Immune monitoring in mesothelioma patients identifies novel immune-modulatory functions of gemcitabine associating with clinical response.

BACKGROUND: Gemcitabine is a frequently used chemotherapeutic agent but its effects on the immune system are incompletely understood. Recently, the randomized NVALT19-trial revealed that maintenance gemcitabine after first-line chemotherapy significantly prolonged progression-free survival (PFS) compared to best supportive care (BSC) in malignant mesothelioma. Whether these effects are paralleled by changes in circulating immune cell subsets is currently unknown. These analyses could offer improved mechanistic insights into the effects of gemcitabine on the host and guide development of effective combination therapies in mesothelioma. METHODS: We stained peripheral blood mononuclear cells (PBMCs) and myeloid-derived suppressor cells (MDSCs) at baseline and 3 weeks following start of gemcitabine or BSC treatment in a subgroup of mesothelioma patients included in the NVALT19-trial. In total, 24 paired samples including both MDSCs and PBMCs were included. We performed multicolour flow-cytometry to assess co-inhibitory and-stimulatory receptor- and cytokine expression and matched these parameters with PFS and OS. FINDINGS: Gemcitabine treatment was significantly associated with an increased NK-cell- and decreased T-regulatory cell proliferation whereas the opposite occurred in control patients. Furthermore, myeloid-derived suppressor cells (MDSCs) frequencies were lower in gemcitabine-treated patients and this correlated with increased T-cell proliferation following treatment. Whereas gemcitabine variably altered co-inhibitory receptor expression, co-stimulatory molecules including ICOS, CD28 and HLA-DR were uniformly increased across CD4+ T-helper, CD8+ T- and NK-cells. Although preliminary in nature, the increase in NK-cell proliferation and PD-1 expression in T cells following gemcitabine treatment was associated with improved PFS and OS. INTERPRETATION: Gemcitabine treatment was associated with widespread effects on circulating immune cells of mesothelioma patients with responding patients displaying increased NK-cell and PD-1 + T-cell proliferation. These exploratory data provide a platform for future on treatment-biomarker development and novel combination treatment strategies.

Induction of Peripheral Effector CD8 T-cell Proliferation by Combination of Paclitaxel, Carboplatin, and Bevacizumab in Non-small Cell Lung Cancer Patients.

PURPOSE: Chemotherapy has long been the standard treatment for advanced stage non-small cell lung cancer (NSCLC), but checkpoint inhibitors are now approved for use in several patient groups and combinations. To design optimal combination strategies, a better understanding of the immune-modulatory capacities of conventional treatments is needed. Therefore, we investigated the immune-modulatory effects of paclitaxel/carboplatin/bevacizumab (PCB), focusing on the immune populations associated with the response to checkpoint inhibitors in peripheral blood. EXPERIMENTAL DESIGN: A total of 223 patients with stage IV NSCLC, enrolled in the NVALT12 study, received PCB, with or without nitroglycerin patch. Peripheral blood was collected at baseline and after the first and second treatment cycle, proportions of T cells, B cells, and monocytes were determined by flow cytometry. Furthermore, several subsets of T cells and the expression of Ki67 and coinhibitory receptors on these subsets were determined. RESULTS: Although proliferation of CD4 T cells remained stable following treatment, proliferation of peripheral blood CD8 T cells was significantly increased, particularly in the effector memory and CD45RA+ effector subsets. The proliferating CD8 T cells more highly expressed programmed death receptor (PD)-1 and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) compared with nonproliferating CD8 T cells. Immunologic responders (iR; >2 fold increased proliferation after treatment) did not show an improved progression-free (PFS) or overall survival (OS). CONCLUSIONS: Paclitaxel/carboplatin/bevacizumab induces proliferation of CD8 T cells, consisting of effector cells expressing coinhibitory checkpoint molecules. Induction of proliferation was not correlated to clinical outcome in the current clinical setting. Our findings provide a rationale for combining PCB with checkpoint inhibition in lung cancer.

Autologous Dendritic Cell Therapy in Mesothelioma Patients Enhances Frequencies of Peripheral CD4 T Cells Expressing HLA-DR, PD-1, or ICOS.

Introduction: Malignant pleural mesothelioma (MPM) is a malignancy with a very poor prognosis for which new treatment options are urgently needed. We have previously shown that dendritic cell (DC) immunotherapy provides a clinically feasible treatment option. In the current study, we set out to assess the immunological changes induced by DC immunotherapy in peripheral blood of MPM patients. Methods: Peripheral blood was collected from nine patients enrolled in a phase I dose escalation study, before and after treatment with DCs that were pulsed with an allogeneic tumor lysate preparation consisting of a mixture of five cultured mesothelioma cell lines. We used immune profiling by multiplex flow cytometry to characterize different populations of immune cells. In particular, we determined frequencies of T cell subsets that showed single and combinatorial expression of multiple markers that signify T cell activation, maturation and inhibition. Therapy-induced T cell reactivity was assessed in peptide/MHC multimer stainings using mesothelin as a prototypic target antigen with confirmed expression in the clinical tumor lysate preparation. T cell receptor (TCR) diversity was evaluated by TCRB gene PCR assays. Results: We observed an increase in the numbers of B cells, CD4 and CD8 T cells, but not NK cells at 6 weeks post-treatment. The increases in B and T lymphocytes were not accompanied by major changes in T cell reactivity toward mesothelin nor in TCRB diversity. Notably, we did observe enhanced proportions of CD4 T cells expressing HLA-DR, PD-1 (at 2 weeks after onset of treatment) and ICOS (6 weeks) and a CD8 T cell population expressing LAG3 (2 weeks). Discussion: DC immunotherapy using allogeneic tumor lysate resulted in enhanced frequencies of B cells and T cells in blood. We did not detect a skewed antigen-reactivity of peripheral CD8 T cells. Interestingly, frequencies of CD4 T cells expressing activation markers and PD-1 were increased. These findings indicate a systemic activation of the adaptive immune response and may guide future immune monitoring studies of DC therapies.

Autologous Dendritic Cells Pulsed with Allogeneic Tumor Cell Lysate in Mesothelioma: From Mouse to Human.

Purpose: Mesothelioma has been regarded as a nonimmunogenic tumor, which is also shown by the low response rates to treatments targeting the PD-1/PD-L1 axis. Previously, we demonstrated that autologous tumor lysate-pulsed dendritic cell (DC) immunotherapy increased T-cell response toward malignant mesothelioma. However, the use of autologous tumor material hampers implementation in large clinical trials, which might be overcome by using allogeneic tumor cell lines as tumor antigen source. The purpose of this study was to investigate whether allogeneic lysate-pulsed DC immunotherapy is effective in mice and safe in humans.Experimental Design: First, in two murine mesothelioma models, mice were treated with autologous DCs pulsed with either autologous or allogeneic tumor lysate or injected with PBS (negative control). Survival and tumor-directed T-cell responses of these mice were monitored. Results were taken forward in a first-in-human clinical trial, in which 9 patients were treated with 10, 25, or 50 million DCs per vaccination. DC vaccination consisted of autologous monocyte-derived DCs pulsed with tumor lysate from five mesothelioma cell lines.Results: In mice, allogeneic lysate-pulsed DC immunotherapy induced tumor-specific T cells and led to an increased survival, to a similar extent as DC immunotherapy with autologous tumor lysate. In the first-in-human clinical trial, no dose-limiting toxicities were established and radiographic responses were observed. Median PFS was 8.8 months [95% confidence interval (CI), 4.1-20.3] and median OS not reached (median follow-up = 22.8 months).Conclusions: DC immunotherapy with allogeneic tumor lysate is effective in mice and safe and feasible in humans. Clin Cancer Res; 24(4); 766-76. ©2017 AACR.

Depletion of Tumor-Associated Macrophages with a CSF-1R Kinase Inhibitor Enhances Antitumor Immunity and Survival Induced by DC Immunotherapy.

New immunotherapeutic strategies are needed to induce effective antitumor immunity in all cancer patients. Malignant mesothelioma is characterized by a poor prognosis and resistance to conventional therapies. Infiltration of tumor-associated macrophages (TAM) is prominent in mesothelioma and is linked to immune suppression, angiogenesis, and tumor aggressiveness. Therefore, TAM depletion could potentially reactivate antitumor immunity. We show that M-CSFR inhibition using the CSF-1R kinase inhibitor PLX3397 (pexidartinib) effectively reduced numbers of TAMs, circulating nonclassical monocytes, as well as amount of neoangiogenesis and ascites in mesothelioma mouse models, but did not improve survival. When combined with dendritic cell vaccination, survival was synergistically enhanced with a concomitant decrease in TAMs and an increase in CD8+ T-cell numbers and functionality. Total as well as tumor antigen-specific CD8+ T cells in tumor tissue of mice treated with combination therapy showed reduced surface expression of the programmed cell death protein-1 (PD-1), a phenomenon associated with T-cell exhaustion. Finally, mice treated with combination therapy were protected from tumor rechallenge and displayed superior T-cell memory responses. We report that decreasing local TAM-mediated immune suppression without immune activation does not improve survival. However, combination of TAM-mediated immune suppression with dendritic cell immunotherapy generates robust and durable antitumor immunity. These findings provide insights into the interaction between immunotherapy-induced antitumor T cells and TAMs and offer a therapeutic strategy for mesothelioma treatment. Cancer Immunol Res; 5(7); 535-46. ©2017 AACR.

Precision immunotherapy; dynamics in the cellular profile of pleural effusions in malignant mesothelioma patients.

OBJECTIVES: Clinical studies have proven the potential of immunotherapy in malignancies. To increase efficacy, a prerequisite is that treatment is tailored, so precision immune-oncology is the logical next step. In order to tailor treatment, characterization of the patient's tumor environment is key. Pleural effusion (PE) often accompanies malignant pleural mesothelioma (MPM) and is an important part of the MPM environment. Furthermore, the composition of PE is used as surrogate for the tumor. In this study, we provide an insight in the dynamics of the MPM environment through characterization of PE composition over time and show that the immunological characteristics of PE do not necessarily mirror those of the tumor. MATERIALS AND METHODS: From 5 MPM patients, PE and tumor biopsies were acquired at the same time point. From one of these patients multiple PEs were obtained. PEs were acquired performing thoracocenteses and total cell amounts were determined. Immunohistochemistry was performed to quantify immune cell composition (T cells, macrophages) and tumor cells in PE derived cytospins and tumor biopsies. RESULTS: The PE amount and (immune) cellular composition varied considerably over time between multiple (n=10) thoracocenteses. These dynamics could in part be attributed to the treatment regimen consisting of standard chemotherapy and dendritic cell (DC)-based immunotherapy. In addition, the presence of T cells and macrophages in PE did not necessarily mirror the infiltration of these immune cells within tumor biopsies in 4 out of 5 patients. CONCLUSIONS: In this proof-of-concept study with limited sample size, we demonstrate that the composition of PE is dynamic and influenced by treatment. Furthermore, the immune cell composition of PE does not automatically reflect the properties of tumor tissue. This has major consequences when applying precision immunotherapy based on PE findings in patients. Furthermore, it implies a regulated trafficking of immune regulating cells within the tumor environment.

Pleural Effusion of Patients with Malignant Mesothelioma Induces Macrophage-Mediated T Cell Suppression.

INTRODUCTION: Clinical studies have demonstrated beneficial effects of immunotherapy in malignant pleural mesothelioma. The pleural cavity seems an attractive compartment to administer these types of therapies; however, local immunosuppressive mechanisms could hamper their efficacy. Macrophages are abundantly present within the mesothelioma microenvironment. This study investigates the influence of the macrophage phenotype, macrophages' capacity to inhibit local immune responses, and the decisive role of pleural effusion (PE) in this regard. METHODS: We cultured macrophages in the presence of PEs and investigated their phenotype. Macrophages and T cells were cocultured in the presence of PEs and tumor cell line supernatants. The levels of 11 cytokines and the prostanoid prostaglandin E2 were measured in PEs and supernatants. The presence and phenotype of macrophages and T cell subsets was measured in the PE of patients with mesothelioma. RESULTS: PE induced a tumor-promoting M2 phenotype in macrophages, which was confirmed by the suppressive activity of macrophages on T cell proliferation during coculture. Prostanoid prostaglandin E2 was identified as a potential inducer of the suppressive capacity of macrophages in PE. Macrophages isolated from PEs displayed an M2 phenotype and were negatively correlated with T cells in vivo. CONCLUSIONS: The current study demonstrates that macrophages in PE can play a pivotal role in directly hampering the antitumor T cell immune response. This emphasizes the potential of macrophages as a therapeutic target in mesothelioma and indicates that the presence and phenotype of macrophages in PE should be taken into consideration in the application of (intrapleural) immunotherapies.

Extended Tumor Control after Dendritic Cell Vaccination with Low-Dose Cyclophosphamide as Adjuvant Treatment in Patients with Malignant Pleural Mesothelioma.

RATIONALE: We demonstrated previously that autologous tumor lysate-pulsed dendritic cell-based immunotherapy in patients with malignant pleural mesothelioma is feasible, well-tolerated, and capable of inducing immunologic responses against tumor cells. In our murine model, we found that reduction of regulatory T cells with metronomic cyclophosphamide increased the efficacy of immunotherapy. OBJECTIVES: To assess the decrease in number of peripheral blood regulatory T cells during combination therapy of low-dose cyclophosphamide and dendritic cell immunotherapy and determine the induction of immunologic responses with this treatment in patients with mesothelioma. METHODS: Ten patients with malignant pleural mesothelioma received metronomic cyclophosphamide and dendritic cell-based immunotherapy. During the treatment, peripheral blood mononuclear cells were analyzed for regulatory T cells and immunologic responses. MEASUREMENTS AND MAIN RESULTS: Administration of dendritic cells pulsed with autologous tumor lysate combined with cyclophosphamide in patients with mesothelioma was safe, the only side effect being moderate fever. Dendritic cell vaccination combined with cyclophosphamide resulted in radiographic disease control in 8 of the 10 patients. Overall survival was promising, with 7 out of 10 patients having a survival of greater than or equal to 24 months and two patients still alive after 50 and 66 months. Low-dose cyclophosphamide reduced the percentage of regulatory T cells of total CD4 cells in peripheral blood from 9.43 (range, 4.34-26.10) to 4.51 (range, 0.27-10.30) after 7 days of cyclophosphamide treatment (P = 0.02). CONCLUSIONS: Consolidation therapy with autologous tumor lysate-pulsed dendritic cell-based therapy and simultaneously reducing the tumor-induced immune suppression is well-tolerated and shows signs of clinical activity in patients with mesothelioma. Clinical trial registered with www.clinicaltrials.gov (NCT 01241682).

Tumour-derived exosomes as antigen delivery carriers in dendritic cell-based immunotherapy for malignant mesothelioma.

BACKGROUND: In 2001, it was postulated that tumour-derived exosomes could be a potent source of tumour-associated antigens (TAA). Since then, much knowledge is gained on their role in tumorigenesis but only very recently tumour-derived exosomes were used in dendritic cell (DC)-based immunotherapy. For this, DCs were cultured ex-vivo and loaded with exosomes derived from immunogenic tumours such as melanoma or glioma and re-administrated to induce anti-tumour responses in primary and metastatic tumour mouse models. In contrast, malignant mesothelioma (MM) is a non-immunogenic tumour and because only a few mesothelioma-specific TAA are known to date, we investigated whether mesothelioma-derived exosomes could be used as antigen source in DC-based immunotherapy. METHODS: Mouse MM AB1 cells were used to generate tumour lysate and tumour-derived exosomes. Tumour lysate was generated by 5 cycles of freeze-thawing followed by sonication of AB1 cells. Tumour exosomes were collected from the AB1 cell culture supernatant and followed a stepwise ultracentrifugation. Protein quantification and electron microscopy were performed to determine the protein amount and to characterise their morphology. To test whether MM derived exosomes are immunogenic and able to stimulate an anti-tumoral response, BALB/c mice were injected with a lethal dose of AB1 tumour cells at day 0, followed by intraperitoneal injection of a single dose of DCs loaded with tumour exosomes, DCs loaded with tumour lysate, or phosphate buffered saline (PBS), at day 7. RESULTS: Mice which received tumour exosome-loaded DC immunotherapy had an increased median and overall survival compared to mice which received tumour lysate-loaded DC or PBS. CONCLUSION: In this study, we showed that DC immunotherapy loaded with tumour exosomes derived from non-immunogenic tumours improved survival of tumour bearing mice.