Abstract A030: Detection of Clinically Actionable RNA Fusions Across Multiple Cancer Types Using Optimized cfRNA Extraction from Diverse Sample Matrices

Abstract Introduction: Fusion-driven oncogenesis is a hallmark of multiple malignancies, with transcripts such as TMPRSS2-ERG, EML4-ALK, FGFR3-TACC3, ETV6-NTRK3, CCDC6-RET, SLC34A2-ROS1, and BCR-ABL1 representing clinically actionable biomarkers for targeted therapies. Accurate detection of these RNA fusions in plasma requires extraction methods that efficiently recover cfRNA molecules, which are typically present at very low abundance. This study assessed the performance and reproducibility of an optimized cfRNA extraction workflow for fusion transcript detection from plasma samples. Methods: Synthetic RNA standards and fusion-positive cell line reference materials were spiked into plasma to mimic clinically relevant fusion transcript abundance. cfRNA was extracted from 10 mL plasma aliquots using a magnetic–capture–based workflow designed to maximize RNA yield. Extracted cfRNA was reverse transcribed and analyzed by singleplex PCR (aggregated to panel coverage) targeting the seven representative RNA fusions listed above. Extraction performance was evaluated by RNA yield, consistency across replicates, and quantitative recovery of fusion-specific transcripts relative to known input copies. Results: The extraction workflow demonstrated high efficiency and consistency in recovering low-abundance RNA from plasma. Total cfRNA yield per 10 mL of plasma demonstrated consistent recovery across replicates, with low variability. All seven target fusions (TMPRSS2-ERG, EML4-ALK, FGFR3-TACC3, ETV6-NTRK3, CCDC6-RET, SLC34A2-ROS1, BCR-ABL1) were successfully detected using singleplex PCR from cfRNA eluates. Extraction reproducibility was maintained across plasma samples with variable background RNA content, and no inhibition was observed in downstream amplification reactions. The data indicate that the majority of fusion transcripts in plasma cfRNA are captured, highlighting the importance of preserving cfRNA species during extraction. Conclusion: Efficient cfRNA extraction from plasma is essential for accurate and sensitive detection of RNA fusions that define actionable cancer subtypes. This study demonstrates a robust and reproducible extraction workflow capable of recovering low-copy fusion transcripts spanning multiple gene families relevant to prostate, lung, bladder, pediatric, and hematologic malignancies. High-quality cfRNA recovery from plasma enables reliable downstream singleplex PCR/ddPCR or sequencing-based detection of TMPRSS2-ERG, EML4-ALK, FGFR3-TACC3, ETV6-NTRK3, CCDC6-RET, SLC34A2-ROS1, and BCR-ABL1, supporting the use of liquid biopsy–derived RNA as a viable substrate for clinical and translational research applications. ChatGPT was used to improve the clarity of the abstract. Citation Format: Nafiseh Jafari, Yabin Lu, Jason Saenz, Carlos Hernandez, Daniel Cedeno, Cameron Van Dieren, Mayer Saidian. Detection of Clinically Actionable RNA Fusions Across Multiple Cancer Types Using Optimized cfRNA Extraction from Diverse Sample Matrices [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr A030.