Background: Urothelial carcinoma (UC) is the most common malignant tumor of the canine lower urinary tract. Current diagnostic approaches rely on invasive sampling and urine-based testing limited by sample volume or quality. MicroRNAs and cell-free DNA are minimally invasive biomarkers that may improve detection of canine UC when integrated. Hypothesis/
Objectives: Assess whether integrating microRNA and cell-free DNA biomarkers can distinguish dogs with UC from controls. Animals: Urine samples included 27 control samples and 34 samples from dogs with UC confirmed by commercially available BRAF test.
Methods: Cell-free small nucleic acids were extracted from 0.2mL of urine using a new extraction protocol with a commercially available kit. The expression of an initial panel of 24 UC-associating microRNAs was assessed by qPCR. The most stable reference microRNA was used to calculate relative expression with a ΔCt approach. Individual ROC analyses were used to assess the importance of each microRNA to UC status, highlighting one that was able to discriminate. The presence of a previously described UC-associating BRAF V595E mutation was evaluated using an established qPCR protocol.
Results: V595E BRAF cell-free DNA alone demonstrated an area under the curve (AUC) of 0.746. Combining the newly identified microRNA with mutant BRAF detection improved diagnostic performance (AUC 0.881), with a sensitivity of 88.2% and a specificity of 77.8%. Conclusions and Clinical Importance: Integration of a microRNA biomarker with V595E BRAF improved detection of canine UC compared with single-analyte approaches and may support minimally invasive diagnosis when invasive sampling or adequate urine volume are limited.
