Vascular E-selectin expression correlates with CD8 lymphocyte infiltration and improved outcome in Merkel cell carcinoma

Abstract

Merkel cell carcinoma (MCC) is an aggressive, polyomavirus-linked skin cancer. Although 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.

Fig 1. Vascular adhesion molecules and CD8 infiltration on representative serial tumor sections

(a) Left to right: Serial sections stained as indicated from 3 patients (cases w453, w456, w236) with the specified intratumoral CD8 and E-selectin scores. Red arrowheads indicate areas of positive staining on immunohistochemistry for the indicated antibody. Scale bar: 100µm. (b) Left to right: Serial sections from an MCC tumor (case w532) with both stromal and tumor components stained with specified antibodies. Black dashed line indicates junction between tumor and stroma. The sections shown are representative of staining patterns in the stroma and tumor. Scale bar: 100µm.

Fig 2. MCC tumors often have decreased E-selectin-positive vessels, which correlate with worse survival

(a) Percent of MCC tumors with low (<1%), moderate (1–5%) or high (>5%) fraction of intratumoral (black bars) or peritumoral (white bars) E-selectin-positive vessels. *p<0.05, Fisher’s exact test. (b) Kaplan-Meier curves showing MCC-specific survival of patients with low (n=29), moderate (n=18) or high (n=8) fractions of intratumoral vessels that were E-selectin-positive. P-value determined by logrank test for trend.

Fig 3. Intratumoral E-selectin is associated with CD8 lymphocytic infiltration into MCC tumors

(a) Correlation between intratumoral E-selectin-positive vessels and CD8 lymphocyte infiltration in 56 MCCs. E-selectin was scored as a percent of all vessels and stratified as low (<1%, n=29), moderate (1–5%, n=19) or high (>5%, n=8). CD8s were scored on a 0–5 scale (Paulson et al, 2011). Black bar indicates median. *p<0.05, Wilcoxon’s ranksum test. (b) Comparison of intratumoral (filled circles) versus peritumoral (empty circles) E-selectin-positive vessels among tumors with CD8 infiltrates characterized as stalled (intratumoral CD8 score ≤1 and peritumoral CD8 score ≥3, n=14), sparse (intratumoral CD8 score ≤2, n=42) or brisk (intratumoral CD8 score ≥3, n=14). Black dots in schematic = CD8 lymphocytes. Black bar indicates mean. **p<0.01, Student’s t-test.

Fig 4. CLA expression on MCC-specific lymphocytes

(a) CLA/CD8 co-expression as evaluated by immunofluorescence with the indicated stains in a tumor (high CLA/CD8 co-expression; case w588). Right: Fraction of MCCs with CLA/CD8 coexpression categorized as none/low (n=4, ≤5% CLA-positive CD8 cells), moderate (n=9, 5–50% CLA-positive CD8s) or high (n=7; ≥50% CLA-positive CD8s). Scale bar: 50µm (b) CLA expression in blood as evaluated by flow cytometry. Top left: Summary data of CLA expression among CD3⁺CD8⁺ cells from control subjects (n=10) and MCC patients (n=8). Top right: CLA expression among CD3⁺CD8⁺Tetramer⁺ cells specific for MCPyV (n=4), CMV (n=4), and EBV (n=3). The red dot on each graph indicates the representative sample selected for flow plot display below. Black bar indicates mean. Tet⁺, tetramer-positive. *p<0.05, Wilcoxon’s ranksum test.

Fig 5. High levels of tissue nitrotyrosine are associated with a reduced fraction of E-selectin-positive vessels and poor CD8 lymphocyte infiltration

(a) Representative MCC tumor cores stained for nitrotyrosine (brown). Nitrotyrosine scores took into account both intensity and proportion and were categorized as: none (n=14), low (n=121), moderate (n=80) and high (n=21) expression. Scale bar: 50µm. (b) Correlation between nitrotyrosine levels and intratumoral E-selectin (left) and CD8 (right) scores among 45 MCC tumors. Black bar represents median. P-value determined by Cuzick’s nonparametric test for trend.