sensitive, capable of detecting an event in test specimens containing 0.5% positive tumors.
Conclusion:
The novel multiplexed assay is robustly capable of detecting 15 different clinically relevant RET and ROS1 fusion variants. The benefits of this detection method include exceptionally low sample input, high cost efficiency, flexibility, and rapid turnover.
Insights
A new multiplex assay efficiently detects ROS1 and RET gene fusions in non-small-cell lung cancer (NSCLC) using mass spectrometry. This method requires minimal sample input and offers high cost efficiency for targeted therapy screening.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- ROS1 and RET gene fusions are key therapeutic targets in non-small-cell lung cancer (NSCLC).
- Conventional screening methods for these fusions are often time-consuming and require substantial sample amounts.
- Developing efficient and sensitive detection methods is crucial for personalized NSCLC treatment.
Purpose of the Study:
- To develop and validate a novel, efficient multiplex assay for detecting ROS1 and RET gene fusions.
- To overcome the limitations of conventional screening methods in terms of speed and sample requirements.
- To enable rapid and accurate identification of actionable gene fusions in NSCLC patients.
Main Methods:
- A multiplex mass spectrometry platform was developed to simultaneously test for 15 ROS1 and RET fusion genes.
- The assay incorporates specific detection of wild-type exon junctions to prevent false negatives.
- Independent assays for each fusion gene junction were included to mitigate false positives.
Main Results:
- The novel assay accurately detected specific ROS1 and RET gene fusions (ROS1-SLC34A2, CCDC6-RET) in NSCLC cell lines.
- Validation using engineered constructs and healthy lung tissue confirmed assay specificity.
- The assay demonstrated high sensitivity, detecting positive events in samples with as little as 0.5% positive tumor cells.
Conclusions:
- The developed multiplexed assay robustly detects 15 clinically relevant RET and ROS1 fusion variants.
- This method offers significant advantages including low sample input, cost-efficiency, flexibility, and rapid results.
- The assay provides a valuable tool for efficient NSCLC patient stratification for targeted therapies.
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