George A. Dominguez

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients

PMN-MDSC in cancer patients can be distinguished from neutrophils by a genomic signature and by expression of the LOX-1 receptor. Stressing myeloid-derived suppressor cells in cancer Immunotherapies for cancer have shown promising results in part because they overcome the suppressive effects of the tumor microenvironment on immune cells. Condamine et al. now report that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) can be distinguished from neutrophils in the same cancer patient by the expression of the lipid metabolism–related molecule lectin-type oxidized LDL receptor-1 (LOX-1). LOX-1–expressing neutrophils were nearly undetectable in healthy individuals but were found prominently in tumor tissues. Moreover, exposing neutrophils from healthy individuals to endoplasmic reticulum stress resulted in up-regulation of LOX-1 and increased suppressive function. These data support the specific targeting of LOX-1–expressing PMN-MDSC for cancer immunotherapy. Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are important regulators of immune responses in cancer and have been directly implicated in the promotion of tumor progression. However, the heterogeneity of these cells and the lack of distinct markers hamper the progress in understanding the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation, we determined that low-density PMN-MDSC and high-density neutrophils from the same cancer patients had a distinct gene profile. The most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Unexpectedly, low-density lipoprotein (LDL) was one of the most increased regulators, and its receptor oxidized LDL receptor 1 (OLR1) was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor-1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5 to 15% of total neutrophils in cancer patients and 15 to 50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1− counterparts, LOX-1+ neutrophils had gene signature, potent immunosuppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSCs. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSCs. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insights into the biology and potential therapeutic targeting of these cells.

Selective Targeting of Myeloid-Derived Suppressor Cells in Cancer Patients Using DS-8273a, an Agonistic TRAIL-R2 Antibody

Purpose: Myeloid-derived suppressor cells (MDSC) are one of the major contributors to immune suppression in cancer. We recently have demonstrated in preclinical study that MDSCs are sensitive to TRAIL receptor 2 (TRAIL-R2) agonist. The goal of this study was to clinically test the hypothesis that targeting TRAIL-R2 can selectively eliminate MDSCs. Experimental Design: The TRAIL-R2 agonistic antibody (DS-8273a) has been tested in 16 patients with advanced cancers enrolled in a phase I trial. The antibody (24 mg/kg) was administered intravenously once every 3 weeks till disease progression, unacceptable toxicities, or withdrawal of consent. The safety and the presence of various populations of myeloid and lymphoid cells in peripheral blood and tumor tissues were evaluated. Results: The treatment was well tolerated with only mild to moderate adverse events attributable to the study drug. Treatment with DS-8273a resulted in reduction of the elevated numbers of MDSCs in the peripheral blood of most patients to the levels observed in healthy volunteers. However, in several patients, MDSCs rebounded back to the pretreatment level by day 42. In contrast, DS-8273a did not affect the number of neutrophils, monocytes, and other populations of myeloid and lymphoid cells. Decrease in MDSCs inversely correlated with the length of progression-free survival. In tumors, DS-8273a treatment resulted in a decrease of MDSCs in 50% of the patients who were able to provide pre- and on-treatment biopsies. Conclusions: Targeting TRAIL-R2 resulted in elimination of different populations of MDSCs without affecting mature myeloid or lymphoid cells. These data support the use of this antibody in combination immmunotherapy of cancer. Clin Cancer Res; 23(12); 2942–50. ©2016 AACR.

Unique pattern of neutrophil migration and function during tumor progression

Although neutrophils have been linked to the formation of the pre-metastatic niche, the mechanism of their migration to distant, uninvolved tissues has remained elusive. We report that bone marrow neutrophils from mice with early-stage cancer exhibited much more spontaneous migration than that of control neutrophils from tumor-free mice. These cells lacked immunosuppressive activity but had elevated rates of oxidative phosphorylation and glycolysis, and increased production of ATP, relative to that of control neutrophils. Their enhanced spontaneous migration was mediated by autocrine ATP signaling through purinergic receptors. In ectopic tumor models and late stages of cancer, bone marrow neutrophils demonstrated potent immunosuppressive activity. However, these cells had metabolic and migratory activity indistinguishable from that of control neutrophils. A similar pattern of migration was observed for neutrophils and polymorphonuclear myeloid-derived suppressor cells from patients with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer and demonstrate the mechanism of neutrophils’ contribution to early tumor dissemination.Neutrophils are linked to tumor progression. Gabrilovich and colleagues demonstrate that neutrophils have tumor-stage-dependent alterations in motility, function and metabolism: in early phases, they are highly motile with altered metabolism, whereas at later stages, they become highly suppressive.

CD38+ M-MDSC expansion characterizes a subset of advanced colorectal cancer patients

BACKGROUND Myeloid-derived suppressor cells (MDSCs) are a population of immature immune cells with several protumorigenic functions. CD38 is a transmembrane receptor-ectoenzyme expressed by MDSCs in murine models of esophageal cancer. We hypothesized that CD38 could be expressed on MDSCs in human colorectal cancer (CRC), which might allow for a new perspective on therapeutic targeting of human MDSCs with anti-CD38 monoclonal antibodies in this cancer. METHODS Blood samples were collected from 41 CRC patients and 8 healthy donors, followed by peripheral blood mononuclear cell (PBMC) separation. Polymorphonuclear (PMN-) and monocytic (M-) MDSCs and CD38 expression levels were quantified by flow cytometry. The immunosuppressive capacity of M-MDSCs from 10 CRC patients was validated in a mixed lymphocyte reaction (MLR) assay. RESULTS A significant expansion of CD38+ M-MDSCs and a trend of expansion of CD38+ PMN-MDSCs (accompanied by a trend of increased CD38 expression on both M- and PMN-MDSCs) were observed in PBMCs of CRC patients when compared with healthy donors. The CD38+ M-MDSCs from CRC patients were found to be immunosuppressive when compared with mature monocytes. CD38+ M- and PMN-MDSC frequencies were significantly higher in CRC patients who previously received treatment when compared with treatment-naive patients. CONCLUSIONS This study provides a rationale for an attempt to target M-MDSCs with an anti-CD38 monoclonal antibody in metastatic CRC patients. FUNDING NCI P01-CA14305603, the American Cancer Society, Scott and Suzi Lustgarten Family Colon Cancer Research Fund, Hansen Foundation, and Janssen Research and Development.

Abstract 1582: The coupling of MDSCs with a computational neural network (NN) to detect solid tumors

The goal of this study was to create a non-invasive cancer detection assay that analyzes flow cytometry data in an objective method. By using an artificial neural network (NN), we were able to distinguish between cancer patients (CPs) and benign/healthy donors (HDs) based upon the flow cytometry profiles of MDSCs and other leukocytes. Myeloid-derived suppressor cells (MDSCs) are known to be key contributors in supporting tumor progression and tumor escape through their ability to suppress anti-tumor responses mediated through T cell and natural killer (NK) cell activity. Several studies have demonstrated their utility as indicators of tumor progression and possible predictors of clinical outcomes, but there is significant overlap with healthy individuals preventing discrete and accurate calls. We used standard multiparametric flow cytometry techniques to immunophenotype MDSCs and other leukocytes found in the peripheral blood of 65 biopsy-confirmed CPs with solid tumors and 84 HDs. A series of NNs utilizing pattern recognition computational algorithms are then created using three data sets: 1) the training set - this ‘teaches9 the two output categories of cancer and not cancer, 2) the validation set - this uses backpropagation to improve the accuracy of the trained networks, and 3) the testing set - this is used to rank the trained networks against each other. Finally, a naive testing set is then used to determine the overall sensitivity and specificity for the top-ranking networks. Using traditional flow cytometry gating methods to analyze MDSCs as a biomarker for cancer detection, it is difficult to achieve high sensitivity and specificity due to the substantial overlap with healthy individuals. Here, we incorporated a standard 12 marker flow cytometry assay with NN technology to achieve a sensitivity of 92% and a specificity of 89%. Pairing the advanced analytical capabilities of our NN with surface biomarker based analysis of MDSCs and certain leukocytes measured in peripheral blood has enabled us the ability to objectively identify patterns indicative for the existence of a solid tumor. Citation Format: George A. Dominguez, Kristen Maslar, Alexander Polo, Cyrus Sholevar, Anthony Campisi, John Roop, Dmitry Gabrilovich, Frank Rauscher, Amit Kumar. The coupling of MDSCs with a computational neural network (NN) to detect solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1582.

Abstract CT095: The selective targeting of myeloid-derived suppressor cells in cancer patients using an agonistic TRAIL-R2 antibody

The goal of this current study was to clinically test the hypothesis that by targeting TRAIL-R2, myeloid-derived suppressor cells (MDSCs) can be selectively eliminated. MDSC) are one of the major contributors to immune suppression in cancer. We previously demonstrated in a preclinical study that MDSCs are sensitive to a TRAIL receptor 2 (TRAIL-R2) agonist. The TRAIL-R2 agonistic antibody (DS-8273a; provided by Daiichi Sankyo, Inc.) was tested in 16 patients with advanced solid cancers or lymphomas enrolled in a phase 1 trial. The antibody (24 mg/kg) was administered IV once every 3 weeks till disease progression, unacceptable toxicities, or withdrawal of consent. The safety and the presence of various populations of myeloid and lymphoid cells in peripheral blood and tumor tissues were evaluated using flow cytometry and immunohistochemistry. Overall, the treatment was well tolerated with only mild to moderate adverse events attributable to the study drug. Furthermore, treatment with DS-8273a resulted in reduction of the elevated numbers of MDSC in the peripheral blood of most patients to the levels observed in healthy volunteers. However, in several patients, MDSC rebounded back to the pre-treatment level by day 42. In contrast, DS-8273a did not affect the number of neutrophils, monocytes, and other populations of myeloid and lymphoid cells with decreases in MDSC levels inversely correlating with the length of progression-free survival. In tumors, DS-8273a treatment resulted in a decrease of MDSC in 50% of the patients who were able to provide pre- and on-treatment biopsies. In conclusion, targeting TRAIL-R2 using DS-8273a resulted in a temporary elimination of MDSCs without affecting mature myeloid or lymphoid cells, and these data support further use of this antibody in combination with current immmunotherapies of cancer. Citation Format: George A. Dominguez, Thomas Condamine, Sridevi Mony, Ayumi Hashimoto, Fang Wang, Qin Liu, Andres Forero, Johanna C. Bendell, Robert Witt, Neil Hockstein, Prasanna Kumar, Dmitry I. Gabrilovich. The selective targeting of myeloid-derived suppressor cells in cancer patients using an agonistic TRAIL-R2 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT095. doi:10.1158/1538-7445.AM2017-CT095

Abstract 667: Immunoprofiling circulating blood as a means to early detection of solid tumors

Our goal was to evaluate whether profiling Myeloid Derived Suppressor Cells (MDSCs) in circulation correlated with the existence and stage of multiple, biopsy-verified solid tumor types and to evaluate whether such analyses could enable early detection. The tumor micro-environment (TME) is replete with numerous immune cells, and the type and concentration of such cells can provide prognostic information. The link between high concentrations of MDSCs and poor prognosis is most likely due to the immuno-suppressive effects of such cells. Some fraction of these cells spill into the blood stream. We utilized flow cytometry to phenotypically quantify subsets of MDSCs and other leukocytes in the circulation of biopsy-verified cancer patients, as well as in age and sex matched healthy donors. Our results indicate a marked increase in MDSC levels in the circulation of tumor patients relative to healthy donors. We have analyzed patients presenting over a dozen solid tumor types (lung, breast, ovarian, colon, melanoma, liver, thyroid, pancreatic, uterine, osteosarcoma, appendiceal, leiomyosarcoma, liposarcoma, and vulvar) and found notable differences in the immune-profiles of circulating blood in these patients. It appears that the immune response, as measured by our flow cytometry technique, is general for most tumor types. We will present correlations and inter-relations between different cell types and evidence of tumors. While our studies to date have been performed in an unblinded manner, we will present performance data (specificity and sensitivity) for our approach. Citation Format: Amit Kumar, Dimitry Gabrilovich, Frank J. Rauscher, George Dominguez, Cyrus Sholevar, John Roop, Anthony Campisi, Alexander Polo. Immunoprofiling circulating blood as a means to early detection of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 667. doi:10.1158/1538-7445.AM2017-667