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Updated: Nov 23, 2025
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
RNA-Based Multiplexing Assay for Routine Testing of Fusion and Splicing Variants in Cytological Samples of NSCLC
Cristina Aguado1, Ana Giménez-Capitán1, Ruth Román1
1Laboratorio de Oncología, Pangaea Oncology, Hospital Quirón Dexeus, 08028 Barcelona, Spain.
Abstract:
The detection of ALK receptor tyrosine kinase (ALK), ROS proto-oncogen1, receptor tyrosine kinase (ROS1), ret proto-oncogen (RET), and MET proto-oncogen exon 14 skipping (METΔex14) allows for the selection of specific kinase inhibitor treatment in patients with non-small cell lung cancer (NSCLC). Multiplex technologies are recommended in this setting. We used nCounter, a multiplexed technology based on RNA hybridization, to detect ALK, ROS1, RET, and METΔex14 in RNA purified from cytological specimens (n = 16) and biopsies (n = 132). Twelve of the 16 cytological samples (75.0%) were evaluable by nCounter compared to 120 out of 132 (90.9%) biopsies. The geometrical mean (geomean) of the housekeeping genes of the nCounter panel, but not the total amount of RNA purified, was significantly higher in biopsies vs. cytological samples. Among cytological samples, we detected ALK (n = 3), METΔex14 (n = 1) and very high MET expression (n = 1) positive cases. The patient with METΔex14 had a partial response to tepotinib, one of the patients with ALK fusions was treated with crizotinib with a complete response. Cell blocks and cytological extensions can be successfully used for the detection of fusions and splicing variants using RNA-based methods such as nCounter.
Insights
Multiplex RNA analysis using nCounter successfully detects key gene alterations like ALK, ROS1, RET, and MET exon 14 skipping in non-small cell lung cancer. This method is effective for both cytological specimens and biopsies, guiding targeted kinase inhibitor therapies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Specific gene alterations, including anaplastic lymphoma receptor tyrosine kinase (ALK), ROS proto-oncogen1, receptor tyrosine kinase (ROS1), ret proto-oncogen (RET), and MET proto-oncogen exon 14 skipping (METΔex14), are crucial for selecting targeted kinase inhibitor treatments in non-small cell lung cancer (NSCLC).
- Multiplex technologies are recommended for detecting these genetic alterations efficiently.
Purpose of the Study:
- To evaluate the efficacy of the nCounter multiplex technology for detecting ALK, ROS1, RET, and METΔex14.
- To assess the utility of RNA extracted from cytological specimens and biopsies for this detection.
Main Methods:
- Utilized nCounter, an RNA hybridization-based multiplexed technology.
- Analyzed RNA from 16 cytological samples and 132 biopsies.
- Compared the evaluability and RNA quality metrics between cytological samples and biopsies.
Main Results:
- nCounter successfully detected ALK, METΔex14, and high MET expression in cytological samples.
- Evaluability rates were 75.0% for cytological samples and 90.9% for biopsies.
- A patient with METΔex14 showed partial response to tepotinib; a patient with ALK fusion achieved complete response to crizotinib.
Conclusions:
- nCounter is a viable multiplex technology for detecting clinically relevant gene fusions and splicing variants in NSCLC.
- Cell blocks and cytological extensions are suitable specimens for RNA-based detection methods like nCounter.
- This approach aids in personalized medicine by enabling targeted therapy selection for NSCLC patients.
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