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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2015
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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. |
format | Online Article Text |
id | pubmed-4674145 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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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