Detection of PD-L1 immune marker expression in cases of mammary tumors in dogs

Canine mammary gland tumors are among the most frequently diagnosed neoplasms in female dogs and are characterized by a multifactorial etiology. In addition to clinical examination, histopathological evaluation is essential for definitive diagnosis, while various imaging modalities are employed for metastasis assessment. The presence of micrometastases may compromise treatment efficacy; therefore, their detection prior to or following surgical intervention is of critical importance. In this context, tumor biomarkers offer potential utility for early detection and screening of micrometastases. Biomarkers are biological molecules measured in body fluids such as blood and urine, or in tissues, that provide valuable insights into the presence and progression of disease. In this study, 62 female dogs that came to Istanbul University-Cerrahpaşa Veterinary Faculty Animal Hospital with complaints of a breast mass were examined for PDL-1, CTLA-4, ER-β, PR and HER-2 biomarkers. Based on histopathological findings, the cases were classified into three main groups: malignant mammary tumors (Group I, n=49), benign mammary tumors (Group II, n=4), and mammary hyperplasia/dysplasia (Group III, n=9). Furthermore, the malignant tumors in Group I were subclassified according to histological grade as Grade I (n=7), Grade II (n=26), and Grade III (n=16). In all groups, the expression levels of the biomarkers PDL-1, CTLA-4, ER-β, PR, and HER-2 were determined. The relationship between these biomarkers and tumor macroscopic size (n=29) was evaluated, as well as the interrelationships among the biomarkers themselves. Additionally, triple-negative mammary tumors were identified within the study population, and the expression of immune checkpoint markers PDL-1 and CTLA-4 in these tumors was xix assessed. Furthermore, preoperative complete blood counts—including neutrophil (NEU), lymphocyte (LYM), monocyte (MON), eosinophil (EOS), and basophil (BAS) counts, along with the neutrophil-to-lymphocyte ratio (NLR)—were analyzed for their association with the dogs' post-mastectomy survival times (24 months) and with biomarker expression levels. Although no statistically significant correlations were found among the biomarkers themselves (P>0.05), a statistically significant difference was observed in CTLA-4 expression between Group I and both Group II and Group III (P<0.05). These findings suggest that CTLA-4 expression in canine mammary tumor tissue may serve as a potential marker for differentiating malignant tumors from benign tumors and hyperplasia/dysplasia. Furthermore, a statistically significant difference was detected in HER-2 expression between Grade II and Grade III tumors (P<0.05), indicating that HER-2 may have prognostic relevance in Grade III malignant tumors. No significant association was found between tumor macroscopic size and biomarker expression levels (P>0.05). In triple-negative mammary tumors, PDL-1 and CTLA-4 immune markers were found to be highly expressed. No significant correlation was identified between the hematological parameters evaluated (including neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts) and CTLA-4 expression (P>0.05). Although no linear trend was observed between PDL-1 expression scores (0–2.5 vs. 2.5–3.0), a statistically significant association was found between PDL-1 expression and the neutrophil-to-lymphocyte ratio (NLR) (P<0.05), suggesting a potential link between NLR, immune response, and the tumor microenvironment. No significant correlation was observed between biomarker expression levels and postoperative survival times following mastectomy (P>0.05). The findings of this study are expected to contribute to a better understanding of the immunological characteristics of canine mammary tumors and may provide valuable insights for the development of novel therapeutic strategies, particularly in dogs with triple-negative mammary tumors.