Development and Validation of an RNA Sequencing Assay for Gene Fusion Detection in Formalin-Fixed, Paraffin-Embedded Tumors.

RNA sequencing (RNA-seq) is a well-validated tool for detecting gene fusions in fresh frozen tumors, whereas it is much more challenging to use it with formalin-fixed paraffin-embedded (FFPE) tumor samples. We evaluated the performance of RNA-seq to detect gene fusions in clinical FFPE tumor samples. Our assay identified all 15 spiked-in NTRK fusions from RNA reference material and 6 known fusions from 5 cancer cell lines. Limit of detection for the assay was assessed with a series of dilutions of RNA from the cell line H2228. These fusions can be detected when the dilution is down to 10%. Good intra- and inter-assay reproducibility was observed in three specimens. For clinical validation, the assay detected 10 of 12 fusions initially identified by a DNA panel (covering 23 gene fusions) in clinical specimens (83.3% sensitivity), whereas one fusion (MET fusion) was identified in another 34 fusion-negative tumor specimens as determined by the DNA panel (negative prediction value of 94.3%). This MET fusion was confirmed by RT-PCR and Sanger sequencing, which demonstrated that this is a false negative for DNA panel. The assay also identified 26 extra fusions not covered by the DNA panel, and 20 of them (76.9%) were validated by RT-PCR and Sanger sequencing. Therefore, this RNA assay has reasonable performance and could be a complement to DNA-based next-generation sequencing assays.