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Background:Adrenal gland metastases (AGMs) are common in advanced-stage melanoma, occurring in up to 50% of patients. The introduction of immune checkpoint inhibitors (ICIs) has markedly altered the outcome of patients with melanoma. However, despite significant successes, anecdotal evidence has suggested that treatment responses in AGMs are significantly lower than in other metastatic sites. We sought to investigate whether having an AGM is associated with altered outcomes and whether ICI responses are dampened in the adrenal glands.Patients and Methods:We retrospectively compared ICI responses and overall survival (OS) in 68 patients with melanoma who were diagnosed with an AGM and a control group of 100 patients without AGMs at a single institution. Response was determined using RECIST 1.1. OS was calculated from time of ICI initiation, anti–PD-1 initiation, initial melanoma diagnosis, and stage IV disease diagnosis. Tumor-infiltrating immune cells were characterized in 9 resected AGMs using immunohistochemical analysis.Results:Response rates of AGMs were significantly lower compared with other metastatic sites in patients with AGMs (16% vs 22%) and compared with those without AGMs (55%). Patients with AGMs also had significantly lower median OS compared with those without AGMs (3.1 years vs not reached, respectively). We further observed that despite this, AGMs exhibited high levels of tumor-infiltrating immune cells.Conclusions:In this cohort of patients with melanoma, those diagnosed with an AGM had lower ICI response rates and OS. These results suggest that tissue-specific microenvironments of AGMs present unique challenges that may require novel, adrenal gland–directed therapies or surgical resection.

BackgroundAdjuvant therapy for high-risk resected melanoma with programmed cell-death 1 blockade results in a median relapse-free survival (RFS) of 5 years. The addition of low dose ipilimumab (IPI) to a regimen of adjuvant nivolumab (NIVO) in CheckMate-915 did not result in increased RFS. A pilot phase II adjuvant study of either standard dose or low dose IPI with NIVO was conducted at two centers to evaluate RFS with correlative biomarker studies.MethodsPatients with resected stages IIIB/IIIC/IV melanoma received either IPI 3 mg/kg and NIVO 1 mg/kg (cohort 4) or IPI 1 mg/kg and NIVO 3 mg/kg (cohorts 5 and 6) induction therapy every 3 weeks for 12 weeks, followed by maintenance NIVO. In an amalgamated subset of patients across cohorts, peripheral T cells at baseline and on-treatment were assessed by flow cytometry and RNA sequencing for exploratory biomarkers.ResultsHigh rates of grade 3–4 adverse events precluded completion of induction therapy in 50%, 35% and 7% of the patients in cohorts 4, 5 and 6, respectively. At a median of 63.9 months of follow-up, 16/56 patients (29%) relapsed. For all patients, at 5 years, RFS was 71% (95% CI: 60 to 84), and overall survival was 94% (95% CI: 88 to 100). Expansion of CD3+CD4+CD38+CD127−GARP− T cells, an on-treatment increase in CD39 expression in CD8+ T cells, and T-cell expression of phosphorylated signal-transducer-and-activator-of-transcription (STAT)2 and STAT5 were associated with relapse.ConclusionsAdjuvant IPI/NIVO at the induction doses used resulted in promising relapse-free and overall survival, although with a high rate of grade 3–4 adverse events. Biomarker analyses highlight an association of ectoenzyme-expressing T cells and STAT signaling pathways with relapse, warranting future validation.Trial registration numberNCT01176474andNCT02970981.

MotivationHigh-dimension cytometry assays can simultaneously measure dozens of markers, enabling the investigation of complex phenotypes. However, as manual gating relies on previous biological knowledge, few marker combinations are often assessed. This results in complex phenotypes with potential for biological relevance being overlooked. Here we present PhenoComb, an R package that allows agnostic exploration of phenotypes by assessing all combinations of markers.DesignPhenoComb uses signal intensity thresholds to assign markers to discrete states (e.g. negative, low, high) and then counts the number of cells per sample from all possible marker combinations in a memory-safe manner. Time and disk space are the only constraints on the number of markers evaluated. PhenoComb also provides several approaches to perform statistical comparisons, evaluate the relevance of phenotypes, and assess the independence of identified phenotypes. PhenoComb allows users to guide analysis by adjusting several function arguments such as identifying parent populations of interest, filtering of low-frequency populations, and defining a maximum complexity of phenotypes to evaluate. We have designed PhenoComb to be compatible with local computer or server-based use.ResultsIn testing of PhenoComb’s performance on synthetic datasets, computation on 16 markers was completed in the scale of minutes and up to 26 markers in hours. We applied PhenoComb to two publicly available datasets: an HIV flow cytometry dataset (12 markers and 421 samples) and the COVIDome CyTOF dataset (40 markers and 99 samples). In the HIV dataset, PhenoComb identified immune phenotypes associated with HIV seroconversion, including those highlighted in the original publication. In the COVID dataset, we identified several immune phenotypes with altered frequencies in infected individuals relative to healthy individuals. Collectively, PhenoComb represents a powerful discovery tool for agnostically assessing high-dimension, single-cell data.AvailabilityThe PhenoComb R package can be downloaded from https://github.com/SciOmicsLab/PhenoComb

Objectives While much of the research concerning factors associated with responses to immune checkpoint inhibitors (ICIs) has focussed on the contributions of conventional peptide‐specific T cells, the role of unconventional T cells, such as mucosal‐associated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are an abundant population of innate‐like T cells expressing a semi‐invariant T‐cell receptor restricted to the MHC class I‐like molecule, MR1, presenting vitamin B metabolites derived from bacteria. We sought to characterise MAIT cells in melanoma patients and determined their association with treatment responses and clinical outcomes. Methods In this prospective clinical study, we analysed the frequency and functional profile of circulating and tumor‐infiltrating MAIT cells in human melanoma patients. Using flow cytometry, we compared these across metastatic sites and between ICI responders vs. non‐responders as well as healthy donors. Results We identified tumor‐infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients compared to healthy donors. However, circulating MAIT cell frequencies are restored by ICI treatment in responding patients, correlating with treatment responses, in which patients with high frequencies of MAIT cells exhibited significantly improved overall survival. Conclusion Our results suggest that MAIT cells may be a potential predictive marker of responses to immunotherapies and provide rationale for testing MAIT cell‐directed therapies in combination with current and next‐generation ICIs., While the number of circulating MAIT cells is reduced in melanoma patients, circulating MAIT cell frequency is restored by immune checkpoint inhibitor treatment in responding patients, correlating with treatment responses, in which patients with high frequencies of MAIT cells exhibited significantly improved overall survival.

Motivation High-dimensional cytometry assays can simultaneously measure dozens of markers, enabling the investigation of complex phenotypes. However, as manual gating relies on previous biological knowledge, few marker combinations are often assessed. This results in complex phenotypes with the potential for biological relevance being overlooked. Here, we present PhenoComb, an R package that allows agnostic exploration of phenotypes by assessing all combinations of markers. PhenoComb uses signal intensity thresholds to assign markers to discrete states (e.g. negative, low, high) and then counts the number of cells per sample from all possible marker combinations in a memory-safe manner. Time and disk space are the only constraints on the number of markers evaluated. PhenoComb also provides several approaches to perform statistical comparisons, evaluate the relevance of phenotypes and assess the independence of identified phenotypes. PhenoComb allows users to guide analysis by adjusting several function arguments, such as identifying parent populations of interest, filtering of low-frequency populations and defining a maximum complexity of phenotypes to evaluate. We have designed PhenoComb to be compatible with a local computer or server-based use. Results In testing of PhenoComb’s performance on synthetic datasets, computation on 16 markers was completed in the scale of minutes and up to 26 markers in hours. We applied PhenoComb to two publicly available datasets: an HIV flow cytometry dataset (12 markers and 421 samples) and the COVIDome CyTOF dataset (40 markers and 99 samples). In the HIV dataset, PhenoComb identified immune phenotypes associated with HIV seroconversion, including those highlighted in the original publication. In the COVID dataset, we identified several immune phenotypes with altered frequencies in infected individuals relative to healthy individuals. Collectively, PhenoComb represents a powerful discovery tool for agnostically assessing high-dimensional single-cell data. Availability and implementation The PhenoComb R package can be downloaded from https://github.com/SciOmicsLab/PhenoComb. Supplementary information Supplementary data are available at Bioinformatics Advances online.

Uveal melanoma (UM) is a subtype of melanoma. Although they share a melanocytic origin with cutaneous melanoma (CM), patients with UM have few treatment options. BCL2 homologous 3 mimetics are small-molecule drugs that mimic proapoptotic BCL2 family members. We compared BCL2 family member expression between UM and CM using immunoblot and The Cancer Genome Atlas transcriptomic analysis. UM has a unique signature of low BFL1 and high PUMA proteins compared with CM and 30 other cancer types, making them an attractive candidate for BCL2 homologous 3 protein mimetics. We tested the efficacy of a BCL2 inhibitor and MCL1 inhibitor (MCL1i) in UM, with viability assays, live-cell imaging, sphere assays, and mouse xenograft models. UM had a higher sensitivity to MCL1i than CM. Overexpression of BFL1 or knockdown of PUMA made the UM more resistant to MCL1i. In contrast, MAPK/extracellular signal‒regulated kinase inhibitor treatment in CM made them more sensitive to MCL1i. However, MCL1i-alone treatment was not very effective to reduce the UM initiating cells; to overcome this, we employed a combination of MCL1i with BCL2 inhibitor that synergistically inhibited UM initiating cell's capacity to expand. Overall, we identify a distinct expression profile of BCL2 family members for UM that makes them susceptible to BCL2 homologous 3 mimetics.

Immune suppression is one of the 10 hallmarks of cancer. Interleukin-37 (IL-37), a member of the IL-1 family, inhibits both innate and adaptive immunity, and has been shown to modulate immune responses in various disease conditions. Yet, IL-37 has rarely been investigated in cancer patients, and its biological role in cancer remains to be elucidated. In this study, we investigated the gene expression of IL-37 in age- and sex-matched blood samples of healthy individuals and melanoma patients, and demonstrated upregulation of IL-37 messenger RNA (mRNA) in the blood samples of melanoma patients. By further analyzing immune cell subsets responsible for the upregulated IL-37 expression, we discovered that IL-37 mRNA was highly expressed in T cells and granulocytes, with the highest expression in regulatory T (Treg ) cells in healthy individuals, and that IL-37 mRNA was upregulated in lymphocytes (T, B, and natural killer cells) in melanoma patient blood. Among all cell subsets, Treg cells from melanoma patients exhibited the highest IL-37 gene expression levels. We provided evidence that melanoma-conditioned media induces IL-37 mRNA and protein expression in multiple lymphocyte populations, particularly in Treg cells. We further confirmed that the IL-1-mediated secretome from human melanoma cells, specifically transforming growth factor-β, induces IL-37 mRNA expression in human Treg cells. Our results suggest a potential immunosuppressive role for IL-1 and IL-37 in melanoma tumorigenesis. Highly elevated IL-37 in specific lymphocyte populations could serve as a biomarker for tumor-induced immunosuppression.

Although treatment options for melanoma patients have expanded in recent years with the approval of immunotherapy and targeted therapy, there is still an unmet need for new treatment options for patients that are ineligible for, or resistant to these therapies. BH3 mimetics, drugs that mimic the activity of pro-apoptotic BCL2 family proteins, have recently achieved remarkable success in the clinical setting. The combination of BH3 mimetic ABT-199 (venetoclax) plus azacitidine has shown substantial benefit in treating acute myelogenous leukemia. We evaluated the efficacy of various combinations of BH3 mimetic + azacitidine in fourteen human melanoma cell lines from cutaneous, mucosal, acral and uveal subtypes. Using a combination of cell viability assay, BCL2 family knockdown cell lines, live cell imaging, and sphere formation assay, we found that combining inhibition of MCL1, an anti-apoptotic BCL2 protein, with azacitidine had substantial pro-apoptotic effects in multiple melanoma cell lines. Specifically, this combination reduced cell viability, proliferation, sphere formation, and induced apoptosis. In addition, this combination is highly effective at reducing cell viability in rare mucosal and uveal subtypes. Overall, our data suggest this combination as a promising therapeutic option for some patients with melanoma and should be further explored in clinical trials.

There is a clear need to identify targetable drivers of resistance and potential biomarkers for salvage therapy for patients with melanoma refractory to the combination of BRAF and MEK inhibition. In this study, we performed whole-exome sequencing on BRAF-V600E–mutant melanoma patient tumors refractory to the combination of BRAF/MEK inhibition and identified acquired oncogenic mutations in NRAS and loss of the tumor suppressor gene CDKN2A. We hypothesized the acquired resistance mechanisms to BRAF/MEK inhibition were reactivation of the MAPK pathway and activation of the cell-cycle pathway, which can both be targeted pharmacologically with the combination of a MEK inhibitor (trametinib) and a CDK4/6 inhibitor (palbociclib). In vivo, we found that combination of CDK4/6 and MEK inhibition significantly decreased tumor growth in two BRAF/MEK inhibitor–resistant patient-derived xenograft models. In vitro, we observed that the combination of CDK4/6 and MEK inhibition resulted in synergy and significantly reduced cellular growth, promoted cell-cycle arrest, and effectively inhibited downstream signaling of MAPK and cell-cycle pathways in BRAF inhibitor–resistant cell lines. Knockdown of CDKN2A in BRAF inhibitor–resistant cells increased sensitivity to CDK4/6 inhibition alone and in combination with MEK inhibition. A key implication of our study is that the combination of CDK4/6 and MEK inhibitors overcomes acquired resistance to BRAF/MEK inhibitors, and loss of CDKN2A may represent a biomarker of response to the combination. Inhibition of the cell-cycle and MAPK pathway represents a promising strategy for patients with metastatic melanoma who are refractory to BRAF/MEK inhibitor therapy.

Subungual melanomas (SUM) arise beneath the nails of the hands and feet, and account for 0.7-3.5% of all malignant melanomas. Most studies include SUM in the category of acral melanoma, but understanding the specific features of SUM is critical for improving patient care. In this study, we performed a site-specific comparison of the clinical and molecular features between 54 cases of SUM and 78 cases of nonsubungual acral melanoma. Compared to patients with acral melanoma, patients with SUM were younger at diagnosis, had a higher prevalence of primary melanomas on the hand, and had more frequent reports of previous trauma at the tumor site. SUM was deeper than acral melanoma at diagnosis, which correlated with an increased frequency of metastases. Analysis of common melanoma driver genes revealed KIT and KRAS mutations were predominantly found in SUM, whereas BRAF and NRAS mutations occurred almost exclusively in acral melanoma. We also discovered molecular differences in the cell cycle pathway, where CDK4/CCND1 amplifications were more frequent in SUM and CDKN2A/B loss occurred mostly in acral melanoma, and in the PI3K/mTOR pathway, where RICTOR amplification and TSC1 K587R mutations were exclusively in SUM and PTEN loss and AKT1 mutations were exclusively in acral melanoma. Comparison of hand versus foot tumors revealed more frequent ulceration of SUM foot tumors, which correlated with more distal metastases and poorer overall survival. In summary, we find SUM are both clinically and molecularly distinct from acral melanoma, and our data suggest KIT, CDK4/6, and mTOR inhibitors may be particularly relevant and effective treatments for patients with SUM.

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 are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are 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 of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options., Acral melanoma occurs on the soles of the feet, palms of the hands and in nail beds. Here, the authors reports the genomic landscape of 87 acral melanomas and find that some tumors harbor a UV signature and that the tumors are diverse at the levels of mutational signatures, structural aberrations and copy number signatures.

While much of the research concerning factors associated with responses to immunotherapies focuses on the contributions of conventional peptide-specific T cells, the role of unconventional T cells, such as mucosalassociated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are innate-like T cells expressing a semi-invariant T cell receptor restricted to the non-classical MHC class I molecule MR1 presenting vitamin metabolites derived from bacteria. In this prospective clinical study, we sought to characterize MAIT cells in melanoma patients and determine their association with clinical outcomes. We identified tumor-infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients. However, circulating MAIT cell frequency is restored by anti-PD1 treatment in responding patients, correlating with treatment responses in which patients with high frequencies of MAIT cells exhibited improved overall survival. These data provide evidence for leveraging MAIT cells and their functions as novel targets for future therapies.

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.

Current treatment for patients with metastatic melanoma include molecular-targeted therapies and immune checkpoint inhibitors. However, a subset of melanomas are difficult-to-treat. These melanomas include those without the genetic markers for targeted therapy, non-responsive to immunotherapy, and those who have relapsed or exhausted their therapeutic options. Therefore, it is necessary to understand and explore other biological processes that may provide new therapeutic approaches. One of most appealing is targeting the apoptotic/anti-apoptotic system that is effective against leukemia. We used genetic knockdown and pharmacologic approaches of BH3 mimetics to target anti-apoptotic BCL2 family members and identified MCL1 and BCLXL as crucial pro-survival members in melanoma. We then examined the effects of combining BH3 mimetics to target MCL1 and BCLXL in vitro and in vivo. These include clinical-trial-ready compounds such as ABT-263 (Navitoclax) and S63845/S64315 (MIK655). We used cell lines derived from patients with difficult-to-treat melanomas. In vitro, the combined inhibition of MCL1 and BCLXL resulted in significantly effective cell killing compared to single-agent treatment (p p p > 0.40). Taken together, this study suggests that dual targeting of MCL1 and BCLXL should be considered as a treatment option for difficult-to-treat melanoma patients.

Despite the recent advancement in treating melanoma, options are still limited for patients without BRAF mutations or in relapse from current treatments. BH3 mimetics against members of the BCL-2 family have gained excitement with the recent success in hematological malignancies. However, single drug BH3 mimetic therapy in melanoma has limited effectiveness due to escape by the anti-apoptotic protein MCL-1 and/or survival of melanoma-initiating cells (MICs). We tested the efficacy of the BH3 mimetic combination of A-1210477 (an MCL-1 inhibitor) and ABT-263 (a BCL-2/BCL-XL/BCL-W inhibitor) in killing melanoma, especially MICs. We also sought to better define Dynamin-Related Protein 1 (DRP-1)’s role in melanoma; DRP-1 is known to interact with members of the BCL-2 family and is a possible therapeutic target for melanoma treatment. We used multiple assays (cell viability, apoptosis, bright field, immunoblot, and sphere formation), as well as the CRISPR/Cas9 genome-editing techniques. For clinical relevance, we employed patient samples of different mutation status, including some relapsed from current treatments such as anti-PD-1 immunotherapy. We found the BH3 mimetic combination kill both the MICs and non-MICs (bulk of melanoma) in all cell lines and patient samples irrespective of the mutation status or relapsed state (p p

With the advancement of next generation sequencing technology, researchers are now able to identify important variants and structural changes in DNA and RNA in cancer patient samples. With this information, we can now correlate specific variants and/or structural changes with actionable therapeutics known to inhibit these variants. We introduce the creation of the IMPACT Web Portal, a new online resource that connects molecular profiles of tumors to approved drugs, investigational therapeutics and pharmacogenetics associated drugs. IMPACT Web Portal contains a total of 776 drugs connected to 1326 target genes and 435 target variants, fusion, and copy number alterations. The online IMPACT Web Portal allows users to search for various genetic alterations and connects them to three levels of actionable therapeutics. The results are categorized into 3 levels: Level 1 contains approved drugs separated into two groups; Level 1A contains approved drugs with variant specific information while Level 1B contains approved drugs with gene level information. Level 2 contains drugs currently in oncology clinical trials. Level 3 provides pharmacogenetic associations between approved drugs and genes. IMPACT Web Portal allows for sequencing data to be linked to actionable therapeutics for translational and drug repurposing research. The IMPACT Web Portal online resource allows users to query genes and variants to approved and investigational drugs. We envision that this resource will be a valuable database for personalized medicine and drug repurposing. IMPACT Web Portal is freely available for non-commercial use at http://tanlab.ucdenver.edu/IMPACT .

Oncogenic ALK fusions occur in several types of cancer and can be effectively treated with ALK inhibitors; however, ALK fusions and treatment response have not been characterized in malignant melanomas. Recently, a novel isoform of ALK (ALKATI) was reported in 11% of melanomas but the response of melanomas expressing ALKATI to ALK inhibition has not been well characterized. We analyzed 45 melanoma patient-derived xenograft models for ALK mRNA and protein expression. ALK expression was identified in 11 of 45 (24.4%) melanomas. Ten melanomas express wild-type (wt) ALK and/or ALKATI and one mucosal melanoma expresses multiple novel EML4-ALK fusion variants. Melanoma cells expressing different ALK variants were tested for response to ALK inhibitors. Whereas the melanoma expressing EML4-ALK were sensitive to ALK inhibitors in vitro and in vivo, the melanomas expressing wt ALK or ALKATI were not sensitive to ALK inhibitors. In addition, a patient with mucosal melanoma expressing ALKATI was treated with an ALK/ROS1/TRK inhibitor (entrectinib) on a phase I trial but did not respond. Our results demonstrate ALK fusions occur in malignant melanomas and respond to targeted therapy, whereas melanomas expressing ALKATI do not respond to ALK inhibitors. Targeting ALK fusions is an effective therapeutic option for a subset of melanoma patients, but additional clinical studies are needed to determine the efficacy of targeted therapies in melanomas expressing wt ALK or ALKATI. Mol Cancer Ther; 17(1); 222–31. ©2017 AACR.

Dielectric spectroscopy is conducted in terahertz frequency range to differentiate human normal tissues from the cancerous ones. Due to sensitivity of terahertz waves on water content of th...

Genomic rearrangements resulting in activating kinase fusions have been increasingly described in a number of cancers including malignant melanoma, but their frequency in specific melanoma subtypes has not been reported. We used break-apart fluorescence in situ hybridization (FISH) to identify genomic rearrangements in tissues from 59 patients with various types of malignant melanoma including acral lentiginous, mucosal, superficial spreading, and nodular. We identified four genomic rearrangements involving the genes BRAF, RET, and ROS1. Of these, three were confirmed by Immunohistochemistry (IHC) or sequencing and one was found to be an ARMC10-BRAF fusion that has not been previously reported in melanoma. These fusions occurred in different subtypes of melanoma but all in tumors lacking known driver mutations. Our data suggest gene fusions are more common than previously thought and should be further explored particularly in melanomas lacking known driver mutations.

Major limitations of current melanoma treatments are for instances of relapse and the lack of therapeutic options for BRAF wild-type patients who do not respond to immunotherapy. Many studies therefore focus on killing resistant subpopulations, such as Melanoma Initiating Cells (MICs) to prevent relapse. Here we examined whether combining a GSI (γ-Secretase Inhibitor) with ABT-737 (a small molecule BCL-2/BCL-XL/BCL-W inhibitor) can kill both the non-MICs (bulk of melanoma) and MICs. To address the limitations of melanoma therapies, we included multiple tumor samples of patients relapsed from current treatments, with a diverse genetic background (with or without the common BRAF, NRAS or NF1 mutations) in these studies. Excitingly, the combination treatment reduced cell viability and induced apoptosis of the non-MICs; disrupted primary spheres, decreased the ALDH+ cells, and inhibited the self-renewability of the MICs in multiple melanoma cell lines and relapsed patient samples. Using a low-cell-number mouse xenograft model, we demonstrated that the combination significantly reduced the tumor initiating ability of MIC-enriched cultures from relapsed patient samples. Mechanistic studies also indicate that cell death is NOXA-dependent. In summary, this combination may be a promising strategy to address treatment relapse and for triple wild-type patients who do not respond to immunotherapy.

Subungual melanoma (SUM) is a type of malignant melanoma that arises beneath the nails of the hands and feet. Subungual melanomas represent a small subset of melanoma patients and have not been well characterized by current molecular techniques. In most studies they have been included in the category of acral melanoma (AM), but there are numerous reasons to believe that they may be different. New effective melanoma treatments rely on both clinical and molecular data to select therapy. In this study, we reviewed characteristics in 29 patients with subungual melanomas seen at our institution in an attempt to provide better understanding and treatment. The median age was 56, almost equally distributed between males and females, with a higher percentage of non-Caucasians than seen in cutaneous melanoma. The most common sites were the thumb (28%), big toe (21%), and index finger (17%). Most were deep at diagnosis (mean Breslow depth 4.21 mm), and 72% had, or developed, metastases and responded poorly to therapy of all kinds. In 12 of the 29 cases, molecular testing via targeted sequencing through Foundation Medicine was done on either a primary or metastatic lesion. These 12 cases were compared to 20 cases of AM seen at our institution. One BRAF mutation was found (1/12, 4%) in SUM, whereas BRAF mutations occurred in about 20% of patients with AM. No patients with SUM had an NRAS mutation, compared to a 45% mutation rate in AM. Two SUM patients had KRAS mutations (2/12, 17%), whereas no patients with AM had a KRAS mutation. Three SUM patients had mutations in KIT (3/12, 25%) compared to one (1/20, 5%) in AM. In addition, 3 patient tumors had a recurrent variant (K587R) in the tuberous sclerosis related gene TSC-1, which has not been described in patients with AM. While our profile of subungual melanoma has some similarities with non-subungual acral melanomas, the high number of KRAS, KIT and TSC1 mutations and lack of BRAF and NRAS mutations suggests that these melanoma may have unique characteristics, different causation, and need specific therapy different from acral melanomas. Citation Format: Blair N. Holman, Robert J. Van Gulick, Carol Amato, Kasey Couts, William A. Robinson. Subungual melanoma: It may be a distinct entity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5431.

Treatment options for melanoma have progressed in recent years, with the rise of targeted therapy and immunotherapy. Options are still limited for patients that are BRAF wildtype, treatment refractory, and patients with non-sun exposed melanoma subtypes that typically do not respond to standard therapies. Therefore, it is necessary to explore other biological processes that may provide new therapeutic approaches. One of most appealing is the BCL-2 family of apoptotic/anti-apoptotic system that is effective in leukemias, with BH3 mimetics venetoclax alone or in combination with the hypomethylating agent 5-azacitidine. We examined similar combinations on the group of eleven patient-derived and commercially available melanoma cell lines. The BH3 mimetics we tested include MCL1 inhibitors (S63845, AMG 176, AZD5991), an inhibitor of BCL2, BCLXL, and BCLW (navitoclax/ABT-263), a BCL2 inhibitor (venotoclax/ABT-199), and a BCLXL inhibitor (A1331852). Our data showed that treatments with MCL1 inhibitors plus 5-azacitidine are effective in reducing melanoma cell viability. S63845 was the most potent compound when combined with 5-azacitidine, with nine out of eleven (~80%) melanoma cell lines achieving less than 50% viability at the highest dose. There was an overall weaker response to treatment with 5-azacitidine in combination with ABT-263 or A1331852, with only 54% and 45% of cell lines responding at the highest dose, respectively. Response to ABT-199 with 5-Azacitidine was highly limited, 18% of cell lines responded at the highest concentration. Our data demonstrate that treatment with S63845 in combination with 5-azacitidine is highly effective in cell lines from a variety of melanoma subtypes, including the rare uveal and mucosal subtypes. In vivo and mechanistic experiments are planned. The novel combination of the MCL1 inhibitor S63845 and 5-azacitidine may be a promising alternative therapeutic target in melanoma. Citation Format: Chiara Dart, Nabanita Mukherjee, Carol Amato, Robert J. Van Gulick, Morgan MacBeth, Kasey Couts, Jacqueline Turner, Stacey Bagby, Mayumi Fujita, William Robinson, Yiqun Shellman. The therapeutic potential of 5-Azacitidine plus various BH3 mimetics in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1805.

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.

The RET proto-oncogene encodes a receptor tyrosine kinase that is activated by glial cell derived neutrotrophic factor (GDNF). Previous studies have found that a single nucleotide polymorphism (SNP), RETp (G691S), in the juxtamembrane domain enhances the signaling pathway and promotes tumor growth by GDNF in pancreatic and thyroid cancer in addition to melanoma. It is uncertain however whether this SNP is a germline variant or somatic mutation. A prior study reported that the RETp variant was a germline SNP in desmoplastic and non-desmoplastic melanomas. In the present study, we examined both melanoma tissue samples and matching peripheral blood DNA to determine if RETp was 1) a germline or somatic variant, 2) more frequent in certain melanoma subtypes, and 3) frequency in brain metastasis. We examined the peripheral blood of 197 melanoma patients whom had at least one matched tumor, and 42 patients with brain metastasis. RETp was present as a germline SNP in 33% of patients. There were no significant differences in RETp frequency among the different melanoma subtypes, and RETp was not correlated with brain metastasis.

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P

A majority of cutaneous melanomas harboring oncogenic driver BRAF-V600E gene mutations initially demonstrate superior responses to targeted therapy with BRAF inhibitors (BRAFi), MEK inhibitors (MEKi), or the combination of both. Unfortunately, tumor regrowth and acquired resistance to BRAF and MEK inhibitors is inevitable, and new therapies are required to treat these patients. To determine acquired resistance mechanisms, we performed Whole Exome Sequencing (WES) on BRAF-V600 mutant cutaneous melanomas from two patients each at two timepoints; (1) prior to BRAFi/MEKi combination therapy, and (2) upon tumor regrowth following therapy. Following BRAFi/MEKi resistance, both patients acquired an activating mutation in NRAS and loss of the tumor suppressor CDKN2A. We hypothesized the acquired mechanism of resistance was through reactivation of MAPK pathway, via NRAS mutations, and activation of the cell cycle pathway, via loss of CDKN2A. Further, we predicted that these resistant patents samples would be sensitive to the combination of inhibiting the MAPK and cell cycle pathway. Both BRAFi/MEKi resistant patient samples were established as patient-derived xenograft (PDX) models to test the in vivo efficacy of trametinib (MEKi) and palbociclib (CDK4/6i) as single agents and in combination. In both PDX models, the combination of MEKi and CDK4/6i resulted in significant tumor reduction as compared to the vehicle control and single agents. Using immunohistochemistry, we observed that only in the combination treated tumors there was a significant decrease in tumor proliferation, measured by Ki-67, and MAPK pathway activity, measured by phos-ERK. To study the mechanism of response to the combination of MEKi and CDK4/6i in the context of BRAFi acquired resistance, we generated an in vitro resistance model using a panel of BRAF-V600 mutant cutaneous melanoma cell lines constantly treated with the BRAF inhibitor, vemurafenib, to generate a resistant cell population. We observed that parental cells are more sensitive to single agent MEKi, and more resistant to single agent CDK4/6i, as compared to BRAFi resistant cells. In line with this, we found that MEKi in parental cells decreased both phos-ERK and phos-RB, suggesting that the parental cell lines are dependent on the MAPK pathway, and that the cell cycle pathway is under MAPK pathway regulation. However, in BRAFi resistant lines, MEKi only slightly reduced phos-ERK, and did not significantly decrease phos-RB levels. Suggesting that, in BRAFi resistant lines, the cell cycle pathway is not regulated by the MAPK pathway. Decreased phos-ERK and phos-RB levels are only observed with the combination of MEKi and CDK4/6i. Furthermore, we found that the BRAFi resistant cells are more sensitive to a low concentration of CDK4/6i in combination with MEKi, as compared to the parental cells. We found that parental cells required a higher concentrations of CDK4/6i in combination with MEKi to reduce cell proliferation. Whereas, the BRAFi resistance cells are the opposite and require a lower concentration of CDK4/6i, and a higher concentration of MEKi, to have a durable response. Taken together, this data supports the observation that upon BRAFi resistance, the cell cycle pathway is uncoupled from the MAPK pathway, and cell cycle reactivation may represent a common acquired resistance mechanism to MEKi. Citation Format: Kelsey W. Nassar, Jennifer Hintzsche, Stacey Bagby, Christophe Langouet-Astrie, Jenny Samson, Carol Amato, Nicholas Gordon, Mayumi Fujita, Rebecca Schweppe, William Robinson, Aik-Choon Tan. Uncoupling of the cell cycle pathway from the MAPK pathway in BRAF/MEK inhibitor resistant melanoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B076. doi:10.1158/1535-7163.TARG-19-B076

Immune checkpoint blockade (ICB) treatments, such as anti-PD1, improves survival in patients with metastatic melanoma. Despite durable response rates, 40-50% of patients show no benefit. Implicated in acquired resistance is the downregulation of antigen presentation, via dysfunction of the interferon-receptor pathway. However, little is known about intrinsic response mechanisms to ICB. The NF-κB-pathway is important for immune response as a key transcription factor for several antigen presentation proteins and cytokine/chemokine production. This study explored NF-κB signaling in patients with favorable, or unfavorable responses to anti-PD1 therapy. Fifty-two patients with metastatic melanoma were grouped into complete or partial responders (CR/PR; n = 6 and 15, respectively), and stable or progressive disease (SD/PD; n = 16 and 15, respectively). Consistent with other studies, tumor mutational burden assessed by whole exome sequencing (WES) was higher in the CR/PR group (p = 0.03). WES analysis also showed an enrichment of variants in NF-κB-pathway related genes within the CR/PR group (14/21, 67%) compared with SD/PD patients (6/31, 19%), p = 0.001. Notably, the hotspot variants G34E/R and G41E in NFKBIE, a negative regulator of NF-κB, were found exclusively in patients with complete or partial response to ICB (5/21, 24%). RNA-seq analysis of 19 tumors showed that the TNFα/NF-κB signaling pathway is upregulated in the CR/PR group, and highly expressed genes included CD83, HLA-DQB, and NFATC1. Therefore we hypothesized that when activated, particularly within the melanoma cells, this pathway contributes to an anti-tumor immune response. Using HEK293 cells, we demonstrated NFKBIEG34E increased NF-κB activity compared with the wildtype, upon TNFα stimulation. Consistently, flow cytometry also show an increase of targets of NF-κB transcription such as CD83, MHC class II, and PDL1. Similarly, the melanoma cell line A375, which harbors the NFKBIEG34E mutation, showed increases in protein expression when stimulated with TNFα and INFγ. Furthermore, progression free survival increased in patients that had tumors with an NF-κB-pathway variant compared with patients that were pathway wildtype (p = 0.03). Patients harboring tumors with NF-κB-pathway variants also had a greater response by RECIST criteria compared with wildtype. Taken together, our results suggest that an increase of pro-immunogenic NF-κB signaling in the tumor may contribute to the favorable response to ICB, highlighting a role for CD83 in the tumor microenvironment that merits further examination. Citation Format: Keith Wells, Jennifer Hintzsche, Carol M. Amato, Richard Tobin, Victoria Vorwald, Martin McCarter, Yiqun Shellman, Aik Choon Tan, William Robinson. Investigating the role of NF- κB signaling and immune checkpoint blockade therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5002.

131 Background: Immune-related adverse events (IRAE) occur commonly with immune checkpoint inhibitor therapy for the treatment of cancer, although the specific event and severity can vary widely. Little is known regarding factors that may predict which patients will develop an IRAE. The goal of this study is to identify blood biomarkers predictive of IRAE associated with immune checkpoint inhibitor therapy. Methods: Blood samples collected from patients with melanoma prior to receiving therapy with immune checkpoint inhibitors were obtained from the University of Colorado Skin Cancer Biorepository. Testing for a panel of autoantibodies and cytokines (ANA, CCP 3.1, IL-1 beta, IL-2, IL-6, IL-10, IL-12, IP-10, MCP-1, TNF alpha, IFN alpha 2, IFN gamma) in serum samples from patients who had at least one documented IRAE was performed by Exsera BioLabs. Descriptive statistics were used to evaluate biomarker levels in relation to type, grade, and number of adverse events. Results: Pre-treatment samples from 45 patients were evaluated. Median age was 55; 26 were male and 19 were female. The most common IRAEs were colitis (n = 22), thyroid dysfunction (n = 21), and dermatitis (n = 20). Most IRAEs were grade 2 in severity, and the majority of patients (n = 36) experienced more than 1 IRAE. TNF alpha was elevated in 60% of patient samples, while IFN alpha 2 was elevated in 44%. Borderline ANA was detected in 27% of samples and ANA was positive in 11%. No samples had elevation of IL-2. Between 9% and 18% of samples had elevation of the other immune markers tested (IFN gamma, IL-1 beta, IL-6, IL-10, IL-12, and CCP 3.1). Elevation of TNF alpha and IFN alpha 2 were associated with higher grades of IRAEs. No associations between immune markers and the number or type of adverse events in an individual patient were noted. Results from 15 patients who did not have a documented IRAE on immune checkpoint inhibitor therapy are currently pending to confirm these findings are unique to patients developing IRAE. Conclusions: This preliminary data suggests that baseline elevations of TNF alpha and IFN alpha 2 may predict development of IRAEs with immune checkpoint inhibitor therapy. Results from samples from patients who did not develop an IRAE on therapy will be reported at the meeting.

Recovery of microRNAs (miRs) from tissue samples of many kinds has become an area of increasing interest and importance in recent years. We isolated and then amplified and quantitated miRs from human peripheral blood samples stored in the University of Colorado Denver Skin Cancer Biorepository to determine whether miR recovery was possible and consistent over time in storage. Forty-five blood samples from patients with different stages of malignant melanoma were collected in PAXgene Blood RNA tubes and then stored at -80°C prior to RNA preparation. The samples examined had been stored from 4 weeks to 3 years. Total RNA was prepared, followed by miR isolation, amplification, and quantitation using real-time polymerase chain reaction. A widely expressed miR, miR-221, was used as a standard for comparison across samples and storage time. miR-221 was recovered from all samples, with no differences observed with longer storage time. These studies show that miRs can be recovered and quantified from human blood samples stored for up to 3 years.

Cyclin D1 (CCND1) is a well-known regulator of cell-cycle progression. It is overexpressed in several types of cancer including breast, lung, squamous, neuroblastoma, and lymphomas. The most well-known mechanism of overexpression is the t(11;14)(q13;q32) translocation found in mantle cell lymphoma (MCL). It has previously been shown that truncated CCND1 mRNA in MCL correlates with poor prognosis. We hypothesized that truncations of the CCND1 mRNA alter its ability to be down-regulated by microRNAs in MCL. MicroRNAs are a new class of abundant small RNAs that play important regulatory roles at the posttranscriptional level by binding to the 3′ untranslated region (UTR) of mRNAs blocking either their translation or initiating their degradation. In this study, we have identified the truncation in CCND1 mRNA in MCL cell lines. We also found that truncated CCND1 mRNA leads to increased CCND1 protein expression and increased S-phase cell fraction. Furthermore, we demonstrated that this truncation alters miR-16-1 binding sites, and through the use of reporter constructs, we were able to show that miR-16-1 regulates CCND1 mRNA expression. This study introduces the role of miR-16-1 in the regulation of CCND1 in MCL.

Clear cell sarcoma (CCS) is a rare tumor classically associated with the tendons and aponeuroses of distal extremities of young adults. CCS and malignant melanoma (MM) share immunohistochemical profiles and ultrastructural features, but classic CCS has characteristic morphology with low mitotic activity and minimal pleomorphism. Occasional cases show pleomorphism, high mitotic index, and/or melanin pigmentation, making CCS indistinguishable from MM based on morphology. However, CCS is genetically distinct owing to its consistent association with a t(12;22)(q13;q12) chromosomal translocation, leading to the formation of the EWS/ATF1 fusion transcript. This translocation has never been documented in cutaneous melanoma, and thus is regarded as specific for CCS. Recent evidence suggests that primary "malignant melanomas" in unusual anatomic sites, most notably the gastrointestinal (GI) tract, may be CCS. This is supported by 11 cases of primary GI CCS with the t(12;22) translocation. We used reverse-transcription polymerase chain reaction and fluorescence in situ hybridization to examine whether a proportion of cases diagnosed as MM of the GI tract in patients without a history of cutaneous MM actually represent primary GI CCS. In total, we examined 7 cases: Four with no prior history of MM, 2 with histories of cutaneous MM, and 1 with an anal MM. All 4 cases for which there was no history of cutaneous/mucosal MM harbored the EWS/ATF1 fusion transcript. We report the largest series of GI CCS and have shown that molecular studies may be warranted in cases that otherwise seem to represent MM of unusual primary locations.

Objective Currently, there is a disconnect between finding a patient’s relevant molecular profile and predicting actionable therapeutics. Here we develop and implement the Integrating Molecular Profiles with Actionable Therapeutics (IMPACT) analysis pipeline, linking variants detected from whole-exome sequencing (WES) to actionable therapeutics.Methods and materials The IMPACT pipeline contains 4 analytical modules: detecting somatic variants, calling copy number alterations, predicting drugs against deleterious variants, and analyzing tumor heterogeneity. We tested the IMPACT pipeline on whole-exome sequencing data in The Cancer Genome Atlas (TCGA) lung adenocarcinoma samples with known EGFR mutations. We also used IMPACT to analyze melanoma patient tumor samples before treatment, after BRAF-inhibitor treatment, and after BRAF- and MEK-inhibitor treatment.Results IMPACT Food and Drug Administration (FDA) correctly identified known EGFR mutations in the TCGA lung adenocarcinoma samples. IMPACT linked these EGFR mutations to the appropriate FDA-approved EGFR inhibitors. For the melanoma patient samples, we identified NRAS p.Q61K as an acquired resistance mutation to BRAF-inhibitor treatment. We also identified CDKN2A deletion as a novel acquired resistance mutation to BRAFi/MEKi inhibition. The IMPACT analysis pipeline predicts these somatic variants to actionable therapeutics. We observed the clonal dynamic in the tumor samples after various treatments. We showed that IMPACT not only helped in successful prioritization of clinically relevant variants but also linked these variations to possible targeted therapies.Conclusion IMPACT provides a new bioinformatics strategy to delineate candidate somatic variants and actionable therapies. This approach can be applied to other patient tumor samples to discover effective drug targets for personalized medicine.IMPACT is publicly available at http://tanlab.ucdenver.edu/IMPACT.

The jet fuel jet propulsion fuel 8 (JP-8) has been shown to cause an inflammatory response in the skin, which is characterized histologically by erythema, edema, and hyperplasia. Studies in laboratory animal skin and cultured keratinocytes have identified a variety of changes in protein levels related to inflammation, oxidative damage, apoptosis, and cellular growth. Most of these studies have focused on prolonged exposures and subsequent effects. In an attempt to understand the earliest responses of the skin to JP-8, we have investigated changes in gene expression in the epidermis for up to 8 h after a 1-h cutaneous exposure in rats. After exposure, we separated the epidermis from the rest of the skin with a cryotome and isolated total mRNA. Gene expression was studied with microarray techniques, and changes from sham treatments were analyzed and characterized. We found consistent twofold increases in gene expression of 27 transcripts at 1, 4, and 8 h after the beginning of the 1-h exposure that were related primarily to structural proteins, cell signaling, inflammatory mediators, growth factors, and enzymes. Analysis of pathways changed showed that several signaling pathways were increased at 1 h and that the most significant changes at 8 h were in metabolic pathways, many of which were downregulated. These results confirm and expand many of the previous molecular studies with JP-8. Based on the 1-h changes in gene expression, we hypothesize that the trigger of the JP-8-induced, epidermal stress response is a physical disruption of osmotic, oxidative, and membrane stability which activates gene expression in the signaling pathways and results in the inflammatory, apoptotic, and growth responses that have been previously identified.

e21048 Background: Approximately 2-10% of metastatic melanoma patients present without an identifiable primary melanocytic lesion. These melanomas of unknown primary (MUP) are clinically classified into stage III or stage IV based upon extent of spread of metastatic disease. Previous studies, evaluating MUP using a limited sequencing of BRAF, NRAS, and KIT, have shown a similar frequency of mutations as cutaneous samples. Methods: We identified and performed retrospective chart review on 43 patients treated at the University of Colorado who presented with a melanoma of unknown primary. We excluded patient who presented with a preceding pigmented lesion treated with excision or cryotherapy without a histological diagnosis. After informed consent, DNA was extracted from the tumor samples of these 43 patients. Whole exome sequencing (WES) was performed with exome capture on an Illumina HiSeq sequencer. Results: Our cohort showed a male predominance at 58.1%. The median age at diagnosis was 50.6 years. In our cohort, 93% were Caucasian, while 4.7% were Hispanic and 2.3% were of unknown ethnic origin. 9.3% of patient had a family history of melanoma. Sun exposure was documented in 39.5% of patients and its presence was associated with an increased survival (Median OS of 3.7 vs 1.3 years, p = 0.026). Patients with stage III melanoma had an increased survival (Median OS of 6.8 vs 1.3 years, p = 0.013). Genomic analysis showed mutational pattern similar to cutaneous melanoma with 50% harboring a BRAF mutation, 12% with an NRAS mutation, and 19% carrying an NF1 variant. We found mutational signatures for each tumor and classified each against a larger cohort of cutaneous, mucosal and acral melanomas. Conclusions: We present the genomic data of a large cohort of MUP. The mutational profile of MUP is consistent with other cutaneous melanomas.

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.

Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

The RET protooncogene was originally identified in 1985. It encodes for a receptor tyrosine kinase. The RET receptor is activated by its ligand glial cell-derived neurotrophic factor. A polymorphism, RETp (G691S), in the intracellular juxtamembrane domain of RET, which enhances signaling by glial cell-derived neurotrophic factor has been described and studied previously in pancreatic cancer, medullary thyroid cancer, the multiple endocrine neoplasia 2 syndromes, and recently in cutaneous malignant melanoma. In particular, it has been shown that desmoplastic melanomas, which have neurotrophic features, have a high frequency of this polymorphism. In previous studies, however, it was not clear whether this was a germline or somatic change. Previous studies on pancreatic cancer indicated that both mechanisms may occur. To clarify this further we examined peripheral blood cell DNA from 30 patients with desmoplastic melanomas and 30 patients with nondesmoplastic melanoma for the RETp. In this study, a germline polymorphism was found in 30% of the patients with desmoplastic melanomas and 21% of the patients with nondesmoplastic melanoma. These findings indicate that the RETp may be a genetic risk factor for the development of desmoplastic melanoma.

Micropthalmia-associated transcription factor (MITF) is the master regulator of melanocyte development, survival, and function. Frequent alteration in the expression of MITF is detected in melanoma, but the mechanism(s) underlying the alteration in expression have not been completely determined. In these studies, we have identified microRNA-137 (miR-137) as a regulator of MITF expression. The genomic locus of miR-137 at chromosome 1p22 places it in a region of the human genome previously determined to harbor an allele for melanoma susceptibility. Here, we show that expression of mature miR-137 in melanoma cell lines down-regulates MITF expression. Further, we have identified a 15-bp variable nucleotide tandem repeat located just 5′ to the pre-miR-137 sequence, which alters the processing and function of miR-137 in melanoma cell lines. [Cancer Res 2008;68(5):1362–8]

Desmoplastic malignant melanomas (DMM) are clinically, pathologically, and molecularly different from other melanomas. Patients with this subset of melanoma are generally older, with tumors exhibiting deeper levels of invasion, a low incidence of nodal involvement, and tend to track along nerves. Previous studies indicate that DMM tumors have a low prevalence of BRAFV600E mutations making them ineligible for targeted therapies with BRAF inhibitors. The oncogene RET is a tyrosine kinase receptor mainly associated with neuroendocrine cancers and is activated by the ligand glial cell-derived neurotrophic factor (GDNF). Numerous studies suggest that given the neurotrophic features of DMM, a variant of RET (G691S, designated RETp) may be a potential molecular target. We examined human melanoma cell lines for RETp and the BRAFV600E mutation and demonstrated that these molecular changes were usually discordant suggesting that RETp might be an attractive molecular target in BRAFwt cells. Further we hypothesized that in tumors with the RETp genotype, certain TKIs may be more effective in inhibiting cell growth in environments rich with GDNF. We screened various melanoma cell lines with and without the RETp genotype and determined their response to clinically available small molecular weight inhibitors. The cell viability effects were accessed in the presence and/or absence of the RET co-receptor, GFRa1, and ligand, GDNF. We show that in cell lines carrying the RETp genotype, GDNF alone increases cell proliferation, and certain small molecular weight inhibitors may be more effective at inhibiting melanoma cell growth in environments rich in GDNF. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C78.

8558 Background: Desmoplastic malignant melanoma (DMM) has a unique pathological and clinical behavior which distinguishes it from other melanomas. DMM has a low incidence of nodal involvement, a s...