Related Experiment Video
Updated: Jun 27, 2026
Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Published on: August 4, 2016
Use of 3' Rapid Amplification of cDNA Ends (3' RACE)-Based Targeted RNA Sequencing for Profiling of Druggable Genetic
Natalia V Mitiushkina1, Vladislav I Tiurin1, Aleksandra A Anuskina1
1Department of Tumor Growth Biology, N.N. Petrov Institute of Oncology, 197758 St. Petersburg, Russia.
Abstract:
Targeted treatment of advanced or metastatic urothelial carcinomas (UCs) requires the identification of druggable mutations. This study describes the development of a 3' Rapid Amplification of cDNA Ends (3' RACE)-based targeted RNA sequencing panel which accounts for the status of all genes relevant to UC treatment, namely, FGFR1-4, KRAS, NRAS, BRAF, ERBB2 (HER2), CD274 (PD-L1) and PIK3CA. FGFR2/3-activating point mutations or fusions were found in 54/233 (23.2%) tumors. FGFR3 rearrangements were identified in 11 patients, with eight of them being undetectable by commonly used PCR kits. In addition, one tumor contained a high-copy FGFR2 gene amplification accompanied by strong overexpression of the gene. Mutations in RAS/RAF genes were present in 30/233 (12.9%) UCs and were mutually exclusive with alterations affecting FGFR2/3 genes. On the contrary, activating events in the HER2 oncogene (point mutations and overexpression), as well as PIK3CA mutations, which were relatively common, occurred with similar frequencies in RAS/RAF- or FGFR2/3-positive vs. negative samples. High PD-L1 mRNA expression was associated with advanced disease stage and was not observed in tumors with increased HER2 mRNA expression or in UCs with evidence for FGFR2/3 activation. Three of the studied carcinomas had high-level microsatellite instability (MSI). Overall, more than half of the UCs had potentially druggable genetic alterations. The proposed NGS panel permits comprehensive and cost-efficient analysis of UC-specific molecular targets and may be considered in clinical routine.
Insights
A new targeted RNA sequencing panel identifies actionable mutations in over half of advanced urothelial carcinomas (UCs). This comprehensive approach detects key gene alterations, including FGFR and RAS/RAF mutations, crucial for guiding targeted therapy in UC treatment.
Area of Science:
- Genomics and Molecular Oncology
- Urothelial Carcinoma Research
- Cancer Biomarker Discovery
Background:
- Targeted therapies for advanced or metastatic urothelial carcinomas (UCs) depend on identifying actionable mutations.
- Existing diagnostic methods may not comprehensively detect all relevant genetic alterations in UCs.
- A need exists for efficient and broad molecular profiling to guide treatment decisions in UC.
Purpose of the Study:
- To develop and validate a targeted RNA sequencing panel for comprehensive analysis of UC-associated genes.
- To identify the frequency and spectrum of druggable mutations in a cohort of UCs.
- To assess the utility of the panel for clinical routine in UC molecular profiling.
Main Methods:
- Development of a 3' Rapid Amplification of cDNA Ends (3' RACE)-based targeted RNA sequencing panel.
- The panel covers key genes: FGFR1-4, KRAS, NRAS, BRAF, ERBB2 (HER2), CD274 (PD-L1), and PIK3CA.
- Analysis of 233 urothelial carcinoma samples to determine the prevalence of genetic alterations.
Main Results:
- FGFR2/3 activating mutations or fusions were detected in 23.2% of UCs, with novel FGFR3 rearrangements found in 8 patients.
- RAS/RAF gene mutations occurred in 12.9% of UCs and were mutually exclusive with FGFR2/3 alterations.
- Over half of UCs exhibited potentially druggable genetic alterations, including HER2 and PIK3CA mutations, and high PD-L1 expression.
Conclusions:
- The developed NGS panel provides a comprehensive and cost-effective method for analyzing UC molecular targets.
- More than 50% of UCs harbor actionable genetic alterations, highlighting the potential for targeted therapy.
- This panel may be integrated into clinical practice for improved patient management and treatment selection in UCs.
