JoVE - Journal of Visualized Experiments
JoVE Visualize
Contact Us

Analytical validation (accuracy, reproducibility, limit of detection) and gene expression analysis of FoundationOneRNA assay for fusion detection in 189 clinical tumor specimens

  • 0Foundation Medicine, Inc., Boston, Massachusetts, United States of America.
Plos One +

|

September 12, 2025

|

September 12, 2025

View abstract on PubMed

Summary

This summary is machine-generated.

A new targeted RNA sequencing assay, FoundationOne®RNA, demonstrates high accuracy and reproducibility for detecting cancer fusions. This assay complements DNA-based genomic profiling, improving fusion detection in clinical practice.

Area Of Science

  • Oncology
  • Genomics
  • Molecular Diagnostics

Background

  • Comprehensive genomic profiling (CGP) using DNA sequencing is standard for advanced cancer.
  • RNA sequencing may enhance the detection of gene fusions, which are critical cancer drivers.
  • Current RNA-based methods require validation for clinical utility.

Purpose Of The Study

  • To develop and analytically validate a targeted RNA sequencing assay, FoundationOne®RNA, for detecting gene fusions and measuring gene expression.
  • To assess the assay's accuracy, reproducibility, and limit of detection (LoD) in clinical solid tumor specimens.

Main Methods

  • Developed a hybrid-capture based targeted RNA sequencing assay (FoundationOne®RNA) covering 318 fusion and 1521 expression genes.
  • Conducted analytical validation in a CAP-accredited and CLIA-certified laboratory.
  • Evaluated assay performance using 189 clinical solid tumor specimens, comparing results to orthogonal DNA and RNA sequencing methods.

Main Results

  • FoundationOne®RNA demonstrated high concordance with orthogonal assays: 98.28% positive percent agreement (PPA) and 99.89% negative percent agreement (NPA).
  • The assay identified a low-level BRAF fusion missed by whole transcriptome RNA sequencing, confirmed by FISH.
  • Achieved 100% reproducibility for 10 target fusions and established an LoD ranging from 1.5ng to 30ng RNA input.

Conclusions

  • FoundationOne®RNA is a robust assay for detecting oncogenic fusions with high accuracy, reproducibility, and sensitivity.
  • The assay serves as a valuable supplement to tissue DNA-based CGP for advanced cancer patients.
  • Further research is needed to define optimal clinical applications and utilize gene expression biomarkers.