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

Abstract

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.

Rationale For Immune-Based Therapies in Merkel Polyomavirus-Positive and -Negative Merkel Cell Carcinomas

“…All of these PD-L1 expressing tumors had TILs while TILs were detected in only 47% of PD-L1 negative tumors [58]. Similarly, in another study PD-L1 protein and mRNA expression correlated with the presence of intratumoral CD8 T cells [49]. Therefore, while increased PD-L1 expression may be preventing a complete antitumor response, detection of intratumoral PD-L1 indicates some degree of immune activity against MCC and suggests that PD-1 blockade may be a promising therapeutic approach for this disease [58].…”

“…Furthermore, expression of genes encoding granzyme A, B, H and K, CCL19, lymphocyte activation genes (SLAMF1 and NKG2D) and CD8α are associated with favorable prognoses, independent of stage [46]. To date, 17 MCPyV-specific CD8 epitopes have been identified, for which 14 HLA-I tetramers have been generated, enabling functional and phenotypic analysis [43,45,49,50]. Importantly, while robust CD8 responses have been associated with improved outcome in MCC, only 4-18% of MCC patients present with significant CD8 infiltration, suggesting that most MCCs block intratumoral CD8 infiltration as a means of evading immune detection [46,51].…”

“…A hallmark of exhausted T cells is the increased expression of various inhibitory receptors including PD-1, TIM-3, Lag-3 and 2B4 [68]. MCPyV-specific T cells isolated from MCC tumors and peripheral blood have been shown to express elevated levels of the inhibitory markers PD-1 and TIM-3 relative to control CMV-or EBV-specific cells (Figure 2: process 'E') [49]. Additionally, MCPyV-specific CD8 T cells often have a limited ability to secrete the effector cytokine IFN-γ following antigenic stimulation and TILs within MCC tumors have markedly lower expression of the early activation marker CD69 relative to T cells isolated from normal skin, supporting the notion that these cells are dysfunctional and exhausted [43,69,70].…”

“…MCPyV-specific T cells express elevated 4-1BB on their surface relative to other virusspecific cells suggesting that these cells may be particularly responsive to 4-1BB agonism [49]. A Phase-I trial in solid tumors (including MCC) and B-cell non-Hodgkin's lymphoma using a 4-1BB agonist (PF-05082566) has completed enrollment.…”

Merkel Cell Carcinoma Masquerading As Cellulitis: A Case Report and Review of the Literature

“…Recent studies have also shown that mccs are CK20-positive, which helps in distinguishing it from other small cell carcinomas 16 . Expression of p63 has been shown to be associated with poor survival and can be useful as a prognostic tool 17 .…”