Integrating tumor intraepithelial CD8+ and stromal FOXP3+ T cell densities as an enhanced immune prognostic index in colorectal cancer.

Abstract

High immune cell infiltration is generally associated with better survival in colorectal cancer (CRC). Recently, a prognostic score called CD8^IE-FOXP3^IS, 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^IE-FOXP3^IS score and assessed its prognostic value in comparison to 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^IE and FOXP3^IS densities in two independent cohorts, comprising 1,724 CRC patients. Multivariable Cox regression models were employed to evaluate the prognostic value of the CD8^IE-FOXP3^IS score. We found that a low CD8^IE-FOXP3^IS score was associated with higher disease stage, more frequent lymphovascular invasion, and mismatch repair (MMR) proficient status. In addition, a low CD8^IE-FOXP3^IS 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: HR for low vs. high CD8^IE-FOXP3^IS score 3.08 95% CI 1.54-6.15; p_trend=6.0E-4; Cohort 2: HR 4.30 95%CI 2.58-7.17; p_trend=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^IE-FOXP3^IS score. The CD8^IE-FOXP3^IS score shows strong prognostic value, which appears superior to overall CD3⁺ and CD8⁺ T cell density measurement.