BRAFV595E detection, uroplakin II expression and PAS staining for diagnosing canine urothelial carcinoma

The gold standard for diagnosing canine urothelial carcinoma (CaUC) is histopathological examination of an incisional/excisional biopsy which is an invasive technique. The risk of tumor seeding by performing invasive techniques in CaUC is known for reason to explore minimally invasive diagnostic tools. Recently, accurate urine-based screening tests, particularly for BRAFV595E detection, have emerged with promising utility. In addition, uroplakin (UPII) is a specific urothelial cell marker for immunohistochemistry (IHC) that is widely used in humans and considered superior to uroplakin III (UPIII) particularly in higher diagnostic sensitivity of poorly differentiated or metastatic carcinoma; however, its expression in CaUC has not been evaluated. This retrospective study aimed to assess UPII and Ki-67 expression in 20 CaUCs (19 urinary bladder and 1 prostate gland) using special stain and IHC. Glycoprotein accumulation in neoplastic urothelial cells was also evaluated using periodic acid–Schiff (PAS) staining to confirm the location of uroplakin plaques. Tumor proliferation was evaluated using the Ki-67 score by IHC. Additionally, BRAFV595E detection by real-time polymerase chain reaction (qPCR) was investigated in three specimen types: urinary cell pellet (UCP), fresh tumor tissue (FT), and formalin-fixed paraffin-embedded tissue (FFPE), and the suitability of these for CaUC screening was compared in 23 CaUCs and 34 controls. Overall, BRAFV595E was detected in 47.8% (11/23) of CaUCs and 2.9% (1/34) of controls. UCP and FT showed consistent BRAF genotypes (k = 0.865, p < 0.001), and heterozygous BRAF mutations were confirmed by chromatogram analysis. UPII was expressed in all CaUCs (100%, 20/20). PAS staining was positive in 86.7% of CaUC cases (13/15). Ki-67 score was not related to histological grade. No association was observed between histological grade, BRAF variant, Ki-67, and UPII expression. In conclusion, qPCR is a promising tool for BRAFV595E detection, and combined IHC for UPII with PAS staining may enhance diagnostic accuracy for CaUC.