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Immune-checkpoint protein V-domain immunoglobulin suppressor of T-cell activation (VISTA) controls antitumor immunity and is a valuable target for cancer immunotherapy. This study identified a role of VISTA in regulating Toll-like receptor (TLR) signaling in myeloid cells and controlling myeloid cell–mediated inflammation and immunosuppression. VISTA modulated the polyubiquitination and protein expression of TRAF6. Consequently, VISTA dampened TLR-mediated activation of MAPK/AP-1 and IKK/NF-κB signaling cascades. At cellular levels, VISTA regulated the effector functions of myeloid-derived suppressor cells and tolerogenic dendritic cell (DC) subsets. Blocking VISTA augmented their ability to produce proinflammatory mediators and diminished their T cell–suppressive functions. These myeloid cell–dependent effects resulted in a stimulatory tumor microenvironment that promoted T-cell infiltration and activation. We conclude that VISTA is a critical myeloid cell–intrinsic immune-checkpoint protein and that the reprogramming of tolerogenic myeloid cells following VISTA blockade promotes the development of T cell–mediated antitumor immunity.

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A growing body of evidence suggests that BRAF inhibitors, in addition to their acute tumor growth–inhibitory effects, can also promote immune responses to melanoma. The present study aimed to define the immunologic basis of BRAF-inhibitor therapy using the Braf/Pten model of inducible, autochthonous melanoma on a pure C57BL/6 background. In the tumor microenvironment, BRAF inhibitor PLX4720 functioned by on-target mechanisms to selectively decrease both the proportions and absolute numbers of CD4+Foxp3+ regulatory T cells (Treg) and CD11b+Gr1+ myeloid-derived suppressor cells (MDSC), while preserving numbers of CD8+ effector T cells. In PLX4720-treated mice, the intratumoral Treg populations decreased significantly, demonstrating enhanced apopotosis. CD11b+ myeloid cells from PLX4720-treated tumors also exhibited decreased immunosuppressive function on a per-cell basis. In accordance with a reversion of tumor immune suppression, tumors that had been treated with PLX4720 grew with reduced kinetics after treatment was discontinued, and this growth delay was dependent on CD8 T cells. These findings demonstrate that BRAF inhibition selectively reverses two major mechanisms of immunosuppression in melanoma and liberates host-adaptive antitumor immunity. Cancer Immunol Res; 2(11); 1044–50. ©2014 AACR.

Supplementary Figure 1: Vemurafenib arrests growth of Braf/Pten melanomas. Supplementary Figure 2: PLX4720 does not alter the character of CD4 helper T cells in the tumor microenvironment. Supplementary Figure 3: PLX4720 does not induce apoptosis of intratumoral myeloid cells. Supplementary Figure 4: Anti-CD8 depletes CD8 T cells in Braf/Pten tumors.

Supplementary Figure Legends from BRAF Inhibition Alleviates Immune Suppression in Murine Autochthonous Melanoma

Treatment significantly affects the circulating immune compartment.

Supplementary Methods, Tables 1-5, Figures 1-4. Contains additional Methods and Materials on the coding of variables in the risk model, recruitment of cases to the Leeds Case-Control study, imputation, comparing self reported and nurse derived mole counts and calculations for estimating absolute risk. Contains Tables 1-5 on missingness of pooled data, risk assessment for the Leeds dataset including imputed cases, comparisons of self classification and nurse classification of mole count/freckling in the Leeds controls. Contains Figures 1-4 showing the distribution of risk scores in the pooled and Leeds data, a bland-altman plot comparing nurse and self assessment of mole count on the back in Leeds controls and a cartoon used in the Leeds Case-Control study to determine mole count.

Percentage of cells (m-MDSC, PMN-MDSC, T regulatory cells, T helper cells, cytotoxic T-cells, PD-L1+ melanoma cells, activated, partially exhausted and exhausted CD8+ T-cells) in archival tissue biopsies for 8/9 patients in the regions of interest selected for the analysis

Heat map showing relative levels of mRNA expression for each gene, specifying the gene represented by each row.

Purpose:Increased β-adrenergic receptor (β-AR) signaling has been shown to promote the creation of an immunosuppressive tumor microenvironment (TME). Preclinical studies have shown that abrogation of this signaling pathway, particularly β2-AR, provides a more favorable TME that enhances the activity of anti–PD-1 checkpoint inhibitors. We hypothesize that blocking stress-related immunosuppressive pathways would improve tumor response to immune checkpoint inhibitors in patients. Here, we report the results of dose escalation of a nonselective β-blocker (propranolol) with pembrolizumab in patients with metastatic melanoma.Patients and Methods:A 3 + 3 dose escalation study for propranolol twice a day with pembrolizumab (200 mg every 3 weeks) was completed. The primary objective was to determine the recommended phase II dose (RP2D). Additional objectives included safety, antitumor activity, and biomarker analyses. Responders were defined as patients with complete or partial response per immune-modified RECIST at 6 months.Results:Nine patients with metastatic melanoma received increasing doses of propranolol in cohorts of 10, 20, and 30 mg twice a day. No dose-limiting toxicities were observed. Most common treatment-related adverse events (TRAEs) were rash, fatigue, and vitiligo, observed in 44% patients. One patient developed two grade ≥3 TRAEs. Objective response rate was 78%. While no significant changes in treatment-associated biomarkers were observed, an increase in IFNγ and a decrease in IL6 was noted in responders.Conclusions:Combination of propranolol with pembrolizumab in treatment-naïve metastatic melanoma is safe and shows very promising activity. Propranolol 30 mg twice a day was selected as RP2D in addition to pembrolizumab based on safety, tolerability, and preliminary antitumor activity.

Biomarker changes over time. A, Variations in chemokines IP-10, eotaxin and TNF-β in pg/ml. B, Variation in CD8+T-cells, m-MDSC, PMN-MDSC, regulatory T-cells (Tregs) expressed as percentage of total CD45+ cells, ratio of CD8+/ PMN-MDSC. C, Relative changes in peripheral blood cells (PMN-MDSC, CD8+ T-cell, CD8+ T cells/T-reg) and IL-12p70. Dose level 1 is propranolol 10 mg BID, dose level 2 is propranolol 20 mg BID and dose level 3 is propranolol 30 mg BID. Asterix show significant values at different time points compared to baseline in different dose levels.

Comparison of patient demographics in validation cohort to original publication discovery and test cohorts.

Figure legends for supplementary figures.

Representative gating strategy illustrating (A) CD4 and CD8 T cell identification (B) expression of ICOS on CD4 positive T cells (C) expression of PD-1 and Ki-67 within PD-1 positive cells and (D) expression of CD38/HLA-DR, PD-1 and KI-67 within CD8+ PD-1+ T cells is shown.

Supplementary Figure 1-3 Legends

Purpose: IL15 induces the activation and proliferation of natural killer (NK) and memory CD8+ T cells and has preclinical antitumor activity. Given the superior activity and favorable kinetics of ALT-803 (IL15N72D:IL15RαSu/IgG1 Fc complex) over recombinant human IL15 (rhIL15) in animal models, we performed this first-in-human phase I trial of ALT-803 in patients with advanced solid tumors.Patients and Methods: Patients with incurable advanced melanoma, renal cell, non–small cell lung, and head and neck cancer were treated with ALT-803 0.3 to 6 μg/kg weekly intravenously or 6 to 20 μg/kg weekly subcutaneously for 4 consecutive weeks, every 6 weeks. Immune correlates included pharmacokinetics, immunogenicity, and lymphocyte expansion and function. Clinical endpoints were toxicity and antitumor activity.Results: Twenty-four patients were enrolled; 11 received intravenous and 13 received subcutaneous ALT-803. Of these patients, nine had melanoma, six renal, three head and neck, and six lung cancer. Although total lymphocyte and CD8+ T-cell expansion were modest, NK cell numbers rose significantly. Neither anti–ALT-803 antibodies nor clinical activity were observed. Overall, ALT-803 was well tolerated, with adverse effects including fatigue and nausea most commonly with intravenous administration, whereas painful injection site wheal was reported most commonly with subcutaneous ALT-803.Conclusions: Subcutaneous ALT-803 produced the expected NK cell expansion and was well tolerated with minimal cytokine toxicities and a strong local inflammatory reaction at injection sites in patients with advanced cancer. These data, together with compelling evidence of synergy in preclinical and clinical studies, provide the rationale for combining ALT-803 with other anticancer agents. Clin Cancer Res; 24(22); 5552–61. ©2018 AACR.

Supplemental Figure 1. Effect of ALT-803 treatment on white blood cell and lymphocyte counts in i.v. dose cohorts Supplemental Figure 2. Circulating NK cell expansion during ALT-803 treatment Supplemental Figure 3. Effect of subcutaneous ALT-803 on circulating T cell expression of HLA-DR

Treatment significantly affects the circulating immune compartment.

Purpose:Neoadjuvant immunotherapy may improve the clinical outcome of regionally advanced operable melanoma and allows for rapid clinical and pathologic assessment of response. We examined neoadjuvant pembrolizumab and high-dose IFNα-2b (HDI) therapy in patients with resectable advanced melanoma.Patients and Methods:Patients with resectable stage III/IV melanoma were treated with concurrent pembrolizumab 200 mg i.v. every 3 weeks and HDI 20 MU/m2/day i.v., 5 days per week for 4 weeks, then 10 MU/m2/day subcutaneously 3 days per week for 2 weeks. Definitive surgery followed, as did adjuvant combination immunotherapy, completing a year of treatment. Primary endpoint was safety of the combination. Secondary endpoints included overall response rate (ORR), pathologic complete response (pCR), recurrence-free survival (RFS), and overall survival (OS). Blood samples for correlative studies were collected throughout. Tumor tissue was assessed by IHC and flow cytometry at baseline and at surgery.Results:A total of 31 patients were enrolled, and 30 were evaluable. At data cutoff (October 2, 2019), median follow-up for OS was 37.87 months (range, 33.2–43.47). Median OS and RFS were not reached. Radiographic ORR was 73.3% [95% confidence interval (CI): 55.5–85.8], with a 43% (95% CI: 27.3–60.1) pCR rate. None of the patients with a pCR have had a recurrence. HDI and pembrolizumab were discontinued in 73% and 43% of patients, respectively. Correlative analyses suggested that intratumoral PD-1/PD-L1 interaction and HLA-DR expression are associated with pCR (P = 0.002 and P = 0.008, respectively).Conclusions:Neoadjuvant concurrent HDI and pembrolizumab demonstrated promising clinical activity despite high rates of treatment discontinuation. pCR is a prognostic indicator.See related commentary by Menzies et al., p. 4133

Purpose:Biomarkers are needed to stratify patients with stage II–III melanoma for clinical trials of adjuvant therapy because, while immunotherapy is protective, it also confers the risk of severe toxicity. We previously defined and validated a 53-immune gene melanoma immune profile (MIP) predictive both of distant metastatic recurrence and of disease-specific survival (DSS). Here, we test MIP on a third independent population.Experimental Design:A retrospective cohort of 78 patients with stage II–III primary melanoma was analyzed using the NanoString assay to measure expression of 53 target genes, and MIP score was calculated. Statistical analysis correlating MIP with DSS, overall survival, distant metastatic recurrence, and distant metastasis-free interval was performed using ROC curves, Kaplan–Meier curves, and standard univariable and multivariable Cox proportional hazards models.Results:MIP significantly distinguished patients with distant metastatic recurrence from those without distant metastatic recurrence using ROC curve analysis (AUC = 0.695; P = 0.008). We defined high- and low-risk groups based on the cutoff defined by this ROC curve and find that MIP correlates with both DSS and overall survival by ROC curve analysis (AUC = 0.719; P = 0.004 and AUC = 0.698; P = 0.004, respectively). Univariable Cox regression reveals that a high-risk MIP score correlates with DSS (P = 0.015; HR = 3.2).Conclusions:MIP identifies patients with low risk of death from melanoma and may constitute a clinical tool to stratify patients with stage II–III melanoma for enrollment in clinical trials.

Myeloid-derived suppressor cells (MDSC) producing arginase I are increased in the peripheral blood of patients with renal cell carcinoma (RCC). MDSC inhibit T-cell function by reducing the availability of l-arginine and are therefore considered an important tumor escape mechanism. We aimed to determine the origin of arginase I–producing MDSC in RCC patients and to identify the mechanisms used to deplete extracellular l-arginine. The results show that human MDSC are a subpopulation of activated polymorphonuclear (PMN) cells expressing high levels of CD66b, CD11b, and VEGFR1 and low levels of CD62L and CD16. In contrast to murine MDSC, human MDSC do not deplete l-arginine by increasing its uptake but instead release arginase I into the circulation. Activation of normal PMN induces phenotypic and functional changes similar to MDSC and also promotes the release of arginase I from intracellular granules. Interestingly, although activation of normal PMN usually ends with apoptosis, MDSC showed no increase in apoptosis compared with autologous PMN or PMN obtained from normal controls. High levels of VEGF have been shown to increase suppressor immature myeloid dendritic cells in cancer patients. Treatment of RCC patients with anti-VEGF antibody bevacizumab, however, did not reduce the accumulation of MDSC in peripheral blood. In contrast, the addition of interleukin-2 to the treatment increased the number of MDSC in peripheral blood and the plasma levels of arginase I. These results may provide new insights on the mechanisms of tumor-induced anergy/tolerance and may help explain why some immunotherapies fail to induce an antitumor response. [Cancer Res 2009;69(4):1553–60]

Supplementary Figure 2 from Arginase I–Producing Myeloid-Derived Suppressor Cells in Renal Cell Carcinoma Are a Subpopulation of Activated Granulocytes

PDF file - 20KB, The suppressive effect of VISTA is stable. Bulk CD4 T cells were purified from human PBMCs by magnetic bead selection. Cells were CFSE labeled and stimulated for 3 days with 2.5ug/ml anti-CD3 co-coated with 0, 2.5, 5 or 10ug/ml of VISTA-Ig. Control-Ig was used to bring the concentration of fusion protein to 10ug/ml in all wells. Cells were then moved to anti-CD3 in the absence of fusion protein. Percentage CFSE low cells are shown as mean +/- SD. Data is representative of at least two separate experiments.

V-domain Ig suppressor of T cell activation (VISTA) is a potent negative regulator of T-cell function that is expressed on hematopoietic cells. VISTA levels are heightened within the tumor microenvironment, in which its blockade can enhance antitumor immune responses in mice. In humans, blockade of the related programmed cell death 1 (PD-1) pathway has shown great potential in clinical immunotherapy trials. Here, we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells and blunted the production of T-cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T-cell activation, induces Foxp3 expression, and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer. Cancer Res; 74(7); 1924–32. ©2013 AACR.

PDF file - 90KB, Human and mouse VISTA-Ig show titratable suppressive activity on human CD4 T cells. Bulk CD4 T cells were purified from human PBMCs by magnetic bead selection. Cells were labeled with CFSE, and stimulated for 5 days with 1, 2.5 or 5ug/ml of anti-CD3 co-coated with 0, 2.5, 5, or 10ug/ml of human (a and c) or mouse (b and d) VISTA-Ig. Control-Ig was added to equalize the amount of fusion protein. (a and b) Overlays showing representative CFSE profiles. Cells are gated on live, singlet CD4 positive cells. (c and d) Percentage CFSE low cells are shown as mean +/- SD. *p

PDF file - 77KB, VISTA does not affect B cell proliferation. CD19 positive B cells were purified from human PBMCs by magnetic bead negative selection. (a and b) cells were stimulated in the presence of VISTA-Ig (a) or control Ig (b) at 5ug/ml for 4 days with 250ng/ml of CD40 agonist. Plots show representative CFSE profiles gated on live CD19 positive events. C) percent proliferation in the presence of decreasing concentrations of agonist in the presence of VISTA-Ig (filled bars) or control Ig (open bars) at 5ug/ml for 4 days. Data is representative of 3 separate experiments.

Supplementary Figure 1 from Arginase I–Producing Myeloid-Derived Suppressor Cells in Renal Cell Carcinoma Are a Subpopulation of Activated Granulocytes

Supplementary Figure Legends 1-2 from Arginase I–Producing Myeloid-Derived Suppressor Cells in Renal Cell Carcinoma Are a Subpopulation of Activated Granulocytes

The broad activity of agents blocking the programmed cell death protein 1 and its ligand (the PD-(L)1 axis) revolutionized oncology, offering long-term benefit to patients and even curative responses for tumors that were once associated with dismal prognosis. However, only a minority of patients experience durable clinical benefit with immune checkpoint inhibitor monotherapy in most disease settings. Spurred by preclinical and correlative studies to understand mechanisms of non-response to the PD-(L)1 antagonists and by combination studies in animal tumor models, many drug development programs were designed to combine anti-PD-(L)1 with a variety of approved and investigational chemotherapies, tumor-targeted therapies, antiangiogenic therapies, and other immunotherapies. Several immunotherapy combinations improved survival outcomes in a variety of indications including melanoma, lung, kidney, and liver cancer, among others. This immunotherapy renaissance, however, has led to many combinations being advanced to late-stage development without definitive predictive biomarkers, limited phase I and phase II data, or clinical trial designs that are not optimized for demonstrating the unique attributes of immune-related antitumor activity—for example, landmark progression-free survival and overall survival. The decision to activate a study at an individual site is investigator-driven, and generalized frameworks to evaluate the potential for phase III trials in immuno-oncology to yield positive data, particularly to increase the number of curative responses or otherwise advance the field have thus far been lacking. To assist in evaluating the potential value to patients and the immunotherapy field of phase III trials, the Society for Immunotherapy of Cancer (SITC) has developed a checklist for investigators, described in this manuscript. Although the checklist focuses on anti-PD-(L)1-based combinations, it may be applied to any regimen in which immune modulation is an important component of the antitumor effect.

It is unclear why some melanomas aggressively metastasize while others remain indolent. Available studies employing multi-omic profiling of melanomas are based on large primary or metastatic tumors. We examine the genomic landscape of early-stage melanomas diagnosed prior to the modern era of immunological treatments. Untreated cases with Stage II/III cutaneous melanoma were identified from institutions throughout the United States, Australia and Spain. FFPE tumor sections were profiled for mutation, methylation and microRNAs. Preliminary results from mutation profiling and clinical pathologic correlates show the distribution of four driver mutation sub-types: 31% BRAF; 18% NRAS; 21% NF1; 26% Triple Wild Type. BRAF mutant tumors had younger age at diagnosis, more associated nevi, more tumor infiltrating lymphocytes, and fewer thick tumors although at generally more advanced stage. NF1 mutant tumors were frequent on the head/neck in older patients with severe solar elastosis, thicker tumors but in earlier stages. Triple Wild Type tumors were predominantly male, frequently on the leg, with more perineural invasion. Mutations in TERT, TP53, CDKN2A and ARID2 were observed often, with TP53 mutations occurring particularly frequently in the NF1 sub-type. The InterMEL study will provide the most extensive multi-omic profiling of early-stage melanoma to date. Initial results demonstrate a nuanced understanding of the mutational and clinicopathological landscape of these early-stage tumors.

Purpose:Neoadjuvant immunotherapy may improve the clinical outcome of regionally advanced operable melanoma and allows for rapid clinical and pathologic assessment of response. We examined neoadjuvant pembrolizumab and high-dose IFNα-2b (HDI) therapy in patients with resectable advanced melanoma.Patients and Methods:Patients with resectable stage III/IV melanoma were treated with concurrent pembrolizumab 200 mg i.v. every 3 weeks and HDI 20 MU/m2/day i.v., 5 days per week for 4 weeks, then 10 MU/m2/day subcutaneously 3 days per week for 2 weeks. Definitive surgery followed, as did adjuvant combination immunotherapy, completing a year of treatment. Primary endpoint was safety of the combination. Secondary endpoints included overall response rate (ORR), pathologic complete response (pCR), recurrence-free survival (RFS), and overall survival (OS). Blood samples for correlative studies were collected throughout. Tumor tissue was assessed by IHC and flow cytometry at baseline and at surgery.Results:A total of 31 patients were enrolled, and 30 were evaluable. At data cutoff (October 2, 2019), median follow-up for OS was 37.87 months (range, 33.2–43.47). Median OS and RFS were not reached. Radiographic ORR was 73.3% [95% confidence interval (CI): 55.5–85.8], with a 43% (95% CI: 27.3–60.1) pCR rate. None of the patients with a pCR have had a recurrence. HDI and pembrolizumab were discontinued in 73% and 43% of patients, respectively. Correlative analyses suggested that intratumoral PD-1/PD-L1 interaction and HLA-DR expression are associated with pCR (P = 0.002 and P = 0.008, respectively).Conclusions:Neoadjuvant concurrent HDI and pembrolizumab demonstrated promising clinical activity despite high rates of treatment discontinuation. pCR is a prognostic indicator.See related commentary by Menzies et al., p. 4133

Introduction: The development and FDA approval of drugs targeting the PD-1/PD-L1 axis has greatly shifted the paradigm of immunotherapy and has improved the clinical outcome for patients diagnosed with advanced melanoma. Despite the recent advancement of immune checkpoint inhibitors, more than 50% of patients still fail to show a sustained response to therapy. Therefore, there is a critical need to develop therapies specific to novel immune targets as well as combination drug regimens. Current immunocompetent preclinical models do not adequately model the responses to immunotherapy, reflecting the urgent need to develop models with greater translational utility. To address this need, we developed a humanized xenograft melanoma model engrafted with autologous peripheral blood lymphocytes (PBLs) to evaluate the utility of nemvaleukin alfa, an engineered IL-2 cytokine which preferentially activates and expands cytotoxic NK cells and antigen-experienced CD8 T cells. Methods: Melanoma-bearing mice were injected intravenously with matched melanoma patient PBLs and treated for four cycles (1 cycle/week) with anti-PD-1, nemvaleukin, or rhIL-2. Tumor growth was monitored, and correlative analyses were done on blood, spleen, and tumors by flow cytometry after 4 cycles of treatment. Results: This humanized melanoma mouse model exhibited good T cell engraftment and was characterized by expression of the proliferative marker Ki-67 in 50-60% of CD8+ and CD4+ T cell populations. Treatment with nemvaleukin resulted in significant tumor growth inhibition rates (TGIs) of an average of 48% compared to vehicle-treated mice. In addition, nemvaleukin promoted expansion of CD8+ and CD4+ T cells, without expansion of CD4+ regulatory T cells, which are known to suppress immunological responses. Compared to rhIL-2 and vehicle, nemvaleukin treatment delayed tumor growth and enhanced survival with a median survival time of 56 days compared to 21 and 50 days, respectively. Nemvaleukin-treated mice had higher CD45+ cell engraftment in spleens and tumors compared to anti-PD-1 treatment, indicating that nemvaleukin boosts T cell proliferation and expansion. Of note, we observed better antitumor efficacy in response to nemvaleukin or anti-PD-1 when mice were engrafted with tumors from patients who responded to checkpoint immunotherapy with TGIs of 36% and 51%, respectively, compared to those who did not with TGIs of 16% and 12%, respectively. These data indicate that treatment responses in our humanized model correlates with clinical outcomes. Conclusion: In this all autologous humanized melanoma mouse model, nemvaleukin treatment not only enhances T cell engraftment, it significantly augments anti-tumor efficacy. These data support the use of this personalized melanoma xenograft model to analyze the underlying immune responses in responders and non-responders to novel immunotherapies. Citation Format: Yee Peng Phoon, Jared E. Lopes, Lukas W. Pfannenstiel, C. Marcela Diaz-Montero, Ye F. Tian, Marc S. Ernstoff, Pauline Funchain, XiaoXia Li, Heather C. Losey, Raymond J. Winquist, Brian R. Gastman. Developing an autologous humanized mouse model to assess responses to novel immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1818.

The melanoma treatment landscape changed in 2011 with the approval of the first anti-cytotoxic T-lymphocyte-associated protein (CTLA)-4 checkpoint inhibitor and of the first BRAF-targeted monoclonal antibody, both of which significantly improved overall survival (OS). Since then, improved understanding of the tumor microenvironment (TME) and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. The approval of new immune and targeted therapies has further improved outcomes for patients with advanced melanoma and other combination modalities are also being explored such as chemotherapy, radiotherapy, electrochemotherapy and surgery. In addition, different strategies of drugs administration including sequential or combination treatment are being tested. Approaches to overcome resistance and to potentiate the immune response are being developed. Increasing evidence emerges that tissue and blood-based biomarkers can predict the response to a therapy. The latest findings in melanoma research, including insights into the tumor microenvironment and new biomarkers, improved understanding of tumor immune response and resistance, novel approaches for combination strategies and the role of neoadjuvant and adjuvant therapy, were the focus of discussions at the Melanoma Bridge meeting (5–7 December, 2019, Naples, Italy), which are summarized in this report.