B and T Tumor-Infiltrating Lymphocyte Subtypes According to Subsite: A Colon Cancer Immunophenotyping Map

<b>Background:</b> Accumulating evidence regarding the association between tumor-infiltrating lymphocyte (TIL) subtypes and prognosis in colorectal cancer has emerged recently in the literature. Whether the prognostic impact of TIL subsets is different according to tumor location remains unknown, despite genetic, epigenetic and molecular differences between the proximal and distal colon. Our study aimed to investigate the value of CD3⁺ lymphocytes, reflecting overall T-cell infiltration, CD8⁺ cells identifying cytotoxic effector T-cells and CD73⁺ cells acting as a modulator of immunosuppression, stratified by primary tumor location. <b>Methods:</b> The density of CD73⁺, CD3⁺ and CD8⁺ tumor-infiltrating B- and T-cells was determined in colon cancer patients using whole-section tissue sampling, heat-induced epitope retrieval, primary antibodies and DAB visualization. QuPath Cell counter function quantified nucleated cells and immune-positive percentages; ImageJ assessed staining intensity via color deconvolution and optical density. An Immunoreactive Score combined intensity and positivity for immune profiling. The Receiver Operating Characteristic (ROC) curve analysis was used to determine the optimal cut-off values for CD3⁺, CD8⁺ and CD73⁺ lymphocytes. Statistical analysis was performed in order to identify potential associations between TILs expression and pathological characteristics, according to the location of the primary tumor. Survival analysis was carried out using the Kaplan-Meier method. <b>Results:</b> A total of 100 patients were included in the study. CD3⁺ T-cells were the most abundantly expressed and were more predominantly encountered in the right colon. Total CD3⁺ numbers were correlated with T stage and the presence of perineural invasion in left-sided tumors, as well as with tumor grading in the right colon. Correlation analysis based on CD3⁺ threshold values according to tumor location demonstrated a statistically significant association between a higher N stage and low CD3⁺ cell values (<i>p</i> value = 0.0306), and higher perineural invasion and low CD3⁺ TILs values in the left colon (<i>p</i> value = 0.0123). In addition, low CD8⁺ values were associated with a higher T stage in the left colon (<i>p</i> value = 0.0382). Survival analysis did not demonstrate statistically significant differences between the investigated groups. <b>Conclusions:</b> TIL subtypes in colon cancer patients demonstrate significant variability according to the location of the primary tumor and are associated with different clinical and pathological characteristics. This exploratory study requires larger validation before TIL densities can guide therapy.