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 Multi-parameter flow cytometry panels and HLA/peptide tetramers were used to identify and characterize T cells from tumors (n=7) and blood (n=18) of MCC patients and control subjects (n=10). PD-1 ligand (PD-L1) and CD8 expression within tumors were determined using mRNA profiling (n=35) and immunohistochemistry (n=13). Results MCPyV-specific CD8 T cells were detected directly ex vivo from the blood of 7 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 and MCC-infiltrating lymphocytes expressed higher levels of therapeutically targetable PD-1 and Tim-3 inhibitory receptors compared to 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.

PURPOSE Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCCs) and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy. METHODS Intracellular cytokine cytometry, IFN-γ-ELISPOT, and a novel HLA-A*2402-restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects. RESULTS We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6), but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402-restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor-infiltrating lymphocytes as compared to blood, implying intact T-cell trafficking into the tumor. While tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide. CONCLUSIONS Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients.

Merkel cell carcinoma (MCC) is an aggressive cutaneous malignancy linked to a contributory virus (Merkel cell polyomavirus/MCPyV). Multiple epidemiologic studies have established an increased incidence of MCC among persons with systemic immune suppression. Several forms of immune suppression are associated with increased MCC incidence, including hematologic malignancies, HIV/AIDS, and immunosuppressive medications for autoimmune disease or transplant. Indeed, immune suppressed persons represent approximately 10% of the MCC patients, a significant over-representation relative to the general population. We hypothesized that immune suppressed patients may have a poorer MCC-specific prognosis and examined a cohort of 471 patients with a combined follow-up of 1427 years (median 2.1 years). Immune suppressed persons (n=41) demonstrated reduced MCC-specific survival (40% at 3 years) compared to persons with no known systemic immune suppression (n=430; 74% MCC-specific survival at 3 years). By competing risk regression analysis, immune suppression was a stage-independent predictor of worsened MCC-specific survival (hazard ratio 3.8, p < 0.01). Immune-suppressed persons thus have both an increased chance of developing MCC and poorer MCC-specific survival. It may be appropriate to follow these higher-risk individuals more closely, and, when clinically feasible, there may be benefit of diminishing iatrogenic systemic immune suppression.

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-associated skin cancer. Robust cellular immune responses are associated with excellent outcomes in MCC patients, but these responses are typically absent. We determined the prevalence and reversibility of class I MHC (MHC-I) downregulation in MCC, a potentially reversible immune evasion mechanism. Cell surface MHC-I expression was assessed on 5 MCC cell lines using flow cytometry as well as immunohistochemistry on tissue microarrays representing 114 patients. Three additional patients were included that had received intralesional interferon treatment and had evaluable specimens before and after treatment. mRNA expression analysis of antigen presentation pathway genes from 35 MCC tumors was used to examine mechanisms of downregulation. 84% of MCCs (total n=114) demonstrated reduced MHC-I expression as compared to surrounding tissues, and 51% had poor or undetectable MHC-1 expression. Expression of MHC-I was lower in polyomavirus-positive MCCs as compared to virus-negative MCCs (p<0.01). The MHC-I downregulation mechanism was multifactorial and did not depend solely on HLA gene expression. Treatment of MCC cell lines with ionizing radiation, etoposide, or interferon (IFN) resulted in MHC-I upregulation, with IFNs strongly upregulating MHC-I expression in vitro and in 3 of 3 patients treated with intralesional IFNs. MCC tumors may be amenable to immunotherapy, but downregulation of MHC-I is frequently present in these tumors, particularly those that are polyomavirus-positive. This downregulation is reversible with any of several clinically available treatments that may thus promote the effectiveness of immune stimulating therapies for MCC.

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-linked skin cancer. While CD8 lymphocyte infiltration into the tumor is strongly correlated with improved survival, these cells are absent or sparse in most MCCs. We investigated whether specific mechanisms of T-cell migration may be commonly disrupted in MCC tumors with poor CD8 lymphocyte infiltration. Intratumoral vascular E-selectin, critical for T-cell entry into skin, was downregulated in the majority (52%) of MCCs (n=56), and its loss was associated with poor intratumoral CD8 lymphocyte infiltration (p<0.05; n=45). Importantly, survival was improved in MCC patients whose tumors had higher vascular E-selectin expression (p<0.05). Local nitric oxide (NO) production is one mechanism of E-selectin downregulation and it can be tracked by quantifying nitrotyrosine, a stable biomarker of NO-induced reactive nitrogen species (RNS). Indeed, increasing levels of nitrotyrosine within MCC tumors were associated with low E-selectin expression (p<0.05; n=45) and decreased CD8 lymphocyte infiltration (p<0.05, n=45). These data suggest that one mechanism of immune evasion in MCC may be restriction of T cell entry into the tumor. Existing therapeutic agents that modulate E-selectin expression and/or RNS generation may restore T cell entry and could potentially synergize with other immune-stimulating therapies.

No abstract

p63 expression has been identified in several cohorts as a predictor of poorer prognosis in Merkel cell carcinoma. We used multivariate analysis in a large independent cohort to determine the clinical utility of this parameter. Immunohistochemistry was used to determine p63 expression on MCC tumors from 128 patients. Of these, 33% had detectable p63 expression. p63 positivity was associated with an increased risk of death from MCC (hazard ratio 2.05, p = 0.02) in a multivariate Cox regression model considering stage at presentation, age at diagnosis, and gender. Although p63 expression correlated with diminished survival in this largest cohort reported thus far, the effect was weaker than that observed in prior studies. Indeed, within a given stage, p63 status did not predict survival in a clinically or statistically significant manner. It thus remains unclear whether this test should be integrated into routine MCC patient management.

The histogenesis of Merkel cell carcinoma (MCC) has remained unresolved. Moreover, one of the questions is whether pure MCC and combined MCC represent the same histogenesis and entity. The existence of combined MCC suggests that MCC likely arise from pluripotent stem cells. Merkel cells (MC) localize within the bulge area, which is populated by hair follicle stem cells. We used hair follicle stem cell markers to investigate whether MCC share certain characteristics of these stem cells. Fourteen MCC specimens were examined histologically and immunohistochemically. There were six pure MCC and eight combined MCC. In six combined MCC, both MCC components and squamous components at least focally shared the expression of one or more of cytokeratin (CK)15, CK19 and CD200, which are hair follicle stem cell markers. On the other hand, four cases of pure MCC showed partially distinct CK19 expression, but did not show CK15 and/or CD200 expression. There was a distinct difference between pure MCC and combined MCC on the expression of hair follicle stem cell markers. The normal skin expressed CK15, CK19 and CD200 in the bulge area, whereas CK15 and CD200 were absent in the MC-rich glabrous skin and touch domes. The results led us to hypothesize that combined MCC originate from the hair follicle stem cells. We postulate that combined MCC undergo multidirectional differentiation into squamous, glandular, mesenchymal and Merkel cells. Further investigation is warranted to confirm the histogenesis of pure MCC and combined MCC.