RNA Sequencing for Solid Tumor Fusion Gene Detection

• Context.—Next-generation sequencing–based approaches using RNA have increasingly been used by clinical laboratories for the detection of fusion genes, intragenic rearrangements, and exon-skipping events. Correspondingly, the College of American Pathologists (CAP) has advanced RNA sequencing proficiency testing (PT) to ensure optimal performance of these assays. Objective.—To report on laboratory performance and practices of RNA sequencing for the detection of fusion genes, intragenic rearrangements, and exon-skipping events using CAP PT data from 8 mailings (2018-A through 2021-B). Design.—CAP PT RNA sequencing program results from 153 laboratories across 24 proficiency test specimens, interrogating 22 distinct engineered fusion transcripts, were analyzed for correct identification of the fusion event, associated performance variables, and laboratory practices. Results.—Overall, the 4-year program detection rate (sensitivity) was 95.5% (1486 of 1556 results). False-negative rates were 3.6% (53 of 1463) and 18.3% (17 of 93) for fusion gene and intragenic rearrangement/exon-skipping events, respectively. Only 19 false-positive results were reported among the 8 PT mailings, and most were likely the result of preanalytical or postanalytical errors. There were no practice characteristics (eg, instrumentation, sequencing method) significantly associated with the fusion detection results. Conclusions.—These data reveal a high overall sensitivity and specificity for fusion gene detection by participating laboratories using clinical RNA sequencing. Performance was comparable across all laboratories, regardless of methodology. The fraction of false-negative results for intragenic rearrangement/exon-skipping events was greater than that for the chimeric fusion genes. False-negative results could not be attributed to any specific practice characteristics. (Arch Pathol Lab Med. 2024;148:538–544; doi: 10.5858/ arpa.2023-0047-CP)