Variant calling and fusion gene detection in formalin-fixed paraffin-embedded tumor tissue using RNA capture sequencing

Post-doctoral fellow Anneleen Decock presents her recent work on variant calling and fusion gene detection on FFPE tumors" abstract: “RNA sequencing is widely used to study gene expression, but less frequently to assess structural alterations such as fusion transcripts or sequence variants. In this study, we applied mRNA capture sequencing to identify such aberrations in formalin-fixed paraffin-embedded tissue of different types of solid tumors. To validate our workflow, we used the TruSight RNA Pan-Cancer Panel to analyze a first cohort of 16 positive control tumors for which the diagnostic workup demonstrated the presence of a specific single nucleotide variant (SNV), small insertion or deletion (indel), or chromosomal rearrangement. RNA capture sequencing enabled the identification of both fusion gene partners and fusion breakpoint in the patients diagnosed with a chromosomal rearrangement, and confirmed all known sequence variants. Next, whole transcriptome TruSeq RNA Access sequencing was applied to a second cohort of 17 patients, diagnosed with fusion gene-negative alveolar rhabdomyosarcoma (ARMS) or undifferentiated round cell sarcoma (URCS), for whom fluorescence in situ hybridization did not identify the classical pathognomonic rearrangements. Remarkably, for 6/17 patients, RNA capture sequencing readily detected the pathognomonic fusion transcript, i.e. PAX3-FOXO1 in 2 ARMS patients, and EWSR1-FLI1, EWSR1-ERG or EWSR1-NFATC2 in 4 URCS patients. These data show that RNA capture sequencing may enhance the detection rate of pathognomonic fusion genes in sarcoma. For the 11 patients with no known pathognomonic fusion transcript, 20 newly identified fusion transcripts were prioritized for RT-qPCR validation and SNV/indel analysis was performed, of which results will be presented. Our results may provide new insights into the underlying genetic causes of these malignancies.