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Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma

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. Su...

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Autores principales: Mochizuki, Hiroyuki, Shapiro, Susan G., Breen, Matthew
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674145/
https://www.ncbi.nlm.nih.gov/pubmed/26649430
http://dx.doi.org/10.1371/journal.pone.0144170
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author Mochizuki, Hiroyuki
Shapiro, Susan G.
Breen, Matthew
author_facet Mochizuki, Hiroyuki
Shapiro, Susan G.
Breen, Matthew
author_sort Mochizuki, Hiroyuki
collection PubMed
description 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.
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spelling pubmed-46741452015-12-23 Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma Mochizuki, Hiroyuki Shapiro, Susan G. Breen, Matthew PLoS One Research Article 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. Public Library of Science 2015-12-09 /pmc/articles/PMC4674145/ /pubmed/26649430 http://dx.doi.org/10.1371/journal.pone.0144170 Text en © 2015 Mochizuki et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Mochizuki, Hiroyuki
Shapiro, Susan G.
Breen, Matthew
Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma
title Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma
title_full Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma
title_fullStr Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma
title_full_unstemmed Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma
title_short Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma
title_sort detection of braf mutation in urine dna as a molecular diagnostic for canine urothelial and prostatic carcinoma
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674145/
https://www.ncbi.nlm.nih.gov/pubmed/26649430
http://dx.doi.org/10.1371/journal.pone.0144170
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