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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

Most patients with advanced melanoma ultimately fail immune checkpoint inhibitor (ICI) therapy because of primary or acquired resistance. There remains a critical unmet need for new therapies that function via alternative immune activation mechanisms to safely trigger an antitumor immune response in patients with ICI-refractory disease. This commentary discusses the recent failures and hope for novel intratumoral therapies under development in the advanced refractory melanoma setting, outlining key mechanistic differences that may be critical to yielding success in this difficult-to-treat population.

Background The pivotal E1684, E1690, E1694, and E2696 trials of adjuvant high-dose interferon-α (HDI) enrolled nearly 2000 patients, and established HDI as the standard of care in adjuvant therapy for patients with resected high-risk melanoma. Herein, the authors present an updated analysis of these 4 trials. Methods Survival and disease status were updated in September 2016. These data represent a median follow-up of 17.9 years for the E1684 trial, 12.2 years for the E1690 trial, 16.0 years for the E1694 trial, and 16.5 years for the E2696 trial. Results The current analysis confirmed the benefit to recurrence-free survival (RFS) of HDI in the E1684 trial at a median follow-up of 17.9 years. The RFS benefit in the E1694 trial remained evident at a median follow-up of 16 years. Furthermore, the results of the current study confirmed the RFS benefit of adjuvant HDI compared with observation in a pooled analysis of the E1684 and E1690 trials. No overall survival benefit was apparent in this pooled analysis. Updated results for the E1690 and E2696 trials did not differ from those previously reported. In addition, to the authors' knowledge, the current study is the first to report a significant difference in melanoma-specific survival (MSS) between patients treated with HDI compared with the ganglioside GM2/keyhole limpet hemocyanin (GMK) vaccine in the E1694 trial. Conclusions In patients with resected high-risk melanoma, adjuvant HDI demonstrated improved RFS in the E1684 and E1694 trials, and improved MSS in a pooled analysis of HDI in the E1694 trial. To the authors' knowledge, these findings represent the most mature level of evidence for the benefit of HDI with respect to RFS and MSS. HDI is the only approved adjuvant treatment for which there are data available in patients with resected stage IIB/IIC melanoma, and remains a reasonable treatment option in this population.

Over the last few years, numerous clinical trials and real-world experience have provided a large amount of evidence demonstrating the potential for long-term survival with immunotherapy agents across various malignancies, beginning with melanoma and extending to other tumours. The clinical success of immune checkpoint blockade has encouraged increasing development of other immunotherapies. It has been estimated that there are over 3000 immuno-oncology trials ongoing, targeting hundreds of disease and immune pathways. Evolving topics on cancer immunotherapy, including the state of the art of immunotherapy across various malignancies, were the focus of discussions at the Immunotherapy Bridge meeting (4–5 December, 2019, Naples, Italy), and are summarised in this report.

Background Immunotherapy has changed the treatment landscape for melanoma, although many patients (pts) do not respond to treatment. While there are likely multiple mechanisms of resistance at play, one key mechanism is the generation of an immunosuppressive and metabolically harsh tumor microenvironment (TME).1 This is likely the result of an altered angiogenic pattern along with dysregulated metabolism of the tumor itself, which leads to hypoxia.2 CD8+ tumor infiltrating lymphocytes (TIL) isolated from tumors with high oxidative metabolism have an exhausted phenotype and decreased functionality (decreased IFN-У and TNF-α production).3 Thus, TIL may be blunted due to failure to meet their metabolic needs. Vascular endothelial growth factor (VEGF) is a critical mediator of angiogenesis and is overexpressed in many solid tumors, including melanoma. Axitinib has high inhibitory activity for VEGF receptors1, 2, and 3. In a preclinical B16 melanoma model, we found that anti-PD1 plus axitinib provided an improved and durable response compared to monotherapy with either agent. We hypothesize that by modulating angiogenesis, axitinib will reduce intra-tumoral hypoxia and resultant T cell dysfunction, which will re-sensitize melanoma to anti-PD1 therapy. Methods This is an investigator-initiated, phase II trial of nivolumab plus axitinib for pts with unresectable stage III or IV melanoma who have progressed on prior anti-PD1 therapy with or without concomitant anti-CTLA4. Prior treatment with BRAF/MEK inhibitors is permitted. Pts with brain metastases are permitted if they are asymptomatic and have stable disease 2 weeks after CNS-directed treatment. Pts will receive nivolumab 480 mg IV every 4 weeks and axitinib PO 5 mg twice daily for up to two years or until progression or unacceptable toxicity. Timing of biopsies is reported in figure 1, with an optional biopsy at progression. Pts will receive an oral dose of pimonidazole 0.5 mg/m2 before each biopsy to permit in vivo evaluation of intra-tumoral hypoxia. Primary endpoint: overall response rate (ORR). Secondary endpoints: safety, progression-free survival, overall survival, and correlative analyses (evaluation of hypoxia in the TME, TIL function, immune phenotype, and tumor cell metabolism). Statistical analysis includes Simon’s minimax two-stage design. The null hypothesis is that the true ORR is 10%, tested against a one-sided alternative of 25% or higher. N=31 patients with a type I error rate of 0.08 and power 0.81 when the true response rate is 0.25. Results N/A Conclusions N/A Trial Registration NCT04493203 Ethics Approval The study was approved by the University of Pittsburgh Institutional Review Board, approval number HCC 20-101. References Romero IL, Mukherjee A, Kenny HA, Litchfield LM, Lengyel E. Molecular pathways: trafficking of metabolic resources in the tumor microenvironment. Clin Cancer Res 2015;21(4):680–6. doi: 10.1158/1078-0432.CCR-14-2198. PubMed PMID: 25691772. Justus CR, Sanderlin EJ, Yang LV. Molecular connections between cancer cell metabolism and the tumor microenvironment. Int J Mol Sci 2015;16(5):11055–86. doi: 10.3390/ijms160511055. PubMed PMID: 25988385. Najjar YG, Menk AV, Sander C, Rao U, Karunamurthy A, Bhatia R, et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI insight. 2019;4(5). Epub 2019/02/06. doi: 10.1172/jci.insight.124989. PubMed PMID: 30721155; PubMed Central PMCID: PMCPMC6483505.

Background Immune checkpoint blockade (ICB) has revolutionized cancer treatment. However, long-term benefits are only achieved in a small fraction of patients. Understanding the mechanisms underlying ICB activity is key to improving the efficacy of immunotherapy. A major limitation to uncovering these mechanisms is the limited number of responders within each ICB trial. Integrating data from multiple studies of ICB would help overcome this issue and more reliably define the immune landscape of durable responses. Towards this goal, we formed the TimIOs consortium, comprising researchers from the Society for Immunotherapy of Cancer Sparkathon TimIOs Initiative, the Parker Institute of Cancer Immunotherapy, the University of North Carolina-Chapel Hill, and the Institute for Systems Biology. Together, we aim to improve the understanding of the molecular mechanisms associated with defined outcomes to ICB, by building on our joint and multifaceted expertise in the field of immuno-oncology. To determine the feasibility and relevance of our approach, we have assembled a compendium of publicly available gene expression datasets from clinical trials of ICB. We plan to analyze this data using a previously reported pipeline that successfully determined main cancer immune-subtypes associated with survival across multiple cancer types in TCGA.1 Methods RNA sequencing data from 1092 patients were uniformly reprocessed harmonized, and annotated with predefined clinical parameters. We defined a comprehensive set of immunogenomics features, including immune gene expression signatures associated with treatment outcome,1,2 estimates of immune cell proportions, metabolic profiles, and T and B cell receptor repertoire, and scored all compendium samples for these features. Elastic net regression models with parameter optimization done via Monte Carlo cross-validation and leave-one-out cross-validation were used to analyze the capacity of an integrated immunogenomics model to predict durable clinical benefit following ICB treatment. Results Our preliminary analyses confirmed an association between the expression of an IFN-gamma signature in tumor (1) and better outcomes of ICB, highlighting the feasibility of our approach. Conclusions In line with analysis of pan-cancer TCGA datasets using this strategy (1), we expect to identify analogous immune subtypes characterizing baseline tumors from patients responding to ICB. Furthermore, we expect to find that these immune subtypes will have different importance in the model predicting response and survival. Results of this study will be incorporated into the Cancer Research Institute iAtlas Portal, to facilitate interactive exploration and hypothesis testing. References Thorsson V, Gibbs DL, Brown SD, Wolf D, Bortone DS, Yang T-H O, Porta-Pardo E. Gao GF, Plaisier CL, Eddy JA, et al. The Immune Landscape of Cancer. Immunity 2018; 48(4): 812–830.e14. https://doi.org/10.1016/j.immuni.2018.03.023. Auslander N, Zhang G, Lee JS, Frederick DT, Miao B, Moll T, Tian T, Wei Z, Madan S, Sullivan RJ, et al. Robust Prediction of Response to Immune Checkpoint Blockade Therapy in Metastatic Melanoma. Nat. Med 2018; 24(10): 1545. https://doi.org/10.1038/s41591-018-0157-9.

Background: Monoclonal antibodies (mAb) targeting the programmed cell death protein 1 (PD-1) receptor provide durable long-term benefit in a subset of patients (pts) with advanced melanoma with response rates of 35-42% and 4-year progression-free survival (PFS) rate of 27%. Separately, the composition of the gut microbiota has been shown to correlate with anti-PD-1 efficacy in human cancer pts with melanoma, renal cell cancer and non-small cell lung cancer (NSCLC) although the precise organisms differ considerably across various studies. In preclinical models, responder-derived fecal microbiome and microbiome consortia produce anti-tumor responses. The effect of microbiome modulation in pts with anti-PD-1 refractory melanoma has not been evaluated. Methods: To evaluate whether primary resistance to anti-PD-1 immunotherapy could be overcome by intestinal microbiome modulation, we designed and conducted a phase II study (NCT03341143). We enrolled pts with primary refractory metastatic melanoma with best response of short-term stable disease (≤6 months) or progressive disease (PD) to prior anti-PD-1 based immunotherapy. Pts received single-administration of responder-derived fecal microbiota transplantation (R-FMT) together with pembrolizumab. Candidate donors were pts with advanced melanoma treated with anti-PD-1 immunotherapy with durable partial or complete response (PR, CR). Pembrolizumab was continued till intolerable toxicity or disease progression. Safety and clinical activity (based on RECIST v1.1) were main objectives; while progression-free survival (PFS) was a key secondary endpoint. Results: As of December 1, 2020, 16 pts with primary refractory melanoma were enrolled, of whom 15 were evaluable. LDH was elevated in 14/15 pts; and the median number of prior therapies was 2. Recipient pts were seromatched to receive a single R-FMT from one of eight candidate donors (5 CR; 3 PR; median PFS 58 months, range 43-70). R-FMT was administered via colonoscopy after bowel preparation with no use of antibiotics. Pembrolizumab was administered IV per label. R-FMT/pembrolizumab was well-tolerated, with no unusual toxicity signals. R-FMT induced rapid and durable microbiota perturbation in most pts; while 6 of 15 evaluable pts had evidence of clinical benefit. Response to R-FMT/pembrolizumab was associated with an increased abundance of taxa previously shown to be associated with response to anti-PD-1, increased CD8+ T cell activation, and decreased frequency of IL-8 expressing myeloid cells. Responders had distinct proteomic and metabolomic signatures, and transkingdom network analyses confirmed that the gut microbiome regulated these changes. Conclusions: In pts with anti-PD-1 primary refractory melanoma, R-FMT/pembrolizumab changed the gut microbiome and reprogrammed the tumor microenvironment to overcome resistance to anti-PD-1 immunotherapy. Response was associated with CD8 T cell induction and reduction of IL-8 expressing myeloid cells. Citation Format: Diwakar Davar, Amiran Dzutsev, John A. McCulloch, Richard R. Rodrigues, Joe-Marc Chauvin, Robert M. Morrison, Richelle N. Deblasio, Carmine Menna, Quanquan Ding, Ornella Pagliano, Bochra Zidi, Shuowen Zhang, Jonathan H. Badger, Marie Vetizou, Alicia M. Cole, Miriam R. Fernandes, Stephanie Prescott, Raquel G. Costa, Ascharya K. Balaji, Andrey Morgun, Ivan Vujkovic-Cvijin, Hong Wang, Amir A. Borhani, Marc B. Schwartz, Howard M. Dubner, Scarlett J. Ernst, Amy Rose, Yana G. Najjar, Yasmine Belkaid, John M. Kirkwood, Giorgio Trinchieri, Hassane M. Zarour. Efficacy of Responder-derived Fecal Microbiota Transplant (R-FMT) and Pembrolizumab in Anti-PD-1 Refractory Patients with Advanced Melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB062.

The tumor microenvironment presents physical, immunologic, and metabolic barriers to durable immunotherapy responses. We have recently described roles for both T cell metabolic insufficiency as well as tumor hypoxia as inhibitory mechanisms that prevent T cell activity in murine tumors, but whether intratumoral T cell activity or response to immunotherapy varies between patients as a function of distinct metabolic profiles in tumor cells remains unclear. Here, we show that metabolic derangement can vary widely in both degree and type in patient-derived cell lines and in ex vivo analysis of patient samples, such that some cells demonstrate solely deregulated oxidative or glycolytic metabolism. Further, deregulated oxidative, but not glycolytic, metabolism was associated with increased generation of hypoxia upon implantation into immunodeficient animals. Generation of murine single-cell melanoma cell lines that lacked either oxidative or glycolytic metabolism showed that elevated tumor oxygen consumption was associated with increased T cell exhaustion and decreased immune activity. Moreover, melanoma lines lacking oxidative metabolism were solely responsive to anti-PD-1 therapy among those tested. Prospective analysis of patient sample immunotherapy revealed that oxidative, but not glycolytic, metabolism was associated with progression on PD-1 blockade. Our data highlight a role for oxygen as a crucial metabolite required for the tumor-infiltrating T cells to differentiate appropriately upon PD-1 blockade, and suggest that tumor oxidative metabolism may be a target to improve immunotherapeutic response.

e21506 Background: Management of patients (pts) with advanced melanoma includes anti-PD1 with or without anti-CTLA4, and for pts with a BRAF mutation, the additional option of targeted therapy. Preclinical and translational evidence suggest BRAF/MEK inhibitors (i) modulate the tumor microenvironment, providing rationale for combination with immune checkpoint inhibitors. Phase 3 IMspire data reported improved progression-free survival (PFS) with triplet therapy (atezolizumab/vemurafenib/cobimetinib), yielding regulatory approval. However, 79% of pts experienced grade 3/4 adverse events (AE) in the triplet arm. Methods: This is an investigator-initiated, phase I trial of pembrolizumab (pembro) plus vemurafenib (vem) and cobimetinib (cobi) for pts with advanced melanoma in the first line setting. The first 4 pts received vem/pembro. The protocol was subsequently amended, and the next 5 pts received vem/cobi/pembro. Vem/cobi had an escalating dosing regimen. Pembro was 200 mg q3 weeks. Primary endpoints: safety and maximum tolerated dose of vem/cobi when administered with pembro. Secondary endpoints: overall response rate (ORR), PFS, overall survival (OS), and quality of life (QoL). We planned to accrue 30 pts; however, the trial was closed after enrollment of 9 pts due to dose-limiting toxicity (DLT). This study NCT02818023 was approved by the IRB, and all pts provided informed consent. Results: Pts received a median of 6 cycles of triplet therapy. 8 of 9 pts experienced drug-related grade 3/4 AEs, most commonly dermatitis (89%). In the vem/pembro group, DLTs included hepatitis (n = 1), dermatitis (n = 3), and arthralgias (n = 1). In the vem/cobi/pembro group, DLTs included dermatitis (n = 5), QTc prolongation (n = 1), and arthralgias (n = 1). QoL assessments identified a clinically significant decrease in average health utility at 1 year compared to baseline (0.38 v 0.43). Median PFS was 20.7 months and median OS was 23.8 months for vem/pembro, and neither was reached for vem/cobi/pembro. Overall, 4 pts had ongoing responses at the time of data analysis. 2 pts experienced a complete response, 5 had a partial response, 1 had stable disease, and 1 had progressive disease at first restaging. Peripheral blood flow cytometry identified significantly decreased PD1 expression on CD4+ T-cells at 3 and 9 weeks compared to baseline. This did not correspond to clinical response. PD-L1 testing was also performed on 6 paired tumor samples, and no significant association was identified between PD-L1 expression and clinical outcomes. Conclusions: Despite preclinical and translational evidence for tumor immunomodulation with BRAF/MEKi and improved PFS noted in IMspire150, toxicity incurred with the triplet is challenging from a practical standpoint. Our study highlights clinical efficacy of the combination and adds additional toxicity data for triplet therapy, with 8 of 9 pts experiencing at least a grade 3 AE. Clinical trial information: NCT02818023.

Simple Summary For decades nearly all cancer patients were treated with cytotoxic chemotherapy, killing any rapidly dividing cell in the body. More recently, researchers have been studying the immune system’s response to cancer and have developed a novel class of drugs that stimulate the body’s own response to tumors. This class of immunotherapy drugs primarily involve T-cells, immune cells that target and destroy cancer cells. While these drugs can lead to remarkable and sustained response, most patients do not respond. Understanding the resistance mechanisms in non-responding tumors is now an active area of investigation. In this review, we explore a host of factors in the tumor microenvironment, the cellular and molecular space within tumor tissue, to identify possible culprits of immunotherapy resistance. Specifically, the downstream effects of low oxygen and metabolic byproducts in the tumor microenvironment have been associated with immune cell dysfunction. Importantly, targeting these pathways may offer promising therapies to improve the response to current immunotherapy. Abstract Immunotherapy (IMT) is now a core component of cancer treatment, however, many patients do not respond to these novel therapies. Investigating the resistance mechanisms behind this differential response is now a critical area of research. Immune-based therapies, particularly immune checkpoint inhibitors (ICI), rely on a robust infiltration of T-cells into the tumor microenvironment (TME) for an effective response. While early efforts relied on quantifying tumor infiltrating lymphocytes (TIL) in the TME, characterizing the functional quality and degree of TIL exhaustion correlates more strongly with ICI response. Even with sufficient TME infiltration, immune cells face a harsh metabolic environment that can significantly impair effector function. These tumor-mediated metabolic perturbations include hypoxia, oxidative stress, and metabolites of cellular energetics. Primarily through HIF-1-dependent processes, hypoxia invokes an immunosuppressive phenotype via altered molecular markers, immune cell trafficking, and angiogenesis. Additionally, oxidative stress can promote lipid peroxidation, ER stress, and Treg dysfunction, all associated with immune dysregulation. Finally, the metabolic byproducts of lipids, amino acids, glucose, and cellular energetics are associated with immunosuppression and ICI resistance. This review will explore these biochemical pathways linked to immune cell dysfunction in the TME and highlight potential adjunctive therapies to be used alongside current IMT.

Aim: To describe treatment patterns among patients with stage III melanoma who underwent surgical excision in years 2011–2016, and assess outcomes among patients who subsequently received systemic adjuvant therapy versus watch-and-wait. Methods: Chart review of 380 patients from 17 melanoma centers in North America, South America and Europe. Results: Of 129 (34%) patients treated with adjuvant therapy, 85% received interferon α-2b and 56% discontinued treatment (mostly due to adverse events). Relapse-free survival was significantly longer for patients treated with adjuvant therapy versus watch-and-wait (hazard ratio = 0.63; p < 0.05). There was considerable heterogeneity in adjuvant treatment schedules and doses. Similar results were found in patients who received interferon-based adjuvant therapy. Conclusion: Adjuvant therapies with better safety/efficacy profiles will improve clinical outcomes in patients with stage III melanoma.

9572 Background: Retrospective studies suggest that various med could dichotomous effects in regards to immunotherapy. These include adverse (antibiotics) and positive (aspirin, beta-blockers) influences. To evaluate potential additive or detrimental effects of various med in patients (pts) receiving PD-1 immunotherapy, we performed a retrospective evaluation of med intake in 172 pts with stage IV cutaneous MEL focusing on aspirin (asp), antacid (ant), antibiotic (abx), bisphosphonate (bisp), metformin (met) and statin (stat) intake. Methods: Pts with stage IV cutaneous MEL who received anti PD-1 therapy at the University of Pittsburgh between 2014-2018 were included in this analysis. PD-1 blockade was continued until progression or intolerable toxicity. Tumor assessment was performed at baseline and every 12 weeks and response classified per RECIST v1.1. Clinical and demographic data were obtained. Med intake was documented based on chart review in all pts. Intake was confirmed by analyzing at least one other note from a non-oncological provider. Descriptive statistics were created for all covariates. Kaplan Meier and Cox proportional hazard regression were performed to assess how categorical variables related to response (ORR), overall survival (OS) and progression free survival (PFS) measured in months (mths). Results: 172 pts with advanced MEL were evaluated. Asp, ant, abx, bisp, met and stat use was documented in 62, 82, 29, 4, 15 and 57 pts respectively. ORR was not significantly related to intake of asp, ant, bisp, met and stat use; although ORR was lower in pts who received abx (p=0.0328). There was no significant difference in PFS and OS in pts who received asp, ant, bisp, met and stat. In patients who received abx compared to those who did not, median PFS (16.6 mths vs. 19.8 mths) and median OS (23.8 mths vs. 35.4) were both lower. Abx use did not interact with other meds. Conclusions: In this retrospective series of advanced MEL pts treated with PD-1 blockade, abx use was adversely associated with response to PD-1 blockade. Abx use was also associated with poorer PFS and OS. Conversely, neither a positive nor negative association with ORR, PFS and/or OS was seen with asp, ant, bisp, met and stat use. These results validate prior studies suggesting that abx use is associated with worse outcomes in pts receiving PD-1 blockade possibly by mediating intestinal dysbiosis.

Background In a previously reported study, patients with regionally advanced melanoma were treated with neoadjuvant ipilimumab (ipi) (Tarhini in PLoS ONE 9(2): e87705, 3). Significant changes in circulating myeloid derived suppressor cells (MDSC), regulatory T cells (Treg) and peptide specific type I CD4+ and CD8+ T cells were noted at week 6 that correlated with clinical outcome. Characterization of antigen-specific effector T cell secreted cytokines may shed insights into ipi associated T cell activation and function. Methods Patients were treated with neoadjuvant ipi (10 mg/kg every 3 weeks ×2) administered intravenously before and after surgery. Peripheral blood mononuclear cells (PBMC) that were collected at baseline and week 6 (after ipi) were tested here. Each sample was divided into 5 groups and stimulated with controls or shared melanoma antigen overlapping peptide pools (NY-ESO 1, gp-100, MART-1). Secreted cytokines, chemokines and growth factors were assessed using Luminex. Cytokine expression levels between the 3 antigen groups were compared using the Wilcox rank-sum test. Results Seventeen cytokines were differentially expressed with stimulation by each antigen at baseline (p value

There will be an estimated 76,100 new cases of melanoma diagnosed in 2015 and 9710 deaths. Patients with stage I/II disease have excellent outcomes, and the treatment landscape for patients with metastatic disease has been transformed by the approval of several immune checkpoint inhibitors and molecular targeted therapies. Patients with stage III disease, however, continue to have very limited options, as the only agent shown to improve survival in the adjuvant setting is high-dose IFN-α. Neoadjuvant trials of chemotherapy and chemobiotherapy have not been successful, and while neoadjuvant ipilimumab and high-dose interferon have shown promise in small trials, neither agent has been approved. Current trials are testing immune therapy and targeted therapy combinations in the neoadjuvant setting.

The discovery of the molecular mechanisms underlying development of renal cell carcinoma have allowed for the development of novel targeted therapy for treatment of this disease. Recently, multiple agents have become approved by regulatory authorities for the treatment of advanced renal cell carcinoma, including sunitinib, sorafenib, bevacizumab (with interferon alpha), pazopanib, temsirolimus and everolimus. While these therapies have generated excitement and have clearly altered the treatment paradigm, multiple limitations have been elucidated over time. These include but are not limited to the fact that treatment is not associated with complete responses, a significant number of patients are primarily refractory to treatment, and clinical trials mostly include clear cell histology. Furthermore, the role of these therapies in the treatment of brain metastases remains unclear and therapies can have considerable toxicities. RECIST criteria (Response Evaluation Criteria In Solid Tumors) can be inadequate for the assessment of these modalities’ treatment efficacy, and biomarkers predictive of individual patient benefit have been elusive. This review summarizes the major clinical data and discusses these limitations.

e21507Background: Prior reports have described an association with hepatic metastases and total TB with reduced response rates to anti-PD-1 treatment in subjects with advanced MEL. We explored base...

Myeloid derived suppressor cells (MDSC, CD33+CD11b+ HLA-DR low/-) play a major role in tumor-mediated immune evasion and are composed of at least 3 subsets PMN (CD15+), monocytic (CD14+) and lineage-negative (CD15-CD14-), and each has been shown to be significantly increased in some human tumor types and to correlate with metastatic burden, clinical cancer stage and outcome. Less in known about the MDSC subsets that accumulate in tumors such as renal cell carcinoma (RCC) and the cytokines/chemokines involved in their recruitment. Flow cytometry analysis of peripheral blood mononuclear cells (PBMC, n = 20) and nephrectomy samples (n = 39, stage 1-4) showed increased levels of total MDSC in RCC patients compared to normal controls (n = 15), with PMN- and Lin- MDSC subsets dominating in the blood and tumor of RCC patients. Blood levels of total MDSC, PMN-MDSC and Lin-MDSC correlated with tumor grade (p = 0.026, p = 0.006 and p = 0.045, respectively), while blood levels of total MDSC and Lin-MDSC correlated with progression free survival (PFS) in patients with limited stage disease (n = 16, stages 1-3) (HR = 1.35, p = 0.03; HR = 1.45, p = 0.02, respectively). In the tumor, higher PMN-MDSC levels were significantly associated with decreased PFS (n = 29, HR = 1.09, p = 0.011). To assess the role of select chemokines (IL-8, CXCL5, Mip-1α, MCP-1 and Rantes) and of the pro-inflammatory cytokine IL-1β in promoting the accumulation of MDSC within the tumor, these proteins were quantitated in tumor lysates by ELISA and correlated to MDSC frequencies (Spearman correlations). We found a direct correlation between the frequency of PMN-MDSC in the parenchyma and the levels of IL-8 (p < 0.001), CXCL-5 (p < 0.001), and IL-1β (p = 0.029). Frequency of parenchymal Lin- MDSC directly correlated with levels of IL-8 (p = 0.033) and CXCL-5 (p = 0.008), but not IL-1β. In circulation, frequency of total MDSCs directly correlated with IL-1β plasma levels (p = 0.003). To further define the role of IL-1β in MDSC accumulation within tumors, we overexpressed IL-1β in RENCA and CT26 tumors and compared them to untransfected tumors. Overexpression of IL-1β resulted in enhanced tumor growth and increased frequency of intratumor PMN-MDSC (10.3X in RENCA and 26X in CT26), with a modest increase in intratumor M-MDSC. A large fraction of tumor infiltrating PMN-MDSC expressed CXCR2 (84% in RENCA and 55% in CT26), which is associated with a significant increase in expression of CXCR2 ligands (KC, CXCL5, and MIP2). These results support the idea that IL-1β-mediated induction of select chemokines promotes the accumulation of MDSC, particularly PMN-MDSC, within tumors, resulting in enhanced immune suppression and angiogenesis.

Tumors escape immune recognition by several mechanisms, and induction of myeloid derived suppressor cells (MDSC) is thought to play a major role in tumor mediated immune evasion. MDSC arise from myeloid progenitor cells that do not differentiate into mature dendritic cells, granulocytes, or macrophages, and are characterized by the ability to suppress T cell and natural killer cell function. They are increased in patients with cancer including renal cell carcinoma (RCC), and their levels have been shown to correlate with prognosis and overall survival. Multiple methods of inhibiting MDSCs are currently under investigation. These can broadly be categorized into methods that (a) promote differentiation of MDSC into mature, non-suppressive cells (all trans retinoic acid, vitamin D), (b) decrease MDSC levels (sunitinib, gemcitabine, 5-FU, CDDO-Me), or (c) functionally inhibit MDSC (PDE-5 inhibitors, cyclooxygenase 2 inhibitors). Recently, several pre-clinical tumor models of combination therapy involving sunitinib plus vaccines and/or adoptive therapy have shown promise in MDSC inhibition and improved outcomes in the tumor bearing host. Current clinical trials are underway in RCC patients to assess not only the impact on clinical outcome, but how this combination can enhance anti-tumor immunity and reduce immune suppression. Decreasing immune suppression by MDSC in the cancer host may improve outcomes and prolong survival in this patient population.

Programmed death-1 receptor (PD-1) is expressed on T cells following TCR activation. Binding of this receptor to its cognate ligands, programmed death ligand (PDL)-1 and PDL-2, down-regulates signals by the TCR, promoting T-cell anergy and apoptosis, thus leading to immune suppression. Here, we find that using an anti-PD-1 antibody (CT-011) with Treg-cell depletion by low-dose cyclophosphamide (CPM), combined with a tumor vaccine, induces synergistic antigen-specific immune responses and reveals novel activities of each agent in this combination. This strategy led to complete regression of established tumors in a significant percentage of treated animals, with survival prolongation. We show for the first time that combining CT-011 and CPM significantly increases the number of vaccine-induced tumor-infiltrating CD8(+) T cells, with simultaneous decrease in infiltrating Treg cells. Interestingly, we find that CT-011 prolongs Treg-cell inhibition induced by CPM, leading to a sustainable significant synergistic decrease of splenic and tumor-infiltrated Treg cells. Surprisingly, we find that the anti-tumor effect elicited by the combination of CT-011 and CPM is dependent on both CD8(+) and CD4(+) T-cell responses, although the antigen we used is a class I MHC-restricted peptide. Thus, we describe a novel and effective therapeutic approach by combining multiple strategies to target several tumor-mediated immune inhibitory mechanisms.

A 55-year-old woman was admitted to our hospital with a 10-day history of right arm weakness and numbness. The patient's medical history was notable for lobular carcinoma in situ of the right breast in 2008 and stage I diffuse large B-cell lymphoma of the left axilla. The patient had completed treatment with chemotherapy and radiation 2 months prior to presentation. Blood counts, metabolic panel and lumbar puncture were unremarkable. MRI of the brain revealed multiple enhancing masses. The patient was started on dexamethasone, with rapid symptom improvement. A stereotactic brain biopsy revealed CD20 diffuse large B-cell lymphoma. The patient was started on high-dose intravenous methotrexate. She has received 11 cycles and has regained near normal function of the right arm. The patient's most recent brain MRI showed near complete resolution of all previously seen abnormal foci of enhancement.

A significant barrier to tumor regression in the setting of a successful immune response is tumor-mediated immunosuppression. Programmed death receptor-1 (PD-1) is an important signaling molecule and in the context of the tumor microenvironment may inhibit T cell immunity when bound to its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), by inducing T cell apoptosis and anergy. Recently we demonstrated that combination of anti-PD-1 antibody with vaccine and low-dose of cyclophosphamide (CPM), that reduces the number and function of Treg cells, significantly enhances antigen-specific immune responses, decreases tumor burden and increases survival of treated mice, suggesting that targeting multiple immune checkpoint inhibitors is a promising strategy for tumor immunotherapy. Here we tested the anti-tumor immune effect of B7-DC-Ig, a recombinant protein that binds and target PD-1. It is composed of the extracellular domain of B7-DC, fused to the Fc portion of murine IgG2a. We demonstrated that B7-DC-Ig can enhance the therapeutic efficacy of a peptide based cancer vaccine when combined with CPM. We showed that this combination significantly enhances antigen-specific immune responses and leads to complete eradication of established tumors in 60% of mice and that this effect is CD8 dependent. Importantly, we found that unlike anti-PD-1 antibody, B7-DC-Ig does not block the suppressive PD-1/PD-L1 interaction as its primary mechanism, and identified a novel mechanism by which B7-DC-Ig exerts its therapeutic effect. We demonstrate, for the first time, that there are significant differences between levels and timing of surface PD-1 expression on different T cell subsets. We found that these differences play a critical role in the anti-tumor effects exhibited by B7-DC-Ig through inhibiting proliferation of dysfunctional PD-1high CD4 T cells, for both Treg and FoxP3−T cells, leading to a significant decrease in levels of tumor infiltration by dysfunctional T cells. In addition, we showed that treatment with B7-DC-Ig also leads to a decrease in PD-1highCD8 T cells, tipping the balance towards non-exhausted, functional PD-1lowCD8 T cells. Aside from presenting a promising anti-tumor immunotherapeutic approach, we demonstrated that the level of PD-1 expression on different T cells subsets determines the “effective target zone” of B7-DC-Ig. We believe that this represents an important parameter that needs to be considered to favorably alter the tumor microenvironment when designing PD-1 targeting immune therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-491. doi:1538-7445.AM2012-LB-491