Fusion Detection in Microsecretory Adenocarcinoma and Mucoepidermoid Carcinoma Using Chromogenic RNA In Situ Hybridization: A Promising Alternative to DNA‐Based Fluorescence In‐Situ Hybridization

Recent advances in molecular genetics have dramatically improved our understanding of the pathophysiology and classification of salivary gland tumors. The identification of recurrent oncogenic fusions has been especially helpful in distinguishing entities with overlapping histomorphology. Here, we provide the first assessment of chromogenic RNA in situ hybridization (RNA‐ISH) using BaseScope TM to detect gene fusions associated with microsecretory adenocarcinoma (MSA), MEF2C :: SS18 , and mucoepidermoid carcinoma (MEC), CRTC1 :: MAML2 , using probes specific to the exon junctions of the MEF2C :: SS18 (exon 7 of MEF2C to exon 4 of SS18 ) and CRTC1 :: MAML2 (exon 1 of CRTC1 to exon 2 of MAML2 ) fusion transcripts. Sixteen cases of MEF2C :: SS18 fusion‐positive MSA, six cases of CRTC1 :: MAML2 fusion‐positive MEC, three cases of fusion‐unknown MEC, and one case of MAML2 ‐negative MEC were included in the test cohort. Positive signal strength was assessed using a semi‐quantitative scoring method as per manufacturer guidelines. Fusion transcripts were detected by RNA‐ISH results in 14/16 cases (88%) of fusion‐positive MSAs, 3/6 cases (50%) of fusion‐positive MEC, and one case of MAML2 ‐negative MEC. Interestingly, 2 cases (67%) of fusion‐unknown MEC were also positive by RNA‐ISH for CRTC1 :: MAML2 . Positivity ranged between 1+ (one dot per cell in ≥ 5% of tumor cells in one 40× field) and 2+ (two to three dots per cell in ≥ 5% of tumor cells in one 40× field). Our results highlight the potential for ultrasensitive RNA‐ISH to be used as an alternative method of fusion detection for salivary gland malignancies with highly conserved fusion transcript exon junctions. While additional studies are needed to validate the clinical utility of the assay and to determine optimal testing conditions, RNA‐ISH may provide a means for restricted fusion analysis in cases with limited material and for pathologists without easy access to conventional molecular diagnostic testing.