Whole exome sequencing analysis of canine urothelial carcinomas without BRAF V595E mutation: Short in-frame deletions in BRAF and MAP2K1 suggest alternative mechanisms for MAPK pathway disruption

Abstract

Molecular profiling studies have shown that 85% of canine urothelial carcinomas (UC) harbor an activating BRAF V595E mutation, which is orthologous to the V600E variant found in several human cancer subtypes. In dogs, this mutation provides both a powerful diagnostic marker and a potential therapeutic target; however, due to their relative infrequency, the remaining 15% of cases remain understudied at the molecular level. We performed whole exome sequencing analysis of 28 canine urine sediments exhibiting the characteristic DNA copy number signatures of canine UC, in which the BRAF V595E mutation was undetected (UDV595E specimens). Among these we identified 13 specimens (46%) harboring short in-frame deletions within either BRAF exon 12 (7/28 cases) or MAP2K1 exons 2 or 3 (6/28 cases). Orthologous variants occur in several human cancer subtypes and confer structural changes to the protein product that are predictive of response to different classes of small molecule MAPK pathway inhibitors. DNA damage response and repair genes, and chromatin modifiers were also recurrently mutated in UDV595E specimens, as were genes that are positive predictors of immunotherapy response in human cancers. Our findings suggest that short in-frame deletions within BRAF exon 12 and MAP2K1 exons 2 and 3 in UDV595E cases are alternative MAPK-pathway activating events that may have significant therapeutic implications for selecting first-line treatment for canine UC. We developed a simple, cost-effective capillary electrophoresis genotyping assay for detection of these deletions in parallel with the BRAF V595E mutation. The identification of these deletion events in dogs offers a compelling cross-species platform in which to study the relationship between somatic alteration, protein conformation, and therapeutic sensitivity.

Canine on the Couch: The New Canary in the Coal Mine for Environmental Health Research

“…A cohort of humans with high-risk urothelial carcinoma, defined as a rate of 55% with a 2-year metastasis, had BRAF mutations on 25% of cases . A recent study found that dogs with urothelial carcinoma that are BRAF V595E undetected have other alterations within the MAPK pathway, including short in-frame deletions within BRAF exon 12 and MAPK1 exons 2 and 3 …”

Leading the pack: Best practices in comparative canine cancer genomics to inform human oncology

“…Standard pipelines are used for routine analyses such as mutation discovery with genomic sequencing reads (e.g., WGS or WES) and gene expression quantification with RNA-seq reads. [26][27][28] Typical pipelines include sequence quality control, alignment to the reference genome, mutation calling and annotation, and transcript/gene assembly and expression level quantification, with some of the commonly used software tools listed in Table 2. For sequence quality control, sequence reads are typically cleaned up with Trimmomatic, which removes adapter sequences and low-quality bases, and examined by FastQC.…”

Genome-wide analyses reveals an association between invasive urothelial carcinoma in the Shetland sheepdog and NIPAL1

“…As with humans, there are multiple environmental factors that reportedly increase risk of canine iUC 18 . Approximately 80% of canine iUC tumors carry a somatic BRAF V595E mutation, which has been shown to increase activity of the MAPK signaling pathway 19 , 20 . Similarly, more than one third of human MIBC tumors carry activating mutations in the MAPK pathway 21 .…”