Koen Bezemer

Tumor-associated macrophages in thoracic malignancies

Tumor-associated macrophages (TAMs) can be abundantly present in numerous cancer types. Under influence of various stimuli in the tumor microenvironment TAMs develop into a tumor-inhibitory (M1) or tumor-promoting (M2) phenotype. Recently, the role of TAMs in tumor biology and their prognostic value in cancer has become a major topic of interest. In this review we will discuss the importance of TAMs in the pathogenesis and clinical outcome of lung cancer and mesothelioma patients. In addition, the potential of TAMs as therapeutic targets will be discussed.

Arginase-1 mRNA expression correlates with myeloid-derived suppressor cell levels in peripheral blood of NSCLC patients

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature and progenitor myeloid cells with immunosuppressive activity that are increased in cancer patients. Until now, the characterization of MDSC in humans was very challenging. The aim of this study was to determine the characterization and optimal assessment of MDSC and to investigate their presence and function in blood of advanced-stage NSCLC patients. We determined MDSC and lymphocyte populations in peripheral blood mononuclear cells (PBMC) samples of 185 treatment-naïve NSCLC patients and 20 healthy controls (HC). NSCLC patients had an increased population of PMN-MDSC compared to HC (p < 0.0001). Frequencies of CD4(+) and CD8(+) T-cells were significantly decreased in NSCLC patients (p < 0.0001 and p = 0.05). We found that PMN-MDSC were able to suppress T-cell proliferation in vitro. qRT-PCR showed that arginase-1 (Arg-1) mRNA is mainly expressed by MDSC and that the level of Arg-1 in PBMC correlates with the frequency of MDSC in PBMC (Spearmans rho: 0.797). There were significant differences in MDSC and lymphocyte populations between NSCLC patients and HC. We found that MDSC frequencies are stable up to six hours at room temperature after blood was drawn and that cryopreservation leads to a strong decrease of MDSC in PBMC. We show that Arg-1 mRNA expression is a valuable method to determine the levels of MDSC in peripheral blood of cancer patients. This method is therefore a useful alternative for the complex flowcytometric analysis in large multicenter patient studies.

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).

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

Mesothelioma has a poor prognosis and resists conventional therapies. Although tumor-associated macrophage depletion alone cannot restore antitumor immunity, its combination with dendritic cell–based therapy enhanced immune activation and survival in mesothelioma tumor models. 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.

Immunoglobulin-like transcript 3 is expressed by myeloid-derived suppressor cells and correlates with survival in patients with non-small cell lung cancer

Myeloid-derived suppressor cells (MDSCs) play an important role in immune suppression and accumulate under pathologic conditions such as cancer and chronic inflammation. They comprise a heterogeneous population of immature myeloid cells that exert their immunosuppressive function via a variety of mechanisms. Immunoglobulin-like transcript 3 (ILT3) is a receptor containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can be expressed on antigen-presenting cells and is an important regulator of dendritic cell tolerance. ILT3 exists in a membrane-bound and a soluble form and can interact with a yet unidentified ligand on T cells and thereby induce T-cell anergy, regulatory T cells, or T suppressor cells. In this study, we analyzed freshly isolated peripheral blood mononuclear cells (PBMCs) of 105 patients with non-small cell lung cancer and 20 healthy controls and demonstrated for the first time that ILT3 is expressed on MDSCs. We show that increased levels of circulating MDSCs correlate with reduced survival. On the basis of ILT3 cell surface expression, an ILT3low and ILT3high population of polymorphonuclear (PMN)-MDSCs could be distinguished. Interestingly, in line with the immunosuppressive function of ILT3 on dendritic cells, patients with an increased proportion of PMN-MDSCs and an increased fraction of the ILT3high subset had a shorter median survival than patients with elevated PMN-MDSC and a smaller ILT3high fraction. No correlation between the ILT3high subset and other immune variables was found. ILT3 expressed on MDSCs might reflect a previously unknown mechanism by which this cell population induces immune suppression and could therefore be an attractive target for immune intervention.

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 patients 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.

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.

Stereotactic Ablative Radiotherapy Induces Peripheral T-Cell Activation in Patients with Early-Stage Lung Cancer

Affiliations: 1. Erasmus MC, Cancer Institute, Department of Pulmonary Medicine, Rotterdam, The Netherlands 2. Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Department of Thoracic Oncology, Amsterdam, The Netherlands 3. Amphia Hospital, Department of Pulmonary Diseases, Breda, The Netherlands 4. Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Department of Surgical Oncology, Amsterdam, The Netherlands 5. Erasmus MC, Department of Radiation Oncology, Rotterdam, The Netherlands 6. Erasmus MC, Department of Cardio-thoracic Surgery, Rotterdam, The Netherlands 7. VU Medical Center, Department of Radiation Oncology, Amsterdam, The Netherlands * Current affiliation: Academic Medical Center, Laboratory for Experimental Oncology and Radiobiology, Amsterdam, The Netherlands

Abstract CT229: Autologous dendritic cells loaded with allogeneic tumor cell lysate in patients with mesothelioma: A phase I study

Malignant Mesothelioma (MM) is an aggressive disease without curative treatment options. Treatment with chemotherapy and surgery is accompanied with high incidences of local recurrences. Novel therapeutic treatment options are urgently needed and immunotherapy may be a new player in the field of MM. We have previously shown the feasibility of dendritic cell (DC) immunotherapy in MM (Hegmans, et al. Am J Respir Crit Care Med 2010). In these studies DC were loaded with autologous tumor cell lysate. Apart from safety also radiographical responses were found and survival was promising. However autologous tumor cell lysate has major drawbacks both in availability, quality and logistics. Therefore a new concept was developed, based on DC loading with a tumor cell lysate derived from human MM cell lines. In a murine MM model allogenic loading of DC9s was equally safe, and effective as autologous (data submitted). We have developed a clinical grade allogenic batch of tumorcell lysate which is now used in a phase I study where patient diagnosed with MM are treated. This batch has been generated according to GMP and GLP regulations and a patent is pending. Orphan drug designation has been obtained from both FDA and EMA. For the trial, major inclusion criteria are chemonaive patients or patients with a disease controle according to modified RECIST after standard chemotherapy. Patients with a need for high dosages of immunosuppressive therapy are excluded. In the study patients undergo a leukapheresis to collect monocytes. These monocytes are in vitro differentiated to DC9s and pulsed with tumor lysate according to the previously described protocol. DCs are re-injected intravenous and intradermally every 2 weeks to determine toxicity. A 3*3 design study is initiated where chosen dosages are 10, 25 and 50* million cells. Primary endpoint is safety. Secondary endpoints are radiological responses according to modified RECIST for mesothelioma, progression free survival and overall survival. At present the study is open for inclusion. At the meeting results of the first dose cohorts will be presented. This will be the first in human study in MM with allogenic tumor cell loaded DC. In case no safety issues are encountered this may open the field of combination treatments (e.g. immunecheckpoint inhibition combined with DC treatment) to increase the population of patients who benefit from immunotherapeutic treatment options. Citation Format: Joachim G. Aerts, Robin Cornelissen, Cor van der Leest, Ferry Eskens, Koen bezemer, Margaretha Kaijen, Rudi Hendriks, Joost Hegmans, Henk C. Hoogsteden. Autologous dendritic cells loaded with allogeneic tumor cell lysate in patients with mesothelioma: A phase I study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT229. doi:10.1158/1538-7445.AM2015-CT229

Abstract 5394: Tumor-induced immunological changes in peripheral blood of untreated stage IV non-squamous NSCLC patients

Background Lung cancer is the most common cause of cancer mortality world wide. The five-year survival rate of stage III and IV non-small cell lung cancer (NSCLC) is poor with 16%. There is growing evidence that the immune system plays a paradoxical role in the development and progression of lung cancer. It influences tumor incidence, tumor growth, response to therapy, and the prognosis of the disease. Mouse models show that myeloid-derived suppressor cells (MDSCs) play a role in these processes. MDSCs are a heterogeneous population of immature myeloid cells and myeloid progenitor cells, which are present in the tumor micro-environment and the peripheral circulation of cancer patients. These cells are able to suppress features of immune responses, like T-cell proliferation and cytokine production. Aim In the present study we want to determine the role of pivotal immunological populations such as CD4+ T-cells, CD8+ T-cells, and MDSCs. Methods Fresh blood of 60 stage IV non-squamous-NSCLC patients and 15 healthy controls (HC) was studied. Mononuclear cells were purified from peripheral blood by density gradient centrifugation and analysed by flowcytometry. MDSCs were characterized as CD11b, CD15 CD33 positive and low expression of HLA-DR and CD16 cells. Suppressive activity of MDSCs was measured by CD3α-chain expression assays, and reactive-oxygen species (ROS) production using flowcytometry. Also T-cell proliferation assays were performed, in which sorted MDSCs were co-cultured with activated CD8-T-cells to investigate whether the MDSCs could inhibit the T-cell proliferation. Results All patients had a significantly increased MDSC population compared to healthy controls (p = 0.0009). Peripheral blood fractions possessed strong immunosuppressive functions, as shown by a high ROS production by MDSC, a decreased CD3α-chain expression in T cells compared to HC. In addition, the CD8+ T-cells showed no proliferation when co-cultured with MDSCs, indicating that MDSCs could block CD8+ -T cell proliferation. The populations of CD4+ T-cells and CD8+ T-cells were significantly decreased in lung cancer patients compared to healthy controls (p = 0.01 and p = 0.0024, respectively). Conclusion Circulating MDSCs are significantly increased, while CD4+ T-cells and CD8+ T-cells are significantly decreased in stage IV non-squamous-NSCLC patients compared to controls. MDSCs suppress the immune system by ROS production, down regulating the CD3α-chain expression and thereby inhibiting T-cell proliferation. This suggests that MDSCs may play an important role in disease progression and overall survival. Therefore, further investigation is warranted. 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 5394. doi:1538-7445.AM2012-5394