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.

Fig 1. Overview of recurrently mutated genes within the study cohort.

POS^V595E samples (n = 8) are denoted by aqua shading and UD^V595E samples (n = 28) by purple shading. The sex of each dog is indicated by a colored box above the sample code (females shown in pink, males in blue, and dogs of unknown sex shown in grey). Within that box, neuter status is shown as either N (neutered male), S (spayed female) or I (intact). Below this, 26 genes are listed that were mutated in two or more samples. The nature of the alteration in each sample is shown by a colored box (green = missense mutation, red = nonsense mutation, blue = frameshift deletion, brown = in-frame deletion). The plot to the right of the gene list indicates the overall percentage of all samples (n = 36) with alteration of each gene, and the nature of those variants. The plot to the left of the gene list compares the percentage of alterations among POS^V595E and UD^V595E samples. The horizontal plot at the top shows the number and nature of variants of these genes identified in each individual sample.

Fig 2. Summary of BRAF exon 12 and MAP2K1 exon 2 deletions identified in UD^V595E specimens.

a) Partial alignment of the deletion hotspot in canine BRAF exon 12 with its human ortholog shows complete conservation of amino acid sequence and only a single nucleotide difference. Horizontal arrows indicate the deleted regions identified within seven of the UD^V595E specimens, spanning either nine or 15 nucleotides (2/7 and 5/7 specimens, respectively). Deletions resulting in loss of entire codons are shown with dotted lines, and disruptive deletions with solid lines. Each region is annotated to indicate the amino acid sequence change resulting from the deletion (e.g. ∆NVTAP). The VAF is shown beside the left arrowhead for each case. Codon numbering in the dog BRAF gene is assigned relative to Ensembl Transcript ENSCAFT00000006305. b) The canine and human deletion hotspots in MAP2K1 exon 2 also show complete conservation of amino acid sequence, with three nucleotide differences. Five samples showed deletions spanning 15 nucleotides. A sixth sample (UD-102) showed a single base change (A > G) resulting in a K57E alteration. Codon numbering in the dog MAP2K1 gene is assigned relative to Ensembl Transcript ENSCAFT00000043934.

Fig 3. Detection of DNA sequence variants within the BRAF and MAP2K1 genes using capillary electrophoresis.

a) Fluorescent peaks represent amplicons generated for each of the four genomic targets in a normal (non-neoplastic) control sample. Numbers above each peak indicate the size of the amplicon in basepairs, determined by reference to the DNA size ladder shown at the bottom. In the normal control sample, only one peak is evident for each of the four targets, consistent with a normal wild-type sequence. b) Corresponding results obtained from tumor samples. The upper three plots show a second peak, indicating the presence of a smaller amplicon resulting from a deletion event. In the fourth plot, the second peak indicates the presence of a mutant BRAF V595E allele. Note that each of the four plots on the right side of the figure is derived from a different tumor specimen, since studies to date have shown these sequence alterations to be mutually exclusive events. BPS = base pair size RFU = relative florescence units.

Fig 4. Potential opportunities for using molecular subclassification as a mechanism for treatment stratification.

This simplified oncoplot shows the distribution of BRAF and MAP2K1 alterations within the sample cohort, shown in context with the site of action of RAF and MEK in the MAPK pathway. The four categories of variants are annotated to show potential therapeutic strategies, based on extrapolation of data from human studies.