Trinh M.T.K.Ploypetch S.Panyaboriban S.Raksaseri P.Srisuwatanasagul K.Srisuwatanasagul S.Mahidol University2026-05-232026-05-232026-01-01Pakistan Veterinary Journal Vol.46 No.4 (2026) , 952-96002538318https://repository.li.mahidol.ac.th/handle/123456789/116812Canine mammary tumors (CMTs) are the most prevalent neoplasms in female dogs; however, the diagnosis and prognosis of CMTs show a significant challenge in veterinary oncology. This study evaluates alpha-fetoprotein (AFP) and phosphodiesterase 4D (PDE4D) as possible biomarkers for the progression of CMTs, in correlation with the proliferation marker Ki-67. The protein expression was studied across the CMTs spectrum which were normal, adjacent, benign, and malignant tissues by using immunohistochemistry (IHC) and western blotting (WB). Quantitative IHC demonstrated that both AFP and PDE4D levels increased in a stage-dependent manner. AFP levels increased gradually from normal to benign and highest in malignant CMTs, indicating that it is an early and persistent marker of neoplastic transformation. PDE4D exhibited a comparable pattern, indicating its role in sustaining cancer cell viability. WB demonstrated that both proteins exhibited similar higher expression in benign and malignant CMTs, revealing differences between total protein and in situ localization. Correlation analyses showed a significant positive relationship between AFP and Ki-67 only in benign cases, while it disappeared in malignant CMTs. This uncoupling mechanism in malignant CMTs suggested that growth pathways beyond initial cell proliferation were involved. In conclusion, this study demonstrates a new perspective on the novel biomarkers for CMTs. While AFP acts as an early sensitive marker of dedifferentiation of mammary tissue, PDE4D serves as a definitive protein that flips during the transition to aggressive malignant CMTs. These findings establish an integration of AFP-and PDE4D into future precision diagnosis for the development of targeted therapy in veterinary oncology.VeterinaryArticleSCOPUS10.29261/pakvetj/2026.0632-s2.0-10503862918320747764