Abstract 1741: Whole-transcriptome characterisation of NRAS and BRAF mutated primary melanomas associated with immune cell infiltration signatures and differential survival benefit
Authors: Pozniak Joanna , Nsengimana Jérémie , Muralidhar Sathya , Laye Jonathan , Bishop D Timothy , Newton-Bishop Julia 
Published: July 2017

Abstract
In 703 primary melanoma transcriptomes derived from formalin-fixed tumors removed from participants in the Leeds Melanoma Cohort, we used in silico methodology described by Bindea et al.1 to develop immune cell signatures. We showed these to be associated with survival2. There is some reported evidence that patients with NRAS mutated melanoma have a better response to checkpoint therapies3. Our aim was, therefore, to use the transcriptomic data to ask if immune cell infiltration (evidenced by the presence of the immune cell signature) differed between mutation subgroups. We saw that the survival benefit of the immune cell signature was strongest in wild-type (HR=0.31, P=3x10-5), intermediate in BRAF mutated tumors (HR=0.56, P=0.008) but absent in NRAS mutated tumors (HR=1.09, P=0.79). That is that even if the NRAS mutated tumors had a strong T cell signature that signature did not correlate with better survival. The results remained significant after adjusting for sex, tumor site, age at diagnosis, and AJCC stage. We observed no evidence of increased numbers of regulatory T cells (FOXP3), in NRAS mutated tumors, nor PDL1 or PD1 expression. The only difference in checkpoint molecule expression studied was a borderline lower expression of CTLA4 in the NRAS mutated tumors (P=0.09). Our data suggest that even when T cells invade NRAS mutated tumors some functional deficiency may be present in
those tumors such that a survival benefit is not seen. Using genes significantly differentially expressed between NRAS and BRAF mutated tumor groups, we tested biological pathway enrichment using Reactome FIViz plugin in Cytoscape. The most enriched pathways in NRAS mutated melanomas were the PI3K (AKT3, NRAS) and mitochondrial translation (CHCHD1, PTCD3). In the BRAF mutated the most enriched were: direct P53 effectors (TGFA, IGFBP3), β-catenin independent WNT signaling (WNT5A) and MHC class I antigen processing (CYBA). A number of pathways related to cell adhesion involving GRIN1 were also identified. Others have reported that β-catenin signaling inhibits transcription of the chemokine CCL4 which reduces the trafficking of CD141+DC to melanomas and therefore T cell infiltration4. We have evidence from the β-catenin candidate transcriptomic analysis that primary melanomas without immune infiltration signatures have upregulated β-catenin but the comparison between transcriptomic patterns in NRAS and BRAF tumors did not suggest that β-catenin signaling was more active in NRAS mutated tumors. It may be that a different immunosuppressive mechanism is more common in NRAS
mutated tumors than in others.
References
1. Bindea et al. Immunity 39, 782-795 (2013)
2. Newton-Bishop et al. PCMR in press
3. Johnson et al. Cancer Immunol Res. 3, 288-295 (2015)
4. Spranger et al. Nature 523, 231-235 (2015)
Citation Format: Joanna Pozniak, Jérémie Nsengimana, Sathya Muralidhar, Jonathan Laye, D Timothy Bishop, Julia Newton-Bishop. Whole-transcriptome characterisation of NRAS and BRAF mutated primary melanomas associated with immune cell infiltration signatures and differential survival benefit [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 1741. doi:10.1158/1538-7445.AM2017-1741