SUMMARY We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant RAS, mutant NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multidimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

The paradoxical coexistence of spontaneous tumor antigen–specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1–specific CD8+ T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1–specific CD8+ T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1+ NY-ESO-1–specific CD8+ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3+PD-1+ NY-ESO-1–specific CD8+ T cells are more dysfunctional than Tim-3−PD-1+ and Tim-3−PD-1− NY-ESO-1–specific CD8+ T cells, producing less IFN-γ, TNF, and IL-2. Tim-3–Tim-3L blockade enhanced cytokine production by NY-ESO-1–specific CD8+ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3–Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1–specific CD8+ T cells upon prolonged antigen stimulation and acted in synergy with PD-1–PD-L1 blockade. Collectively, our findings support the use of Tim-3–Tim-3L blockade together with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.

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Summary Regulatory T cells (Tregs) are a barrier to anti-tumor immunity. Neuropilin-1 (Nrp1) is required to maintain intratumoral Treg stability and function but is dispensable for peripheral immune tolerance. Treg-restricted Nrp1 deletion results in profound tumor resistance due to Treg functional fragility. Thus, identifying the basis for Nrp1 dependency and the key drivers of Treg fragility could help to improve immunotherapy for human cancer. We show that a high percentage of intratumoral NRP1+ Tregs correlates with poor prognosis in melanoma and head and neck squamous cell carcinoma. Using a mouse model of melanoma where Nrp1-deficient (Nrp1−/−) and wild-type (Nrp1+/+) Tregs can be assessed in a competitive environment, we find that a high proportion of intratumoral Nrp1−/− Tregs produce interferon-γ (IFNγ), which drives the fragility of surrounding WT Tregs, boosts anti-tumor immunity, and facilitates tumor clearance. We also show that IFNγ-induced Treg fragility is required for response to anti-PD1, suggesting that cancer therapies promoting Treg fragility may be efficacious.

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8+ T cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8+ T cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8+ T cell expansion and function in melanoma. BTLA-expressing PD-1+Tim-3− CD8+ T cells represented the largest subset of NY-ESO-1-specific CD8+ T cells in melanoma patients. These cells were partially dysfunctional, producing less IFN-γ than BTLA− T cells, but more IFN-γ, TNF and IL-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation and cytokine production of NY-ESO-1-specific CD8+ T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

BackgroundWe evaluated candidate circulating serum cytokines, chemokines and growth factors in patients with locally/regionally advanced melanoma receiving neoadjuvant ipilimumab with toxicity and clinical outcome.MethodsPatients were treated with ipilimumab (10 mg/kg IV every 3 weeks, 2 doses) before and after surgery. xMAP multiplex serum testing for 36 functionally selected cytokines and chemokines was performed at baseline and at six weeks (following ipilimumab). Based on our prior data, the association of IL-17 and immune related colitis was tested. Serum cytokines were divided into functional groups (Th1, Th2, Regulatory, Proinflammatory) and were assessed at baseline and week 6 using sparse-group Lasso modeling to assess the association of various cytokine groups with progression free survival (PFS). The linear combination of the cytokines/chemokines in this model was then used as a risk score and a Kaplan-Meier curve was generated to examine the association of the dichotomized score and PFS.ResultsThirty-five patients were enrolled whose staging was: IIIB (3; N2b), IIIC (30; N2c, N3), IV (2). Median follow-up was 18 months. Among 33 evaluable patients, median PFS was 11 months (95 % CI = 6.2–19.2). IL-17 was found to correlate significantly with the incidence of grade 3 diarrhea/colitis when measured at baseline (p = 0.02) with a trend towards significance at 6 weeks (p = 0.06). In the modeling analysis, at baseline, the linear combination of 2 regulatory cytokines [TGF- β1 (ρ = 0.19) and IL-10 (ρ = -0.34)] was significantly associated with PFS (HR 2.66; p = 0.035). No significant correlations with clinical outcomes were found in examining the week 6 cytokines.ConclusionsBaseline IL-17 level was significantly associated with the later development of severe diarrhea/colitis while the combination of baseline TGF- β1 and IL-10 levels were associated with therapeutic clinical outcome after neoadjuvant ipilimumab. These findings warrant further investigation and validation.Trial registrationClinicalTrials.gov Identifier NCT00972933.

We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks ×2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2–19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4+CD25hi+Foxp3+) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4+ and CD8+ antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8+ T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69+) CD3+/CD4+ and CD3+/CD8+ T cells with evidence of induction/potentiation of memory T cells (CD45RO+). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin1−/HLA-DR−/CD33+/CD11b+ was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy.

Although melanoma vaccines stimulate tumor antigen (TA)-specific CD8+ T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8+ T cells with regard to the inhibitory T cell co-receptors PD-1 and Tim-3 in metastatic melanoma patients who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant (IFA), CpG oligodeoxynucleotide (CpG) and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid TA-specific CD8+ T-cell responses detected ex vivo, however, TA-specific CD8+ T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8+ T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8+ T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8+ T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T cell responses and increase the likelihood of clinical responses in advanced melanoma patients.