To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes areBRAF, NRAS,NF1,NOTCH2,PTENandTYRP1. Mutations and amplification ofKITare also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification ofTERT,CDK4,MDM2,CCND1,PAK1andGAB2, indicating potential therapeutic options.

Immunotherapy, such as anti-PD1, has improved the survival of patients with metastatic melanoma. However, predicting which patients will respond to immunotherapy remains a significant knowledge gap. In this study we analyzed pre-immunotherapy treated tumors from 52 patients with metastatic melanoma and monitored their response based on RECIST 1.1 criteria. The responders group contained 21 patients that had a complete or partial response, while the 31 non-responders had stable or progressive disease. Whole exome sequencing (WES) was used to identify biomarkers of anti-PD1 response from somatic mutations between the two groups. Variants in codons G34 and G41 in NFKBIE, a negative regulator of NFkB, were found exclusively in the responders. Mutations in NKBIE-related genes were also enriched in the responder group compared to the non-responders. Patients that harbored NFKBIE-related gene mutations also had a higher mutational burden, decreased tumor volume with treatment, and increased progression-free survival. RNA sequencing on a subset of tumor samples identified that CD83 was highly expressed in our responder group. Additionally, Gene Set Enrichment Analysis showed that the TNFalpha signaling via NFkB pathway was one of the top pathways with differential expression in responders vs. non-responders. In vitro NFkB activity assays indicated that the G34E variant caused loss-of-function of NFKBIE, and resulted in activation of NFkB signaling. Flow cytometry assays indicated that G34E variant was associated with upregulation of CD83 in human melanoma cell lines. These results suggest that NFkB activation and signaling in tumor cells contributes to a favorable anti-PD1 treatment response, and clinical screening to include aberrations in NFkB-related genes should be considered.

There is an urgent need to develop treatments for patients with melanoma who are refractory to or ineligible for immune checkpoint blockade, including patients who lack BRAF-V600E/K mutations. This is often the case in patients diagnosed with rare melanoma subtypes such as mucosal and acral melanoma. Here, we analyzed data from the cutaneous melanoma The Cancer Genome Atlas Network (TCGA) transcriptomic and proteomic databases for differential expression of apoptosis molecules between melanomas with or without BRAF hotspot mutations. Our data indicated higher B-cell CLL/lymphoma 2 (BCL2) expression in melanoma without BRAF hotspot mutations, suggesting that BH3 mimetics, such as ABT-199 (venetoclax, a small molecule against BCL2), may be a potential therapeutic option for these patients. We explored the efficacy of combining two BH3 mimetics, ABT-199 and a myeloid cell leukemia sequence 1 (MCL1) inhibitor (S63845 or S64315/MIK665) in cutaneous, mucosal and acral melanomas, in vitro and in vivo. Our data indicate this combination induced cell death in a broad range of melanoma cell lines, including melanoma initiating cell populations, and was more potent in melanoma cells without BRAF-V600E/K mutations. Our knockdown/knockout experiments suggest that several pro-apoptotic BCL2 family members, BCL2-like 11 (apoptosis facilitator) (BIM), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) or BID, play a role in the combination-induced effects. Overall, our study supports the rationale for combining an MCL1 inhibitor with a BCL2 inhibitor as a therapeutic option in patients with advanced melanoma.

Purpose: We previously showed that elevated frequencies of peripheral blood CD3+CD4+CD127−GARP−CD38+CD39+ T cells were associated with checkpoint immunotherapy resistance in patients with metastatic melanoma. In the present study, we sought to further investigate this population of ectoenzyme-expressing T cells (Teee). Experimental Design: Teee derived from the peripheral blood of patients with metastatic melanoma were evaluated by bulk RNA-sequencing (RNA-seq) and flow cytometry. The presence of Teee in the tumor microenvironment was assessed using publically available single-cell RNA-seq datasets of melanoma, lung, and bladder cancers along with multispectral immunofluorescent imaging of melanoma patient formalin-fixed, paraffin-embedded specimens. Suppressive function of Teee was determined by an in vitro autologous suppression assay. Results: Teee had phenotypes associated with proliferation, apoptosis, exhaustion, and high expression of inhibitory molecules. Cells with a Teee gene signature were present in tumors of patients with melanoma, lung, and bladder cancers. CD4+ T cells co-expressing CD38 and CD39 in the tumor microenvironment were preferentially associated with Ki67− CD8+ T cells. Co-culture of patient Teee with autologous T cells resulted in decreased proliferation of target T cells. High baseline intratumoral frequencies of Teee were associated with checkpoint immunotherapy resistance and poor overall survival in patients with metastatic melanoma. Conclusions: These results demonstrate that a novel population of CD4+ T cells co-expressing CD38 and CD39 is found both in the peripheral blood and tumor of patients with melanoma and is associated with checkpoint immunotherapy resistance.

19 Background: Immunotherapy has quickly altered the treatment paradigm for advanced melanoma. Prior studies using whole exome sequencing (WES) in immunotherapy have shown limited correlation with clinical response to immunotherapy, but not an evaluation of autoimmune side effects. Understanding mechanisms of immune-related adverse events (irAE) at a genomic level may lead to improved rational treatment design and better patient selection. Methods: DNA was extracted from 87 tumor samples from 49 patients with metastatic melanoma treated with ipilimumab (anti-CTLA-4) or with nivolumab or pembrolizumab (anti-PD-1) immunotherapy. WES was performed with exome capture on an Illumina HiSeq sequencer. Statistical analysis was performed with Chi-squared testing. Computational neoantigen analysis has been performed on the sequencing data. Results: 27 patients with advanced or metastatic melanoma were treated with only ipilimumab, 11 with only anti-PD1, and 11 patients were treated with both anti-CLTA-4 and anti-PD1 either sequentially or in combination. irAE evaluated included cutaneous, colitis, endocrinopathies, hepatitis, and pneumonitis. In 10 patients who developed colitis, 14 mutated genes were enriched in their tumor samples (p=0.01-0.046) compared with patients without colitis. Analyzing all patients with any irAE versus no irAE treated with anti-CTLA-4, anti-PD1, or both (either sequential or combination) revealed 7 mutated genes enriched in patient samples who developed irAE (p=0.02-0.05). Preliminary neoantigen analysis has identified possible irAE associated neoantigens and further analysis of additional samples are ongoing. Overall mutational burden of the tumor samples did not correlate with development of irAE. Conclusions: Autoimmune side effects are frequent and often serious side effects of immunotherapy for melanoma. Genetic analysis may provide insight in prediction of these side effects and help direct therapeutic decisions.