Abstract Introduction: The presence of the t(11;19) chromosomal translocation and its CRTC1-MAML2 (C1/M2) fusion product occurs frequently in salivary mucoepidermoid carcinoma (MEC) and has been correlated with a favorable prognosis. Recently, whole-exome sequencing studies found genomic alterations in MEC to be infrequent, which supports the role of C1/M2 as an early driver event. However, when present, TP53 and PIK3CA alterations may represent alternative driver mutations in fusion-negative cases. To better understand the MEC transcriptional landscape by fusion status and histologic grade, we performed RNA deep sequencing. Methods: 16 parotid MEC (8 low-grade and 8-high grade) tumors, 12 adjacent normal, and 6 age-matched normal parotid tissues were retrospectively analyzed from UNC Surgical Pathology specimens collected from 2000-2019. H&E and mucicarmine stained slides were independently reviewed by pathology for diagnostic confirmation. The Air Force Institute of Pathology (AFIP) classification system by Ellis and Auclair was utilized for histologic grading. RNA was extracted from FFPE tissue sections and quality checked prior to library preparation. RNA-seq (HS2500) was then performed on all samples and computational methods were used to determine C1/M2 fusion status. Gene set variation analyses (GSVA) were performed on genes differentially expressed between MEC tumor versus normal, or according to tumor grade, and/or fusion status. Results: Compared to matched normals, MEC tumors display significant changes in ERBB2 and PI3K signaling cascades (FC=-3.2, p=0.049). We also noted significant upregulation of inflammatory and apoptosis markers including interferon gamma, IL-4, NOS1, and programmed cell death (p<0.05). Moreover, compared to low-grade, high-grade MEC tumors had significant upregulation of angiogenesis pathways, including the VEGF-VEGF receptor signaling network (FC=4, p=0.007) and activation of developmental pathways such as TGF-β signaling (FC=3.62, p=0.015). Notably, we report the expression of C1/M2 fusion transcripts in both low- and high-grade MEC. Further, p53 signaling pathway is downregulated in C1/M2 fusion-positive cases compared to fusion-negative cases (p<0.05). Discussion: This is the first study to perform transcriptome analyses of salivary MEC comparing to normal parotid tissue, while accounting for histologic grade. While C1/M2 fusion status is considered pathognomonic of low-grade MEC, we identified the expression of C1/M2 fusion transcripts in high-grade MEC. This confirms recent reports challenging the prognostic value of fusion status. Our study also confirms that reprogramming TP53 and PI3K signaling is a universal feature of MEC and epigenetic mechanisms may repress p53 in the absence of TP53 driver mutations seen in fusion negative tumors. Lastly, we report that VEGF and TGF-β pathway activation are associated with cancer pathogenesis in high-grade MEC. The evaluation of anticancer therapies targeting these pathways should be evaluated further. Citation Format: Siddharth Sheth, Renee Betancourt, Joel Parker, Kshitij Sharma, Adele Musicant, Wendell Yarbrough, Trevor Hackman, Antonio Amelio. RNA sequencing of salivary mucoepidermoid carcinoma reveals distinct molecular profiles based on histologic grade [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR04.