Fernando Lopez-Diaz
Abstract
Fernando Lopez-Diaza, Steven Riverab, Christophe Magnana, Brad Thomasc, Yanglong Mouc, Segun Jungb, Sally Agersborgb, Vincent Funarib
aNeogenomics Laboratories, Carlsbad, CA, USA; bNeogenomics Laboratories, Aliso Viejo, CA, USA; cNeogenomics Laboratories, Houston, TX, USA
Accurate detection of gene fusions is key for supporting cancer diagnosis and therapy selection. Most assays are limited to select intronic DNA breakpoints capture assays or amplicon RNA panels querying only a small number of genes, while whole exome RNA capture or full transcriptome assays lack sensitivity. We developed a pair-ended, strand-specific hybridization-based RNA sequencing assay targeting 1194 unique known fusions pairs and 1104 genes involving 250 fusion genes clinically relevant to most solid tumors. The capture probes design targets fusion RNA sequences and exon junction regions of aberrant RNAs, including EGFRvIII, MET -exon 14 skipping, ARv7/v9. It also allows to enrich and detect novel fusion partners. Fusions are called by a custom BI pipeline assisted by AI. Commercial, PCR-based Archer FusionPlex assays and RT-PCR +Sanger were used for orthogonal validation. Assay clinical validation was performed on extracted total nucleic acids (TNA) from FFPE and included 113 clinical samples from 15 cancers and 206 unique fusion genes. We detected (116/121) fusions reported by either orthogonal assay. including multiple fusions not detected by the Archer RNA-seq but confirmed by Sanger sequencing. The assay has 95.9% sensitivity and 100% specificity, as all fusions were confirmed by either orthogonal assay. Importantly, the assay detected MET exon 14 skipping not predicted from splicing sites DNA mutation analysis. 55 novel fusion partners were detected by capturing only one of the partner genes.This novel breakpoint targeted fusion detection RNA-seq assay samples can comprehensively detect known and novel clinically actionable aberrant RNAs fusions in solid tumors.