PD-L1, PD-L2 and PD-1 expression in metastatic melanoma: Correlation with tumor-infiltrating immune cells and clinical outcome

Abstract

Therapeutic blockade of PD-1/PD-L1 can have dramatic therapeutic benefit in some patients; however, the prognostic associations of PD-1 and its ligands, in the absence of therapeutic blockade have not been definitively addressed. In particular, associations of PD-L2 with immune infiltrates and with outcome have yet to be explored. We hypothesized that surface expression of both PD-L1 and PD-L2 by melanoma cells would be associated with immune cell infiltration and with overall patient survival, independent of checkpoint blockade therapy. We also characterized the heterogeneity of their distribution within a tumor and within tumors of the same patient. Tissue microarrays of metastatic melanoma samples from 147 patients were quantified for CD8⁺, CD45, CD4⁺, CD3, CD163, CD20, CD138, FoxP3, PD-1, PD-L1 and PD-L2 markers by immunohistochemistry. Relationships between the proportions of PD-L1 and PD-L2 expressing tumor cells with the immune cell count, distribution (immunotype) and patient survival were studied. Expressions of both PD-L1 and PD-L2 correlated significantly with increasing densities of immune cells in the tumor specimens and with immunotype. Positive PD-L2 expression was associated with improved overall survival and the simultaneous positive expression of both PD-1 ligands showed a higher association with survival. Significant heterogeneity of PD-L1 and PD-L2 expressions within tumors were observed, however, they were less pronounced with PD-L2. In conclusion, both are markers of immune infiltration and PD-L2, alone or in combination with PD-L1, is a marker for prognosis in metastatic melanoma patients. Larger tumor samples yield more reliable assessments of PD-L1/L2 expression.

Keywords: Immune checkpoint; PD-1; PD-L1; PD-L2; immune infiltrates; metastatic melanoma; patient outcomes; tumor-infiltrating lymphocytes.

Figure 1.

PD-L1 and PD-L2 staining in the melanoma TME. Representative examples of PD-L1 staining of melanoma cells are shown with membranous (A), membranous and cytoplasmic (B), and negative (C) staining patterns. Immune cells that stained for PD-L1 included lymphocytes (D), histiocytes/macrophages (E) and dendritic cells (F). PD-L1 staining of cells at the tumor-immune cell interface is shown at 100× (G), 200× (H) and 400× (I). Representative examples of PD-L2 of melanoma cells are shown with membranous (J), membranous and cytoplasmic (K), and negative (L) staining patterns. For all images in this figure, the chromogen was brown (3,3′-diaminobenzidene; DAB), and images were acquired at 400× except in (G) and (H). Scale bars measure 50 micrometers.

Figure 2.

Distribution of PD-L1/PD-L2 expression profiles among Immunotypes A, B and C. The distributions of PD-L1 and PD-L2 expressions in tumor cells and PD-L1 expression in lymphocytes among the 42 Immunotype A tumors, 93 Immunotype B tumors and 12 Immunotype C tumors are represented by box plots (median, quartiles). Means are noted by the dark dots. Comparisons of expression profiles of tumors of Immunotype A versus those with Immunotypes B and C and for those with Immunotype B versus C using the Wilcoxon Test, and p-values are reported. A comparison for PD-L1 expression in TILs was not feasible (p:na) as there was only nine tumors of Immunotype A.

Figure 3.

Association of PD-L1 and PD-L2 expression on patient survival. Kaplan–Meier curves illustrating survival for patients whose tumor cells are positive for staining PD-L1, PD-L2 and both and for patients with positive PD-L1 staining in lymphocytes (E). Threshold for positivity was considered at 5% and at 20% of the indicated cells staining positively. Continuous lines represent the population with positive expression and interrupted lines for no expression. Survival was compared using the log-rank test and p-values are reported.

Figure 4.

Distribution of PD-L1 and PD-L2 expression in different cores of the same tumor and of metachronous tumors. The percent tumor cells expressing PD-L1 and PD-L2 are represented on the Y-axis. Each tumor with at least three available cores on the TMA is represented on the X-axis expressing PD-L1 (A, n = 162) and PD-L2 (B, n = 167). The mean % expression of the cores from the same tumor is noted by a cross, and the range of the expression on the cores is represented by a vertical line. The horizontal dotted lines express the 5% and 20% thresholds for positivity. Tumors containing a core that is discordant with the mean are those with lines that cross either the 5% or the 20% expression levels. In (C) and (D), the PD-L1 and PD-L2 expression, respectively, in the first resected metastasis and the second resected metastasis of the same patient are connected with a line, means are denoted by squares. The difference of the mean PD-L1 (and PD-L2) expression in the 25 tumors and that of the 25 subsequent tumors is reported (δ), and p-values are obtained from paired T-tests.