Characterizing the Molecular and Immune Landscape of Canine Bladder Cancer

Abstract

Transitional cell carcinoma (TCC) is the most common bladder cancer in both humans and dogs. The majority of canine TCCs are invasive tumors of intermediate‐ to high‐grade at diagnosis and result in a median survival of less than a year. Approximately 20% of human TCCs are muscle‐invasive tumors, which frequently metastasize and have a poor prognosis. Human and canine TCCs share similarities in risk factors, histopathology, and sites of metastasis. Thus, naturally‐occurring canine TCC has the potential to serve as a model for the assessment of novel therapeutics which may inform treatment modalities in human TCC.In this study, we performed whole exome sequencing of 11 canine TCC tumors and three matched normal tissue samples, identifying 581 variants in known protein coding genes. Protein coding variants showed enrichment of KEGG pathways involved in PI3K‐Akt signaling, axon guidance, focal adhesion, and chemokine signaling. Variants were screened against the COSMIC Cancer Gene Census, identifying 37 variants in 27 cancer‐related genes. BRAF V to E missense mutations were identified in 4/11 samples and later confirmed via Sanger sequencing in 73% of samples (8/11). Other cancer genes mutated in at least 2 samples include LRP1B, CUL3, MSH2, and RNF213. Microarray analysis was utilized to characterize the transcriptome of canine TCC relative to normal bladder. 802 and 1,327 genes were up‐ and down‐regulated in tumor samples, respectively. Up‐regulated genes were enriched for cell cycle, p53 signaling, and metabolic KEGG pathways. Down‐regulated genes include members of focal adhesion, ECM‐receptor interaction, and PI3K‐Akt signaling pathways. Gene set enrichment analysis was performed, identifying four hallmark gene sets enriched in tumors: E2F targets, G2M checkpoint, mitotic spindle, and interferon alpha response.A major advantage of the dog model is the ability to evaluate novel immunotherapies in spontaneous tumors that develop under normal immunosurveilance. However, a basic understanding of the immune landscape of canine cancers, such as TCC, is required. Using immunohistochemical labeling, we quantified CD3+ T cells and MAC387+ myeloid cells in TCC tumors. CD3+ T cell counts were variable, exhibiting a bimodal distribution, ranging from 1 to 415 cells/mm2, suggesting immunologically “hot” and “cold” tumors. MAC387+ cell counts ranged from 3 to 613 cells/mm2 and did not correlate with CD3+ T cell counts. Clustering of tumors using expression of immune‐related genes (GO:0002376) revealed two distinct groups. Group I exhibited a strong type I interferon gene expression signature, showing enrichment of hallmark gene sets for interferon alpha and gamma responses. Group II exhibited enrichment of hallmark gene sets for G2M checkpoint and E2F targets. The type I interferon signature was applied to human muscle‐invasive bladder tumors in the TCGA database and was associated with a better disease‐free survival. Overall, the results of this study establish the utility of canine bladder cancer as a model for identifying novel therapeutics, specifically immunotherapies, for the treatment of human muscle‐invasive bladder cancer.Support or Funding InformationMorris Animal Foundation and Shipley Foundation