Abstract Background: Previous epidemiological and population sequencing studies have established that subjects with melanocortin 1 receptor (MC1R) germline mutations, associated with red hair and light skin phenotypes, have an increased risk of melanoma. However, several conflicting reports about the role of BRAF somatic mutations in MC1R subjects exist in the literature. We hypothesise that this conflict is due to biological process driven heterogeneity within MC1R subjects carrying disruptive alleles. To understand the heterogeneity, we analysed the TCGA cohort at the gene expression level using mRNA sequencing data. Method: From the previously published studies, we identified 68 cutaneous melanoma subjects who were of white ethinicity, had at least one disruptive allele of MC1R, and had BRAF mutations. Associated clinical and mRNA sequencing data were downloaded from the Firebrowse website. An unbiased hierarchical clustering analysis was performed using the preprocessed mRNA data containing 14669 gene expressions. The resultant clusters were then characterised using available clinical variables. A differential expression analysis was performed to identify the genomic signatures of these clusters. The resultant p-values were corrected for false discovery rate (FDR) using the Benjamini-Hochberg approach. Genes with corrected p-values less than 0.01 were further analysed using gene set enrichment analysis (GSEA) preranked lists. Results: An unbiased clustering identified two clear and separable clusters with 40 and 28 subjects respectively. The UV-signature (dipyrimidine C>T load) was correlated with these clusters (p-value = 0.047). However, no significant differences in age at the time of diagnosis, overall survival, and total mutation load between clusters were observed. At the genomic level, 2588 genes were significantly differentially expressed between two clusters (FDR corrected p- value less than 0.05). The top 10 differentially expressed genes included ATF2, MSH6, SP3, MAP3K2, and VEGFB. Further GSEA revealed gene sets playing roles in several pathways and biological processes, including oxidative phosphorylation, UV response in keratinocytes, DNA repair, cell cycle, and cellular response to stress. Conclusion: Cutaneous melanoma subjects with disruptive MC1R alleles and BRAF hotspot mutations have heterogeneous gene expression profiles with several key oncogenes differentially expressed. This suggests potential roles of other pathways, either independently or in cooperation with BRAF mutations, in melanomagenesis in MC1R bearing patients. Citation Format: Piyushkumar A. Mundra, Candelaria Bracalente, Lucas Trucco, Nathalie Dhomen, Richard Marais. Gene expression analysis identifies heterogeneity in cutaneous melanoma subjects with disruptive MC1R alleles and BRAF hotspot mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1561. doi:10.1158/1538-7445.AM2017-1561