# Diagnostic whole transcriptome sequencing in a series of 1233 FFPE solid tumor samples

## TL;DR

This study shows that whole transcriptome sequencing is a reliable method for detecting gene fusions in tumors when strict quality controls are applied.

## Contribution

The study establishes QC thresholds for WTS in FFPE tumor samples and demonstrates its clinical utility for fusion detection.

## Key findings

- WTS identified 92% of known fusions in an initial cohort of 64 FFPE tumor samples.
- Validation in 357 samples showed 100% concordance with panel-based results when QC thresholds were met.
- WTS detected 121 fusions in 812 clinical cases and provided additional insights into novel fusions and oncogenic pathways.

## Abstract

Whole Transcriptome Sequencing (WTS) is a comprehensive alternative to targeted panels for detecting gene fusions and splice variants. To integrate WTS into clinical diagnostics, we compared its performance against established fusion assays (Archer FusionPlex and TSO500 RNA).

WTS was evaluated in an initial cohort of 64 FFPE tumor samples, and quality control (QC) thresholds were defined based on missed fusions correlating with low tumor cell content (TCC < 40%). Key QC metrics included TCC ≥ 40%, RNA input ≥50 ng, ≥50 million reads, and median insert size >100 bp.

WTS identified 92% of known fusions in the initial cohort. Validation in 357 samples showed 100% concordance with panel-based results when QC thresholds were met. Subsequent clinical deployment across 812 diverse tumor cases detected 121 fusions, though 423 (34%) required fallback to targeted assays due to low TCC. WTS provided added value by detecting novel fusions, pathogens, and enabling oncogenic pathway analysis.

WTS is a reliable and informative method for fusion and splice variant detection in clinical diagnostics, provided rigorous pre-analytical and sequencing QC metrics are strictly applied.