Diagnostic utility of anchored multiplex polymerase chain reaction‐based fusion assay on sacrificed cytology slides

BACKGROUND: Gene fusion testing, essential for the diagnosis and classification of many salivary gland and soft tissue neoplasms, is typically performed on formalin-fixed paraffin-embedded material, which may be limited or unavailable in cytology specimens. Although cytology smears have been validated for single-gene and DNA-based assays, evidence supporting routine diagnostic use of RNA-based next-generation sequencing fusion testing on smears remains limited. METHODS: The authors evaluated the clinical utility of a clinically validated anchored multiplex polymerase chain reaction (adenosine monophosphate)-based RNA fusion assay performed on digitized, scraped, and sacrificed cytology smears. Alcohol-fixed Papanicolaou-stained and air-dried modified Giemsa-stained smears meeting predefined cellularity thresholds were digitally archived, manually scraped, and processed for RNA extraction. Fusion testing targeted solid tumor-associated genes, and sequencing was performed on an Illumina platform. RESULTS: Of 8706 fusion assays performed during the study period, 807 (9.3%) were applied to cytology specimens, including 14 cases (1.7%) using sacrificed direct smears. Adequate nucleic acid was obtained in all cases. Gene fusions were detected in 64% (9/14), including MYB::NFIB, ACTB::PLAG1, NCALD::PLAG1, CRTC1::MAML2, COL1A1::PDGFB, COL1A2::USP6, and FGFR1::NOL4. Fusion results enabled definitive diagnosis for adenoid cystic carcinoma, mucoepidermoid carcinoma, pleomorphic adenoma, nodular fasciitis, and dermatofibrosarcoma protuberans. CONCLUSIONS: Adenosine monophosphate-based RNA fusion testing on sacrificed cytology smears is feasible, reliable, and diagnostically impactful, expanding the role of cytology specimens in definitive tumor classification.