Comparative Gene Expression Analyses Identify Luminal and Basal Subtypes of Canine Invasive Urothelial Carcinoma That Mimic Patterns in Human Invasive Bladder Cancer.

More than 160,000 people are expected to die from invasive urothelial carcinoma (iUC) this year worldwide. Research in relevant animal models is essential to improving iUC management. Naturally-occurring canine iUC closely resembles human iUC in histopathology, metastatic behavior, and treatment response, and could provide a relevant model for human iUC. The molecular characterization of canine iUC, however, has been limited. Work was conducted to compare gene expression array results between tissue samples from iUC and normal bladder in dogs, with comparison to similar expression array data from human iUC and normal bladder in the literature. Considerable similarities between enrichment patterns of genes in canine and human iUC were observed. These included patterns mirroring basal and luminal subtypes initially observed in human breast cancer and more recently noted in human iUC. Canine iUC samples also exhibited enrichment for genes involved in P53 pathways, as has been reported in human iUC. This is particularly relevant as drugs targeting these genes/pathways in other cancers could be repurposed to treat iUC, with dogs providing a model to optimize therapy. As part of the validation of the results and proof of principal for evaluating individualized targeted therapy, the overexpression of EGFR in canine bladder iUC was confirmed. The similarities in gene expression patterns between dogs and humans add considerably to the value of naturally-occurring canine iUC as a relevant and much needed animal model for human iUC. Furthermore, the finding of expression patterns that cross different pathologically-defined cancers could allow studies of dogs with iUC to help optimize cancer management across multiple cancer types. The work is also expected to lead to a better understanding of the biological importance of the gene expression patterns, and the potential application of the cross-species comparisons approach to other cancer types as well

Immunohistochemical detection of EGFR expression in normal and iUC canine tissues.

<p>Photomicrographs of canine normal bladder (A and B) and canine iUC samples (C and D) demonstrating immunoreactivity to EGFR. Paired negative controls were used for each specimen (B and D). Please note membrane immunostaining of tumor cells (C) and normal urothelium (A).</p

Analyses of canine iUC samples reveal enrichment for basal and luminal subtypes and also for genes in <i>P53</i> pathways.

<p>A list of genes representing basal and luminal subtypes and those involved in <i>P53</i> pathways was manually generated from published human iUC dataset (Cancer Genome Atlas Research Network. 2014a; Choi et al 2014a and Damrauer et al 2014). A second list was generated, using these genes as a reference, which were also significantly expressed in canine iUC samples and hierarchical clustering was performed using Euclidean distance metrix. Heat maps indicate genes enriched for basal (A) and luminal (B) subtypes of breast cancer in the canine iUC samples and also indicate enriched genes in the <i>P53</i> pathways (C). Red and blue bars above the heat map indicate the clean separation of samples into the previously observed groups. There was no clear segregation of basal and luminal genes.</p

Flow chart and summary of cross-species analyses.

<p>Step-wise depiction of sample processing and cross-species analyses that were performed.</p

Principal Component Analyses (PCA) plot of normal canine bladder and iUC samples.

<p>A PCA plot was generated using normalized data. The PCA plot shows clear separation between normal samples and canine iUC samples. In addition, the PCA plot also demonstrates clear segregation of the canine iUC samples into two groups i.e., group I and group II.</p

Canine iUC samples cluster as two distinct groups.

<p>Hierarchical clustering illustrates the differential expression of genes (p<0.05, FC2) between canine iUC samples vs. normal canine bladder. Furthermore, the canine iUC samples clustered in two distinct groups.</p

Canine and human iUC samples cluster together.

<p>A list of genes that are commonly annotated and significantly expressed (between normal and iUC, p<0.05, FC2) in dogs and humans, was generated. Hierarchical clustering was performed on these genes (n = 436) using Euclidean distance metrix. Figure illustrates that canine and human normal controls cluster together and these cluster separately from canine and human iUC samples. The iUC samples from dogs and humans clustered together. The color codes are: (1) red bar denoting canine normal bladder, (2) brown bar denoting normal human bladder, (3) blue bar denoting canine iUC samples, and (4) grey bar denoting human iUC samples.</p