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.

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There have been major developments in targeted therapeutics with the clinical development of selective BRAF inhibitors (BRAFi) for patients with metastatic BRAF V600E mutant melanoma. Objective response rate of almost 50% has been witnessed in BRAFi clinical trials. Frequent side effects range from squamoproliferative lesions, including hyperplasia, keratoacanthomas, and squamous cell carcinomas to second primary melanomas. We describe a 50-year-old Hispanic woman with BRAF V600E mutant metastatic melanoma who was treated with surgery, radiation therapy, interleukin-2, and was enrolled on a BRAFi (dabrafenib) trial. Two months after initiation, she developed multiple erythematous, indurated, tender subcutaneous nodules bilaterally on the anterior thighs, posterior arms, and left dorsal forearm without overlying epidermal change. Punch biopsy revealed panniculitis with necrotizing granulomata. Infectious and other causes for panniculitis were excluded. We believe the histology likely represents a reaction to BRAFi therapy based on the temporal relationship of its onset to initiation of BRAFi therapy and previously reported cases of neutrophilic panniculitis associated with BRAFi therapy. Panniculitis has been emerging as an important unusual side effect of BRAFi therapy. Our case illustrates a unique presentation of BRAFi-associated panniculitis demonstrating necrotizing granulomata.

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.

Also referred to as “osteoclast-rich, clear cell sarcoma–like tumor of the gastrointestinal tract (CCSLGT),” malignant gastrointestinal neuroectodermal tumor is a newly described, rare, aggressive sarcoma that commonly arises in the small bowel, stomach, and colon. Histogenesis is likely from an autonomous nervous system–related primitive cell of neural crest origin. The hallmark genetic finding of EWS-CREB1 or EWS-ATF1 fusion transcripts clinches the diagnosis. Annular constrictive lesions tend to be smaller, show homogenous contrast enhancement on computed tomography, and may present with bowel obstruction. Larger, expansile masses tend to be exophytic and show heterogeneous contrast enhancement. Surgical resection is the mainstay of treatment. Frequent recurrences, metastases, and death from disease in 75% of patients portend a poor prognosis. Targeted chemotherapy based on specific tumor pathways is being developed.