Fusion genes are commonly found in cancer and drive an estimated 20% of all cases. As such, precise fusion gene identification is essential for accurate cancer diagnosis, prognosis and treatment. However, current diagnostic techniques are only capable of detecting single fusion events, and misdiagnosis is a leading cause of mortality. To address these limitations, we developed two novel fusion gene assays, based on targeted RNA sequencing.
We designed two sets of biotinylated oligonucleotide probes targeting genes involved in fusion events in blood cancers (Blood FuSeq) and solid tumours (Solid FuSeq). Each set “captures” the full range of oncogenic fusion RNA transcripts, providing enriched sequencing coverage of the target regions to improve diagnostic performance. Utilising well-characterised cancer cell lines and spike-in sequencing standards, we demonstrated the ability of the assays to accurately detect and quantify known fusion genes.
Following refinement of laboratory and bioinformatic protocols, we analysed 135 clinical patient samples from 15 cancer subtypes covering a range of sample sources and RNA qualities, from shelf archived formalin-fixed tissues to cryopreserved snap-frozen liquid and solid specimens. Applying the FuSeq assays improved the overall fusion gene diagnostic rate from 55% to 64% and demonstrated high concordance for samples with previous diagnoses. In addition to identifying 58 different fusion genes across these samples, these assays had the capacity to identify multiple isoforms for several of the fusion genes (clinical significance to be determined), simultaneously measure parental target gene expression levels and report on immune receptor profiling.
These targeted RNA sequencing assays provide an advanced sequencing-based approach to fusion gene diagnosis and have the potential to both accurately determine fusion presence and highlight novel therapeutic targets. Following further clinical validation, we believe the assays can be implemented as accredited clinical tests, representing a significant advancement for fusion gene molecular diagnostics in cancer.