Factors Impacting Clinically Relevant RNA Fusion Assays Using Next-Generation Sequencing.

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.