Clinical and analytical validation of a combined RNA and DNA exome assay across a large tumor cohort.

Background: Combining RNA sequencing (RNA-seq) with whole exome sequencing (WES) from a single tumor sample can substantially improve the detection of clinically relevant alterations in cancer. However, routine clinical adoption of this integrated approach remains limited, especially for RNA-seq, due to the absence of standardized validation frameworks.

Methods: We developed and validated an assay that integrates RNA-seq and WES for evaluating gene expression, gene fusions, tumor microenvironment signatures, somatic single nucleotide variants (SNVs), insertions/deletions (INDELs), and copy number variations (CNVs). Exome-wide somatic reference standards were generated to support analytical validation using multiple sequencing runs of cell lines at varying purities.

Results: Assay validation involves 3 steps: (1) analytical validation using custom reference samples containing 3042 SNVs and 47,466 CNVs; (2) orthogonal testing in patient samples; and (3) assessment of clinical utility in real-world cases. Applied to 2230 clinical tumor samples, the integrated assay enables direct correlation of somatic alterations with gene expression, recovery of variants missed by DNA-only testing, and improves detection of gene fusions. In addition to uncovering clinically actionable alterations in 98% of cases, the assay also reveals complex genomic rearrangements that would likely have remained undetected without RNA data.

Conclusions: This study provides practical validation guidelines for integrated RNA and DNA sequencing in clinical oncology. The combined assay enhances the detection of actionable alterations, thereby facilitating personalized treatment strategies for cancer patients.