Purpose To determine the role of the CCL2/CCR2 axis and inflammatory monocytes (IM; CCR2+/CD14+) as immunotherapeutic targets in the treatment of pancreatic cancer (PC). Experimental Design Survival analysis was performed to determine if the prevalence of pre-operative blood monocytes correlates with survival in PC patients following tumor resection. IM prevalence in the blood and bone marrow of PC patients and controls was compared. The immunosuppressive properties of IM and macrophages in the blood and tumors, respectively, of PC patients were assessed. CCL2 expression by human PC tumors was compared to normal pancreas. A novel CCR2 inhibitor (PF-04136309) was tested in an orthotopic model of murine PC. Results Monocyte prevalence in the peripheral blood correlates inversely with survival, and low monocyte prevalence is an independent predictor of increased survival in PC patients with resected tumors. IM are increased in the blood and decreased in the bone marrow of PC patients compared to controls. An increased ratio of IM in the blood versus the bone marrow is a novel predictor of decreased patient survival following tumor resection. Human PC produces CCL2, and immunosuppressive CCR2+ macrophages infiltrate these tumors. Patients with tumors that exhibit high CCL2 expression/low CD8 T cell infiltrate have significantly decreased survival. In mice, CCR2 blockade depletes IM and macrophages from the primary tumor and premetastatic liver resulting in enhanced anti-tumor immunity, decreased tumor growth, and reduced metastasis. Conclusions IM recruitment is critical to PC progression, and targeting CCR2 may be an effective immunotherapeutic strategy in this disease.

Background Pancreatic ductal adenocarcinoma utilizes the CCL2/CCR2 chemokine axis to facilitate recruitment of tumor associated macrophages to sculpt an immunosuppressive tumor microenvironment. This pathway has prognostic implications in pancreas cancer, and blockade of CCR2 restores anti-tumor immunity in pre-clinical models. This provided the rationale for a clinical study in pancreatic adenocarcinoma to determine the safety and recommended phase 2 oral dosage of the CCR2 inhibitor PF-04136309 in combination with chemotherapy (FOLFIRINOX). Methods In this single-center, open label, phase Ib clinical trial patients age ≥ 18 years with treatment naïve borderline resectable or locally advanced, biopsy-proven pancreatic ductal adenocarcinoma, Eastern Cooperative Oncology Group performance status <2, measurable disease by Response Evaluation Criteria in Solid Tumors Version 1.1, and normal end organ function were eligible for enrollment. FOLFIRINOX (oxaliplatin, 85 mg/m2; irinotecan, 180 mg/m2; leucovorin, 400 mg/m2, and bolus fluorouracil 400 mg/m2 followed by 2,400 mg/m2 46 hour continuous infusion) was administered every 2 weeks for a total of six treatment cycles. To determine the recommended phase 2 dose, PF-04136309 was orally administered at a starting dose of 500 mg twice daily in a standard 3+3 dose de-escalation design with an expansion phase planned at the recommended phase 2 dose. Both FOLFIRINOX and PF-04136309 were simultaneously initiated with a total treatment duration of 3 months. The primary endpoints were to determine the recommended phase 2 dose and toxicity of PF-04136309 in combination with FOLFIRINOX. All patients in the dose de-escalation and expansion phase received the recommended phase 2 dose of PF-04136309 were combined for assessment of treatment toxicity by an intention to treat analysis. For tissue specimen comparison in corollary studies, a group of patients receiving FOLFIRINOX alone were enrolled and evaluated for treatment related toxicity. This study has been completed and is registered at ClinicalTrials.gov; number NCT01413022. Results From April 19th, 2012 through November 12th, 2014 a total of 47 patients were enrolled. The dose de-escalation group (n=6) received PF-04136309 at 500 mg administered orally twice daily. No dose-limiting toxicities were observed and this was established as the recommended phase 2 dose. The expansion phase cohort (n=33) and patients in the dose de-escalation arm receiving PF-04136309 at the recommended phase 2 dose (n=6) were combined for assessment of treatment related toxicity. No therapy related deaths occurring during the study interval. Early termination as the result of treatment related toxicity occurred in 2 of the 39 patients (5%) in the FOLFIRINOX plus PF-04136309 arm. Grade ≥3 adverse events reported in ≥10% of the patients receiving PF-04136309 included neutropenia in 27 patients (69%), febrile neutropenia in 7 patients (18%), lymphopenia in 4 patients (10%), diarrhea in 6 patients (15%), and hypokalemia in 7 patients (18%). Among...

ObjectiveChemokine pathways are co-opted by pancreatic adenocarcinoma (PDAC) to facilitate myeloid cell recruitment from the bone marrow to establish an immunosuppressive tumour microenvironment (TME). Targeting tumour-associated CXCR2+neutrophils (TAN) or tumour-associated CCR2+ macrophages (TAM) alone improves antitumour immunity in preclinical models. However, a compensatory influx of an alternative myeloid subset may result in a persistent immunosuppressive TME and promote therapeutic resistance. Here, we show CCR2 and CXCR2 combined blockade reduces total tumour-infiltrating myeloids, promoting a more robust antitumour immune response in PDAC compared with either strategy alone.MethodsBlood, bone marrow and tumours were analysed from PDAC patients and controls. Treatment response and correlative studies were performed in mice with established orthotopic PDAC tumours treated with a small molecule CCR2 inhibitor (CCR2i) and CXCR2 inhibitor (CXCR2i), alone and in combination with chemotherapy.ResultsA systemic increase in CXCR2+ TAN correlates with poor prognosis in PDAC, and patients receiving CCR2i showed increased tumour-infiltrating CXCR2+ TAN following treatment. In an orthotopic PDAC model, CXCR2 blockade prevented neutrophil mobilisation from the circulation and augmented chemotherapeutic efficacy. However, depletion of either CXCR2+ TAN or CCR2+ TAM resulted in a compensatory response of the alternative myeloid subset, recapitulating human disease. This was overcome by combined CCR2i and CXCR2i, which augmented antitumour immunity and improved response to FOLFIRINOX chemotherapy.ConclusionDual targeting of CCR2+ TAM and CXCR2+ TAN improves antitumour immunity and chemotherapeutic response in PDAC compared with either strategy alone.

Purpose: Molecular subtyping for pancreatic cancer has made substantial progress in recent years, facilitating the optimization of existing therapeutic approaches to improve clinical outcomes in pancreatic cancer. With advances in treatment combinations and choices, it is becoming increasingly important to determine ways to place patients on the best therapies upfront. While various molecular subtyping systems for pancreatic cancer have been proposed, consensus regarding proposed subtypes, as well as their relative clinical utility, remains largely unknown and presents a natural barrier to wider clinical adoption. Methods:We assess three major subtype classification schemas in the context of results from two clinical trials and by meta-analysis of publicly available expression data to assess statistical criteria of subtype robustness and overall clinical relevance. We then developed a Single Sample Classifier (SSC) using penalized logistic regression based on the most robust and replicable schema.

Pancreatic cancer (PC) mobilizes myeloid cells from the bone marrow to the tumor where they promote tumor growth and proliferation. Cancer stem cells (CSCs) are a population of tumor cells that are responsible for tumor initiation. Aldehyde dehydrogenase-1 activity in PC identifies CSCs, and its activity has been correlated with poor overall prognosis in human PC. Myeloid cells have been shown to impact tumor stemness, but the impact of immunosuppressive tumor-infiltrating granulocytic and monocytic myeloid-derived suppressor cells (Mo-MDSC) on ALDH1Bright CSCs and epithelial to mesenchymal transition is not well understood. In this study, we demonstrate that Mo-MDSC (CD11b+/Gr1+/Ly6G−/Ly6Chi) significantly increase the frequency of ALDH1Bright CSCs in a mouse model of PC. Additionally, there was significant upregulation of genes associated with epithelial to mesenchymal transition. We also found that human PC converts CD14+ peripheral blood monocytes into Mo-MDSC (CD14+/HLA-DRlow/−) in vitro, and this transformation is dependent on the activation of the STAT3 pathway. In turn, these Mo-MDSC increase the frequency of ALDH1Bright CSCs and promote mesenchymal features of tumor cells. Finally, blockade of STAT3 activation reversed the increase in ALDH1Bright CSCs. These data suggest that the PC tumor microenvironment transforms monocytes to Mo-MDSC by STAT3 activation, and these cells increase the frequency of ALDH1Bright CSCs. Therefore, targeting STAT3 activation may be an effective therapeutic strategy in targeting CSCs in PC.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-014-1527-x) contains supplementary material, which is available to authorized users.