Related Experiment Video
Updated: Mar 26, 2026
Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample
Published on: November 21, 2023
Multiplex Diagnosis of Oncogenic Fusion and MET Exon Skipping by Molecular Counting Using Formalin-Fixed Paraffin
Kuniko Sunami1, Koh Furuta2, Koji Tsuta3
1Division of Genome Biology, National Cancer Center Research Institute, Tokyo, Japan; Course of Advanced Clinical Research of Cancer, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Introduction:
Fusions of the anaplastic lymphoma receptor tyrosine kinase gene (ALK), ret proto-oncogene (RET), ROS proto-oncogene 1, receptor tyrosine kinase gene (ROS1), B-Raf proto-oncogene, serine/threonine kinase gene (BRAF), and neuregulin 1 gene (NRG1) and intronic MMNG HOS Transforming gene (MET) mutations are druggable oncogene alterations in lung adenocarcinoma that cause expression of aberrant transcripts. Because these aberrant transcripts are both infrequent (incidence <5%) and mutually exclusive, multiplex assays are required to detect them in tumor samples.
Methods:
Aberrant transcripts of the six aforementioned oncogenes (36 transcripts in total) were examined in a molecular counting (MC) assay, which counts RNA molecules by simultaneous hybridization of several probes. Forty-one samples of surgically resected lung adenocarcinoma tissue found to express one of these aberrant oncogenic transcripts upon whole transcriptome sequencing (test cohort: n = 22) or reverse transcription polymerase chain reaction (validation cohort: n = 19) analyses were subjected to MC, after which biopsies were performed on tumor tissue samples.
Results:
Threshold values for the diagnosis of each of the 36 transcripts were determined in frozen and formalin-fixed paraffin-embedded samples from the test cohort. On the basis of these threshold values, the MC assay diagnosed expression of oncogenic transcripts in the validation cohort samples with 100% accuracy. The assay also accurately detected oncogenic fusions in bronchial lavage fluid and transbronchial biopsy samples.
Conclusions:
The MC assay allows multiplex detection of oncogenic fusion and exon-skipped transcripts in tumor samples, including in formalin-fixed paraffin-embedded samples obtained in the clinic.
Insights
A new molecular counting (MC) assay accurately detects rare, actionable oncogenic fusions in lung adenocarcinoma. This multiplex assay is crucial for identifying these specific gene alterations in tumor samples for targeted therapy.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Druggable oncogene alterations, including fusions and mutations in ALK, RET, ROS1, BRAF, NRG1, and MET, are present in lung adenocarcinoma.
- These alterations result in aberrant transcripts, are infrequent (<5% incidence), and occur mutually exclusively.
- Multiplex assays are necessary for detecting these rare oncogenic transcripts in tumor samples.
Purpose of the Study:
- To evaluate a novel molecular counting (MC) assay for the multiplex detection of aberrant transcripts.
- To assess the accuracy of the MC assay in identifying oncogenic fusions and exon-skipped transcripts in lung adenocarcinoma.
Main Methods:
- Examined 36 aberrant transcripts from six oncogenes (ALK, RET, ROS1, BRAF, NRG1, MET) using a molecular counting (MC) assay.
- Analyzed 41 lung adenocarcinoma samples (22 in test cohort, 19 in validation cohort) using MC assay.
- Validated MC assay performance on formalin-fixed paraffin-embedded samples and biopsy specimens.
Main Results:
- Established diagnostic threshold values for 36 aberrant transcripts in both frozen and formalin-fixed paraffin-embedded samples.
- Achieved 100% accuracy in diagnosing oncogenic transcripts in the validation cohort using the MC assay.
- Demonstrated accurate detection of oncogenic fusions in bronchial lavage fluid and transbronchial biopsy samples.
Conclusions:
- The MC assay enables multiplex detection of oncogenic fusion and exon-skipped transcripts in lung adenocarcinoma.
- The assay is effective for clinical samples, including formalin-fixed paraffin-embedded tissues.
- This method facilitates the identification of actionable targets for personalized lung cancer therapy.
