Association of tumor-infiltrating T-cell density with molecular subtype, racial ancestry and clinical outcomes in prostate cancer

Abstract

The inflammatory microenvironment plays an important role in the pathogenesis and progression of tumors and may be associated with somatic genomic alterations. We examined the association of tumor-infiltrating T-cell density with clinical-pathologic variables, tumor molecular subtype, and oncologic outcomes in surgically treated primary prostate cancer occurring in patients of European-American or African-American ancestry. We evaluated 312 primary prostate tumors, enriched for patients with African-American ancestry and high grade disease. Tissue microarrays were immunostained for CD3, CD8, and FOXP3 and were previously immunostained for ERG and PTEN using genetically validated protocols. Image analysis for quantification of T-cell density in tissue microarray tumor spots was performed. Automated quantification of T-cell densities in tumor-containing regions of tissue microarray spots and standard histologic sections were correlated (r = 0.73, p < 0.00001) and there was good agreement between visual and automated T-cell density counts on tissue microarray spots (r = 0.93, p < 0.00001). There was a significant correlation between CD3+, CD8+, and FOXP3+ T-cell densities (p < 0.00001), but these were not associated with most clinical or pathologic variables. Increased T-cell density was significantly associated with ERG positivity (median 309 vs. 188 CD3+ T cells/mm²; p = 0.0004) and also with PTEN loss (median 317 vs. 192 CD3+ T cells/mm²; p = 0.001) in the combined cohort of matched European-American and African-American ancestry patients. The same association or a similar trend was present in patients of both ancestries when analyzed separately. When the African-American patients from the matched race set were combined with a separate high grade set of African-American cases, there was a weak association of increased FOXP3+ T-cell densities with increased risk of metastasis in multivariable analysis. Though high T-cell density is associated with specific molecular subclasses of prostate cancer, we did not find an association of T-cell density with racial ancestry.

Figure 1. Validation of automated T-cell density measurements on tissue microarray

A) Representative H&E staining and lymphocyte immunostaining in prostate tumor on tissue microarray cores. Immunostaining for CD3, CD8 and FOXP3 identifies respective specific subsets of tumor infiltrating lymphocytes in prostate tumor (top). Aperio image software identifies CD3+ T-cells, CD8+ T-cells and FOXP3+ T-cells (red) in selected tumor regions and surrounding tumor and stromal nuclei (blue; bottom). B) CD3+ T-cell density (cells/mm²) on standard histologic sections correlates significantly with CD3+ T-cell density (cells/mm²) on tissue microarray (TMA) spots, when assessed by automated digital quantification. (Pearson’s correlation coefficient r= 0.73, p<0.00001). C) Automated digitally scored T-cell densities (cells/mm²) correlate significantly with manual visually scored T-cell densities (cells/mm²) on tissue microarray spots, thus validating the methodology used in this study (Pearson’s correlation coefficient r= 0.93, p<0.00001). D) CD3+ T-cell density (cells/mm²) on tissue microarray spot correlates significantly with CD3+ T-cell density (cells/mm²) on deeper level of the same tissue microarray spot, when assessed by automated digital quantification. (Pearson’s correlation coefficient r= 0.83, p<0.00001). E) CD8+ T-cell density (cells/mm²) on tissue microarray spot correlates significantly with CD8+ T-cell density (cells/mm²) on deeper level of the same tissue microarray spot, when assessed by automated digital quantification (Pearson’s correlation coefficient r= 0.79, p<0.00001)

Figure 2. Correlation of T-cell density measurements across various subsets in matched race tissue microarray (TMA) cohort

A) Density of CD8+ T-cells (cells/mm²) correlates with density of CD3+ T-cells (cells/mm²) in prostate tumor on tissue microarray spots (Pearson’s correlation coefficient r= 0.66, p<0.00001) B) Density of FOXP3+ T-cells (cells/mm²) correlates with density of CD3+ T-cells (cells/mm²) in prostate tumor on tissue microarray spots (Pearson’s correlation coefficient r= 0.59, p<0.00001) C) Density of FOXP3+ T-cells (cells/mm²) correlates with density of CD8+ infiltrating lymphocytes (cells/mm²) in prostate tumor on tissue microarray spots (Pearson’s correlation coefficient r= 0.45, p<0.00001)

Figure 3. Kaplan-Meier analysis stratified by T-cell density for combined African-American set

Kaplan-Meier analysis for (A) biochemical recurrence (BCR)-free survival and (B) metastasis-free survival stratified by T-cell density for CD3, CD8, FOXP3. Patients in the top tertile of T-cell densities are shown by the hashed line, while those in the bottom two tertiles are shown by the unbroken line. P-values by log-rank test.