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Context.--RNA-based next-generation sequencing (NGS) assays are being used with increasing frequency for comprehensive molecular profiling of solid tumors. Objective.--To evaluate factors that might impact clinical assay performance. Design.--A 4-month retrospective review of cases analyzed by a targeted RNA-based NGS assay to detect fusions was performed. RNA extraction was performed from formalin-fixed, paraffin-embedded tissue sections and/or cytology smears of 767 cases, including 493 in-house and 274 outside referral cases. The types of samples included 422 core needle biopsy specimens (55%), 268 resection specimens (35%), and 77 cytology samples (10%). Results.--Successful NGS fusion testing was achieved in 697 specimens (90.9%) and correlated positively with RNA yield (P < .001) and negatively with specimen necrosis (P = .002), decalcification (P < .001), and paraffin block age of more than 2 years (P = .001). Of the 697 cases that were successfully sequenced, 50 (7.2%) had clinically relevant fusions. The testing success rates and fusion detection rates were similar between core needle biopsy and cytology samples. In contrast, RNA fusion testing was often less successful using resection specimens (P = .007). Testing success was independent of the tumor percentage in the specimen, given that at least 20% tumor cellularity was present. Conclusions.--The success of RNA-based NGS testing is multifactorial and is influenced by RNA quality and quantity. Identification of preanalytical factors affecting RNA quality and yield can improve NGS testing success rates. (Arch Pathol Lab Med. 2021;145:1405-1412; doi: 10.5858/arpa.2020-0415-OA), With the increasing use of targeted therapeutic agents in solid tumors, RNA-based next-generation sequencing (NGS) assays are progressively used for comprehensive molecular analysis. (1) Chromosome translocations and/or gene fusions are [...]

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. Cytology specimens provide a reliable source for RNA‐based next‐generation sequencing fusion assays. The addition of cytology smears significantly improves the testing success rate of cytologic samples for RNA‐based next‐generation sequencing and is an appropriate alternative for cases in which the cell block preparation is inadequate or suboptimal. [ABSTRACT FROM AUTHOR]