Clinical utility of circulating tumor RNA (ctRNA) in a combined circulating tumor DNA (ctDNA) and ctRNA next-generation sequencing (NGS) pan-cancer liquid biopsy assay.

3016 Background: Liquid biopsies are increasingly used in the real world for cancer therapy selection, disease monitoring, and early detection. Combining RNA with DNA sequencing for the detection of FDA-actionable gene fusions is already recommended as standard of care in guidelines as fusions are inherently challenging to detect by DNA-only methods. However, the clinical utility of profiling plasma ctRNA in addition to ctDNA has not yet been quantified in large studies. Here we report results from the first large real-world study establishing the clinical utility of combining ctRNA and ctDNA in a single liquid biopsy assay. Methods: A total of 1,007 consecutive plasma samples from 979 cancer patients across the USA and Asia underwent real-world liquid biopsy testing with a combined ctDNA and ctRNA assay (LiquidHALLMARK) in two CAP-accredited CLIA-certified laboratories from Jun 2021 to Dec 2024. The combined amplicon-based assay profiles genomic alterations in 80 genes on ctDNA and up to 37 genes on ctRNA. Only gene fusions (including MET exon 14 skipping) were included in this analysis. The limit of detection for gene fusions of the ctDNA and ctRNA panel were validated as 0.5% and 10 copies. Results: Plasma ctRNA was successfully analyzed in 99.6% (1003/1007) of samples across 30 cancer types. The top 5 cancer types (lung, prostate, breast, colorectal, and pancreas) comprised 84.2% of the clinical volume. Gene fusions were detected across 11 genes ( ALK , RET , ROS1 , MET , NRG1 , NTRK1 , FGFR2 , FGFR3 , ESR1 , ERG , ETV4 ) in 7.8% (78/1003) of cases, primarily in prostate and lung, but also in breast, bile duct, thyroid, liver, and bladder cancers. A total of 80 fusions were detected, of which 25 were detected by both ctDNA and ctRNA, while ctDNA and ctRNA each exclusively detected 27 and 28 fusions. Among the 28 ctRNA-only fusions, 5 were not covered by the ctDNA panel (1 ATP1B1 - NRG1 , 1 ESR1-CCDC170 , 1 ESR1 - AKAP12 , and 2 SLC45A3 - ERG fusions). Eleven (11/28) ctRNA-only fusions were actionable; all 11 were found in lung cancer. Two of these co-occurred with another lung driver mutation, highlighting potential resistance mechanisms to targeted therapy. Of the remaining 9, 3 were treated with fusion-matched targeted therapy. Two patients had real-world response rates available; both exhibited partial response to treatment. Overall, inclusion of ctRNA analysis increased the diagnostic yield of all fusions by 53.8% and actionable fusions by 36.7%. Conclusions: This is the first large study showing that adding ctRNA to ctDNA liquid biopsy increases total actionable diagnostic yield by 36.7%, highlights potential resistance mechanisms, and can broaden panel coverage to include gene fusions not amenable to detection by conventional DNA-based methods. These findings support recommendations for combined DNA/RNA testing of fusions in both tissue and liquid samples.