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Updated: May 24, 2025
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
An accurate DNA and RNA based targeted sequencing assay for clinical detection of gene fusions in solid tumors
Gang Ji1,2,3, Qianlan Yao1,2,3, Min Ren1,2,3
1Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China.
Abstract:
Gene fusions are one of the most important molecular biomarkers for tumor diagnosis, classification and targeted therapy. How to accurately detect them is a key issue in clinical work. In this study, a custom-designed integration of DNA and RNA-based next generation sequencing (NGS) assay including 16 targeted therapy related genes was developed and validated to identify gene fusions in solid tumors. This assay accurately identified all 10 different types of fusion in 8 commercial fusion spiked-in reference standards and 29 fusions including 16 different fusion forms in 60 clinical solid tumor samples previously identified by clinical testing methods. In addition, a TPM3::NTRK1 fusion was additionally identified and validated by Sanger sequencing, which showed a false-negative result for the previous result. Mutational abundance limit of detection for the assay was assessed with a series of dilution experiments. These fusions can be stably detected when the mutational abundance is down to 5% for DNA and 250-400 copies/100 ng for RNA. The intra-assay and inter-assay reproducibility was observed in three samples and three replicates. This integration of DNA and RNA-based NGS assay shows excellent performance on formalin-fixed, paraffin-embedded samples, results at different levels can complement each other, thereby facilitating precise diagnosis and treatment.
Insights
A novel DNA and RNA next-generation sequencing (NGS) assay accurately detects gene fusions in solid tumors. This integrated approach improves tumor diagnosis and targeted therapy selection.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genomics
Background:
- Gene fusions are critical molecular biomarkers for cancer diagnosis, classification, and targeted therapy.
- Accurate detection of gene fusions remains a significant challenge in clinical practice.
Purpose of the Study:
- To develop and validate a custom-designed, integrated DNA and RNA-based next-generation sequencing (NGS) assay for identifying gene fusions in solid tumors.
- To assess the assay's performance, including accuracy, sensitivity, and reproducibility.
Main Methods:
- Development of a targeted NGS assay covering 16 genes relevant to targeted therapy.
- Validation using commercial fusion standards and clinical solid tumor samples.
- Assessment of limit of detection using dilution experiments and reproducibility studies.
Main Results:
- The assay accurately identified all 10 fusion types in reference standards and 29 fusions (16 forms) in 60 clinical samples.
- A TPM3::NTRK1 fusion was newly identified, highlighting potential for discovering missed fusions.
- Stable detection limits were established at 5% mutational abundance for DNA and 250-400 copies/100 ng for RNA.
- High intra-assay and inter-assay reproducibility was demonstrated.
Conclusions:
- The integrated DNA and RNA-based NGS assay demonstrates excellent performance for detecting gene fusions in formalin-fixed, paraffin-embedded solid tumors.
- The assay's complementary DNA and RNA results facilitate precise cancer diagnosis and treatment selection.
- This approach enhances the clinical utility of molecular biomarkers for personalized oncology.
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