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In patients with melanoma brain metastases (MBM), a combination of radiotherapy (RT) with immune checkpoint inhibitors (ICI) is routinely used. However, the best sequence of radio-immunotherapy (RIT) remains unclear. In an exploratory phase 2 trial, MBM patients received RT (stereotactic or whole-brain radiotherapy depending on the number of MBM) combined with ipilimumab (ipi) ± nivolumab (nivo) in different sequencing (Rad-ICI or ICI-Rad). Comparators arms included patients treated with ipi-free systemic treatment or without RT (in MBM-free patients). The primary endpoints were radiological and immunological responses in the peripheral blood. Secondary endpoints were progression-free survival (PFS) and overall survival (OS). Of 106 screened, 92 patients were included in the study. Multivariate analysis revealed an advantage for patients starting with RT (Rad-ICI) for overall response rate (RR: p = .007; HR: 7.88 (95%CI: 1.76-35.27)) and disease control rate (DCR: p = .036; HR: 6.26 (95%CI: 1.13-34.71)) with a trend for a better PFS (p = .162; HR: 1.64 (95%CI: 0.8-3.3)). After RT plus two cycles of ipi-based ICI in both RIT sequences, increased frequencies of activated CD4, CD8 T cells and an increase in melanoma-specific T cell responses were observed in the peripheral blood. Lasso regression analysis revealed a significant clinical benefit for patients treated with Rad-ICI sequence and immunological features, including high frequencies of memory T cells and activated CD8 T cells in the blood. This study supports increasing evidence that sequencing RT followed by ICI treatment may have better effects on the immunological responses and clinical outcomes in MBM patients., Competing Interests: TES, HS, JM, DM, KL, MFH, IH, CK, RS, and PB declare no competing interests. JCH received scientific grant support from BMS; honoraria for talks from Almirall, Amgen, BMS, GSK, MSD, Novartis, Pfizer, Pierre Fabre, Roche, Sanofi; advisory board member for MSD, Pierre Fabre; travel grants from BMS, Immunocore, 4SC. MS received honoraria for talks from Novartis; travel grants from Merck, Abbvie, Novartis, Sanofi-Aventis, BMS, Merck Sharp & Dome, Pfizer. CM received a fellowship from the German Research Foundation (DFG) (ME 5482/1-1) 2/2020-5/2021. LK reports personal fees from Accuray Inc., and Novocure GmbH. AE received honoraria for Biotest AG, Meet the Experts, Janssen-Cilag, Klinikum Minden; consulting fees for Biotest AG, MSD, Galderma Laboratorium, Janssen-Cilag, Roche, AbbVie; advisory board member for MSD, Biotest. JD received grants from Viewray Inc, Accuray International Sari, RaySearch Laboratories AB, Vision RT Limited, Astellas Pharma GmbH, Siemens Healthcare GmbH, Solution Akademie GmbH, Egomed PLC Surrey Research Park, Quintiles GmbH, Pharmaceutical Research Associates GmbH, Boehringer Ingelheim Pharma GmbH&CoKG, PTW-Freiburg Dr. Pychlau GmbH, Nanobiotix S.A., Accuray Incorporated., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Purpose: Immunotherapy with ipilimumab improves the survival of patients with metastatic melanoma. Because only around 20% of patients experience long-term benefit, reliable markers are needed to predict a clinical response. Therefore, we sought to determine if some myeloid cells and related inflammatory mediators could serve as predictive factors for the patients' response to ipilimumab., Experimental Design: We performed an analysis of myeloid cells in the peripheral blood of 59 stage IV melanoma patients before the treatment and at different time points upon the therapy using a clinical laboratory analysis and multicolor flow cytometry. In addition, the production of related inflammatory factors was evaluated by ELISA or Bio-Plex assays., Results: An early increase in eosinophil count during the treatment with ipilimumab was associated with an improved clinical response. In contrast, elevated amounts of monocytic myeloid-derived suppressor cells (moMDSC), neutrophils, and monocytes were found in nonresponders (n = 36) as compared with basal levels and with responding patients (n = 23). Moreover, in nonresponders, moMDSCs produced significantly more nitric oxide, and granulocytic MDSCs expressed higher levels of PD-L1 than these parameters at baseline and in responders, suggesting their enhanced immunosuppressive capacity. Upon the first ipilimumab infusion, nonresponders displayed elevated serum concentrations of S100A8/A9 and HMGB1 that attract and activate MDSCs., Conclusions: These findings highlight additional mechanisms of ipilimumab effects and suggest levels of eosinophils, MDSCs, as well as related inflammatory factors S100A8/A9 and HMGB1 as novel complex predictive markers for patients who may benefit from the ipilimumab therapy. Clin Cancer Res; 21(24); 5453-9. ©2015 AACR., (©2015 American Association for Cancer Research.)

Endogenous tumor-specific T cells are detectable in patients with different tumor types including malignant melanoma (MM). They can control tumor growth, have impact on patient survival and correlate with improved clinical response to immune checkpoint therapy. Thus, they may represent a potent biomarker for respective treatment decisions. So far, major target antigens of endogenous MM-reactive T cells have not been determined systematically. Instead, autoantibodies are discussed as surrogate parameter for MM-specific T cells. Throughout a period of more than 60 days after tumor resection, we therefore determined in 38 non-metastasized primary MM patients and in healthy individuals by IFNγ ELISpot and bead-based fluorescent multiplex assay major target antigens of spontaneous T cell and humoral responses using a broad panel of MM antigens and assessed the presence and suppressive impact of MM-reactive regulatory T cells (Tregs). We show that MM-reactive T cells are frequent in MM patients, transiently increase after tumor removal and are mostly directed against Melan-A/MART-1, Tyrosinase, NA17-A and p53. MM-specific Tregs were only detected in few patients and inhibited MM-reactive T cells particularly early after tumor resection. Tumor-specific autoantibodies occurred in most patients, but did not correlate with T cell responses. Thus, endogenous antibodies may not be reliable surrogate parameters of MM-reactive T cells.

Tumor-associated eosinophilia is frequently observed in cancer. However, despite numerous studies of patients with cancer and mouse models of cancer, it has remained uncertain if eosinophils contribute to tumor immunity or are mere bystander cells. Here we report that activated eosinophils were essential for tumor rejection in the presence of tumor-specific CD8(+) T cells. Tumor-homing eosinophils secreted chemoattractants that guided T cells into the tumor, which resulted in tumor eradication and survival. Activated eosinophils initiated substantial changes in the tumor microenvironment, including macrophage polarization and normalization of the tumor vasculature, which are known to promote tumor rejection. Thus, our study presents a new concept for eosinophils in cancer that may lead to novel therapeutic strategies.

Chronic inflammation is considered to be one of the hallmarks for tumor initiation and progression. Moreover, a long-term production and accumulation of inflammatory factors lead to a local and systemic immunosuppression associated with cancer progression. However, the correlation between inflammatory mediators, immunosuppressive cells and the clinical outcome of malignant melanoma patients was poorly investigated. In this study, we performed a complex analysis of various inflammatory factors, myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in the peripheral blood of patients suffering from malignant melanoma of different stages. We demonstrated that levels of serum IL-1β, IFN-γ and CXCL10 were significantly increased in advanced melanoma patients. In addition, these factors were found to be associated with an increased frequency of MDSCs and Tregs as compared to age- and gender-matched healthy donors. Importantly, advanced melanoma patients with signs of progression displayed markedly elevated concentrations of IL-1β and CXCL10 as compared to patients with stable disease. Moreover, an enrichment of circulating monocytic (Mo)-MDSCs significantly correlated with a decreased progression free survival of these patients. Our data highlight a complex association between circulating inflammatory mediators, Mo-MDSCs and the clinical outcome as well as suggest that their levels in patients with advanced melanoma are of important prognostic value allowing the identification of those with high risk of disease progression., (© 2014 UICC.)

Tumor-mediated procoagulatory activity leads to venous thromboembolism and supports metastasis in cancer patients. A prerequisite for metastasis formation is the interaction of cancer cells with endothelial cells (ECs) followed by their extravasation. Although it is known that activation of ECs and the release of the procoagulatory protein von Willebrand factor (VWF) is essential for malignancy, the underlying mechanisms remain poorly understood. We hypothesized that VWF fibers in tumor vessels promote tumor-associated thromboembolism and metastasis. Using in vitro settings, mouse models, and human tumor samples, we showed that melanoma cells activate ECs followed by the luminal release of VWF fibers and platelet aggregation in tumor microvessels. Analysis of human blood samples and tumor tissue revealed that a promoted VWF release combined with a local inhibition of proteolytic activity and protein expression of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type I repeats 13) accounts for this procoagulatory milieu. Blocking endothelial cell activation by the low-molecular-weight heparin tinzaparin was accompanied by a lack of VWF networks and inhibited tumor progression in a transgenic mouse model. Our findings implicate a mechanism wherein tumor-derived vascular endothelial growth factor-A (VEGF-A) promotes tumor progression and angiogenesis. Thus, targeting EC activation envisions new therapeutic strategies attenuating tumor-related angiogenesis and coagulation., (© 2015 by The American Society of Hematology.)

The frequency and function of regulatory T cells (Tregs) were studied in stage II-III melanoma patients who were enrolled in a phase II randomized trial of vaccination with HLA-A*0201-modified tumor peptides versus observation. The vaccinated patients received low-dose cyclophosphamide (CTX) and low-dose interleukin-2 (IL-2). Tregs were analyzed in the lymph nodes (LNs) of stage III patients who were undergoing complete lymph node dissection and in peripheral blood mononuclear cells (PBMCs) collected before vaccination and at different time points during the vaccination period. The LNs of the vaccinated patients, which were surgically removed after two rounds of vaccination and one dose of CTX, displayed a low frequency of Tregs and a less immunosuppressive environment compared with those of the untreated patients. The accurate time-course analysis of the PBMCs of patients enrolled in the vaccination arm indicated a limited and transient modulation in the frequencies of Tregs in PBMCs collected after low-dose CTX administration and a strong Treg boost in those PBMCs collected after low-dose IL-2 administration. However, a fraction of the IL-2-boosted Tregs was functionally modulated to a Th-1-like phenotype in the vaccinated patients. Moreover, low-dose IL-2 promoted the concomitant expansion of conventional activated CD4(+) T cells. Despite the amplification of Tregs, IL-2 administration maintained or further increased the number of antigen-specific CD8(+) T cells that were induced by vaccination as demonstrated by the ex vivo human leukocyte antigen-multimer staining and IFN-γ ELISpot assays. Our study suggests that the use of CTX as a Treg modulator should be revised in terms of the administration schedule and of patients who may benefit from this drug treatment. Despite the Treg expansion that was observed in this study, low-dose IL-2 is not detrimental to the functional activities of vaccine-primed CD8(+) T cell effectors when used in the inflammatory environment of vaccination.

The antitumor effects of paclitaxel are generally attributed to the suppression of microtubule dynamics resulting in defects in cell division. New data demonstrated that in ultralow noncytotoxic concentrations, paclitaxel modulated in immune cells in vitro the activity of small Rho GTPases, the key regulators of intracellular actin dynamics. However, the immunomodulatory properties of paclitaxel in vivo have not been evaluated. In this study, using the ret transgenic murine melanoma model, which mimics human cutaneous melanoma, we tested effects of ultralow noncytotoxic dose paclitaxel on functions of myeloid-derived suppressor cells (MDSCs), chronic inflammatory mediators, and T cell activities in the tumor microenvironment in vivo. Administration of paclitaxel significantly decreased accumulation and immunosuppressive activities of tumor-infiltrating MDSCs without alterations of the bone marrow hematopoiesis. This was associated with the inhibition of p38 MAPK activity, TNF-α and production, and S100A9 expression in MDSCs. The production of mediators of chronic inflammation in the tumor milieu also was diminished. Importantly, reduced tumor burden and increased animal survival upon paclitaxel application was mediated by the restoration of CD8 T cell effector functions. We suggest that the ability of paclitaxel in a noncytotoxic dose to block the immunosuppressive potential of MDSCs in vivo represents a new therapeutic strategy to downregulate immunosuppression and chronic inflammation in the tumor microenvironment for enhancing the efficacy of concomitant anticancer therapies.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Using IFN-gamma enzyme-linked immunospot, we investigated reactivity of T cells from bone marrow and peripheral blood to melanoma lysate-pulsed autologous dendritic cells in 40 melanoma patients. Melanoma-reactive T cells were present in the bone marrow of seven patients and in peripheral blood of four patients. In the bone marrow, melanoma-reactive T cells were present in 6 of 21 stage IV patients and in 1 of 10 stage III patients, whereas none were detected in stage I to II patients (0 of 9). The occurrence of tumor-reactive bone marrow T cells in melanoma patients was associated with advanced disease stage, disease duration and tumor load, and independent of treatment. These findings provide new insights into the generation of T-cell responses in melanoma patients.