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

Abstract

ABSTRACTBackgroundRecent 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.MethodsChromogenic RNA in situ hybridization (RNA-ISH) using BaseScope™ technology was performed 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 theMEF2C::SS18(exon 7 ofMEF2Cto exon 4 ofSS18) andCRTC1::MAML2(exon 1 ofCRTC1to exon 2 ofMAML2) fusion transcripts. Sixteen cases ofMEF2C::SS18fusion-positive MSA, six cases ofCRTC1::MAML2fusion-positive MEC, three cases of fusion-unknown MEC, and one case of fusion-negative MEC were included in the test cohort. Positive signal strength was assessed using a semi-quantitative scoring method as per manufacturer guidelines.ResultsFusion transcripts were detected by RNA-ISH results in 14/16 cases (88%) of fusion-positive MSAs and 3/6 cases (50%) of fusion-positive MEC. Interestingly, 2 cases (67%) of fusion-unknown MEC were also positive by RNA-ISH forCRTC1::MAML2while the fusion-negative MEC was also negative by RNA-ISH. Positivity ranged between 1+ (one dot per cell in ≥5% of tumor cells in one 40X field) and 2+ (two to three dots per cell in ≥5% of tumor cells in one 40X field).ConclusionHere, we provide the first assessment of chromogenic RNA-ISH to detect gene fusions associated with microsecretory adenocarcinoma,MEF2C::SS18, and mucoepidermoid carcinoma,CRTC1::MAML2. 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.