@article{mochizuki_shapiro_breen_pathology_2016, title={Detection of Copy Number Imbalance in Canine Urothelial Carcinoma With Droplet Digital Polymerase Chain Reaction}, volume={53}, ISSN={["1544-2217"]}, url={http://europepmc.org/abstract/med/26574558}, DOI={10.1177/0300985815614975}, abstractNote={Urothelial carcinoma (UC) is the most common neoplasm of the canine urinary tract. Clinical presentation of UC is shared with several other, more common urinary tract disorders, and this often delays diagnosis of the UC. Definitive diagnosis of UC requires histopathologic examination of a biopsy specimen, but the cost and invasiveness for these diagnostic tests often result in most diagnoses being made on the basis of clinical findings, diagnostic imaging, and cytologic examination of urine sediment. Regardless of the diagnostic process used, most UCs currently are not diagnosed until they are at an advanced clinical stage and so are associated with poor prognosis. Improved methods for earlier and less invasive detection are needed. In a previous study, the authors demonstrated the presence of highly recurrent DNA copy number aberrations (CNAs) in canine UC and hypothesized that detection of these CNAs in tumor cells can be used as a molecular diagnostic for UC. In this study, a multiplexed droplet digital polymerase chain reaction (ddPCR) assay was detected to detect and quantify CNAs of specific regions of canine chromosomes 8, 13, 19, and 36. The assay was effective at differentiating 31 neoplastic and 25 nonneoplastic bladder tissues based on copy number, with 100% sensitivity and specificity in tissue samples. CNAs were also detected by ddPCR in 67% (12 of 18) of urine DNA specimens derived from UC patients. The findings show that ddPCR is a useful molecular technique to detect CNAs and may be used as a noninvasive molecular diagnostic test for canine UC.}, number={4}, journal={VETERINARY PATHOLOGY}, author={Mochizuki, Hiroyuki and Shapiro, S.G. and Breen, M. and pathology, Veterinary}, year={2016}, month={Jul}, pages={764–772} }
 @article{mochizuki_kennedy_shapiro_breen_2015, title={BRAF Mutations in Canine Cancers}, volume={10}, ISSN={["1932-6203"]}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000355955300139&KeyUID=WOS:000355955300139}, DOI={10.1371/journal.pone.0129534}, abstractNote={Activating mutations of the BRAF gene lead to constitutive activation of the MAPK pathway. Although many human cancers carry the mutated BRAF gene, this mutation has not yet been characterized in canine cancers. As human and canine cancers share molecular abnormalities, we hypothesized that BRAF gene mutations also exist in canine cancers. To test this hypothesis, we sequenced the exon 15 of BRAF, mutation hot spot of the gene, in 667 canine primary tumors and 38 control tissues. Sequencing analysis revealed that a single nucleotide T to A transversion at nucleotide 1349 occurred in 64 primary tumors (9.6%), with particularly high frequency in prostatic carcinoma (20/25, 80%) and urothelial carcinoma (30/45, 67%). This mutation results in the amino acid substitution of glutamic acid for valine at codon 450 (V450E) of canine BRAF, corresponding to the most common BRAF mutation in human cancer, V600E. The evolutional conservation of the BRAF V600E mutation highlights the importance of MAPK pathway activation in neoplasia and may offer opportunity for molecular diagnostics and targeted therapeutics for dogs bearing BRAF-mutated cancers.}, number={6}, journal={PLOS ONE}, author={Mochizuki, Hiroyuki and Kennedy, Katherine and Shapiro, Susan G. and Breen, Matthew}, year={2015}, month={Jun} }
 @article{mochizuki_shapiro_breen_2015, title={Detection of BRAF Mutation in Urine DNA as a Molecular Diagnostic for Canine Urothelial and Prostatic Carcinoma}, volume={10}, ISSN={["1932-6203"]}, url={http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=ORCID&SrcApp=OrcidOrg&DestLinkType=FullRecord&DestApp=WOS_CPL&KeyUT=WOS:000366903300038&KeyUID=WOS:000366903300038}, DOI={10.1371/journal.pone.0144170}, abstractNote={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.}, number={12}, journal={PLOS ONE}, author={Mochizuki, Hiroyuki and Shapiro, Susan G. and Breen, Matthew}, year={2015}, month={Dec} }