Characterisation of Gene Expression in Canine Invasive Urothelial Carcinoma Using a <scp>NanoString</scp>‐Based Urine Assay

For human and canine invasive urothelial carcinoma (InvUC), there is growing interest in using the molecular characteristics of a tumour to guide individualised treatment strategies. The objective of this study was to use a non-invasive, urine-based method to characterise gene expression signatures in dogs with InvUC. RNA was isolated from canine InvUC tumour samples, urine sediment from dogs with InvUC, normal canine bladder mucosa, and normal canine urine sediment and queried using the nCounter Canine Immuno-Oncology Panel. Differential gene expression profiles were characterised for tissue and urine samples, and nCounter results were compared to bulk RNA-seq gene expression profiles. The effect of spiking normal urine with white blood cells (WBCs) from the same dog was also assessed. Key genes involved in antitumor immune responses and oncogenic signalling pathways, including potential small molecule inhibitor targets, were differentially expressed in tumour and urine samples from dogs with InvUC, compared to normal samples. nCounter-generated gene expression profiles for tumour tissue and urine from dogs with InvUC were highly correlated, whereas the correlation between the nCounter IO panel and bulk RNA-seq results for InvUC tissue was moderate. The addition of WBCs to normal urine affected the gene expression profiles. Analysis of canine urine using the nCounter canine IO panel has good potential for revealing gene expression patterns in InvUC. Additional studies are warranted to determine the extent to which WBC infiltration affects the results related to immune response patterns and the expression of other genes.