Background: Penile squamous cell carcinoma (PSCC) remains a relatively rare but formidable malignancy, especially in regions with limited access to preventive measures. The tumor microenvironment (TME) -specifically, the balance between CD8+ cytotoxic T cells (CTLs) and FOXP3+ regulatory T cells (Tregs)- has emerged as a pivotal determinant of tumor progression and immune evasion. This study aimed to evaluate CD8+/FOXP3+ ratios in both tumor and stromal compartments across different PSCC subtypes and grades. Methods: This retrospective study analyzed tissue microarray (TMA) samples from 108 patients with invasive PSCC. Immunohistochemical staining for CD8+ and FOXP3+ was performed. Tumor and stromal compartments were assessed separately. Ratios of CD8+/FOXP3+ were categorized as CD8 > FOXP3 or CD8 ≤ FOXP3. Associations with histologic subtype and grade were examined using Chi-Square or Fisher's Exact tests, with Cramér's V indicating effect size. Results: Eighty TMA spots (15.2% of the total) had quantifiable data for both markers. We observed a significant association between CD8+/FOXP3+ ratio and histologic grade in both tumor (P=0.03) and stromal compartments (P=0.02), with moderate effect sizes (Cramér's V ~ 0.3). Although no statistically significant associations emerged for histologic subtype, effect size measures suggested potential immune-infiltration differences across subtypes. Descriptive analyses indicated that tumor compartments often contained fewer T cells overall, while stromal areas demonstrated robust infiltration patterns. Conclusions: Tumor grade appears to influence the relative infiltration of cytotoxic and regulatory T cells in PSCC, underscoring the need for compartment-specific immune profiling. These observations highlight the potential utility of CD8+/FOXP3+ ratios as prognostic markers and in guiding future immunotherapeutic strategies. Prospective studies incorporating larger cohorts and HPV stratification could further clarify the immunobiology of PSCC and inform personalized treatment approaches.

The authors have declared no competing interest.

The authors received no financial support for the research, authorship, and/or publication of this article.

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This study received ethical approval from the Institutional Review Board at the Johns Hopkins School of Medicine (Baltimore, MD). This is an IRB-approved retrospective study, all patient information was deidentified and patient consent was not required. Patient data will not be shared with third parties.

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We have made the entire dataset used openly accessible to the scientific community. Interested researchers and collaborators can access this resource through the following link: https://doi.org/10.6084/m9.figshare.28110671.v1.

https://doi.org/10.6084/m9.figshare.28110671.v1