Integrating Tumor Intraepithelial CD8 and Stromal FOXP3 T-Cell Densities as an Enhanced Immune Prognostic Index in Colorectal Cancer.

High immune cell infiltration is generally associated with better survival in colorectal cancer (CRC). Recently, a prognostic score called CD8-FOXP3, which integrates the densities of tumor intraepithelial CD8 and intrastromal FOXP3 cells, was introduced using multiplex immunofluorescence. In this study, we developed a triple chromogenic immunohistochemistry assay to evaluate the CD8-FOXP3 score and assessed its prognostic value in comparison with the CD3-CD8 T-cell density score (based on the principles of the Immunoscore) and conventional prognostic parameters. Multiplex immunohistochemistry combined with machine learning-assisted image analysis was used to quantify CD8 and FOXP3 densities in 2 independent cohorts comprising 1724 CRC patients. Multivariable Cox regression models were used to evaluate the prognostic value of the CD8-FOXP3 score. We found that a low CD8-FOXP3 score was associated with higher disease stage, more frequent lymphovascular invasion, and mismatch repair proficient status. In addition, a low CD8-FOXP3 score was associated with higher CRC-specific mortality independent of the CD3-CD8 T-cell density score and other tumor and patient characteristics (cohort 1: hazard ratio [HR] for low vs high CD8-FOXP3 score, 3.08; 95% CI, 1.54-6.15; P = 6.0E-4; cohort 2: HR, 4.30; 95% CI, 2.58-7.17; P = 3.2E-9). These findings indicate that triple chromogenic immunohistochemistry combined with digital pathology is an applicable method for quantifying tumor intraepithelial CD8 and stromal FOXP3 cell densities, allowing for the determination of the CD8-FOXP3 score. The CD8-FOXP3 score shows a strong prognostic value, which appears superior to overall CD3 and CD8 T-cell density measurement.