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

Background 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. Methods Chromogenic 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 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 fusion-negative MEC were included in the test cohort. Positive signal strength was assessed using a semi-quantitative scoring method as per manufacturer guidelines. Results Fusion 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 for CRTC1 :: MAML2 while 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). Conclusion Here, 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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement American Cancer Society (RSG-18-058-0) Mary Kay Foundation Cancer Research Grant (RW) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The procedures performed in this retrospective study were approved by UT Southwestern Institutional Review Board (IRB 112017-073) (Dallas, TX, USA). This study of anonymized specimens did not require informed consent. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript