Abstract 617: Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib

Abstract

Abstract Transitional cell carcinoma (TCC) is the most common bladder cancer in humans and their canine companions. Canine TCCs are usually papillary infiltrative TCCs of intermediate to high grade. Genetic defects identified in human TCCs aid in the diagnosis and therapy of human bladder cancer. We utilized whole exome sequencing to screen canine TCCs for gene mutations that contribute to pathogenesis. Genomic DNA was isolated from 11 canine TCCs, 3 matched normal tissue samples, and 2 canine TCC cell lines. Whole exome capture was conducted using the Canine Agilent Sure-select in-solution capture system, and captured fragments were sequenced using an Illumina HiSeq2000 platform. The sequences were mapped to the CanFam3.1 canine reference genome and somatic mutations were identified using Freebayes. Somatic mutations were characterized and compared to the Cancer Gene Census (COSMIC). Nonsense, missense, and insertion/deletion mutations were identified in 126 genes shown to be drivers or repressors in human cancer. Missense mutations were further screened using SIFT to identify alterations deleterious to protein function. The genes exhibiting in/dels, nonsense, or missense mutations with SIFT scores < 0.45 in at least 2 sites or 2 samples were: BRAF, RPL5, RANBP2, EWSR1, NONO, PTPRB, LYL1, JAK1, MSH2, PER1, PIM1, and WRN. In fact, an activating BRAF V to E mutation was identified in 5 of the tumors and both cell lines. The RPL5 and RANBP2 mutations were also hotspot mutations identified in 5 and 3 of the samples, respectively. Mutations were confirmed using Sanger sequencing of amplified genomic DNA. Interestingly, deleterious RANBP2 mutations were only observed in non-BRAF mutant samples. Drug sensitivity assays using the BRAF V600E targeting drug, Vemurafenib, were conducted in the BRAF mutant canine cell lines as well as the BRAF mutant human A375 melanoma cell line. As previously described, the IC50 for the sensitive A375 line was approximately 100 nM, while each of the BRAF mutant canine lines had an IC50 value ≥ 10 μM. Reverse transcriptase PCR was used to amplify the coding sequence for BRAF from the Bliley canine TCC cell line. The amplified transcript (2125 bp) was sequenced, confirming the heterozygous expression of the V548 to E mutant form of BRAF in this cell line. The sequence also indicated that the predicted expressed protein (AA 10 - 715 of XP_013975364.1, corresponding to predicted exons 1 through 20 of XM_014119889.1) exhibited 99% homology to human BRAF AA53 - 763 (NP_004324.2). BRAF protein expression in these cell lines was confirmed by Western blot analysis. Thus, insensitivity to Vemurafenib is not due to differences in canine BRAF, reduced expression, or alternative splicing of the expressed transcript as previously observed in some Vemurafenib resistant human melanomas. These data indicate that although constitutively active BRAF is expressed in canine TCC, other factors may contribute to pathogenesis. Citation Format: Belen Hernandez, Kathryn Cronise, James C. Costello, Rodney Page, Susan Lana, Kenneth L. Jones, Dawn L. Duval. Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 617.