The Immune Contexture in Canine Anal Sac Adenocarcinoma: Immunohistochemical Quantification of Tumor-Infiltrating Lymphocytes and Tumor-Associated Macrophages with Image Analysis

Canine anal sac gland adenocarcinomas (ASACs) are locally aggressive and highly metastatic to regional lymph nodes. Tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) can be effective prognostic and predictive markers in numerous human neoplasms and are increasingly investigated in dogs. The aim of this study was to characterize immune cells in canine ASACs and their relationship with tumor size, histologic metastatic status, and tumor clinical stage. Thirty ASACs with known tumor size, metastatic status, and clinical stage were immunolabeled for Iba1 (macrophages), CD20 (B cells), CD3 (T cells), and Foxp3 (regulatory T cells). With image analysis, two areas of 1 mm² were analyzed for each case at the tumor core (TC) and invasive margin (IM) and immune cells were counted. Eighteen patients had metastasis at the time of diagnosis, of which fifteen were nodal only, and three were both distant and nodal. The median tumor size was 32.5 mm (range 11-70). The clinical stage was I in five cases, II in seven cases, III in fifteen cases, and IV in three cases. T cells and macrophages were the most abundant immune cells in all tumors. Tumor size did not influence the number or type of infiltrating immune cells. By contrast, significantly higher numbers of TC T lymphocytes were found in patients without metastasis, while significantly higher numbers of TC macrophages were found in dogs with metastasis. Immune cell infiltrate did not differ according to clinical stage. The results indicate that the tumor immune microenvironment, specifically TILs and TAMs, contribute to tumor behavior and may influence metastatic potential; in particular, high CD3 infiltration may prevent tumor progression, while increased macrophage infiltration could promote it.