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Updated: Sep 15, 2025
Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Published on: August 4, 2016
Clinical utility of targeted RNA sequencing in cancer molecular diagnostics
Robert Siddaway1, Aida I Glembocki2, Anthony Arnoldo2
1Division of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada. robert.siddaway@sickkids.ca.
Abstract:
Molecular diagnostics in oncology uses next-generation sequencing to improve diagnostic precision, prognostic prediction and therapeutic target selection. To date, most approaches have focused on DNA-based sequencing. However, RNA sequencing (RNA-seq) enables superior fusion detection and functional characterization of other alterations including splice site mutations and expression changes. Here we report our prospective, real-world, clinical experience using a single, targeted RNA-seq assay on 2,310 solid, central nervous system and hematopoietic neoplasms from children and adults (range 0-90 years). RNA-seq performed as well as DNA-based diagnostics, with a 4.8% failure rate despite the fact that most samples were formalin fixed and paraffin embedded, and provided valuable molecular data for 87% of patients, including diagnostic alterations that resulted in revised diagnoses and the identification of clinically actionable alterations that led to a change in treatment for patients, including the administration of targeted therapies. Our experience supports the use of RNA-seq as a stand-alone tool for precision clinical diagnostics, minimizing cost, tissue requirement and turnaround time.
Insights
Targeted RNA sequencing (RNA-seq) offers a powerful, stand-alone tool for molecular diagnostics in oncology. This approach provides valuable data for precision medicine, improving diagnoses and guiding targeted therapies in diverse cancers.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genomics
Background:
- Next-generation sequencing (NGS) advances oncology diagnostics, prognostics, and treatment selection.
- Current NGS methods predominantly utilize DNA sequencing.
- RNA sequencing (RNA-seq) offers advantages in detecting gene fusions, splice variants, and gene expression changes.
Purpose of the Study:
- To evaluate the clinical utility of a targeted RNA-seq assay in a large, real-world cohort.
- To compare RNA-seq performance against established DNA-based diagnostic methods.
- To assess the impact of RNA-seq findings on patient diagnoses and treatment decisions.
Main Methods:
- Prospective analysis of 2,310 solid, central nervous system, and hematopoietic neoplasms.
- Utilized a single, targeted RNA-seq assay across a wide age range (0-90 years).
- Included formalin-fixed, paraffin-embedded (FFPE) samples, common in clinical practice.
Main Results:
- RNA-seq demonstrated comparable performance to DNA-based diagnostics with a low failure rate (4.8%).
- Valuable molecular data was obtained for 87% of patients.
- Identified diagnostic alterations leading to revised diagnoses and actionable alterations guiding targeted therapy selection.
Conclusions:
- Targeted RNA-seq is a robust, stand-alone diagnostic tool for precision oncology.
- RNA-seq can minimize costs, tissue requirements, and turnaround times.
- Clinical implementation of RNA-seq supports improved patient care through precise molecular profiling.
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