Human Cancer Biology Merkel Polyomavirus-Specific T Cells Fluctuate with Merkel Cell Carcinoma Burden and Express Therapeutically Targetable PD-1 and Tim-3 Exhaustion Markers

Purpose: The persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins in Merkel cell carcinoma (MCC) provides a unique opportunity to characterize immune evasion mechanisms in human cancer. We isolated MCPyV-specific T cells and determined their frequency and functional status. Experimental Design: Multiparameter flow cytometry panels and HLA/peptide tetramers were used to identify and characterize T cells from tumors (n1⁄4 7) and blood (n1⁄4 18) of patients with MCC and control subjects (n 1⁄4 10). PD-1 ligand (PD-L1) and CD8 expression within tumors were determined using mRNA profiling (n 1⁄4 35) and immunohistochemistry (n 1⁄4 13). Results:MCPyV-specific CD8 T cells were detected directly ex vivo from the blood samples of 7 out of 11 (64%) patients with MCPyV-positive tumors. In contrast, 0 of 10 control subjects had detectable levels of these cells in their blood (P < 0.01). MCPyV-specific T cells in serial blood specimens increased with MCC disease progression and decreased with effective therapy. MCPyV-specific CD8 T cells andMCC-infiltrating lymphocytes expressed higher levels of therapeutically targetable PD-1 and Tim-3 inhibitory receptors compared with T cells specific to other human viruses (P < 0.01). PD-L1 was present in 9 of 13 (69%)MCCs and its expression was correlated with CD8-lymphocyte infiltration. Conclusions: MCC-targeting T cells expand with tumor burden and express high levels of immune checkpoint receptors PD-1 and Tim-3. Reversal of these inhibitory pathways is therefore a promising therapeutic approach for this virus-driven cancer. Clin Cancer Res; 19(19); 5351–60. 2013 AACR. Introduction Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancerwith adisease-associatedmortality three times that of malignant melanoma ( 46% vs. 15%, respectively; ref. 1). MCC is increasingly common with an estimated 1,600 cases per year in the United States (2), and the reported incidence has more than tripled over the past 20 years (3). This increasing incidence is partly due to improved detection using a specific immunohistochemical marker, cytokeratin-20 (4), but may also be due to the higher prevalence of known risk factors for MCC: chronic T-cell immune suppression and the number of Caucasians more than 50 years of age with extensive prior sun exposure (5). Furthermore, the recent discovery of the Merkel cell polyomavirus (MCPyV) and its causal association with at least 80% of MCCs (6–8) has provided insight into MCC pathogenesis and underscores the importance of characterizing MCPyV-specific immune responses. The necessary and persistent (7) expression of MCPyV Tantigen (T-Ag) oncoproteins in MCC tumors provides an opportunity to study antitumor immunity by assessing responses against a viral, tumor-specific antigen. Although the role of T cells is variable amongdifferent human cancers, multiple lines of evidence suggest that cellular immune function is unusually important for survival in MCC. We have previously shown that intratumoral CD8 lymphocyte infiltration (9) and lack of systemic immune suppression (10) are each significantly associated with improved survival. Furthermore, recent evidence suggests that patients with MCC have T cells that are specific for persistently expressed viral oncoproteins (11). In this study, we made use of an extensive collection of clinically annotated longitudinally collectedblood specimens to track the frequency and function ofMCPyV-specific CD8 T cells. It is hoped that characterizing the molecular pathways involved in the inhibition of MCPyV-specific T-cell responses may guide the design of rational therapies to overcome tumor immune escape. Authors' Affiliations: Departments of Medicine/Dermatology, Pathology, Medicine, Laboratory Medicine, Global Health, University of Washington; Fred Hutchinson Cancer Research Center; Benaroya Research Institute, Seattle, Washington; and Department of Medicine/ Dermatology, Saga University, Nabeshima, Japan Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). CorrespondingAuthor:Paul Nghiem, 850RepublicanStreet, University of Washington Dermatology, Seattle, WA 98109. Phone: 206-221-2632; Fax: 206-221-4364; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-13-0035 2013 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 5351 on July 14, 2017. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst August 6, 2013; DOI: 10.1158/1078-0432.CCR-13-0035