Validation of a Liquid Biopsy Protocol for Canine BRAFV595E Variant Detection in Dog Urine and Its Evaluation as a Diagnostic Test Complementary to Cytology

Abstract

A significant proportion of canine urothelial carcinomas carry the driver valine to glutamic acid variation (V595E) in BRAF kinase. The detection of V595E may prove suitable to guide molecularly targeted therapies and support non-invasive diagnosis of the urogenital system by means of a liquid biopsy approach using urine. Three cohorts and a control group were included in this multi-step validation study which included setting up a digital PCR assay. This was followed by investigation of preanalytical factors and two alternative PCR techniques on a liquid biopsy protocol. Finally, a blind study using urine as diagnostic sample has been carried out to verify its suitability as diagnostic test to complement cytology. The digital PCR (dPCR) assay proved consistently specific, sensitive, and linear. Using the dPCR assay, the prevalence of V595E in 22 urothelial carcinomas was 90.9%. When compared with histopathology as gold standard in the blind-label cases, the diagnostic accuracy of using the canine BRAF (cBRAF) variation as a surrogate assay against the histologic diagnosis was 85.7% with 92.3% positive predictive value and 80.0% negative predictive value. In all the cases, in which both biopsy tissue and the associated urine were assayed, the findings matched completely. Finally, when combined with urine sediment cytology examination in blind-label cases with clinical suspicion of malignancy, the dPCR assay significantly improved the overall diagnostic accuracy. A liquid biopsy approach on urine using the digital PCR may be a valuable breakthrough in the diagnostic of urothelial carcinomas in dogs.

Neoangiogenesis markers in canine urothelial carcinomas: A cross‐sectional study

“…The V595E BRAF variation detection was carried out using a digital PCR (dPCR) assay previously validated (Gentilini et al., 2022 ). Briefly, using the following procedures: The dPCRs were carried out using a mixture containing 1× of master mix (QuantStudio 3D Digital PCR Master Mix v2; Thermo Fisher Scientific), 1× of primer mix (TaqMan SNP Genotyping Assay, Thermo Fisher) and 2 μL of gDNA template and molecular biology grade water to reach a final volume of 20 μL.…”

“…The V595E BRAF variation detection was carried out using a digital PCR (dPCR) assay previously validated (Gentilini et al, 2022). Briefly, using the following procedures: The dPCRs were carried out using a…”

“…Although there are marker similarities between human UC and its canine counterpart, differences also exist. For example, although activating mutations in the MAPK pathway occur in invasive UC of both species, BRAF mutations are common in dogs (79.0%–90.9%) but rare in humans (Decker et al., 2015 ; Dhawan et al., 2018 ; Fulkerson et al., 2017 ; Gentilini et al., 2022 ; Mochizuki et al., 2015 ; Tagawa et al., 2020 ) BRAF is part of the MAPK signalling pathway and its activating mutations lead to uncontrolled cell growth (Ghosh & Chin, 2009 ; Haluska et al., 2006 ; Quintero‐Fabián et al., 2019 ).…”

Effective detection of BRAFV595E mutation in canine urothelial and prostate carcinomas using immunohistochemistry

“…20,27 Both PCR techniques are highly specific, however, dPCR outperforms qPCR due to its higher sensitivity. 28 PCR can be performed using liquid or tissue biopsies, including urine, cytologic smears or formalin-fixed paraffin-embedded (FFPE) material. 29 However, as with any diagnostic tool, there are certain limitations.…”

“…In contrast, in dogs, PCR, either quantitative (qPCR) or digital (dPCR), is currently the only well‐established method to detect the BRAF V595E mutation 20,27 . Both PCR techniques are highly specific, however, dPCR outperforms qPCR due to its higher sensitivity 28 . PCR can be performed using liquid or tissue biopsies, including urine, cytologic smears or formalin‐fixed paraffin‐embedded (FFPE) material 29 .…”