Clinical Use of Molecular Biomarkers in Canine and Feline Oncology: Current and Future.

Molecular biomarkers are central to personalised medicine for human cancer patients. It is gaining traction as part of standard veterinary clinical practice for dogs and cats with cancer. Molecular biomarkers can be somatic or germline genomic alterations and can be ascertained from tissues or body fluids using various techniques. This review discusses how these genomic alterations can be determined and the findings used in clinical settings as diagnostic, prognostic, predictive, and screening biomarkers. We showcase the somatic and germline genomic alterations currently available to date for testing dogs and cats in a clinical setting, discussing their utility in each biomarker class. We also look at some emerging molecular biomarkers that are promising for clinical use. Finally, we discuss the hurdles that need to be overcome in going 'bench to bedside', i.e., the translation from discovery of genomic alterations to adoption by veterinary clinicians. As we understand more of the genomics underlying canine and feline tumours, molecular biomarkers will undoubtedly become a mainstay in delivering precision veterinary care to dogs and cats with cancer.

Increased susceptibility to Mycobacterium avium complex infection in miniature Schnauzer dogs caused by a codon deletion in CARD9.

Mammals are generally resistant to Mycobacterium avium complex (MAC) infections. We report here on a primary immunodeficiency disorder causing increased susceptibility to MAC infections in a canine breed. Adult Miniature Schnauzers developing progressive systemic MAC infections were related to a common founder, and pedigree analysis was consistent with an autosomal recessive trait. A genome-wide association study and homozygosity mapping using 8 infected, 9 non-infected relatives, and 160 control Miniature Schnauzers detected an associated region on chromosome 9. Whole genome sequencing of 2 MAC-infected dogs identified a codon deletion in the CARD9 gene (c.493_495del; p.Lys165del). Genotyping of Miniature Schnauzers revealed the presence of this mutant CARD9 allele worldwide, and all tested MAC-infected dogs were homozygous mutants. Peripheral blood mononuclear cells from a dog homozygous for the CARD9 variant exhibited a dysfunctional CARD9 protein with impaired TNF-α production upon stimulation with the fungal polysaccharide ß-glucan that activates the CARD9-coupled C-type lectin receptor, Dectin-1. While CARD9-deficient knockout mice are susceptible to experimental challenges by fungi and mycobacteria, Miniature Schnauzer dogs with systemic MAC susceptibility represent the first spontaneous animal model of CARD9 deficiency, which will help to further elucidate host defense mechanisms against mycobacteria and fungi and assess potential therapies for animals and humans.

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

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.

Review of Molecular Technologies for Investigating Canine Cancer.

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.

NONDOMESTIC FELID ABC BLOOD PHENOTYPING, GENOTYPING, AND CROSSMATCHING.

Based upon previous clinical experience with domestic cats (Felis catus), the ability to assess ABC blood types and blood (in-)compatibilities of nondomestic felids, and adequately consider and plan for blood transfusions, may be important. Although nondomestic felids appear to have an ABC blood group system similar to domestic cats, typing with point-of-care kits and by CMAH genotyping for domestic cats have not been reported. In this study, 162 blood samples from 18 different nondomestic felid species (cheetah [Acinonyx jubatus, n = 42], lion [Panthera leo, n = 33], tiger [Panthera tigris, n = 23], Canada lynx [Lynx canadensis, n = 11], snow leopard [Uncia uncia, n = 10], puma [Puma concolor, n = 7], clouded leopard [Neofelis nebulosa, n = 6], serval [Leptailurus serval, n = 5], jaguar [Panthera onca, n = 5], fishing cat [Prionailurus viverrinus, n = 4], Pallas cat [Felis manul, n = 3], bobcat [Lynx rufus, n = 3], ocelot [Leopardus pardalis, n = 3], black footed cat [Felis nigripes, n = 2], leopard [Panthera pardus, n = 2], African wildcat [Felis lybica, n = 1], caracal [Caracal caracal, n = 1], and sand cat [Felis margarita, n = 1]) were ABC blood typed by laboratory and point-of-care tests, genotyped for four known CMAH variants for type B and type C (AB) phenotypes, and crossmatched with one another and domestic type A cats. Traditional tube typing identified blood type A (n = 106), type B (n = 8), type C (n = 43), and no discernible ABC type (n = 4). Several discrepancies were found between point-of-care and traditional typing test results. None of the tested felids possessed the four CMAH variants responsible for type B and C (AB) in domestic cats. Crossmatch incompatibilities (≥2+ agglutination) were identified within and between nondomestic felid species and beyond ABC incompatibilities. Of 26 crossmatches performed between domestic cats and various nondomestic felids, only 7 (27%) were compatible. In conclusion, point-of-care typing kits and CMAH genotyping, successfully used in domestic cats, may not identify the correct ABC blood type in nondomestic felids. Prior crossmatching is recommended to increase the likelihood of compatible transfusions between any nondomestic felids.

Spatial variability in herbaceous plant phenology is mostly explained by variability in temperature but also by photoperiod and functional traits.

Whereas temporal variability of plant phenology in response to climate change has already been well studied, the spatial variability of phenology is not well understood. Given that phenological shifts may affect biotic interactions, there is a need to investigate how the variability in environmental factors relates to the spatial variability in herbaceous species' phenology by at the same time considering their functional traits to predict their general and species-specific responses to future climate change. In this project, we analysed phenology records of 148 herbaceous species, which were observed for a single year by the PhenObs network in 15 botanical gardens. For each species, we characterised the spatial variability in six different phenological stages across gardens. We used boosted regression trees to link these variabilities in phenology to the variability in environmental parameters (temperature, latitude and local habitat conditions) as well as species traits (seed mass, vegetative height, specific leaf area and temporal niche) hypothesised to be related to phenology variability. We found that spatial variability in the phenology of herbaceous species was mainly driven by the variability in temperature but also photoperiod was an important driving factor for some phenological stages. In addition, we found that early-flowering and less competitive species characterised by small specific leaf area and vegetative height were more variable in their phenology. Our findings contribute to the field of phenology by showing that besides temperature, photoperiod and functional traits are important to be included when spatial variability of herbaceous species is investigated.

PCYT2 deficiency in Saarlooswolfdogs with progressive retinal, central, and peripheral neurodegeneration.

We investigated a syndromic disease comprising blindness and neurodegeneration in 11 Saarlooswolfdogs. Clinical signs involved early adult onset retinal degeneration and adult-onset neurological deficits including gait abnormalities, hind limb weakness, tremors, ataxia, cognitive decline and behavioral changes such as aggression towards the owner. Histopathology in one affected dog demonstrated cataract, retinal degeneration, central and peripheral axonal degeneration, and severe astroglial hypertrophy and hyperplasia in the central nervous system. Pedigrees indicated autosomal recessive inheritance. We mapped the suspected genetic defect to a 15 Mb critical interval by combined linkage and autozygosity analysis. Whole genome sequencing revealed a private homozygous missense variant, PCYT2:c.4A>G, predicted to change the second amino acid of the encoded ethanolamine-phosphate cytidylyltransferase 2, XP_038402224.1:(p.Ile2Val). Genotyping of additional Saarlooswolfdogs confirmed the homozygous genotype in all eleven affected dogs and demonstrated an allele frequency of 9.9% in the population. This experiment also identified three additional homozygous mutant young dogs without overt clinical signs. Subsequent examination of one of these dogs revealed early-stage progressive retinal atrophy (PRA) and expansion of subarachnoid CSF spaces in MRI. Dogs homozygous for the pathogenic variant showed ether lipid accumulation, confirming a functional PCYT2 deficiency. The clinical and metabolic phenotype in affected dogs shows some parallels with human patients, in whom PCYT2 variants lead to a rare form of spastic paraplegia or axonal motor and sensory polyneuropathy. Our results demonstrate that PCYT2:c.4A>G in dogs cause PCYT2 deficiency. This canine model with histopathologically documented retinal, central, and peripheral neurodegeneration further deepens the knowledge of PCYT2 deficiency.

Clinical Signs in 166 Beagles with Different Genotypes of Lafora.

Lafora disease (LD) is a genetic disease affecting beagles, resulting in seizures in combination with other signs. The aim of this study was to describe the clinical signs of LD in beagles with different NHLRC1 genotypes. One hundred and sixty-six beagles were tested for an NHLRC1 gene defect: L/L (n = 67), N/L (n = 32), N/N (n = 67). Owners were asked to participate in a survey about the clinical signs of LD in their dogs. These were recorded for the three possible genotypes in the two age groups, <6 years and ≥6 years. In all genotypes, nearly all the signs of LD were described. In the age group ≥ 6 years, however, they were significantly more frequent in beagles with the L/L genotype. If the following three clinical signs occur together in a beagle ≥ 6 years-jerking of the head, photosensitivity and forgetting things he/she used to be able to do-98.2% of these dogs are correctly assigned to the L/L genotype. If one or two of these signs are missing, the correct classification decreases to 92.1% and 13.2%, respectively. Only the combination of certain signs truly indicates the L/L genotype. Yet, for many dogs, only genetic testing will provide confirmation of the disease.

Discordance between ABC blood phenotype and genotype in a domestic short-haired cat.

An adult domestic short-haired feline leukemia virus-infected cat was referred for kidney failure and worsening anemia requiring transfusions. ABC blood typing was performed with an immunochromatographic strip assay at different occasions. Gel column systems were used for the major and minor crossmatching tests, and anti-A and anti-B titers were determined. No discrete A or B bands appeared on the immunochromatographic strips at any time point for the recipient cat. The recipient's plasma agglutinated RBCs from tested type A and B cats. The recipient's RBCs appeared compatible with plasma from 1 type A and 2 B donors, and incompatible with plasma from another type A cat. Genotyping of recipient blood revealed a single homozygous c.179G>T CMAH variant predicting a blood type B. These studies suggest an unusual weak type B or missing all ABC antigens. The latter resembles the exceedingly rare Bombay phenotype in the human ABO blood group system.

Hemophilia A in a litter of Border Collies caused by a one base pair deletion in the F8 gene.

In dogs, hemophilia A is known to affect different breeds. This is a case report describing hemophilia A in a litter of Border Collies. A privately owned bitch and her puppies (n = 7) were presented to the referring veterinarian after acute hematoma formation in the male offspring (n = 3) following microchip implantation. Global coagulation testing, as well as determination of factor VIII and IX activity, were carried out. Based on the results, factor VIII deficiency was suspected. Two of the affected male puppies were euthanized within a few days. Genetic testing of the mother and the surviving male puppy resulted in the description of a deletion in exon 14 of the F8 gene. This c.3206delA variant leads to a frameshift in amino acid sequence and a premature stop codon (p.Asn1069IlefsTer7). The detection of the mutation and consequent testing of related dogs revealed that the deletion most likely had occurred spontaneously in the mother and had been transmitted to several of her offspring in different litters. Identified carriers were taken out of the breeding scheme. It is concluded that genetic testing in the context of suspected genetic disease can lead to preventive measures, including timely exclusion of carriers from breeding.

Artificial Intelligence to Predict the BRAF V595E Mutation in Canine Urinary Bladder Urothelial 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.

Immunohistochemical and Molecular Genetic Analysis of Canine Digital Mast Cell Tumours.

Grading, immunohistochemistry and c-kit mutation status are criteria for assessing the prognosis and therapeutic options of canine cutaneous mast cell tumours (MCTs). As a subset, canine digital MCTs have rarely been explored in this context. Therefore, in this retrospective study, 68 paraffin-embedded canine digital MCTs were analysed, and histological grading was assessed according to Patnaik and Kiupel. The immunohistochemical markers KIT and Ki67 were used, as well as polymerase chain reaction (PCR) for mutational screening in c-kit exons 8, 9, 11 and 14. Patnaik grading resulted in 22.1% grade I, 67.6% grade II and 10.3% grade III tumours. Some 86.8% of the digital MCTs were Kiupel low-grade. Aberrant KIT staining patterns II and III were found in 58.8%, and a count of more than 23 Ki67-positive cells in 52.3% of the cases. Both parameters were significantly associated with an internal tandem duplication (ITD) in c-kit exon 11 (12.7%). French Bulldogs, which tend to form well-differentiated cutaneous MCTs, had a higher proportion of digital high-grade MCTs and ITD in c-kit exon 11 compared with mongrels. Due to its retrospective nature, this study did not allow for an analysis of survival data. Nevertheless, it may contribute to the targeted characterisation of digital MCTs.

A loss-of-function variant in canine GLRA1 associates with a neurological disorder resembling human hyperekplexia.

Hereditary hyperekplexia is a rare neuronal disorder characterized by an exaggerated startle response to sudden tactile or acoustic stimuli. In this study, we present a Miniature Australian Shepherd family showing clinical signs, which have genetic and phenotypic similarities with human hereditary hyperekplexia: episodes of muscle stiffness that could occasionally be triggered by acoustic stimuli. Whole genome sequence data analysis of two affected dogs revealed a 36-bp deletion spanning the exon-intron boundary in the glycine receptor alpha 1 (GLRA1) gene. Further validation in pedigree samples and an additional cohort of 127 Miniature Australian Shepherds, 45 Miniature American Shepherds and 74 Australian Shepherds demonstrated complete segregation of the variant with the disease, according to an autosomal recessive inheritance pattern. The protein encoded by GLRA1 is a subunit of the glycine receptor, which mediates postsynaptic inhibition in the brain stem and spinal cord. The canine GLRA1 deletion is located in the signal peptide and is predicted to cause exon skipping and subsequent premature stop codon resulting in a significant defect in glycine signaling. Variants in GLRA1 are known to cause hereditary hyperekplexia in humans; however, this is the first study to associate a variant in canine GLRA1 with the disorder, establishing a spontaneous large animal disease model for the human condition.

Evaluating the Histologic Grade of Digital Squamous Cell Carcinomas in Dogs and Copy Number Variation of KIT Ligand-A Correlation Study.

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.

miRNA Profiles of Canine Intestinal Carcinomas, Lymphomas and Enteritis Analysed by Digital Droplet PCR from FFPE Material.

Most canine intestinal tumours are B-cell or T-cell lymphomas or carcinomas. They have to be distinguished from cases of enteritis. Non-invasive biomarkers such as miRNAs would be a step towards faster diagnosis. The aim of this study was to investigate shifts in miRNA expression in tissue samples collected from cases of enteritis, carcinoma and lymphoma of the small and large intestine to better understand the potential of miRNA as biomarkers for tumour diagnosis and classification. We selected two oncogenic miRNAs (miR-18b and 20b), two tumour suppressive miRNAs (miR-192 and 194) and two potential biomarkers for neoplasms (miR-126 and 214). They were isolated from FFPE material, quantified by ddPCR, normalised with RNU6B and compared with normal tissue values. Our results confirmed that ddPCR is a suitable method for quantifying miRNA from FFPE material. Expression of miR-18b and miR-192 was higher in carcinomas of the small intestine than in those of the large intestine. Specific miRNA patterns were observed in cases of enteritis, B-cell and T-cell lymphoma and carcinoma. However, oncogenic miR-18b and 20b were not elevated in any group and miR-126 and 214 were down-regulated in T-cell and B-cell lymphoma, as well as in carcinomas and lymphoplasmacytic enteritis of the small intestine.

KITLG Copy Number Germline Variations in Schnauzer Breeds and Their Relevance in Digital Squamous Cell Carcinoma in Black Giant Schnauzers.

Copy number variations (CNVs) of the KITLG gene seem to be involved in the oncogenesis of digital squamous cell carcinoma (dSCC). The aims of this study were (1) to investigate KITLG CNV in giant (GS), standard (SS), and miniature (MS) schnauzers and (2) to compare KITLG CNV between black GS with and without dSCC. Blood samples from black GS (22 with and 17 without dSCC), black SS (18 with and 4 without dSSC; 5 unknown), and 50 MS (unknown dSSC status and coat colour) were analysed by digital droplet PCR. The results are that (1) most dogs had a copy number (CN) value > 4 (range 2.5-7.6) with no significant differences between GS, SS, and MS, and (2) the CN value in black GS with dSCC was significantly higher than in those without dSCC (p = 0.02). CN values > 5.8 indicate a significantly increased risk for dSCC, while CN values < 4.7 suggest a reduced risk for dSCC (grey area: 4.7-5.8). Diagnostic testing for KITLG CNV may sensitise owners to the individual risk of their black GS for dSCC. Further studies should investigate the relevance of KITLG CNV in SS and the protective effects in MS, who rarely suffer from dSCC.

Pathological Findings in Gastrointestinal Neoplasms and Polyps in 860 Cats and a Pilot Study on miRNA Analyses.

Background: Gastrointestinal masses in cats are of clinical relevance, but pathological studies with larger case numbers are lacking. Biomarkers such as miRNA have not yet been investigated in feline intestinal neoplasms. Methods: A retrospective analysis of pathology reports included 860 feline gastrointestinal masses. Immunohistochemistry was performed on 91 lymphomas, 10 sarcomas and 7 mast cell tumours (MCT). Analyses of miRNA-20b and miRNA-192 were performed on 11 lymphomas, 5 carcinomas and 5 control tissues by ddPCR. Results: The pathological diagnosis identified 679 lymphomas, 122 carcinomas, 28 sarcomas, 23 polyps, 7 MCT and 1 leiomyoma. Carcinomas and polyps were most commonly found in the large intestine, lymphomas were most commonly found in the stomach and small intestine and MCT only occurred in the small intestine. Besides the well-described small-cell, mitotic count <2 T-cell lymphomas and the large-cell B-cell lymphomas with a high mitotic count, several variants of lymphomas were identified. The values of miRNA-20b were found to be up-regulated in samples of all types of cancer, whereas miRNA-192 was only up-regulated in carcinomas and B-cell lymphomas. Conclusions: The histopathological and immunohistochemical (sub-)classification of feline intestinal masses confirmed the occurrence of different tumour types, with lymphoma being the most frequent neoplasm. Novel biomarkers such as miRNA-20b and miRNA-192 might have diagnostic potential in feline intestinal neoplasms and should be further investigated.

A Common Missense Variant Causing Factor XI Deficiency and Increased Bleeding Tendency in Maine Coon Cats.

Hereditary factor XI (FXI) deficiency is characterized as an autosomal mild to moderate coagulopathy in humans and domestic animals. Coagulation testing revealed FXI deficiency in a core family of Maine Coon cats (MCCs) in the United States. Factor XI-deficient MCCs were homozygous for a guanine to adenine transition resulting in a methionine substitution for the highly conserved valine-516 in the FXI catalytic domain. Immunoblots detected FXI of normal size and quantity in plasmas of MCCs homozygous for V516M. Some FXI-deficient MCCs experienced excessive post-operative/traumatic bleeding. Screening of 263 MCCs in Europe revealed a mutant allele frequency of 0.232 (23.2%). However, V516M was not found among 100 cats of other breeds. Recombinant feline FXI-M516 (fFXI-M516) expressed ~4% of the activity of wild-type fFXI-V516 in plasma clotting assays. Furthermore, fFXIa-M516 cleaved the chromogenic substrate S-2366 with ~4.3-fold lower catalytic efficacy (kcat/Km) than fFXIa-V516, supporting a conformational alteration of the protease active site. The rate of FIX activation by fFXIa-M516 was reduced >3-fold compared with fFXIa-V516. The common missense variant FXI-V516M causes a cross-reactive material positive FXI deficiency in MCCs that is associated with mild-moderate bleeding tendencies. Given the prevalence of the variant in MCCs, genotyping is recommended prior to invasive procedures or breeding.

Molecular Genetic Investigation of Digital Melanoma in Dogs.

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.

BRAF mutation status and its prognostic significance in 79 canine urothelial carcinomas: A retrospective study (2006-2019).

Urothelial carcinoma (UC) is the most common tumour of the canine urinary bladder. Recently, BRAF mutation testing emerged as a diagnostic option, but its prognostic significance is unknown. This study investigates the relationship between BRAF (variant V595E) mutation status and overall survival in UC-bearing dogs. Seventy-nine patients histologically diagnosed with UC of the bladder and/or urethra between 2006 and 2019 were included in this retrospective single-centre-study. Treatment consisted of meloxicam (n = 39, group 1 'Melox'), mitoxantrone and meloxicam (+/- followed by metronomic chlorambucil; n = 23, group 2 'Chemo') or partial cystectomy followed by meloxicam +/- mitoxantrone (n = 17, group 3 'Sx'). Survival was significantly influenced by treatment (p = .0002) and tumour location (p < .001) in both uni- and multivariable analyses. BRAF mutation was identified in 51 tumours (=64.6%) and had no statistically significant influence on overall survival: MST for BRAF-negative patients 359 versus 214 days for BRAF-positive dogs (p = .055). However, in BRAF-positive dogs, survival depended significantly on type of treatment in univariable analysis: MSTs for groups 1-3 were 151, 244 and 853 days, respectively (p = .006); In BRAF-positive group 2 ('Chemo')-patients, adjuvant metronomic chlorambucil after mitoxantrone more than doubled MST compared to patients receiving mitoxantrone alone (588 vs. 216 days; p = .030). In contrast, MSTs were not significantly different in BRAF-negative patients among the three treatment groups (p = .069). Multivariate analysis of these data was not possible due to group size limitations. This study identified tumour location and treatment type, but not BRAF mutation status, as independent prognostic factors for overall survival.