RNA in situ hybridization detection of CRTC1/3::MAML2 fusions and LINC00473 in mucoepidermoid carcinomas and hidradenomas of breast, salivary glands, and skin.

Abstract

Genomic rearrangements involving MAML2 have been reported in mucoepidermoid carcinoma (MEC) arising in various anatomic sites, as well as the benign counterpart of hidradenoma. Depending on the location, MAML2-rearranged neoplasms may share morphologic overlap with additional diagnostic entities including other salivary gland malignancies and cutaneous mimics. In some cases, detection of a CRTC1::MAML2 or less common CRTC3::MAML2 rearrangement by fluorescence in situ hybridization (FISH) or next-generation sequencing (NGS) may be necessary to help confirm the diagnosis. However, such testing can be time consuming, relatively expensive, and unavailable in many pathology laboratories. We describe the development and validation of an in situ hybridization (ISH) custom BaseScope assay targeting the recurrent breakpoints of CRTC1::MAML2 and CRTC3::MAML2 rearrangements. Moreover, we investigated the diagnostic utility of LINC00473 RNAscope as a surrogate marker for MAML2 fusion status. LINC00473 is a long non-coding RNA reportedly downstream of the CRTC1::MAML2 oncoprotein. We evaluated 227 patient cases, including 30 salivary gland and 2 breast MEC, 14 cutaneous and 8 breast hidradenoma, and 173 cases representing >20 potential histologic entities in the differential diagnosis for the presence of each fusion transcript by BaseScope, and a subset of cases (n=205) for the detection of LINC00473 by RNAscope. RNA ISH was directly visualized by chromogenic signal, and the MAML2 fusion partner could be positively identified in the majority of cases. Overall, RNA ISH demonstrates high concordance with orthogonal testing, with CRTC1/3::MAML2 BaseScope showing 93% sensitivity and 100% specificity and LINC00473 RNAscope showing 92% sensitivity and 99% specificity. RNA ISH for CRTC1/3::MAML2 rearrangements and LINC00473 represent reasonable timely and cost-effective alternatives to FISH and NGS. Such markers may provide the means for accurate diagnosis to ensure appropriate therapy of MEC and HA-neoplasms that can arise in multiple anatomic sites and be encountered by a wide range of pathologists.