Immunotype and immunohistologic characteristics of tumor-infiltrating immune cells are associated with clinical outcome in metastatic melanoma

Abstract

Immune cells infiltrating the microenvironment of melanoma metastases may either limit or promote tumor progression, but the characteristics that distinguish these effects are obscure. In this study, we systematically evaluated the composition and organization of immune cells that infiltrated melanoma metastases in human patients. Three histologic patterns of immune cell infiltration were identified, designated immunotypes A, B, and C. Immunotype A was characterized by no immune cell infiltrate. Immunotype B was characterized by infiltration of immune cells limited only to regions proximal to intratumoral blood vessels. Immunotype C was characterized by a diffuse immune cell infiltrate throughout a metastatic tumor. These immunotypes represented 29%, 63%, and 8% of metastases with estimated median survival periods of 15, 23, and 130 months, respectively. Notably, from immunotypes A to C, there were increasing proportions of B cells and decreasing proportions of macrophages. Overall, the predominant immune cells were T cells (53%), B cell lineage cells (33%), and macrophages (13%), with natural killer and mature dendritic cells only rarely present. Whereas higher densities of CD8(+) T cells correlated best with survival, a higher density of CD45(+) leukocytes, T cells, and B cells also correlated with increased survival. Together, our findings reveal striking differences in the immune infiltrate in melanoma metastases in patients, suggesting microenvironmental differences in immune homing receptors and ligands that affect immune cell recruitment. These findings are important, not only by revealing how the immune microenvironment can affect outcomes but also because they reveal characteristics that may help improve individualized therapy for patients with metastatic melanoma.

Figure 1. Immunotypes

TMAs are stained with routine H&E (400x and 100x), immunostained with CD34 and CD45, and scored for immune cell density and location for immunotyping. Metastatic melanomas are characterized as “Immunotype A” when immune cell infiltrate is absent, “Immunotype B” when immune cell infiltrate is perivascular only, and “Immunotype C” when immune cells are infiltrating among tumor cells beyond the vessels.

Figure 2. Immunotype frequency, prognosis, and cellular composition

(A) The proportion of patients with each immunotype, in the first surgical specimen in the TMA, (B) Kaplan-Meier survival estimates for patients with each Immunotype, (C) Mean numbers of each immune cell subset, by Immunotype, and for all patients, (D) Proportion of mean total immune cells infiltrating tumor that are represented by each cell subset, by Immunotype.

Figure 3. Immune cell subsets overall and across patient groups

(A) Box plots of numbers of immune cell subsets, per mm2, in first melanoma metastases for 147 patients. Boxes represent 25^th to 75^th percentiles. Middle bar identifies median; solid circle represents mean; whiskers show minimum and maximum, (B) Percentages of tumor-infiltrating immune cells in pie chart, (C) Mean numbers of immune cell subsets for clinical subgroups. Numbers of tumors represented by each bar are shown below the graph (peritoneum = one), (D) Mean numbers of cells expressing FoxP3 or PD1 for clinical subgroups.

Figure 4. Patient survival and associations with immune cell density

Representative examples of sections with low- or high- numbers of cells staining for CD45, CD3, CD8, CD20, and CD138 are shown beside Kaplan-Meier survival curves for low and high counts of those markers. Magnification, 200×.