Immune cell analysis in equine penile papilloma, in situ squamous cell carcinoma and invasive squamous cell carcinoma: FoxP3+ T regulatory lymphocytes differ according to equine papillomavirus 2 status

Equine penile tumors are common in horses and are often related to infection with equine papillomavirus type 2 (EcPV2). This study investigated the immune cell infiltrate (ICI) of these tumors in horses, focusing on the role of EcPV2. Using multiplex immunohistochemistry (mIHC) for CD3, CD20, and IBA-1 and immunohistochemistry (IHC) for FoxP3, 27 horses with papillomas (5/27), in situ carcinomas (CISs) (3/27), and squamous cell carcinomas (SCCs) (19/27) were evaluated. Eighteen cases tested positive for EcPV2 by either or both in situ hybridization (ISH) and polymerase chain reaction (PCR) (18/27 by PCR, of which 16 were ISH+). The ICIs were more abundant in EcPV2-positive tumors, although differences were not statistically significant. The number of FoxP3+ regulatory T-cells was significantly higher in EcPV2+ tumors, both in intraepithelial and stromal compartments. There were higher IBA-1+ macrophage densities in SCCs than in papillomas or CISs. p53 IHC was performed, and non-basal positivity was associated with malignancy. The TP53 mutational analysis with next-generation sequencing revealed that 13/21 cases had a wild-type TP53, while TP53 variants were detected in 4/21 cases. The ICIs did not vary according to TP53 status. Tumor proliferation was also assessed with Ki67, which indicated progressively higher proliferation from benign to malignant tumors. In conclusion, although the number and distribution of B-cells, T-cells, and macrophages did not vary according to EcPV2 status, FoxP3 regulatory T-cells were observed in significantly higher numbers in EcPV2+ neoplasms, indicating a different immune landscape compared to EcPV2-negative tumors.