Thierry van den Bosch

Clinical significance of immunohistochemistry for detection of BAP1 mutations in uveal melanoma.

Uveal melanoma is a lethal cancer with a strong propensity to metastasize. Limited therapeutic options are available once the disease has disseminated. A strong predictor for metastasis is the loss of chromosome 3. Inactivating mutations in BAP1 encoding the BRCA1-associated protein 1 and located on chromosome 3p21.1, have been described in uveal melanoma and other types of cancer. In this study, we determined the prevalence of somatic BAP1 mutations and examined whether these mutations correlate with the functional expression of BAP1 in uveal melanoma tissue and with other clinical, histopathological and chromosomal parameters. We screened a cohort of 74 uveal melanomas for BAP1 mutations, using different deep sequencing methods. The frequency of BAP1 mutations in our study group was 47%. The expression of BAP1 protein was studied using immunohistochemistry. BAP1 staining was absent in 43% of the cases. BAP1 mutation status was strongly associated with BAP1 protein expression (P<0.001), loss of chromosome 3 (P<0.001), and other aggressive prognostic factors. Patients with a BAP1 mutation and absent BAP1 expression had an almost eightfold higher chance of developing metastases compared with those without these changes (P=0.002). We found a strong correlation between the immunohistochemical and sequencing data and therefore propose that, immunohistochemical screening for BAP1 should become routine in the histopathological work-up of uveal melanoma. Furthermore, our analysis indicates that loss of BAP1 may be particularly involved in the progression of uveal melanoma to an aggressive, metastatic phenotype.

Human monocytes produce interferon-gamma upon stimulation with LPS

Representing a crucial T-helper 1 cytokine, IFN-γ acts as an important bridge between innate and adaptive immunity and is involved in many acute and chronic pathologic states, such as autoimmune diseases and solid organ transplant rejection. At present, debate still prevails about the ability of human monocytes to produce IFN-γ. We aimed to investigate whether human monocytes possess the capacity to produce IFN-γ at mRNA and protein level. Using real time PCR, flow cytometric analysis and ELISA, we investigated the capacity of freshly isolated CD14+ monocytes of healthy individuals and kidney transplant recipients to produce IFN-γ after stimulation with IFN-γ and LPS or LPS alone. We observed increased IFN-γ mRNA levels in CD14+ monocytes after stimulation as compared to the unstimulated controls in both populations. In addition, stimulation with IFN-γ and LPS or LPS alone led to a significant increase in the percentage of CD14+ monocytes producing TNF-α and IFN-γ at protein level (p<0.05). A trend towards increased secreted IFN-γ production in supernatants was also observed after LPS stimulation using ELISA. We conclude that human monocytes from healthy individuals and kidney transplant recipients possess the capacity to produce IFN-γ.

Chemokine receptor CCR7 expression predicts poor outcome in uveal melanoma and relates to liver metastasis whereas expression of CXCR4 is not of clinical relevance.

PURPOSE To examine the prognostic relevance of expression of the chemokine receptors CCR7 and CXCR4 and its ligand CXCL12 in uveal melanoma in nonmetastatic and metastatic patients with correlation to liver metastasis and overall survival. METHODS Primary uveal melanoma specimens from 19 patients with correlating liver metastasis specimens and 30 primary uveal melanoma specimens of patients without metastasis were collected between the years 1988 and 2008. Expression of CCR7, CXCR4, and CXCL12 were studied using immunohistochemistry. Single nucleotide polymorphism (SNP) arrays were used to examine gains or losses of chromosomes 1, 3, 6, and 8 and the regions of CCR7 (17q12-q21.2), CXCR4 (2q21), and CXCL12 (10q11.1) genes. RESULTS Strong cytoplasmic staining for CCR7 correlated with the presence of epithelioid cells (P = 0.037), tumor thickness (P = 0.011), lymphocytic infiltration (P = 0.041), and necrosis (P = 0.045). Nuclear staining for CXCR4 correlated with lymphocytic infiltration (P = 0.017). CXCL12 showed no correlation to histologic parameters. Single nucleotide polymorphism analyses showed no copy number variations in the regions of CCR7, CXCR4, or CXCL12. Strong expression of CCR7 was observed in 76% of the metastatic patients and 0% of nonmetastasis patients. In multivariate analysis, CCR7 staining was inversely correlated to overall survival and disease-free survival, whereas CXCR4 nuclear staining was not. CONCLUSIONS Our data suggest that CCR7 plays a role in uveal melanoma metastasis and is associated with poor survival. CCR7 and its involved related pathways are of prognostic value in uveal melanoma and may prove to be a target for therapeutic intervention.

Targeting the monocyte-macrophage lineage in solid organ transplantation

There is an unmet clinical need for immunotherapeutic strategies that specifically target the active immune cells participating in the process of rejection after solid organ transplantation. The monocyte–macrophage cell lineage is increasingly recognized as a major player in acute and chronic allograft immunopathology. The dominant presence of cells of this lineage in rejecting allograft tissue is associated with worse graft function and survival. Monocytes and macrophages contribute to alloimmunity via diverse pathways: antigen processing and presentation, costimulation, pro-inflammatory cytokine production, and tissue repair. Cross talk with other recipient immune competent cells and donor endothelial cells leads to amplification of inflammation and a cytolytic response in the graft. Surprisingly, little is known about therapeutic manipulation of the function of cells of the monocyte–macrophage lineage in transplantation by immunosuppressive agents. Although not primarily designed to target monocyte–macrophage lineage cells, multiple categories of currently prescribed immunosuppressive drugs, such as mycophenolate mofetil, mammalian target of rapamycin inhibitors, and calcineurin inhibitors, do have limited inhibitory effects. These effects include diminishing the degree of cytokine production, thereby blocking costimulation and inhibiting the migration of monocytes to the site of rejection. Outside the field of transplantation, some clinical studies have shown that the monoclonal antibodies canakinumab, tocilizumab, and infliximab are effective in inhibiting monocyte functions. Indirect effects have also been shown for simvastatin, a lipid lowering drug, and bromodomain and extra-terminal motif inhibitors that reduce the cytokine production by monocytes–macrophages in patients with diabetes mellitus and rheumatoid arthritis. To date, detailed knowledge concerning the origin, the developmental requirements, and functions of diverse specialized monocyte–macrophage subsets justifies research for therapeutic manipulation. Here, we will discuss the effects of currently prescribed immunosuppressive drugs on monocyte/macrophage features and the future challenges.

CD16+ Monocytes and Skewed Macrophage Polarization toward M2 Type Hallmark Heart Transplant Acute Cellular Rejection

Background During acute heart transplant rejection, infiltration of lymphocytes and monocytes is followed by endothelial injury and eventually myocardial fibrosis. To date, no information is available on monocyte–macrophage-related cellular shifts and their polarization status during rejection. Here, we aimed to define and correlate monocyte–macrophage endomyocardial tissue profiles obtained at rejection and time points prior to rejection, with corresponding serial blood samples in 25 heart transplant recipients experiencing acute cellular rejection. Additionally, 33 healthy individuals served as control. Materials and methods Using histology, immunohistochemistry, confocal laser scan microscopy, and digital imaging expression of CD14, CD16, CD56, CD68, CD80, and CD163 were explored to define monocyte and macrophage tissue profiles during rejection. Fibrosis was investigated using Sirius Red stainings of rejection, non-rejection, and 1-year biopsies. Expression of co-stimulatory and migration-related molecules on circulating monocytes, and production potential for pro- and anti-inflammatory cytokines were studied using flow cytometry. Results At tissue level, striking CD16+ monocyte infiltration was observed during rejection (p < 0.001). Significantly more CD68+CD163+ M2 macrophages were documented during rejection compared to barely present CD68+CD80+ M1 macrophages. Rejection was associated with severe fibrosis in 1-year biopsies (p < 0.001). Irrespective of rejection status, decreased frequencies of circulating CD16+ monocytes were found in patients compared to healthy individuals. Rejection was reflected by significantly increased CD54 and HLA-DR expression on CD16+ monocytes with retained cytokine production potential. Conclusion CD16+ monocytes and M2 macrophages hallmark the correlates of heart transplant acute cellular rejection on tissue level and seem to be associated with fibrosis in the long term.

Abstract 2348: Expression of CECR1 by activated M2-type macrophages in glioma

Objectives Glial neoplasms harbor macrophages which are mainly of the M2 type and secrete Th2 cytokines. These M2 macrophages are not only involved in immune suppression but also serve functions in angiogenesis. Recent studies have shown that the adenosine deaminase CECR1 is involved in the maintenance of vascular homeostasis and M2-macrophage differentiation. Here we aim to identify the expressional levels of CECR1 in M1 and M2 macrophage populations in gliomas and investigate the role of these cells in glioma development. Methods To identify the relation between CECR1 expression and the macrophage subtypes, tissue samples of human glioma and normal brain controls were used for Real Time PCR and immunostaining. For in vitro functional studies, we generated M1 and M2-like macrophages by differentiating CD14+ monocytes isolated from human peripheral blood in GM-CSF and M-CSF medium. With and without stimulation of GBM conditioned medium, CECR1 level was measured by RT-PCR and western blot. CECR1 protein was added to the macrophage culture medium and the expression level of the M1 marker CD80 and the M2 marker CD163 were measured by flow cytometry and confocal microscopy. Results The macrophages were predominantly present in perivascular spaces, but also in the tumor tissue and neuropil of the normal brain. At the mRNA level the expression of CECR1 was positively correlated with the M2 markers CD204 and IL-10. Immunohistochemical investigation revealed that CECR1 is localized in microglia and macrophages in low-grade gliomas and control brain and it overlaps and co-localizes with the M2-markers CD204 and CD163, but not the M1-marker CD80. In vitro experiments showed that CECR1 expression is higher in the M-CSF-induced M2-macrophages than in their GM-CSF-induced M1 counterparts. Under the stimulation of GBM conditioned medium, increased CECR1 is detected in both M1 and M2-like macrophages. In addition, exogenous CECR1 greatly affects both M-CSF and GM-CSF induced macrophages’ response by up-regulating CD163 in M1-like macrophages and increasing CD163 positive cells in M2-like macrophages. As a result, CECR1 skews macrophage differentiation towards the M2 phenotype. Conclusion This study is the first to show that the M2-macrophage, not the M1-macrophage in glioma, is the main source of CECR1. In addition, CECR1 is shown to be a potent regulator of M2-macrophages. High levels of CECR1 is able to skew GM-CSF driven M1 macrophage differentiation towards an M2 macrophage phenotype. We propose that CECR1 expression by macrophages promotes M2-macrophage differentiation under the influence of an adenosine-rich glioma microenvironment to support tumor growth and tumor angiogenesis. The findings may well direct the search for new anti-angiogenic target molecules for the treatment of glioma. Citation Format: Changbin Zhu, Marcel M. van der Weiden, Adrea Scchetti, Thierry P.P. van den Bosch, Ihsan Chrifi, Maarten M. Brandt, Dana A.M. Mustafa, Caroline Cheng, Johan M. Kros. Expression of CECR1 by activated M2-type macrophages in glioma. [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 2348. doi:10.1158/1538-7445.AM2015-2348