Abstract 2396: Gene fusion and variant-aware isoform detection with functional prediction from long-read RNA sequencing

Abstract Although most cancer variant profiling is done with short-read-based methods, many cancers are driven by structural variants that are difficult to detect with these methods. Our current approach to understanding driver mutations is also limited to single variants and rarely considers the context in which they are expressed. Alterations in the expression and splicing of genes containing variants can both impact their tumorigenicity and allow them to develop resistance to therapies. We present FLAIR3, which generates a custom transcriptome from long-read RNA sequencing and identifies SNVs, insertions, deletions, and gene fusions from alignment to this transcriptome. We show that this improves accuracy above alignment to the genome or annotated transcripts. FLAIR3 then integrates these variants with the transcripts to predict functional changes to the amino acid sequence. We apply this approach to patient-derived osteosarcoma cell lines, a cancer whose pathology is driven by complex structural variation. In these samples, we identify more complex changes to previously identified amplified drivers such as alternative splicing of MYC and alternative splicing of a gene fusion in CCNE1. We also identify a novel set of actionable cancer gene alterations not previously detected by short-read methods. This includes a large deletion in KEAP1 and a number of novel gene fusions, including TP53 and other genes fused with intergenic regions and a complex 4-locus fusion in NOTCH1 not detected by any preexisting tools. We then validated the protein sequence of novel fusions with Quantum-Si protein sequencing. This analysis shows that long-read RNA sequencing can detect novel variants in actionable cancer genes and that integrating splicing and variant alterations provides the most complete picture of gene alterations in cancer. Citation Format: Colette Felton, Andrea Galvez, Leanne Sayles, Christopher Vollmers, Alejandro Sweet-Cordero, Angela Brooks. Gene fusion and variant-aware isoform detection with functional prediction from long-read RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2396.