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Canine urothelial carcinoma (UC) and prostate carcinoma (PC) frequently exhibit the BRAFV595E mutation, akin to the BRAFV600E mutation common in various human cancers. Since the initial discovery of the BRAF mutation in canine cancers in 2015, PCR has been the standard method for its detection in both liquid and tissue biopsies. Considering the similarity between the canine BRAFV595E and human BRAFV600E mutations, we hypothesized that immunohistochemistry (IHC) using a BRAFV600E‐specific antibody could effectively identify the canine mutant BRAFV595E protein. We tested 122 canine UC (bladder n = 108, urethra n = 14), 21 PC, and benign tissue using IHC and performed digital droplet PCR (ddPCR) on all 122 UC and on 14 IHC positive PC cases. The results from ddPCR and IHC were concordant in 99% (135/136) of the tumours. Using IHC, BRAFV595E was detected in 72/122 (59%) UC and 14/21 (65%) PC. Staining of all benign bladder and prostate tissues was negative. If present, mutant BRAF staining was homogenous, with rare intratumour heterogeneity in three (4%) cases of UC. Additionally, the BRAFV595E mutation was more prevalent in tumours with urothelial morphology, and less common in glandular PC or UC with divergent differentiation. This study establishes that BRAFV600‐specific IHC is a reliable and accurate method for detecting the mutant BRAFV595E protein in canine UC and PC. Moreover, the use of IHC, especially with tissue microarrays, provides a cost‐efficient test for large‐scale screening of canine cancers for the presence of BRAF mutations. This advancement paves the way for further research to define the prognostic and predictive role of this tumour marker in dogs and use IHC to stratify dogs for the treatment with BRAF inhibitors. [ABSTRACT FROM AUTHOR]

Simple Summary: Genetic molecular testing is starting to become part of standard clinical practice for dogs with cancer as it can assist veterinarians with diagnosis, provide information about prognosis and aid with selection of therapeutic options. In this review, we consider the different body fluids and tissues from which tumour cells, DNA, RNA and the relevant proteins can be isolated, and what methods are currently used for characterising the molecular profile of cancer in dogs. We also consider new methods that are currently being developed and look promising. The aim is to provide an overview of molecular technologies for veterinarians without previous experience in molecular biology. Genetic molecular testing is starting to gain traction as part of standard clinical practice for dogs with cancer due to its multi-faceted benefits, such as potentially being able to provide diagnostic, prognostic and/or therapeutic information. However, the benefits and ultimate success of genomic analysis in the clinical setting are reliant on the robustness of the tools used to generate the results, which continually expand as new technologies are developed. To this end, we review the different materials from which tumour cells, DNA, RNA and the relevant proteins can be isolated and what methods are available for interrogating their molecular profile, including analysis of the genetic alterations (both somatic and germline), transcriptional changes and epigenetic modifications (including DNA methylation/acetylation and microRNAs). We also look to the future and the tools that are currently being developed, such as using artificial intelligence (AI) to identify genetic mutations from histomorphological criteria. In summary, we find that the molecular genetic characterisation of canine neoplasms has made a promising start. As we understand more of the genetics underlying these tumours and more targeted therapies become available, it will no doubt become a mainstay in the delivery of precision veterinary care to dogs with cancer. [ABSTRACT FROM AUTHOR]

Simple Summary: In canine urothelial carcinoma, the BRAF gene is frequently mutated (V595E). To detect this mutation, urine or tissue samples are currently tested by PCR. Recent advances in digital pathology and the power of artificial intelligence (AI) have opened up new possibilities for the detection of genetic alterations through AI histology. This new approach offers a wide range of new opportunities in the field of diagnostic and predictive tumour marker detection. The aim of this study was to test the efficacy of AI histology to predict the presence of the BRAF mutation in canine bladder carcinomas and to assess its intratumoral heterogeneity. This is the first study to utilise AI histology to predict BRAF mutational status in canine urothelial cell carcinomas. In dogs, the BRAF mutation (V595E) is common in bladder and prostate cancer and represents a specific diagnostic marker. Recent advantages in artificial intelligence (AI) offer new opportunities in the field of tumour marker detection. While AI histology studies have been conducted in humans to detect BRAF mutation in cancer, comparable studies in animals are lacking. In this study, we used commercially available AI histology software to predict BRAF mutation in whole slide images (WSI) of bladder urothelial carcinomas (UC) stained with haematoxylin and eosin (HE), based on a training (n = 81) and a validation set (n = 96). Among 96 WSI, 57 showed identical PCR and AI-based BRAF predictions, resulting in a sensitivity of 58% and a specificity of 63%. The sensitivity increased substantially to 89% when excluding small or poor-quality tissue sections. Test reliability depended on tumour differentiation (p < 0.01), presence of inflammation (p < 0.01), slide quality (p < 0.02) and sample size (p < 0.02). Based on a small subset of cases with available adjacent non-neoplastic urothelium, AI was able to distinguish malignant from benign epithelium. This is the first study to demonstrate the use of AI histology to predict BRAF mutation status in canine UC. Despite certain limitations, the results highlight the potential of AI in predicting molecular alterations in routine tissue sections. [ABSTRACT FROM AUTHOR]

Simple Summary: Dark-haired dogs are predisposed to the development of digital squamous cell carcinoma (DSCC), suggesting an underlying genetic predisposition which is yet to be explained. Some authors have suggested a correlation between the number of copies of KIT Ligand, a gene associated with cell survival, proliferation, and melanogenesis, among other functions, and the potential predisposition to DSCC in dogs. This was evidenced by the fact that dogs with DSCC had a significantly higher copy number of this gene than those who did not have the neoplasia. For this reason, we evaluated the potential correlation between the number of copies of the KIT Ligand in genomic DNA with the histological grade of malignancy in dogs with DSCC. Our findings reveal a significant correlation between the number of copies of KIT Ligand and DSCC histological grade. This supports previous studies that KIT Ligand may play a role in DSCC development and, additionally, may be involved with the presence of histologically malignant morphological features. This suggests a potential factor in the development of canine DSCC, which may signify a potential advance in personalized veterinary oncological approaches as well as future breeding programs. Dark-haired dogs are predisposed to the development of digital squamous cell carcinoma (DSCC). This may potentially suggest an underlying genetic predisposition not yet completely elucidated. Some authors have suggested a potential correlation between the number of copies KIT Ligand (KITLG) and the predisposition of dogs to DSCC, containing a higher number of copies in those affected by the neoplasm. In this study, the aim was to evaluate a potential correlation between the number of copies of the KITLG and the histological grade of malignancy in dogs with DSCC. For this, 72 paraffin-embedded DSCCs with paired whole blood samples of 70 different dogs were included and grouped according to their haircoat color as follow: Group 0/unknown haircoat color (n = 11); Group 1.a/black non-Schnauzers (n = 15); group 1.b/black Schnauzers (n = 33); group 1.c/black and tan dogs (n = 7); group 2/tan animals (n = 4). The DSCCs were histologically graded. Additionally, KITLG Copy Number Variation (CNV) was determined by ddPCR. A significant correlation was observed between KITLG copy number and the histological grade and score value. This finding may suggest a possible factor for the development of canine DSCC, thus potentially having an impact on personalized veterinary oncological strategies and breeding programs. [ABSTRACT FROM AUTHOR]

Breed predispositions to canine digital neoplasms are well known. However, there is currently no statistical analysis identifying the least affected breeds. To this end, 2912 canine amputated digits submitted from 2014–2019 to the Laboklin GmbH & Co. KG for routine diagnostics were statistically analyzed. The study population consisted of 155 different breeds (most common: 634 Mongrels, 411 Schnauzers, 197 Labrador Retrievers, 93 Golden Retrievers). Non-neoplastic processes were present in 1246 (43%), tumor-like lesions in 138 (5%), and neoplasms in 1528 cases (52%). Benign tumors (n = 335) were characterized by 217 subungual keratoacanthomas, 36 histiocytomas, 35 plasmacytomas, 16 papillomas, 12 melanocytomas, 9 sebaceous gland tumors, 6 lipomas, and 4 bone tumors. Malignant neoplasms (n = 1193) included 758 squamous cell carcinomas (SCC), 196 malignant melanomas (MM), 76 soft tissue sarcomas, 52 mast cell tumors, 37 non-specified sarcomas, 29 anaplastic neoplasms, 24 carcinomas, 20 bone tumors, and 1 histiocytic sarcoma. Predisposed breeds for SCC included the Schnauzer (log OR = 2.61), Briard (log OR = 1.78), Rottweiler (log OR = 1.54), Poodle (log OR = 1.40), and Dachshund (log OR = 1.30). Jack Russell Terriers (log OR = −2.95) were significantly less affected by SCC than Mongrels. Acral MM were significantly more frequent in Rottweilers (log OR = 1.88) and Labrador Retrievers (log OR = 1.09). In contrast, Dachshunds (log OR = −2.17), Jack Russell Terriers (log OR = −1.88), and Rhodesian Ridgebacks (log OR = −1.88) were rarely affected. This contrasted with the well-known predisposition of Dachshunds and Rhodesian Ridgebacks to oral and cutaneous melanocytic neoplasms. Further studies are needed to explain the underlying reasons for breed predisposition or “resistance” to the development of specific acral tumors and/or other sites. [ABSTRACT FROM AUTHOR]

Canine digital melanoma, in contrast to canine oral melanoma, is still largely unexplored at the molecular genetic level. The aim of this study was to detect mutant genes in digital melanoma. Paraffin-embedded samples from 86 canine digital melanomas were examined for the BRAF V595E variant by digital droplet PCR (ddPCR), and for exon 11 mutations in c-kit. Furthermore, exons 2 and 3 of KRAS and NRAS were analysed by Sanger sequencing. Copy number variations (CNV) of KITLG in genomic DNA were analysed from nine dogs. The BRAF V595E variant was absent and in c-kit, a single nucleotide polymorphism was found in 16/70 tumours (23%). The number of copies of KITLG varied between 4 and 6. KRAS exon 2 codons 12 and 13 were mutated in 22/86 (25.6%) of the melanomas examined. Other mutually exclusive RAS mutations were found within the hotspot loci, i.e., KRAS exon 3 codon 61: 2/55 (3.6%); NRAS exon 2 codons 12 and 13: 2/83 (2.4%); and NRAS exon 3 codon 61: 9/86 (10.5%). However, no correlation could be established between histological malignancy criteria, survival times and the presence of RAS mutations. In summary, canine digital melanoma differs from molecular genetic data of canine oral melanoma and human melanoma, especially regarding the proportion of RAS mutations. [ABSTRACT FROM AUTHOR]