CROSS-SPECIES TRANSCRIPTOME INVESTIGATION OF HUMAN AND CANINE BLADDER CANCER Tanjin Xu 1, Stephen A. Ramsey 1,2, Cheri Goodall 3, Jun He 1, Shay Bracha 3 Gene functional enrichment analysis of cancer vs. normal differential transcript abundances Abstract Canine transitional cell carcinoma (TCC) is anatomically similar to human bladder cancer. Correlating the molecular profiles of these canine and human malignancies is of interest for improving diagnosis and treatment of bladder cancer in both species. Based on their anatomic similarities, we hypothesized that human and canine bladder cancers involve dysregulation of common pathways and genes at the level of the transcriptome and somatic mutations. We tested this hypothesis by profiling cancer transcriptomes and somatic variants from canine TCC tumors and by comparing to analogous datasets from human bladder cancer that were published by the Cancer Genome Atlas (TCGA). We found that canine and human bladder cancer are similar at the level of the transcriptome, pathway- level gene expression, and at the level of mutation counts per gene. Methods RNA was extracted from canine TCC tumors (N = 8) and from unmatched normal bladder samples (N = 3) using TRIzol. PolyA mRNA was isolated from total RNA using the PrepX polyA Isolation Kit on the Apollo 324 instrument (Wafergen). Strand-specific mRNA- seq libraries were prepared from polyA mRNA using the PrepX RNA-seq for Illumina Library kit on the Apollo 324 (Wafergen). Libraries were multiplexed and sequenced at 2x100 bp on a HiSeq 2000 instrument (Illumina). An average of 28M mapped reads per sample were obtained. Reads were aligned to the CanFam3.1 genome assembly using STAR. Counts of aligned reads per gene were obtained using htseq-count with canine gene annotations from Ensembl Release 75. Testing for gene differential read counts between normal and cancer sample groups was performed using DESeq2 with the Benjamini-Hochberg method for false discovery (FDR) rate control. Additionally, 50X-coverage exome sequences were obtained for four tumor samples and for matched non-cancerous tissues (Otogenetics). Exonic somatic mutation calls were made using MuTect (SNVs) and samtools (for indels). RNA-seq BAM files for 414 human urothelial carcinoma and 19 normal bladder samples were obtained from the controlled data access tier of the TCGA project [1] and aligned to the GRCh37 genome assembly. Results Canine TCC and normal bladder have highly distinct transcriptome profiles References 1.Cancer Genome Atlas Research Network. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature 507, 315–322 (2014). 2.Decker, B. et al. Homologous Mutation to Human BRAF V600E Is Common in Naturally Occurring Canine Bladder Cancer--Evidence for a Relevant Model System and Urine-Based Diagnostic Test. Molecular Cancer Research (2015). 3.Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2012). 4.Stransky, N., Cerami, E., Schalm, S., Kim, J. L. & Lengauer, C. The landscape of kinase fusions in cancer. Nat Comms 5, 4846 (2014). 5.Yoshihara, K. et al. The landscape and therapeutic relevance of cancer- associated transcript fusions. Oncogene (2014). Canine bladder cancer and normal bladder epithelium are clearly distinguishable by unbiased analysis of global transcriptome measurements. Each of the eleven marks represents a sample (8 cancer and 3 normal bladder). The horizontal and vertical coordinates are assigned using the Multidimensional Scaling (MDS) algorithm applied to log-transformed, normalized counts of RNA-seq reads aligned to genes. The distance between any two data marks corresponds to the overall dissimilarity of the two samples. The analysis is unbiased because no differential expression testing between sample groups is used in the MDS. Gene functional analysis of transcriptome differences of canine TCC and normal bladder reveal both expected and novel gene functions. Each row in the heatmap corresponds to a gene functional annotation term from the Gene Ontology (GO). Each column in the heatmap corresponds to one of 11 samples (8 bladder cancer and 3 normal bladder epithelium). The heatmap color indicates the overall expression level in the indicated sample, of all genes within the indicated gene function category, relative to the average across all samples (yellow indicates higher than the sample-averaged overall expression level, and blue indicates lower than the sample-averaged overall expression level). Gene functional categories were selected to display based on a Welch’s t-test with empirical Bayes variance estimation, with a statistical significance threshold of FDR < Gene functional terms with expected patterns of differential expression include “Regulation of Mitosis”, “Unfolded Protein Response”, and “Mitochondrial Respiratory Chain Complex”. Human and dog bladder cancer have similar differential cancer/normal gene expression Gene expression ratios between bladder cancer and normal bladder epithelium are highly correlated between dog and human. Each mark corresponds to one of 1,590 genes that are differentially expressed (FDR < 0.05) in bladder cancer vs. normal bladder epithelium in both a canine cohort (8 tumors, 3 normal samples) and in a human cohort (414 tumors, 19 normal samples). The probability that the observed enrichment of genes that are consistently differentially expressed (up in cancer vs. normal in both species, or down in cancer vs. normal in both species) would occur by chance is less than (Fisher’s exact test; O.R. = 31.5). Unbiased analysis of gene-function-level- summarized transcriptome profiling of bladder tumors and normal bladder epithelium reveals similarities between human and dog. Each mark represents a sample from one of 11 dog samples (8 cancer and 3 normal) or 433 human samples (414 cancer and 19 normal). Horizontal and vertical coordinates of the data marks were computed by MDS. MDS was applied to log-transformed, quantile- normalized gene expression counts that were previously transformed to representative values for gene functional annotation groups using Gene Set Variation Analysis (GSVA). Cancer samples from both species (blue) generally segregate from the normal samples from both species (red), indicating the similarity of canine TCC and human urothelial carcinoma at the level of gene function. Genes’ propensities for mutations in bladder cancer are correlated between dog and human The number of mutations within a gene in a canine bladder tumor is correlated with the frequency of mutations seen in its ortholog, in human urothelial carcinomas. A single canine TCC tumor, and a blood sample from the same patient, were profiled by exome DNA sequencing at 50X coverage. Variant predictions were obtained by counting of alternate alleles in the cancer vs. blood samples and requiring that the LOD score for a mutation vs. non-mutation be greater than 6.3 (corresponding to the background rate of somatic mutations). Dog genes were categorized by the numbers of mutations that mapped to them in the annotated dog genome; they ranged from 0 to 14 (horizontal bar positions). Genes were mapped to human orthologs and the average frequencies of mutations in the human orthologs across 209 human urothelial carcinomas (obtained from the COSMIC database) are plotted as the bar heights (error bars indicate standard error). Exome sequencing reveals the presence of a somatic mutation p.V588E in BRAF, which corresponds to mutation p.V600E in human BRAF [2]. Top data track in IGV plot: dog cancer exome. Middle data track: matched blood exome. Bottom data track: dog cancer RNA-seq showing expression of a p.V588E- bearing BRAF transcript in the tumor. Fusion gene analysis of canine bladder cancer: Example: ST7-MET fusion transcript Fusion gene analysis of tumor RNA-seq profile identifies candidate fusion transcript ST7-MET that was validated by PCR and sequencing. Shown above is a 500 kb locus in canine Chromosome 14 (56 Mb), containing the proto-oncogene MET and the tumor suppressor gene ST7. Analysis of paired-end mRNA-seq data from one TCC sample using STAR-fusion [3] identified a split read connecting exon 2 of ST7 to exon 2 of MET. PCR analysis of cDNA from the same tumor sample yielded a PCR product, and Sanger sequencing analysis of the PCR product yielded a sequence that mapped to both exons, indicating the presence of a fusion transcript. Efforts to map the genomic fusion breakpoint for this locus are ongoing. A human ST7-MET fusion transcript has been previously reported in a TCGA lung adenocarcinoma sample [4] and in an astrocytoma sample [5]. Conclusions On both the transcriptional and gene functional annotation levels, canine TCC is highly similar to invasive human urothelial carcinoma, and this similarity could be leveraged to improve sensitivity for detecting cancer-driving genomic mutations and pathways. Acknowlegements We acknowledge financial support for the project from the OSU College of Veterinary Medicine and computing support from the OSU Center for Genome Research and Biocomputing. We thank Prof. Matti Nykter (University of Tampere) for advice on fusion gene analysis. We thank Dr. Sheila Reynolds and Prof. Ilya Shmulevich (Institute for Systems Biology) for help and guidance on accessing and analyzing TCGA clinical and RNA-seq data. 1 School of Electrical Engineering and Computer Science, Oregon State University, Corvallis, OR, USA 2 Department of Biomedical Sciences, Oregon State University, Corvallis, OR, USA 3 Department of Clinical Sciences, Oregon State University, Corvallis, OR, USA