Precision Medicine in Veterinary Science.

Precision medicine focuses on the clinical management of the individual patient, not on population-based findings. Successes from human precision medicine inform veterinary oncology. Early evidence of success for canines shows how precision medicine can be integrated into practice. Decreasing genomic profiling costs will allow increased utilization and subsequent improvement of knowledge base from which to make better informed decisions. Utility of precision medicine in canine oncology will only increase for improved cancer characterization, enhanced therapy selection, and overall more successful management of canine cancer. As such, practitioners are called to interpret and leverage precision medicine reports for their patients.

Precision medicine using whole genome sequencing identifies a novel dystrophin (DMD) variant for X-linked muscular dystrophy in a cat.

BACKGROUND: Muscular dystrophies (MDs) are a large, heterogeneous group of degenerative muscle diseases. X-linked dystrophin-deficient MD in cats is the first genetically characterized cat model for a human disease and a few novel forms have been identified. HYPOTHESIS/OBJECTIVES: Muscular dystrophy was suspected in a young male domestic shorthair cat. Clinical, molecular, and genetic techniques could provide a definitive diagnosis. ANIMALS: A 1-year-old male domestic shorthair cat presented for progressive difficulty walking, macroglossia and dysphagia beginning at 6 months of age. The tongue was thickened, protruded with constant ptyalism, and thickening and rigidity of the neck and shoulders were observed. METHODS: A complete neurological examination, baseline laboratory evaluation and biopsies of the trapezius muscle were performed with owner consent. Indirect immunofluorescence staining of muscle cryosections was performed using several monoclonal and polyclonal antibodies against dystrophy-associated proteins. DNA was isolated for genomic analyses by whole genome sequencing and comparison to DNA variants in the 99 Lives Cat Genome Sequencing dataset. RESULTS AND CLINICAL IMPORTANCE: Aspartate aminotransferase (687 IU/L) and creatine kinase (24 830 IU/L) activities were increased and mild hypokalemia (3.7 mmol/L) was present. Biopsy samples from the trapezius muscle confirmed a degenerative and regenerative myopathy and protein alterations identified by immunohistochemistry resulted in a diagnosis of a in dystrophin-deficient form of X-linked MD. A stop gain variant (c.4849C>T; p.Gln1617Ter) dystrophin was identified by genome sequencing. Precision/genomic medicine efforts for the domestic cat and in veterinary medicine support disease variant and animal model discovery and provide opportunities for targeted treatments for companion animals.

Precision medicine using whole genome sequencing in a cat identifies a novel COL5A1 variant for classical Ehlers-Danlos syndrome.

BACKGROUND: Ehlers-Danlos syndromes (EDS) are a heterogeneous group of heritable connective tissue disorders occurring in both human and veterinary patients. The genetics of these disorders are poorly described in small animal patients. HYPOTHESIS/OBJECTIVES: Define the clinical manifestations and genetic cause of a suspected form of EDS in a cat. ANIMALS: A 14-week-old male domestic medium hair cat was presented with skin hyperextensibility and fragility. The classic tragic facial expression was observed as well as chronic pruritus and mild hyperesthesia. METHODS: Blood samples and a skin biopsy sample were collected from the affected cat. Clinical examinations, histology, electron microscopy and whole genome sequencing were conducted to characterize the clinical presentation and identify possible pathogenic DNA variants to support a diagnosis. Criteria defining variant pathogenicity were examined including human disease variant databases. RESULTS: Histology showed sparse, disorganized collagen and an increase in cutaneous mast cells. Electron microscopy identified ultrastructural defects commonly seen in collagen type V alpha 1 chain (COL5A1) variants including flower-like collagen fibrils in cross-section. Whole genome sequencing and comparison with 413 cats in the 99 Lives Cat Genome Sequencing Consortium database identified a novel splice acceptor site variant at exon 4 in COL5A1 (c.501-2A>C). CONCLUSIONS AND CLINICAL IMPORTANCE: Our report broadens the current understanding of EDS in veterinary patients and supports the use of precision medicine techniques in clinical veterinary practice. The classification of variants for pathogenicity should be considered in companion animals.

The Role of Personalized Medicine in Companion Animal Cardiology.

Cardiomyopathies remain one of the most common inherited cardiac diseases in both human and veterinary patients. To date, well over 100 mutated genes are known to cause cardiomyopathies in humans with only a handful known in cats and dogs. This review highlights the need and use of personalized one-health approaches to cardiovascular case management and advancement in pharmacogenetic-based therapy in veterinary medicine. Personalized medicine holds promise in understanding the molecular basis of disease and ultimately will unlock the next generation of targeted novel pharmaceuticals and aid in the reversal of detrimental effects at a molecular level.

Feline precision medicine using whole-exome sequencing identifies a novel frameshift mutation for vitamin D-dependent rickets type 2.

OBJECTIVES: A 14-week-old female domestic longhair kitten presented with shifting lameness and disproportionately smaller size compared with a co-housed littermate. METHODS: Hematology and serum biochemical testing were conducted to investigate causes for delayed growth, and radiographs of the appendicular skeleton were obtained. RESULTS: The afflicted kitten had marked hypocalcemia, mild hypophosphatemia and substantial elevations in alkaline phosphatase activity, as well as pathognomonic radiographic findings consistent with rickets. Skeletal changes and hypocalcemia prompted testing of concentrations of parathyroid hormone (PTH) and vitamin D metabolites. Endocrine testing demonstrated significant increases in serum concentrations of PTH and 1,25-dihydroxycholecalciferol (calcitriol), supporting a diagnosis of vitamin D-dependent rickets type 2. Provision of analgesia, supraphysiologic doses of calcitriol and calcium carbonate supplementation achieved normalization of the serum calcium concentration and restoration of normal growth, although some skeletal abnormalities persisted. Once skeletally mature, ongoing calcitriol supplementation was not required. Whole-exome sequencing (WES) was conducted to identify the underlying DNA variant. A cytosine deletion at cat chromosome position B4:76777621 in VDR (ENSFCAT00000029466:c.106delC) was identified and predicted to cause a stop codon in exon 2 (p.Arg36Glufs*18), disrupting >90% of the receptor. The variant was unique and homozygous in this patient and absent in the sibling and approximately 400 other cats for which whole-genome and whole-exome data were available. CONCLUSIONS AND RELEVANCE: A unique, heritable form of rickets was diagnosed in a domestic longhair cat. WES identified a novel frameshift mutation affecting the gene coding for the vitamin D3 receptor, determining the likely causal genetic variant. Precision medicine techniques, including whole-exome and whole-genome sequencing, can be a standard of care in cats to identify disease etiologies, and to target therapeutics and personalize treatment.

New molecular targets in canine hemangiosarcoma-Comparative review and future of the precision medicine.

Human angiosarcoma and canine hemangiosarcoma reveal similarities not only in their aggressive clinical behaviour, but especially in molecular landscape and genetic alterations involved in tumorigenesis and metastasis formation. Currently, no satisfying treatment that allows for achieving long overall survival or even prolonged time to progression does not exist. Due to the progress that has been made in targeted therapies and precision medicine the basis for a new treatment design is to uncover mutations and their functions as possible targets to provide tailored drugs for individual cases. Whole exome or genome sequencing studies and immunohistochemistry brought in the last few years important discoveries and identified the most common mutations with probably crucial role in this tumour development. Also, despite a lack of mutation in some of the culprit genes, the cancerogenesis cause may be buried in main cellular pathways connected with proteins encoded by those genes and involving, for example, pathological angiogenesis. The aim of this review is to highlight the most promising molecular targets for precision oncology treatment from the veterinary perspective aided by the principles of comparative science. Some of the drugs are only undergoing laboratory in vitro studies and others entered the clinic in the management of other cancer types in humans, but those used in dogs with promising responses have been mentioned as priorities.

Standing in the canine precision medicine knowledge gap: Improving annotation of canine cancer genomic biomarkers through systematic comparative analysis of human cancer mutations in COSMIC.

The accrual of cancer mutation data and related functional and clinical associations have revolutionised human oncology, enabling the advancement of precision medicine and biomarker-guided clinical management. The catalogue of cancer mutations is also growing in canine cancers. However, without direct high-powered functional data in dogs, it remains challenging to interpret and utilise them in research and clinical settings. It is well-recognised that canine and human cancers share genetic, molecular and phenotypic similarities. Therefore, leveraging the massive wealth of human mutation data may help advance canine oncology. Here, we present a structured analysis of sequence conservation and conversion of human mutations to the canine genome through a 'caninisation' process. We applied this analysis to COSMIC, the Catalogue of Somatic Mutations in Cancer, the most prominent human cancer mutation database. For the project's initial phase, we focused on the subset of the COSMIC data corresponding to Cancer Gene Census (CGC) genes. A total of 670 canine orthologs were found for 721 CGC genes. In these genes, 365 K unique mutations across 160 tumour types were converted successfully to canine coordinates. We identified shared putative cancer-driving mutations, including pathogenic and hotspot mutations and mutations bearing similar biomarker associations with diagnostic, prognostic and therapeutic utility. Thus, this structured caninisation of human cancer mutations facilitates the interpretation and annotation of canine mutations and helps bridge the knowledge gap to enable canine precision medicine.

Canine Oncopanel: A capture-based, NGS platform for evaluating the mutational landscape and detecting putative driver mutations in canine cancers.

Canine cancer, a significant cause of mortality in domestic dogs, is a powerful comparative model for human cancers. Revealing genetic alterations driving the oncogenesis of canine cancers holds great potential to deepen our understanding of the cancer biology, guide therapeutic development, and improve cancer management in both dogs and people. Next generation sequencing (NGS) based-diagnostic panels have been routinely used in human oncology for the identification of clinically-actionable mutations, enabling tailored treatments based on the individual's unique mutation profiles. Here, we report the development of a comprehensive canine cancer gene panel, the Canine Oncopanel, using a hybridization capture-based targeted NGS method. The Canine Oncopanel allows deep sequencing of 283 cancer genes and the detection of somatic mutations within these genes. Vigorous optimization was performed to achieve robust, high-standard performance using metrics of similar cancer panels in human oncology as benchmarks. Validation of the Canine Oncopanel on reference tumour samples with known mutations demonstrated that it can detect variants previously identified by alternative methods, with high accuracy and sensitivity. Putative drivers were detected in over 90% of clinical samples, showing high sensitivity. The Canine Oncopanel is suitable to map mutation profiles and identify putative driver mutations across common and rare cancer types in dogs. The data generated by the Canine Oncopanel presents a rich resource of putative oncogenic driver mutations and potential clinically relevant markers, paving the way for personalized diagnostics and precision medicine in canine oncology.

Molecular pathology in the cancer clinic - where are we now and where are we headed?

Molecular pathology is a developing sub-microscopic discipline of pathology that studies the effects of molecular variations and mutations on disease processes. The ultimate goal of molecular pathology in cancer is to predict risk, facilitate diagnosis and improve prognostication based on a complete understanding of the biological impact of specific molecular variations, mutations and dysregulations. This knowledge will provide the basis for customised cancer treatment, so-called precision medicine. Rapid developments in genomics have placed this field at the forefront of clinical molecular pathology and there are already a number of well-established genetic tests available for clinical use including PCR of antigen receptor rearrangement and KIT mutational analysis. Moving beyond tests assessing a single gene, there are significant research efforts utilising genomics to predict cancer risk, forecast aggressive behaviour and identify druggable mutations and therapeutic biomarkers. Researchers are also investigating the use of circulating cells and nucleic acid for clinically useful low morbidity genomic assessments. If we are to realise the full potential of molecular pathology and precision medicine there are a number of challenges to overcome. These include developing our understanding of the underlying biology (in particular intra-tumoural heterogeneity), methodological standardisation of assays, provision of adequate infrastructure and production of novel therapeutics backed by high-quality clinical data supporting the precision medicine approach. The era of molecular pathology holds the potential to revolutionise veterinary cancer care, but its impact on clinical practice will depend upon the extent to which the inherent challenges can be overcome.

3D-culture models as drug-testing platforms in canine lymphoma and their cross talk with lymph node-derived stromal cells.

BACKGROUND: Malignant lymphoma is the most common hematopoietic malignancy in dogs, and relapse is frequently seen despite aggressive initial treatment. In order for the treatment of these recurrent lymphomas in dogs to be effective, it is important to choose a personalized and sensitive anticancer agent. To provide a reliable tool for drug development and for personalized cancer therapy, it is critical to maintain key characteristics of the original tumor. OBJECTIVES: In this study, we established a model of hybrid tumor/stromal spheroids and investigated the association between canine lymphoma cell line (GL-1) and canine lymph node (LN)-derived stromal cells (SCs). METHODS: A hybrid spheroid model consisting of GL-1 cells and LN-derived SC was created using ultra low attachment plate. The relationship between SCs and tumor cells (TCs) was investigated using a coculture system. RESULTS: TCs cocultured with SCs were found to have significantly upregulated multidrug resistance genes, such as P-qp, MRP1, and BCRP, compared with TC monocultures. Additionally, it was revealed that coculture with SCs reduced doxorubicin-induced apoptosis and G2/M cell cycle arrest of GL-1 cells. CONCLUSIONS: SCs upregulated multidrug resistance genes in TCs and influenced apoptosis and the cell cycle of TCs in the presence of anticancer drugs. This study revealed that understanding the interaction between the tumor microenvironment and TCs is essential in designing experimental approaches to personalized medicine and to predict the effect of drugs.

Equine simplified acute physiology score: Personalised medicine for the equine emergency patient.

BACKGROUND: Scoring models are useful tools that guide the attending clinician in gauging the severity of disease evolution and in evaluating the efficacy of treatment. There are few tools available with this purpose for the non-human patient, including horses. We aimed (i) to adapt the simplified acute physiology score 3 (SAPS-3) model for the equine species, reaching a margin of accuracy greater than 75% in the calculation of the probability of survival/death and (ii) to build a decision tree that helps the attending veterinarian in assessment of the clinical evolution of the equine patient. METHODS: From an initial pool of 5568 medical records from University-based Veterinary Hospitals, a final cohort of 1000 was further mined manually for data extraction. A set of 19 variables were evaluated and tested by five machine learning data mining algorithms. RESULTS: The final scoring model, named EqSAPS for equine simplified acute physiology score, reached 91.83% of correct estimates (post hoc) for probability of death within 24 hours upon hospitalization. The area under receiver operating characteristic curve for outcome 'death' was 0.742, while for 'survival' was 0.652. The final decision tree was able to refine prognosis of patients whose EqSAPS score suggested 'death'. CONCLUSION: EqSAPS is a useful tool to gauge the severity of the clinical presentation of the equine patient.

Precision medicine identifies a pathogenic variant of the ITGA2B gene responsible for Glanzmann's thrombasthenia in a cat.

BACKGROUND: A nonpedigreed male cat presented with epistaxis, severe bladder hemorrhage, and secondary urethral obstruction after cystocentesis. OBJECTIVES: To characterize the phenotype of a cat with bleeding diathesis and use a precision medicine approach to identify the molecular genetic defect by whole genome sequencing. METHODS: Adenosine diphosphate (ADP) and arachidonic acid (AA)-induced whole blood platelet aggregometry was performed in the affected cat and a healthy cat. Platelet activation, measured by P-selectin expression, and surface integrin subunit ß3 expression were evaluated by flow cytometry in the affected cat and healthy control. Total integrin subunit αIIb expression was assessed by western blot. Whole genome sequencing at 30× coverage was used to identify genetic variants that segregated in the affected cat compared to 194 cats from the 99 Lives Sequencing Consortium. RESULTS: Platelet aggregometry identified significant impairment in platelet aggregation in response to ADP and AA compared to the control cat. Targeted protein expression analyses by flow cytometry and immunoblot analysis determined that the surface expression and total expression of the integrin, αIIbß3, was absent. Whole genome sequencing identified a homozygous c.1986delC frameshift variant in the integrin subunit αIIb (ITGA2B) gene that was not detected in the control population. The p.Pro662fs (ITGA2B P662X) variant terminates translation of the protein at the extracellular domain of the integrin prematurely, which is predicted to affect expression of the ß3 unit. CONCLUSIONS AND CLINICAL IMPORTANCE: This novel ITGA2B variant and the associated phenotype closely resemble Glanzmann's thrombasthenia, which has never been reported in cats.

Genetics, Genomics, and Emergent Precision Medicine 12 Years After the Equine Reference Genome Was Published.

The first equine reference genome was completed in 2007 and published in 2009. This major accomplishment has enabled equine science to advance in ways that broadly parallel the transformative impact that genomics has had on many animal species including humans. A conceptual overview of reference genomes, genome annotation, and the major implications for equine science is presented. The relationship between genomic sequencing and the accelerating application of precision P4 medicine is discussed in the context of human and equine patients. Emergent technologies built on the foundation of genomic sequencing and rapidly gaining traction in research and clinical settings are introduced.

Precision/Genomic Medicine for Domestic Cats.

The era of Precision / Genomic Medicine has arrived and can improve the veterinary healthcare of companion animals. The goal of Precision / Genomic Medicine is to use an individual's DNA signature / profile to tailor their treatments of their specific health problems. Whole genome sequencing is now a cost-effective diagnostic tool, leading to the discovery of DNA variants associated with health outcomes. These DNA variants become genetic tests and can readily be applied to future cases of individuals with similar symptoms. This article addresses the current state of Precision Medicine in domestic cats and the implications for veterinary care.

Devising a new dialogue for nutrition science: how life course perspective, U-shaped thinking, whole organism thinking, and language precision contribute to our understanding of biological heterogeneity and forge a fresh advance toward precision medicine.

The process of designing and implementing individualized health-promoting interventions, nutritional or otherwise, is fraught with great difficulty owing to the heterogeneity inherent in factors that influence healthy longevity. This article proposes that careful attention to three principles-life course perspective, U-shaped thinking, and whole organism thinking-creates an attitudinal framework that can be used to reframe biological heterogeneity into the clinically relevant question: Who will benefit? The search for tools to cope with the complexity of this heterogeneity has been dominated by technological advances, including state-of-the-art "-omics" approaches and machine-based handling of "big data." Here, it is proposed that language precision and nuanced category usage could provide critical tools for coping with heterogeneity, thereby enabling interventionalists to design and implement strategies to promote healthy longevity with greater precision. The lack of a clear understanding of "Who will benefit?" stands as a major obstacle to the design and implementation of nutritional strategies to optimize healthy longevity. This article opens a new dialogue situating the principles of life course perspective, U-shaped thinking, and whole organism thinking, along with cultivating an attitude of language precision at the very core of accelerating creative discovery and refining practical advance in the field of nutrition science.

Towards the Delivery of Precision Veterinary Cancer Medicine.

We introduce a next phase in the evolution of medicine affecting human and veterinary patients. This evolution, genomic cancer medicine (Pmed), involves expansion of genomic and molecular biology into clinical medicine. The implementation of these new technologies has already begun and is a commercial reality. We introduce the underpinnings for this evolution, and focus on application in complex disease states. Pet owners have begun requesting Pmed technologies. To meet this demand, it is important to be aware of the opportunities and obstacles associated with available Pmed offerings as well as the current state of the field.

Precision medicine validation: identifying the MYBPC3 A31P variant with whole-genome sequencing in two Maine Coon cats with hypertrophic cardiomyopathy.

OBJECTIVES: The objective of this study was to perform a proof-of-concept experiment that validates a precision medicine approach to identify variants associated with hypertrophic cardiomyopathy (HCM). We hypothesized that whole-genome sequencing would identify variant(s) associated with HCM in two affected Maine Coon/Maine Coon cross cats when compared with 79 controls of various breeds. METHODS: Two affected and two control Maine Coon/Maine Coon cross cats had whole-genome sequencing performed at approximately × 30 coverage. Variants were called in these four cats and 77 cats of various breeds as part of the 99 Lives Cat Genome Sequencing Initiative ( http://felinegenetics.missouri.edu/99lives ) using Platypus v0.7.9.1, annotated with dbSNP ID, and variants' effect predicted by SnpEff. Strict filtering criteria (alternate allele frequency >49%) were applied to identify homozygous-alternate or heterozygous variants in the two HCM-affected samples when compared with 79 controls of various breeds. RESULTS: A total of four variants were identified in the two Maine Coon/Maine Coon cross cats with HCM when compared with 79 controls after strict filtering. Three of the variants identified in genes MFSD12, BTN1A1 and SLITRK5 did not segregate with disease in a separate cohort of seven HCM-affected and five control Maine Coon/Maine Coon cross cats. The remaining variant MYBPC3 segregated with disease status. Furthermore, this gene was previously associated with heart disease and encodes for a protein with sarcomeric function. CONCLUSIONS AND RELEVANCE: This proof-of-concept experiment identified the previously reported MYBPC3 A31P Maine Coon variant in two HCM-affected cases. This result validates and highlights the power of whole-genome sequencing for feline precision medicine.

Implantable biosensors and their contribution to the future of precision medicine.

Precision medicine can be defined as the prevention, investigation and treatment of diseases taking individual variability into account. There are multiple ways in which the field of precision medicine may be advanced; however, recent innovations in the fields of electronics and microfabrication techniques have led to an increased interest in the use of implantable biosensors in precision medicine. Implantable biosensors are an important class of biosensors because of their ability to provide continuous data on the levels of a target analyte; this enables trends and changes in analyte levels over time to be monitored without any need for intervention from either the patient or clinician. As such, implantable biosensors have great potential in the diagnosis, monitoring, management and treatment of a variety of disease conditions. In this review, we describe precision medicine and the role implantable biosensors may have in this field, along with challenges in their clinical implementation due to the host immune responses they elicit within the body.

Advanced nutritional and stem cells approaches to prevent equine metabolic syndrome.

Horses metabolic disorders have become an important problem of modern veterinary medicine. Pathological obesity, insulin resistance and predisposition toward laminitis are associated with Equine Metabolic Syndrome (EMS). Based on pathogenesis of EMS, dietary and cell therapy management may significantly reduce development of this disorder. Special attention has been paid to the diet supplementation with highly bioavailable minerals and mesenchymal stem cells (MSC) which increase insulin sensitivity. In nutrition, there is a great interests in natural algae enriched via biosorption process with micro- and macroelements. In the case of cellular therapy, metabolic condition of engrafted cells may be crucial for the effectiveness of the therapy. Although, recent studies indicated on MSC deterioration in EMS individuals. Here, we described the combined nutritional and stem cells therapy for the EMS treatment. Moreover, we specified in details how EMS affects the adipose-derived stem cells (ASC) population. Presented here, combined kind of therapy- an innovative and cutting edge approach of metabolic disorders treatment may become a new gold standard in personalized veterinary medicine.

Comparative effectiveness of individualised homeopathy and antibiotics in the treatment of bovine clinical mastitis: randomised controlled trial.

Based on the widespread use of homeopathy in dairy farm practice when treating mastitis, a blind randomised controlled trial (RCT) was conducted to assess the effectiveness of homeopathic treatment of clinical mastitis on four dairy farms. The study considered specific guidelines for RCTs as well as the basic principles of individualised homeopathy and involved 180 lactating dairy cows. Evaluation of cure rates was based on clinical investigation of the udder and on laboratory analysis of milk samples. In culture-positive cases, the antibiotic treatment provided suboptimal bacteriological cures (60-81 per cent) but was more effective than individualised homeopathy (33-43 per cent) whose effects appeared little different to those of placebos (45-47 per cent) (P≤0.05). On the cytological cure level, all three treatment methods were similarly ineffective: antibiotic being 2-21 per cent, individualised homeopathy 0-8 per cent and placebo 3-13 per cent (P≤0.05; P=0.13). Antibiotics, individualised homeopathy and placebo had similar effects on bacteriological and cytological cure in cases of culture-negative milk samples (P>0.4) and Escherichia coli infections (P=1.0). The study results implied that the effectiveness of individualised homeopathy does not go beyond a placebo effect and successful treatment is highly dependent on the specific mastitis pathogen. Thus, antimicrobial or alternative remedies used should be based on the bacterial culture of the milk sample. TRIAL REGISTRATION NUMBER: NTP-ID: 00008011-1-9, Pre-results.