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.

A pilot study of toceranib/vinblastine therapy for canine transitional cell carcinoma

“…Based on inter-operator variability and the lack of correlation between clinical outcome and imaging, clinical decision-making based upon image findings alone is not recommended. Alternative measures of disease burden, including activating BRAF mutations, survivin and telomerase activity have also been evaluated in TCC [48–51]. …”

Outcome and prognostic factors in medically treated canine prostatic carcinomas: A multi‐institutional study

“…Median age was 9.5 years (range 6‐14 years) and median body weight 23.2 kg (range 5.3‐59 kg). Most patients () were large breed dogs, followed by medium breed (), giant breed () and small breed dogs (). Eighteen patients were cross breed dogs, followed by Labrador (), Jack Russell Terrier (), Staffordshire Bull Terrier (), Boxer dog (), Boston Terrier (), German Short‐Haired Pointer (), Miniature Schnauzer (), Beagle (), English Cocker Spaniel (), Pyrenean Mountain Dog (), Weimaraner (), Rhodesian Ridgeback (), Border Collie () and one each of the following: Scottish Terrier, Airedale Terrier, Bernese Mountain Dog, American Staffordshire Bull Terrier, Shih‐Tzu, Golden Retriever, Tibetan Terrier, Fox Terrier, Welsh Terrier, Old English Sheep Dog, Rottweiler and German Shepherd dog.…”

“…It is of the author's opinion that ultrasound or CT lesions are a poor measure of disease progression or response, and improvements are needed in this area. With further research into the BRAF mutation shed in the urine of canine PCs, this assay may carry potential for monitoring tumour load …”

“…Fine needle aspirates (FNAs) or tissue biopsy can provide a useful diagnostic tool when dealing with PC although percutaneous transabdominal aspirates have been shown to increase the risk of tumour seeding in canine bladder TCC and PC . Molecular detection of the BRAF V595E mutation, found in 80% of canine urothelial and PCs, has been exploited to produce a new non‐invasive and sensitive assay with the use of only free‐catch urine samples …”

Glutathione S‐transferase theta genotypes and environmental exposures in the risk of canine transitional cell carcinoma

“…Dogs with TCC were recruited from the University of Wisconsin‐Madison and from Purdue University, and through collaborations with Colorado State University, the University of Georgia, Texas A&M University, and several private veterinary oncology and primary care practices. The diagnosis of TCC was based on visualization of a bladder or urethral mass on abdominal ultrasound or cystoscopy, combined with positive urine cytology, bladder histopathology, or the BRAF (Raf family serine/threonine‐protein kinase B gene) mutation detection in voided urothelial cells . Confirmed TCC cases could be of any breed, sex, or age.…”