Utilization of cytology smears improves success rates of RNA-based next-generation sequencing gene fusion assays for clinically relevant predictive biomarkers

Background: The use of RNA-based next-generation sequencing (NGS) assays to detect gene fusions for targeted therapy has rapidly become an essential component of comprehensive molecular profiling. For cytology specimens, the cell block (CB) is most commonly used for fusion testing; however, insufficient cellularity and/or suboptimal RNA quality are often limiting factors. In the current study, the authors evaluated the factors affecting RNA fusion testing in cytology and the added value of smears in cases with a suboptimal or inadequate CB. Methods: A 12-month retrospective review was performed to identify cytology cases that were evaluated by a targeted RNA-based NGS assay. Samples were sequenced by targeted amplicon-based NGS for 51 clinically relevant genes on a proprietary platform. Preanalytic factors and NGS quality parameters were correlated with the results of RNA fusion testing. Results: The overall success rate of RNA fusion testing was 92%. Of the 146 cases successfully sequenced, 14% had a clinically relevant fusion detected. NGS testing success positively correlated with RNA yield (P =.03) but was independent of the tumor fraction, the tumor size, or the number of slides used for extraction. CB preparations were adequate for testing in 45% cases, but the inclusion of direct smears increased the adequacy rate to 92%. There was no significant difference in testing success rates between smears and CB preparations. Conclusions: The success of RNA-based NGS fusion testing depends on the quality and quantity of RNA extracted. The use of direct smears significantly improves the adequacy of cytologic samples for RNA fusion testing for predictive biomarkers.