We undertook a comprehensive analysis of circulating myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs) in pancreatic, esophageal and gastric cancer patients and investigated whether MDSCs are an independent prognostic factor for survival. We evaluated a series of plasma cytokines and in particular re-evaluated the Th2 cytokine interleukin-13 (IL-13). Peripheral blood was collected from 131 cancer patients (46 pancreatic, 60 esophageal and 25 gastric) and 54 healthy controls. PBMC were harvested with subsequent flow cytometric analysis of MDSC (HLADR− Lin1low/− CD33+ CD11b+) and Treg (CD4+ CD25+ CD127low/− FoxP3+) percentages. Plasma IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL-17, G-CSF, IFN-γ, TNF-α and VEGF levels were analyzed by the Bio-Plex cytokine assay. Plasma arginase I levels were analyzed by ELISA. MDSCs and Tregs were statistically significantly elevated in pancreatic, esophageal and gastric cancer compared with controls, and MDSC numbers correlated with Treg levels. Increasing MDSC percentage was associated with increased risk of death, and in a multivariate analysis, MDSC level was an independent prognostic factor for survival. A unit increase in MDSC percentage was associated with a 22% increased risk of death (hazard ratio 1.22, 95% confidence interval 1.06–1.41). Arginase I levels were also statistically significantly elevated in upper gastrointestinal cancer patients compared with controls. There was Th2 skewing for cytokine production in all three diseases, and importantly there were significant elevations of the pivotal Th2 cytokine interleukin-13, an increase that correlated with MDSC levels.

In pre-clinical models, the only two chemotherapy drugs which have been demonstrated to directly reduce the number of myeloid-derived suppressor cells (MDSCs) are gemcitabine and 5-fluorouracil. Here we analyze the dynamics of MDSCs, phenotyped as Lin-DR-CD11b+, in patients with advanced pancreatic cancer receiving the combination of gemcitabine and capecitabine, a 5-FU pro-drug. We found no evidence that gemcitabine and capecitabine directly reduce MDSC% in patients. Gemcitabine and capecitabine reduced MDSCs in 42% of patients (n = 19) and MDSC% fell in only 3/9 patients with above-median baseline MDSCs. In 5/8 patients with minimal tumour volume change on treatment, the MDSC% went up: increases in MDSC% in these patients appeared to correlate with sustained cancer-related inflammatory cytokine upregulation. In a separate cohort of 21 patients treated with gemcitabine and capecitabine together with concurrently administered GV1001 vaccine with adjuvant GM-CSF, the MDSC% fell in 18/21 patients and there was a significant difference in the trajectory of MDSCs between those receiving GV1001 and GM-CSF in combination with chemotherapy and those receiving chemotherapy alone. Thus, there was no evidence that the addition of low-dose adjuvant GM-CSF increased Lin-DR-CD11b+ MDSC in patients receiving combination chemoimmunotherapy. 9/21 patients developed an immune response to GV1001 and the MDSCs fell in 8 of these 9 patients, 6 of whom had above-median pre-vaccination MDSC levels. A high pre-vaccination MDSC% does not preclude the development of immunity to a tumour-associated antigen.

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Due to our increased understanding of tumor immunology a number of immunotherapeutic approaches have been successfully introduced into the clinic and further promising therapeutic strategies are being investigated in on-going clinical trials. However, evaluation of anti-tumor immunity in such trials as in animal models has shown that tumor escape from immune recognition and tumor-mediated suppression of anti-tumor immunity can pose a significant obstacle to successful cancer therapy. One of the main cell types that have emerged as potent suppressors of T cell-mediated immunity are myeloid derived suppressor cells (MDSCs). MDSCs are a heterogeneous population of immature cells of myeloid origin with a wide repertoire of effector mechanisms. While there are published data on circulating MDSC in cancer patients with diverse primary tumors, there is little published data investigating MDSCs specifically in prostate cancer. Following on from our recent study looking at HLADR- Lin1low/− CD33+ CD11b+ MDSCs in pancreatic, esophageal and gastric cancer patients we sought to determine whether this population of MDSCs also played a role in prostate cancer. Peripheral blood was collected from 75 prostate cancer patients and 50 healthy controls. PBMC were harvested with subsequent flow cytometric analysis of MDSC. MDSCs were highly statistically significantly elevated in prostate cancer patients compared with healthy controls (p<0.0001). In this cohort there was no evidence of increasing MDSC % with stage of disease. MDSCs exert their immunosuppressive activity through multiple mechanisms, including arginine depletion. MDSC expressing arginase I deplete L-arginine from the microenvironment and profoundly inhibit T-cell function. Analysis of arginase I levels by ELISA on the plasma from the same prostate cancer patient cohort showed that Arginase I levels were also highly statistically significantly elevated in prostate cancer patients compared with controls (p<0.0001). There was a clear association between increasing Arginase I level and increasing MDSC percentage (p=0.0195). In contrast to our finding in upper gastrointestinal cancers where significant elevations of the Th2 cytokine interleukin-13 (IL-13) were found and correlated with MDSC levels, we were unable to detect IL-13 in the plasma of prostate cancer patients. This probably reflects the diverse combinations of cytokines produced by different tumors and the large number of potential MDSC-inducing and MDSC activating factors. These data not only add to our understanding of the immunobiology of prostate cancer but provide the rationale for combining immunotherapy studies with MDSC inhibition strategies. Citation Format: Nicola Annels, Rachel Gabitass, Gary Middleton, Hardev Pandha. Increased circulating HLA-DRneg, Lin1 lo/neg, CD33+ CD11b+ myeloidderived suppressor cells correlate with increased arginase levels in patients with prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A67.