A synonymous germline variant in a gene encoding a cell adhesion molecule is associated with cutaneous mast cell tumour development in Labrador and Golden Retrievers.

Mast cell tumours are the most common type of skin cancer in dogs, representing a significant concern in canine health. The molecular pathogenesis is largely unknown, but breed-predisposition for mast cell tumour development suggests the involvement of inherited genetic risk factors in some breeds. In this study, we aimed to identify germline risk factors associated with the development of mast cell tumours in Labrador Retrievers, a breed with an elevated risk of mast cell tumour development. Using a methodological approach that combined a genome-wide association study, targeted next generation sequencing, and TaqMan genotyping, we identified a synonymous variant in the DSCAM gene on canine chromosome 31 that is associated with mast cell tumours in Labrador Retrievers. DSCAM encodes a cell-adhesion molecule. We showed that the variant has no effect on the DSCAM mRNA level but is associated with a significant reduction in the level of the DSCAM protein, suggesting that the variant affects the dynamics of DSCAM mRNA translation. Furthermore, we showed that the variant is also associated with mast cell tumours in Golden Retrievers, a breed that is closely related to Labrador Retrievers and that also has a predilection for mast cell tumour development. The variant is common in both Labradors and Golden Retrievers and consequently is likely to be a significant genetic contributor to the increased susceptibility of both breeds to develop mast cell tumours. The results presented here not only represent an important contribution to the understanding of mast cell tumour development in dogs, as they highlight the role of cell adhesion in mast cell tumour tumourigenesis, but they also emphasise the potential importance of the effects of synonymous variants in complex diseases such as cancer.

The genomic signature of dog domestication reveals adaptation to a starch-rich diet.

The domestication of dogs was an important episode in the development of human civilization. The precise timing and location of this event is debated and little is known about the genetic changes that accompanied the transformation of ancient wolves into domestic dogs. Here we conduct whole-genome resequencing of dogs and wolves to identify 3.8 million genetic variants used to identify 36 genomic regions that probably represent targets for selection during dog domestication. Nineteen of these regions contain genes important in brain function, eight of which belong to nervous system development pathways and potentially underlie behavioural changes central to dog domestication. Ten genes with key roles in starch digestion and fat metabolism also show signals of selection. We identify candidate mutations in key genes and provide functional support for an increased starch digestion in dogs relative to wolves. Our results indicate that novel adaptations allowing the early ancestors of modern dogs to thrive on a diet rich in starch, relative to the carnivorous diet of wolves, constituted a crucial step in the early domestication of dogs.

Genome-Wide Analysis Identifies Germ-Line Risk Factors Associated with Canine Mammary Tumours.

Canine mammary tumours (CMT) are the most common neoplasia in unspayed female dogs. CMTs are suitable naturally occurring models for human breast cancer and share many characteristics, indicating that the genetic causes could also be shared. We have performed a genome-wide association study (GWAS) in English Springer Spaniel dogs and identified a genome-wide significant locus on chromosome 11 (praw = 5.6x10-7, pperm = 0.019). The most associated haplotype spans a 446 kb region overlapping the CDK5RAP2 gene. The CDK5RAP2 protein has a function in cell cycle regulation and could potentially have an impact on response to chemotherapy treatment. Two additional loci, both on chromosome 27, were nominally associated (praw = 1.97x10-5 and praw = 8.30x10-6). The three loci explain 28.1±10.0% of the phenotypic variation seen in the cohort, whereas the top ten associated regions account for 38.2±10.8% of the risk. Furthermore, the ten GWAS loci and regions with reduced genetic variability are significantly enriched for snoRNAs and tumour-associated antigen genes, suggesting a role for these genes in CMT development. We have identified several candidate genes associated with canine mammary tumours, including CDK5RAP2. Our findings enable further comparative studies to investigate the genes and pathways in human breast cancer patients.

Genome-Wide Association Study of Golden Retrievers Identifies Germ-Line Risk Factors Predisposing to Mast Cell Tumours.

Canine mast cell tumours (CMCT) are one of the most common skin tumours in dogs with a major impact on canine health. Certain breeds have a higher risk of developing mast cell tumours, suggesting that underlying predisposing germ-line genetic factors play a role in the development of this disease. The genetic risk factors are largely unknown, although somatic mutations in the oncogene C-KIT have been detected in a proportion of CMCT, making CMCT a comparative model for mastocytosis in humans where C-KIT mutations are frequent. We have performed a genome wide association study in golden retrievers from two continents and identified separate regions in the genome associated with risk of CMCT in the two populations. Sequence capture of associated regions and subsequent fine mapping in a larger cohort of dogs identified a SNP associated with development of CMCT in the GNAI2 gene (p = 2.2x10-16), introducing an alternative splice form of this gene resulting in a truncated protein. In addition, disease associated haplotypes harbouring the hyaluronidase genes HYAL1, HYAL2 and HYAL3 on cfa20 and HYAL4, SPAM1 and HYALP1 on cfa14 were identified as separate risk factors in European and US golden retrievers, respectively, suggesting that turnover of hyaluronan plays an important role in the development of CMCT.

Genome-wide association study identifies shared risk loci common to two malignancies in golden retrievers.

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10-7 and 2.7×10-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca2+-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps the vesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers.

Amylase activity is associated with AMY2B copy numbers in dog: implications for dog domestication, diet and diabetes.

High amylase activity in dogs is associated with a drastic increase in copy numbers of the gene coding for pancreatic amylase, AMY2B, that likely allowed dogs to thrive on a relatively starch-rich diet during early dog domestication. Although most dogs thus probably digest starch more efficiently than do wolves, AMY2B copy numbers vary widely within the dog population, and it is not clear how this variation affects the individual ability to handle starch nor how it affects dog health. In humans, copy numbers of the gene coding for salivary amylase, AMY1, correlate with both salivary amylase levels and enzyme activity, and high amylase activity is related to improved glycemic homeostasis and lower frequencies of metabolic syndrome. Here, we investigate the relationship between AMY2B copy numbers and serum amylase activity in dogs and show that amylase activity correlates with AMY2B copy numbers. We then describe how AMY2B copy numbers vary in individuals from 20 dog breeds and find strong breed-dependent patterns, indicating that the ability to digest starch varies both at the breed and individual level. Finally, to test whether AMY2B copy number is strongly associated with the risk of developing diabetes mellitus, we compare copy numbers in cases and controls as well as in breeds with varying diabetes susceptibility. Although we see no such association here, future studies using larger cohorts are needed before excluding a possible link between AMY2B and diabetes mellitus.

Danish dog owners' use and the perceived effect of unlicensed cannabis products in dogs.

The interest in the use of medical cannabis has increased in recent years in both human and veterinary fields. In Denmark, there are no veterinary-licensed medical cannabis or cannabinoid supplements, and it is illegal to prescribe or sell cannabinoids intended for the treatment of veterinary patients. This study aimed to explore the unlicensed cannabinoid use in Danish dogs, by questioning dog owners about usage, indication for use, way of purchase, and their perceived effect of the cannabinoid treatment. An anonymous online survey was distributed via social media. The total number of respondents were 2,002, of which 38% indicated using or having administered cannabinoids to their dog. The majority of the respondents confirming the use of cannabinoids (93%) had used cannabidiol drops/oil and only few (4%) reported using Δ9-tetrahydrocannabinol-based products. Most owners (67%) purchased the products online. The three most common indications for use were pain alleviation, behavioural issues, and allergy. When asked about the respondent-perceived effect the majority reported a good or very good effect. The indication with the highest percentage of owner-perceived positive effect (77%) was pain alleviation. This study shows that, despite no licensed veterinary cannabinoid products being available in Denmark, dog owners do supplement their dogs with cannabinoids and the majority of these perceive that the treatment had a positive effect. This supports the need for more evidence-based knowledge in veterinary cannabinoid therapy.

Genome wide association study in Swedish Labrador retrievers identifies genetic loci associated with hip dysplasia and body weight.

Genome wide association studies (GWAS) have been utilized to identify genetic risk loci associated with both simple and complex inherited disorders. Here, we performed a GWAS in Labrador retrievers to identify genetic loci associated with hip dysplasia and body weight. Hip dysplasia scores were available for 209 genotyped dogs. We identified a significantly associated locus for hip dysplasia on chromosome 24, with three equally associated SNPs (p = 4.3 × 10-7) in complete linkage disequilibrium located within NDRG3, a gene which in humans has been shown to be differentially expressed in osteoarthritic joint cartilage. Body weight, available for 85 female dogs, was used as phenotype for a second analysis. We identified two significantly associated loci on chromosome 10 (p = 4.5 × 10-7) and chromosome 31 (p = 2.5 × 10-6). The most associated SNPs within these loci were located within the introns of the PRKCE and CADM2 genes, respectively. PRKCE has been shown to play a role in regulation of adipogenesis whilst CADM2 has been associated with body weight in multiple human GWAS. In summary, we identified credible candidate loci explaining part of the genetic inheritance for hip dysplasia and body weight in Labrador retrievers with strong candidate genes in each locus previously implicated in the phenotypes investigated.

Long-term toxicity and efficacy of FLASH radiotherapy in dogs with superficial malignant tumors.

Introduction: FLASH radiotherapy (RT) has emerged as a promising modality, demonstrating both a normal tissue sparing effect and anticancer efficacy. We have previously reported on the safety and efficacy of single fraction FLASH RT in the treatment of oral tumors in canine cancer patients, showing tumor response but also a risk of radiation-induced severe late adverse effects (osteoradionecrosis) for doses ≥35 Gy. Accordingly, the objective in this study was to investigate if single fraction high dose FLASH RT is safe for treating non-oral tumors. Methods: Privately-owned dogs with superficial tumors or microscopic residual disease were included. Treatment was generally delivered as a single fraction of 15-35 Gy 10 MeV electron FLASH RT, although two dogs were re-irradiated at a later timepoint. Follow-up visits were conducted up to 12 months post-treatment to evaluate treatment efficiency and adverse effects. Results: Fourteen dogs with 16 tumors were included, of which nine tumors were treated for gross disease whilst seven tumors were treated post-surgery for microscopic residual disease. Four treatment sites treated with 35 Gy had ulceration post irradiation, which was graded as severe adverse effect. Only mild adverse effects were observed for the remaining treatment sites. None of the patients with microscopic disease experienced recurrence (0/7), and all patients with macroscopic disease showed either a complete (5/9) or a partial response (4/9). Five dogs were euthanized due to clinical disease progression. Discussion: Our study demonstrates that single fraction high dose FLASH RT is generally safe, with few severe adverse effects, particularly in areas less susceptible to radiation-induced damage. In addition, our study indicates that FLASH has anti-tumor efficacy in a clinical setting. No osteoradionecrosis was observed in this study, although other types of high-grade adverse effects including ulcer-formations were observed for the highest delivered dose (35 Gy). Overall, we conclude that osteoradionecrosis following single fraction, high dose FLASH does not appear to be a general problem for non-oral tumor locations. Also, as has been shown previously for oral tumors, 30 Gy appeared to be the maximum safe dose to deliver with single fraction FLASH RT.

Sarcoma Predisposition in Dogs with a Comparative View to Human Orthologous Disease.

Sarcomas are malignant tumors arising from the embryonic mesodermal cell lineage. This group of cancers covers a heterogenous set of solid tumors arising from soft tissues or bone. Many features such as histology, biological behavior and molecular characteristics are shared between sarcomas in humans and dogs, suggesting that human sarcoma research can be informative for canine disease, and that dogs with sarcomas can serve as relevant translational cancer models, to aid in the understanding of human disease and cancer biology. In the present paper, risk factors for the development of sarcoma in dogs are reviewed, with a particular focus on recent advances in clinical genetics, and on the identification of simple and complex genetic risk factors with a comparison with what has been found in human orthologous disease.

Quality of Life After Partial Mandibulectomy or Maxillectomy in 45 Dogs With Oral Tumors.

Treatment for oral tumors in dogs may involve aggressive surgery, radiation therapy, and/or chemotherapy. It is of utmost importance that veterinarians can document the good quality of life (QoL) for patients during and after cancer treatment. In this retrospective study, medical records from 2 private practices during a 10-year period (2011-2020) were searched to identify dogs with confirmed histopathological diagnosis of an oral tumor. Owners of dogs who underwent surgery received a questionnaire to assess their perception of QoL before and after surgery, clinical signs from the oral tumor, pain before and after surgery, physical appearance, and drinking and eating ability after surgery. Forty-two of 45 (93%) owners answered the questionnaire. Thirty-eight owners (90%) perceived that their dog had not changed its appearance after surgery after the hair had regrown. Thirty owners (71%) reported that their dog prehended food and water normally within 4 weeks after surgery. Forty owners (95%) perceived that their dog had more "good'' than ''bad'' days after surgery. Thirty-eight owners (90%) would choose the same treatment again. Our results strongly support that dog owners perceived that their dogs had good QoL after partial mandibulectomy or maxillectomy.

Evaluation of single-fraction high dose FLASH radiotherapy in a cohort of canine oral cancer patients.

Background: FLASH radiotherapy (RT) is a novel method for delivering ionizing radiation, which has been shown in preclinical studies to have a normal tissue sparing effect and to maintain anticancer efficacy as compared to conventional RT. Treatment of head and neck tumors with conventional RT is commonly associated with severe toxicity, hence the normal tissue sparing effect of FLASH RT potentially makes it especially advantageous for treating oral tumors. In this work, the objective was to study the adverse effects of dogs with spontaneous oral tumors treated with FLASH RT. Methods: Privately-owned dogs with macroscopic malignant tumors of the oral cavity were treated with a single fraction of ≥30Gy electron FLASH RT and subsequently followed for 12 months. A modified conventional linear accelerator was used to deliver the FLASH RT. Results: Eleven dogs were enrolled in this prospective study. High grade adverse effects were common, especially if bone was included in the treatment field. Four out of six dogs, who had bone in their treatment field and lived at least 5 months after RT, developed osteoradionecrosis at 3-12 months post treatment. The treatment was overall effective with 8/11 complete clinical responses and 3/11 partial responses. Conclusion: This study shows that single-fraction high dose FLASH RT was generally effective in this mixed group of malignant oral tumors, but the risk of osteoradionecrosis is a serious clinical concern. It is possible that the risk of osteonecrosis can be mitigated through fractionation and improved dose conformity, which needs to be addressed before moving forward with clinical trials in human cancer patients.

Evaluation of intensity-modulated electron FLASH radiotherapy in a clinical setting using veterinary cases.

PURPOSE: The increased normal tissue tolerance for FLASH radiotherapy (FLASH-RT), as compared to conventional radiotherapy, was first observed in ultra-high dose rate electron beams. Initial clinical trials in companion animals have revealed a high risk of developing osteoradionecrosis following high-dose single-fraction electron FLASH-RT, which may be related to inhomogeneities in the dose distribution. In the current study, we aim to evaluate the possibilities of intensity-modulated electron FLASH-RT in a clinical setting to ensure a homogeneous dose distribution in future veterinary and human clinical trials. METHODS: Our beam model in the treatment planning system electronRT (.decimal, LLC, Sanford, FL, USA) was based on a 10-MeV electron beam from a clinical linear accelerator used to treat veterinary patients with FLASH-RT in a clinical setting. In electronRT, the beam can be intensity-modulated using tungsten island blocks in the electron block cutout, and range-modulated using a customized bolus with variable thickness. Modulations were first validated in a heterogeneous phantom by comparing measured and calculated dose distributions. To evaluate the impact of intensity modulation in superficial single-fraction FLASH-RT, a treatment planning study was conducted, including eight canine cancer patient cases with simulated tumors in the head-and-neck region. For each case, treatment plans with and without intensity modulation were created for a uniform bolus and a range-modulating bolus. Treatment plans were evaluated using a target dose homogeneity index (HI), a conformity index (CI), the near-maximum dose outside the target ( D 2 % , Body - PTV ${D_{2{\mathrm{\% }},{\mathrm{\ Body}} - {\mathrm{PTV}}}}$ ), and the near-minimum dose to the target ( D 98 % ${D_{98\% }}$ ). RESULTS: By adding intensity modulation to plans with a uniform bolus, the HI could be improved (p = 0.017). The combination of a range-modulating bolus and intensity modulation provided a further significant improvement of the HI as compared to using intensity modulation in combination with a uniform bolus (p = 0.036). The range-modulating bolus also improved the CI compared to using a uniform bolus, both with an open beam (p = 0.046) and with intensity modulation (p = 0.018), as well as increased the D 98 % ${D_{98\% }}$ (p = 0.036 with open beam and p = 0.05 with intensity modulation) and reduced the median D 2 % , Body - PTV ${D_{2\% ,{\mathrm{\ Body}} - {\mathrm{PTV}}}}$ (not significant). CONCLUSIONS: By using intensity-modulated electron FLASH-RT in combination with range-modulating bolus, the target dose homogeneity and conformity in canine patients with simulated tumors in complex areas in the head-and-neck region could be improved. By utilizing this technique, we hope to decrease the dose outside the target volume and avoid hot spots in future clinical electron FLASH-RT studies, thereby reducing the risk of radiation-induced toxicity.

PIK3CA is recurrently mutated in canine mammary tumors, similarly to in human mammary neoplasia.

Biological features of neoplastic disease affecting mammary gland tissue are shared between canines and humans. Research performed in either species has translational value and early phase clinical trials performed in canines with spontaneous disease could be informative for human trials. The purpose of this study was to investigate the somatic genetic aberrations occurring in canine mammary neoplasia by exome capture and next generation sequencing. Based on 55 tumor-normal pairs we identified the PIK3CA gene as the most commonly mutated gene in canine mammary tumors, with 25% of samples carrying mutations in this gene. A recurrent missense mutation was identified, p.H1047R, which is homologous to the human PIK3CA hotspot mutation found in different types of breast neoplasia. Mutations homologous to other known human mutation hotspots such as the PIK3CA p.E545K and the KRAS p.G12V/D were also identified. We identified copy number aberrations affecting important tumor suppressor and oncogenic pathways including deletions affecting the PTEN tumor suppressor gene. We suggest that activation of the KRAS or PIK3CA oncogenes or loss of the PTEN suppressor gene may be important for mammary tumor development in dogs. This data endorses the conservation of cancer across species and the validity of studying cancer in non-human species.

Molecular detection of bovine leukosis virus in naturally infected dairy and dual-purpose cattle in Mexico.

The objective of this study was to determine the prevalence of bovine leukosis virus (BLV) in specialized and dual-purpose dairy cows located in the central zone of Veracruz state in Mexico, using endpoint polymerase chain reaction (PCR). The study population consisted of 307 specialized dairy cows and 95 dual-purpose cows from 13 municipalities located in the study area. All cows were apparently healthy and ≥ 3 years old. Cows were stratified by age (3 - 5, 6 - 8 and ≥ 9 years). The overall prevalence of infection was 6.96%; the calculated prevalence in dairy cows was 7.82% and in dual-purpose cows it was 4.21%. The municipality with the highest proportion was Acajete (14.28%), followed by Huatusco and Tomatlán (11.53%). The association analysis confirms the infection's independence to the cows' productive purpose. The results by age strata were 3 - 5 (4.60%), 6 - 8 (8.00%) and ≥ 9 (18.40%) with X2 = 9.96, with an odds ratio of 4.68 for the stratum ≥ 9 years with a significant difference. The present study determined the prevalence of proviral DNA of BLV in dairy and dual-purpose cows in six municipalities in the central zone of Veracruz state, Mexico, using endpoint PCR.

Surface guided electron FLASH radiotherapy for canine cancer patients.

BACKGROUND: During recent years FLASH radiotherapy (FLASH-RT) has shown promising results in radiation oncology, with the potential to spare normal tissue while maintaining the antitumor effects. The high speed of the FLASH-RT delivery increases the need for fast and precise motion monitoring to avoid underdosing the target. Surface guided radiotherapy (SGRT) uses surface imaging (SI) to render a 3D surface of the patient. SI provides real-time motion monitoring and has a large scanning field of view, covering off-isocentric positions. However, SI has so far only been used for human patients with conventional setup and treatment. PURPOSE: The aim of this study was to investigate the performance of SI as a motion management tool during electron FLASH-RT of canine cancer patients. METHODS: To evaluate the SI system's ability to render surfaces of fur, three fur-like blankets in white, grey, and black were used to imitate the surface of canine patients and the camera settings were optimized for each blanket. Phantom measurements using the fur blankets were carried out, simulating respiratory motion and sudden shift. Respiratory motion was simulated using the QUASAR Respiratory Motion Phantom with the fur blankets placed on the phantom platform, which moved 10 mm vertically with a simulated respiratory period of 4 s. Sudden motion was simulated with an in-house developed phantom, consisting of a platform which was moved vertically in a stepwise motion at a chosen frequency. For sudden measurements, 1, 2, 3, 4, 5, 6, 7, and 10 Hz were measured. All measurements were both carried out at the conventional source-to-surface distance (SSD) of 100 cm, and in the locally used FLASH-RT setup at SSD = 70 cm. The capability of the SI system to reproduce the simulated motion and the sampling time were evaluated. As an initial step towards clinical implementation, the feasibility of SI for surface guided FLASH-RT was evaluated for 11 canine cancer patients. RESULTS: The SI camera was capable of rendering surfaces for all blankets. The deviation between simulated and measured mean peak-to-peak breathing amplitude was within 0.6 mm for all blankets. The sampling time was generally higher for the black fur than for the white and grey fur, for the measurement of both respiratory and sudden motion. The SI system could measure sudden motion within 62.5 ms and detect motion with a frequency of 10 Hz. The feasibility study of the canine patients showed that the SI system could be an important tool to ensure patient safety. By using this system we could ensure and document that 10 out of 11 canine patients had a total vector offset from the reference setup position <2 mm immediately before and after irradiation. CONCLUSIONS: We have shown that SI can be used for surface guided FLASH-RT of canine patients. The SI system is currently not fast enough to interrupt a FLASH-RT beam while irradiating but with the short sampling time sudden motion can be detected. The beam can therefore be held just prior to irradiation, preventing treatment errors such as underdosing the target.

Ixovex-1, a novel oncolytic E1B-mutated adenovirus.

There is a great demand for improved oncolytic viruses that selectively replicate within cancer cells while sparing normal cells. Here, we describe a novel oncolytic adenovirus, Ixovex-1, that obtains a cancer-selective replication phenotype by modulating the level of expression of the different, alternatively spliced E1B mRNA isoforms. Ixovex-1 is a recombinant adenovirus that carries a single point mutation in the E1B-93R 3' splice acceptor site that results in overexpression of the E1B-156R splice isoform. In this paper, we studied the characteristics of this novel oncolytic adenovirus by validating its in vitro behaviour in a panel of normal cells and cancer cells. We additionally studied its anti-tumour efficacy in vivo. Ixovex-1 significantly inhibited tumour growth and prolonged survival of mice in an immune-deficient lung carcinoma tumour implantation model. In complementation experiments, overexpression of E1B-156R was shown to increase the oncolytic index of both Ad5wt and ONYX-015. In contrast to prior viruses of similar type, Ixovex-1 includes a functional E3B region for better in vivo efficacy. Throughout this study, the Ixovex-1 virus has been proven to be superior in competency compared to a virus with multiple deletions.

Establishment and Initial Experience of Clinical FLASH Radiotherapy in Canine Cancer Patients.

FLASH radiotherapy has emerged as a treatment technique with great potential to increase the differential effect between normal tissue toxicity and tumor response compared to conventional radiotherapy. To evaluate the feasibility of FLASH radiotherapy in a relevant clinical setting, we have commenced a feasibility and safety study of FLASH radiotherapy in canine cancer patients with spontaneous superficial solid tumors or microscopic residual disease, using the electron beam of our modified clinical linear accelerator. The setup for FLASH radiotherapy was established using a short electron applicator with a nominal source-to-surface distance of 70 cm and custom-made Cerrobend blocks for collimation. The beam was characterized by measuring dose profiles and depth dose curves for various field sizes. Ten canine cancer patients were included in this initial study; seven patients with nine solid superficial tumors and three patients with microscopic disease. The administered dose ranged from 15 to 35 Gy. To ensure correct delivery of the prescribed dose, film measurements were performed prior to and during treatment, and a Farmer-type ion-chamber was used for monitoring. Treatments were found to be feasible, with partial response, complete response or stable disease recorded in 11/13 irradiated tumors. Adverse events observed at follow-up ranging from 3-6 months were mild and consisted of local alopecia, leukotricia, dry desquamation, mild erythema or swelling. One patient receiving a 35 Gy dose to the nasal planum, had a grade 3 skin adverse event. Dosimetric procedures, safety and an efficient clincal workflow for FLASH radiotherapy was established. The experience from this initial study will be used as a basis for a veterinary phase I/II clinical trial with more specific patient inclusion selection, and subsequently for human trials.

The ABCC4 gene is associated with pyometra in golden retriever dogs.

Pyometra is one of the most common diseases in female dogs, presenting as purulent inflammation and bacterial infection of the uterus. On average 20% of intact female dogs are affected before 10 years of age, a proportion that varies greatly between breeds (3-66%). The clear breed predisposition suggests that genetic risk factors are involved in disease development. To identify genetic risk factors associated with the disease, we performed a genome-wide association study (GWAS) in golden retrievers, a breed with increased risk of developing pyometra (risk ratio: 3.3). We applied a mixed model approach comparing 98 cases, and 96 healthy controls and identified an associated locus on chromosome 22 (p = 1.2 × 10-6, passing Bonferroni corrected significance). This locus contained five significantly associated SNPs positioned within introns of the ATP-binding cassette transporter 4 (ABCC4) gene. This gene encodes a transmembrane transporter that is important for prostaglandin transport. Next generation sequencing and genotyping of cases and controls subsequently identified four missense SNPs within the ABCC4 gene. One missense SNP at chr22:45,893,198 (p.Met787Val) showed complete linkage disequilibrium with the associated GWAS SNPs suggesting a potential role in disease development. Another locus on chromosome 18 overlapping the TESMIN gene, is also potentially implicated in the development of the disease.

A novel canine reference genome resolves genomic architecture and uncovers transcript complexity.

We present GSD_1.0, a high-quality domestic dog reference genome with chromosome length scaffolds and contiguity increased 55-fold over CanFam3.1. Annotation with generated and existing long and short read RNA-seq, miRNA-seq and ATAC-seq, revealed that 32.1% of lifted over CanFam3.1 gaps harboured previously hidden functional elements, including promoters, genes and miRNAs in GSD_1.0. A catalogue of canine "dark" regions was made to facilitate mapping rescue. Alignment in these regions is difficult, but we demonstrate that they harbour trait-associated variation. Key genomic regions were completed, including the Dog Leucocyte Antigen (DLA), T Cell Receptor (TCR) and 366 COSMIC cancer genes. 10x linked-read sequencing of 27 dogs (19 breeds) uncovered 22.1 million SNPs, indels and larger structural variants. Subsequent intersection with protein coding genes showed that 1.4% of these could directly influence gene products, and so provide a source of normal or aberrant phenotypic modifications.