Immune Monitoring of the Circulation and the Tumor Microenvironment in Patients with Regionally Advanced Melanoma Receiving Neoadjuvant Ipilimumab Ahmad A. Tarhini 1 *, Howard Edington 2 , Lisa H. Butterfield 1 , Yan Lin 3 , Yongli Shuai 3 , Hussein Tawbi 1 , Cindy Sander 1 , Yan Yin 1 , Matthew Holtzman 4 , Jonas Johnson 5 , Uma N. M. Rao 6 , John M. Kirkwood 1 1 Department of Medicine, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania, United States of America, 2 Department of Surgery, West Penn Allegheny Health System, Pittsburgh, Pennsylvania, United States of America, 3 Department of Biostatistics, University of Pittsburgh Graduate School of Public Health, Pittsburgh, Pennsylvania, United States of America, 4 Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, United States of America, 5 Department of Otolaryngology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America, 6 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America Abstract We evaluated neoadjuvant ipilimumab in patients with surgically operable regionally advanced melanoma in order to define markers of activity in the blood and tumor as assessed at baseline (before ipilimumab) and early on-treatment. Patients were treated with ipilimumab (10 mg/kg intravenously every 3 weeks 6 2 doses) bracketing surgery. Tumor and blood biospecimens were obtained at baseline and at surgery. Flow cytometry and immunohistochemistry for select biomarkers were performed. Thirty five patients were enrolled; IIIB (3; N2b), IIIC (32; N2c, N3), IV (2). Worst toxicities included Grade 3 diarrhea/colitis (5; 14%), hepatitis (2; 6%), rash (1; 3%), elevated lipase (3; 9%). Median follow up was 18 months: among 33 evaluable patients, median progression free survival (PFS) was 11 months, 95% CI (6.2–19.2). There was a significant decrease in circulating myeloid derived suppressor cells (MDSC). Greater decrease in circulating monocyte gate MDSC Lin12/HLA-DR2/CD33+/CD11b+ was associated with improved PFS (p = 0.03). There was a significant increase in circulating regulatory T cells (Treg; CD4+CD25hi+Foxp3+) that, unexpectedly, was associated with improved PFS (HR = 0.57; p = 0.034). Baseline evidence of fully activated type I CD4 + and CD8 + antigen-specific T cell immunity against cancer-testis (NY-ESO-1) and melanocytic lineage (MART-1, gp100) antigens was detected and was significantly potentiated after ipilimumab. In tumor, there was a significant increase in CD8 + T cells after ipilimumab (p = 0.02). Ipilimumab induced increased tumor infiltration by fully activated (CD69 + ) CD3 + /CD4 + and CD3 + /CD8 + T cells with evidence of induction/ potentiation of memory T cells (CD45RO + ). The change in Treg observed within the tumor showed an inverse relationship with clinical benefit and greater decrease in tumor MDSC subset Lin12/HLA-DR2/CD33 + /CD11b + was associated with improved PFS at one year. Neoadjuvant evaluation revealed a significant immunomodulating role for ipilimumab on Treg, MDSC and effector T cells in the circulation and tumor microenvironment that warrants further pursuit in the quest for optimizing melanoma immunotherapy. Citation: Tarhini AA, Edington H, Butterfield LH, Lin Y, Shuai Y, et al. (2014) Immune Monitoring of the Circulation and the Tumor Microenvironment in Patients with Regionally Advanced Melanoma Receiving Neoadjuvant Ipilimumab. PLoS ONE 9(2): e87705. doi:10.1371/journal.pone.0087705 Editor: Lucienne Chatenoud, Universite ´ Paris Descartes, France Received October 30, 2013; Accepted January 2, 2014; Published February 3, 2014 Copyright: ß 2014 Tarhini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This investigator-initiated study was supported by a grant from Bristol-Myers Squibb and in part by National Institutes of Health (NIH) award P50CA121973. UPCI shared resources, including Immunologic Monitoring Laboratory and Tissue Procurement Facility that are supported in part by NIH award P30CA047904 were used for this project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: AAT has acted as a consultant (advisory board participation) for Bristol-Myers Squibb. All other authors have no conflicts of interest to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. The authors confirm that the funding from received from ‘‘Bristol-Myers Squibb’’ along with any other relevant declarations as noted does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: tarhiniaa@upmc.edu Introduction Patients with palpable regional lymphatic involvement with melanoma (AJCC stage IIIB-C) carry a risk of relapse and death that approaches 70% at 5 years [1]. The development of local or regional recurrence after initial surgical management portends an even poorer prognosis [2–4]. In the Melanoma Surgical Trial, a local recurrence was associated with 5 and 10 year survival rates of 9–11% and 5%, respectively [3]. Neoadjuvant therapy has been demonstrated to improve outcome in the management of patients with multiple different solid tumors [5–8]. A further significant advantage of the neoadjuvant approach in relation to translational investigations of new agents is the ability to evaluate the clinical and pathologic responses, and the access to tumor tissue before and after neoadjuvant therapy. This allows a direct assessment of the antitumor mechanisms that may enable selective application of therapeutic agents to those patients most likely to benefit. Ipilimumab is a human immunoglobulin-G (IgG1)k anti- CTLA-4 monoclonal antibody. It was approved by the U.S. FDA for the treatment of advanced inoperable melanoma in March 2011 at a dose of 3 mg/kg given every 3 weeks for 4 doses, PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e87705