Abstract 4668: Targeted, massively parallel sequencing identifies novel genetic subsets of cutaneous melanoma
Authors: Garman Bradley , Krepler Clemens , Sproesser Katrin , Brafford Patrica , Wilson Melissa , Wubbenhorst Bradley , Amaravadi Ravi , Bennett Joseph , Beqiri Marilda , Davies Michael , Elder David , Flaherty Keith , Frederick Dennie , Gangadhar Tara C. , Guarino Michael , Hoon David , Karakousis Giorgos , Mitra Nandita , Petrelli Nicholas J. , Schuchter Lynn , Shannan Batool , Wargo Jennifer , Xiao Min , Xu Wei , Xu Xaiowei , Herlyn Meenhard , Nathanson Katherine 
Published: August 2015

Abstract
Despite the prevalence of recurrent, high activating BRAF V600 mutations in 45% of tumors, cutaneous melanoma (CM) is a heterogeneous malignancy resulting from aberrant signaling in multiple pathways. It has been traditionally characterized by activation of the MAPK and PI3K signaling pathways, as well as cell cycle disruption. In recent years, whole-genome and exome sequencing studies have identified several new genes associated with melanomagenesis. However, a comprehensive understanding of concurrent, and mutually exclusive, mutations in tumors is currently lacking. Using a custom targeted capture of 108 genes previously implicated in melanoma pathogenesis, massively parallel sequencing was performed on 94 human melanoma cell lines, 67 patient-derived xenografts (PDX), and 5 cell lines made from PDX, all untreated. Samples were then clustered into groups based on deleterious mutations. 83% of samples had deleterious mutations in the MAPK signaling pathway, including 92 high activity BRAF (55%), 35 RAS codon 61 (21%), 7 with multiple mutations (e.g. low activity BRAF/RAS codons 12/13) (4%) and 10 NF1 (6%) mutated samples. Likely deleterious NF1 mutations were found in several BRAF or NRAS-mutated samples. PI3K pathway mutations were found in 10% of samples, predominantly associated with BRAF mutations. TP53 mutations were found in 24% of samples and were associated with all MAPK signaling mutations. Mutations in chromatin remodeling genes (ARID1A/1B, ARID2, TRRAP, and BAP1) were mutually exclusive with each other and primarily found in tumors with high activity BRAF or NRAS mutations. The majority of BRAF or RAS-mutated samples with a mutation in a chromatin remodeling gene lacked mutations in cell cycle, TP53, and PI3K signaling genes; however, 100% of deleterious, or likely deleterious, NF1-mutated samples with a chromatin remodeling gene mutation harbored additional mutations in cell cycle, TP53, and/or PI3K signaling genes. Of particular interest, five of the 10 NF1-mutated samples (50%) lacked BRAF, RAS, and MEK1/2 mutations but harbored likely deleterious mutations in MAP3K5 or MAP3K9, suggesting the potential involvement of the JNK signal transduction pathway in this particular cohort. Only 4% of samples did not have a deleterious mutation in any of the genes on the panel. These data reveal novel insights into the genetics of melanomas lacking a canonical BRAF V600 mutation. Functional assays are needed to confirm the biological relevance of likely deleterious mutations, which will further facilitate a more thorough classification of CM subsets.
Citation Format: Bradley Garman, Clemens Krepler, Katrin Sproesser, Patrica Brafford, Melissa Wilson, Bradley Wubbenhorst, Ravi Amaravadi, Joseph Bennett, Marilda Beqiri, Michael Davies, David Elder, Keith Flaherty, Dennie Frederick, Tara C. Gangadhar, Michael Guarino, David Hoon, Giorgos Karakousis, Nandita Mitra, Nicholas J. Petrelli, Lynn Schuchter, Batool Shannan, Jennifer Wargo, Min Xiao, Wei Xu, Xaiowei Xu, Meenhard Herlyn, Katherine Nathanson. Targeted, massively parallel sequencing identifies novel genetic subsets of cutaneous melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4668. doi:10.1158/1538-7445.AM2015-4668