Histopathological and Molecular Characterization of Canine and Feline Neoplasms: An Integrated Approach Using Immunohistochemistry, PCR and qPCR

Abstract

Neoplastic conditions in companion animals are increasingly recognized in clinical veterinary practice, particularly in dogs and cats. These tumors often exhibit diverse biological behaviors, histopathological patterns and clinical outcomes, posing diagnostic and therapeutic challenges. The objective of this study was to comprehensively evaluate four clinical cases of neoplasia-two in dogs and two in cats- using an integrative diagnostic approach that combined histopathology, immunohistochemistry (IHC) and molecular techniques such as polymerase chain reaction (PCR), quantitative real-time PCR (qPCR) and partial sequencing. Here, case 1 featured an 8-year-old female German Shepherd with a chronic mammary gland mass. Histopathology showed ductal and nest-like epithelial arrangements with moderate pleomorphism and stromal desmoplasia. IHC revealed Ki-67 positivity in 13% of tumor cells and P53 expression in 5%. PCR confirmed expression of BCL2, Ki-67, MYC, MMP9 and P53. qPCR demonstrated upregulation of Ki-67, MYC, MMP9 and P53, suggesting transcriptionally active oncogenic pathways. The tumor was diagnosed as an intermediate-grade mammary gland carcinoma. Case 2 was a 9-year-old male local breed dog with a prepubic cutaneous mass. Histological features included squamous epithelial nests with keratin pearls, dyskeratosis and necrosis- typical of well differentiated squamous cell carcinoma. IHC showed 10% Ki-67 positivity and 7% P53 expression, indicative of modest proliferation and limited tumor suppressor involvement. PCR confirmed neoplastic gene expression, but qPCR was not performed. The diagnosis was cutaneous squamous cell carcinoma. Case 3 involved a 1-year-old female cat with a recurrent mammary mass post-surgery. Histopathology revealed papillary and tubular epithelial patterns with low mitotic activity and focal necrosis. IHC results showed only 1% Ki-67 positivity and <1% P53 expression. PCR confirmed the presence of neoplastic markers, but qPCR was not feasible. Despite histological low-grade features, clinical recurrence and rapid progression suggested an aggressive course, possibly due to incomplete resection or multicentric origin and possibly a low grade feline mammary gland carcinoma. Case 4 concerned a 4-year-old male cat with a nasal bridge mass and facial swelling. Histology revealed infiltrative glandular structures with marked atypia and high mitotic activity. Stromal desmoplasia and subcutaneous invasion were also observed. IHC showed 30% Ki-67 positivity, indicating high proliferation, while P53 staining was minimal (<1%). PCR detected all targeted oncogenes. qPCR revealed high expression of Ki-67, MYC and BCL2; MMP9and P53 were also elevated, though less significantly. The diagnosis was high-grade nasal adenocarcinoma. Across all cases, Ki-67 levels strongly correlated with tumor grade. High-grade tumors (Case 4) showed elevated Ki-67 protein and mRNA expression, while low-grade tumors (Cases 2 and 3) showed minimal Ki-67 positivity. MYC expression was notably upregulated in all tumors, indicating its central role in oncogenesis across species. BCL2 was more prominent in feline tumors, particularly in the nasal adenocarcinoma, suggesting species-specific apoptotic regulation. MMP9 was elevated in canine cases, aligning with its known role in tissue invasion and remodeling. P53 expression varied, with discrepancies between IHC and qPCR in some cases (Case 1), potentially reflecting post-translational regulation or degradation. Hematological findings supported these pathological observations. All animals showed elevated erythrocyte sedimentation rates (ESR), indicating chronic inflammation. Lymphocytosis was evident in feline cases, possibly due to prolonged antigenic stimulation from neoplastic processes. Among the amplified genes, only BCL2 produced good-quality sequence data. Several SNPs were detected within the sequenced region of BCL2, with cats showing more variants than dogs. However, as only partial mRNA and CDS regions were obtained, full-length sequencing will be required to confirm and characterize these mutations. Sequencing of other genes (Ki67, MMP9, MYC, and P53) was not successful due to poor-quality reads. This study underscores the utility of a multimodal diagnostic approach in veterinary oncology. Histopathology provided tumor classification and grading; IHC enabled assessment of cell proliferation and tumor suppressor protein involvement; PCR/qPCR offered insights into gene expression and oncogenic activity. In conclusion, integrating histological, immunohistochemical and molecular diagnostics enhances the understanding and classification of tumors in companion animals. This approach supports more precise diagnosis, better prognostication and lays the groundwork for individualized therapeutic strategies. Future research should aim to validate these molecular markers in larger cohorts and further explore their potential in targeted veterinary cancer treatments.