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

Area of Science:

• Genomics
• Molecular Diagnostics
• Cancer Research

Background:

• Whole Transcriptome Sequencing (WTS) offers a comprehensive approach for detecting gene fusions and splice variants.
• Established fusion assays (Archer FusionPlex, TSO500 RNA) are currently used in clinical diagnostics.
• Integrating WTS into clinical workflows requires performance validation against existing methods.

Purpose of the Study:

• To compare the diagnostic performance of Whole Transcriptome Sequencing (WTS) against established targeted gene fusion assays.
• To establish quality control (QC) metrics for reliable WTS implementation in clinical settings.
• To assess the added value of WTS in detecting novel fusions, pathogens, and analyzing oncogenic pathways.

Main Methods:

• WTS was initially evaluated on 64 FFPE tumor samples to define QC thresholds.
• Key QC metrics included tumor cell content (TCC) ≥40%, RNA input ≥50 ng, ≥50 million reads, and median insert size >100 bp.
• Performance was validated in a larger cohort of 357 samples and clinically deployed across 812 diverse tumor cases.

Main Results:

• WTS identified 92% of known fusions in the initial cohort.
• 100% concordance with panel-based results was achieved when QC thresholds were met in 357 samples.
• Despite 34% of cases requiring fallback to targeted assays due to low TCC, WTS detected novel fusions and provided pathway insights.

Conclusions:

• Whole Transcriptome Sequencing (WTS) is a dependable method for detecting gene fusions and splice variants in clinical diagnostics.
• Strict adherence to pre-analytical and sequencing quality control metrics is crucial for WTS reliability.
• WTS demonstrates significant value in comprehensive genomic profiling for cancer diagnostics.