title: Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma.
abstract: Urothelial carcinoma (UC) of the lower urinary tract and prostatic carcinoma (PC) are aggressive genitourinary cancers in dogs, characterized by invasion to surrounding tissues and high metastatic potential. Current diagnosis of canine UC and PC requires histopathological examination of a biopsy. Such specimens require specialized medical equipment and are invasive procedures, limiting the availability of diagnosis by histopathology for many canine patients. Access to a non-invasive means to confirm diagnosis is currently an unmet need. Recently, the canine BRAF V595E mutation was detected in ~80% of canine UCs and PCs. In this study, we developed a droplet digital PCR (ddPCR) assay for detection of the canine BRAF V595E mutation in canine urogenital tumors. The assay was evaluated in DNA samples prepared from biopsy specimens of UC (n = 48) and PC (n = 27), as well and non-neoplastic bladder epithelium (n = 38). In addition the assay was assessed for use with DNA isolated from free catch urine samples derived from canine patients with UC (n = 23), PC (n = 3), as well as from dogs with cystitis and healthy controls (n = 37). In all cases the sensitivity to detect the mutant allele was compared with conventional Sanger sequencing. ddPCR had superior sensitivity for detection of the V595E mutation: 75% of UC, 85% of PC, and 0% of control samples were mutation positive, respectively, and the V595E mutation was detected at a level as low as just 1 in 10,000 alleles (~0.01%). Furthermore, the ddPCR assay identified the mutation in free catch urine samples from 83% of canine UC and PC patients, demonstrating its utility as a non-invasive means of diagnosis. We have shown that ddPCR is a sensitive molecular technique with the potential to facilitate accurate and non-invasive means of canine UC and PC diagnosis.
authors.affiliation: Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Mochizuki
authors.affiliation: Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.
authors.forename: Susan G
authors.initials: SG
authors.lastname: Shapiro
authors.affiliation: Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America.|Comparative Medicine Institute, North Carolina State University, Raleigh, North Carolina, United States of America.|Center for Human Health and the Environment, North Carolina State University, Raleigh, North Carolina, United States of America.|Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Matthew
authors.initials: M
authors.lastname: Breen
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000652:urine*; D004247:DNA / Q000652:urine*; D004285:Dogs; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction; D011471:Prostatic Neoplasms / Q000175:diagnosis* / Q000652:urine; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000652:urine
chemical_list: D014408:Biomarkers, Tumor; D004247:DNA; D048493:Proto-Oncogene Proteins B-raf