Matthew Breen

Avmaquin is a dietary supplement developed by Nutramax Labs, based on sulfurophane, a naturally occulting compound in cruciferous vegetables with an especially high level in broccoli. Clinical trials with human cancer patients have demonstrated that sulforaphane induces chemo-preventive enzymes via Keap1-Nrf2 signaling. For over 20 years, sulfurophane has been studied for anti-cancer properties in in known to inhibit cell growth and promote apoptosis in carcinoma cells. Numerous clinical trials have since been conducted that reinforce the potential for sulfurophane to have beneficial effect for cancer patient outcomes. A survey of the literature reveals in excess of 45 published manuscripts reporting on clinical studies that used broccoli based interventions, including glucoraphanin-rich and/or sulforaphane-rich broccoli beverages, powders and dietary supplements, as well as stabilized formulations of sulforaphane. In addition, with over 40 completed and ongoing clinical trials investigating the benefit of these compounds for treatments, there is a solid body of evidence to indicate the potential for use in cancer bearing dogs. Nutramax Labs developed Avmaquin as a dietary supplement and have developed an appropriate dosing regimen for use with dogs. This study is aimed at investigating the impact of dietary supplementation of Avmaquin on the progression of canine urothelial carcinoma.

Title: Early diagnosis of urothelial carcinoma Rationale: Diagnosis of canine urothelial carcinoma, also known as transitional cell carcinoma, occurs commonly late in the disease process, once a mass has developed and causes discomfort noticeable to owners. With 40,000 to 80,000 dogs diagnosed in the United States each year, earlier diagnosis with the promise for improved survival and quality of life would have a significant impact on canine health. When a single mutation in the canine BRAF gene was identified in canine UC/TCC, the CADETSM BRAF Mutation Detection Assay was launched as a tool for early detection of this cancer. With the technical sensitivity of droplet digital PCR, this assay may become a game-changer in diagnosing UC/TCC early and increasing survival in this devastating disease. Hypothesis/Objectives: Our long-term goal is to achieve increased survival of dogs with UC/TCC through early detection and improved treatment. The central hypothesis is that the CADETSM BRAF Mutation Detection Assay is a valid tool for the early diagnosis of urothelial carcinoma (UC) in dogs. To address this hypothesis, we propose to screen free-catch urine samples of population of dogs to identify a cohort with low levels of the BRAF mutation and evaluate the urinary tract of these dogs by ultrasonography and cystoscopy over time. Once a mass is detected we will correlate tumor volume in dogs with BRAF mutation fractional abundance shed in their urine. Study Design: Prospective clinical study ������������������ Owners of ~51,000 dogs from five breeds at an increased risk of developing UC/TCC will be invited to collect urine for the CADETSM BRAF Mutation Detection Assay. An estimated 1,095 samples will be collected, to identify over 40 dogs with BRAF mutation levels of <5% in their urine. Dogs will be invited to attend a detailed clinical assessment through history, physical examination, complete blood count, serum biochemistry, urinalysis urine cytology, and urogenital 2-D ultrasound with tumor volume measurements. We will continue to recruit cases until we have 30 dogs enrolled. This group will be advanced to cystoscopy and biopsy of any visible lesions. For all 30 dogs, the level of BRAF mutation shed into the urine will be monitored monthly. Once the levels reach 10%, dogs will have repeat diagnostics, including ultrasound, urine cytology, and cystoscopy with biopsy. End point will be definitive diagnosis of UC/TCC through urine cytology or histopathology of tumor biopsies. Preliminary Data: Assessment of over 300 pathology-verified specimens of canine UC/TCC with the CADETSM BRAF Mutation Detection Assay showed that 85% are associated with a V595E BRAF mutation. Analysis of free-catch urine samples from over 100 dogs that had biopsy-verified UC/TCC show 100% agreement between the presence of the BRAF mutation detected and UC/TCC. The BRAF mutation has not been detected in hundreds of specimens from dogs without UC/TCC (e.g. polyps, inflammation etc.). Five dogs with low levels of detectable BRAF mutation (i.e. <3%) had no visible mass on ultrasound and no clinical signs of disease. These dogs received no treatment and developed lower urinary signs consistent with UC/TCC within 3-4 months. Three of these dogs were then imaged and all had developed a trigonal mass on ultrasound. One dog was lost to follow up and the fifth dog developed clinical signs six weeks later, at which time ultrasound revealed generalized bladder wall thickening. Monitoring of free-catch urine of all dogs revealed progressive increases in BRAF mutation level, suggesting that as the UC/TCC advances within the urinary tract, the proportion of cells shed into the urine also increases. Expected Results: If our hypothesis is correct, we will have compelling evidence that detection of the BRAF V595E mutation in the urine of dogs is diagnostic for UC/TCC and can be used to initiate early treatment. Budget and Timeline: We are requesting $203,777 to complete this study over 2 years. Potential Impact for Animal Health: With a strong indication that the CADETSM BRAF Mut

In 2018, bladder cancer (BC) was the 9th most common cancer diagnosed in the US, with 81,000 new diagnoses and 17,000 deaths. BC has three general phenotypes: non-muscle invasive (NMIBC), muscle-invasive (MIBC), and metastatic. Of new BC diagnoses, approximately 75% are NMIBC, and the mainstays of treatment are transurethral resection and intravesical therapy. Unfortunately, despite ����������������adequate��������������� therapy, >50% of NMIBC patients experience tumor recurrences. Due to this high and repeated recurrence rate, care for these patients involves repeated surveillance cystoscopies and surgeries. Patients may also require escalation of care to radical cystectomy, a surgery with high perioperative mortality and permanent long-term quality of life decrements. While patients with NMIBC have good life expectancy, their need for ongoing surveillance, and repeated interventions results in BC being the most costly cancer per patient. Key goals for human NMIBC are identification of these high-risk patients (HR-NMIBC) at the time of diagnosis and prevention of tumor recurrences. With all cancers, prevention is the ultimate goal. Occupational and recreational (principally tobacco smoke) carcinogens are the leading causes of BC, but many patients do not have these exposures. Determining whether other exposures are causing BC remains challenging, but is important because identification and elimination of carcinogens is the first-step in cancer prevention. One innovative approach that might provide an opportunity to identify new environmental risk factors for BC is cross-species comparative oncology. Over 60,000 dogs are diagnosed with BC in the US each year; none are smokers and most do not share the same occupational exposures that humans do. Why then are so many dogs affected by BC? Dogs with BC are usually diagnosed at an advanced stage and consequently most canine BC closely parallels human MIBC. We have developed a non-invasive urine-based test for canine BC that allows the identification of canine BC earlier in its course. Preliminary analyses of earlier canine BCs identified some genomic similarities with human HR-NMIBC, indicating an opportunity to evaluate the dog as a comparative model for human HR-NMIBC. A key next steps to validate our findings is to conduct a rigorous evaluation of genetic mutational burden of these early canine tumors and develop a comprehensive understanding of the clinical and molecular progression of canine BC. We hypothesize that early canine BCs are genetically similar to human HR-NMIBC. Since domestic dogs and their owners share the same home environment, they share many exposures. In several human epidemiological studies, exposure to water disinfection by-proucts (DBPs) has been found to be associated with BC, however, this association has not been fully investigated in the canine model. With their shorter lifespan, dogs have a substantially reduced latency period between carcinogenic exposure and cancer development and can serve as a sentinel species for identification of possible environmental risk factors linked to BC in both species. For this study, we hypothesize that environmental exposures to DBPs, pesticides and carcinogenic organophosphate flame retardants are associated with genomic aberrations that drive carcinogenesis and progression of HR-NMIBC in both dogs and humans.

As part of the Golden Retriever Lifetime Study (GRLS). Morris Animal Foundation (MAF) have initiated a study to collect blood samples from healthy golden retrievers considered seniors of the breed. As part of this 'Golden Oldies' study, blood samples are being collected from across the nation. The blood samples are to be shipped to NC State where they will be stored and subsequently processed to obtain high quality genomic DNA. The DNA will assessed for quality and quantity and then submitted for genomic analysis to a 3rd party laboratory selected by MAF.

Splenic masses comprise ~50% of all canine splenic disease. Despite advances in imaging and pathologic definition, the etiology and medical relevance of splenic lesions in dogs is often ambiguous. While some splenic tumors are benign, ~2/3 are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs most often in several of the most popular large breeds including Golden Retrievers, German Shepherds, and Boxers. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for cardiac and splenic HSA remain unclear, confounding development of preventative strategies. Recently we reported a high prevalence of species of the bacterial genus Bartonella in dogs with HSA from North Carolina, suggesting a potential role in the initiation and/or progression of this cancer. Bartonella species exist worldwide and are transmitted by blood-sucking arthropods (e.g. ticks, fleas). Their presence in splenic tissue could potentially be explained by the fact that the spleen is primarily responsible for removal of blood-borne parasites from the systemic circulation. We therefore propose a comprehensive examination of the potential association between Bartonella infection and HSA by comparing the prevalence of Bartonella DNA in tumor and blood samples from both splenic and cardiac HSA cases, and also within and between distant geographical locations in the US. Ultimately, demonstration of a robust association between Bartonella infection and the development of HSA may lead to new opportunities for improved diagnosis, treatment and prevention of this devastating cancer.

Globally this year, there will be an estimated 650,000 new human head and neck cancer (HNC) diagnoses.1 Half of those people will be cured with intensive combinations of surgery, radiation therapy and chemotherapy. But along their path towards a cure, essentially all of those patients will experience oral mucositis (OM) and discomfort. Indeed, significant treatment-associated pain is reported by 70% of HNC patients undergoing definitive radiotherapy, and 60% of patients report persistent pain for at least six months following treatment.2,3 OM occurs in nearly all patients who receive radiotherapy, and, in 10-25%, an interruption or modification in treatment is required. These treatment delays worsen local tumor control and overall survival time.4 To improve comfort and avoid treatment delays, effective analgesics are needed. Development of effective and appropriately targeted analgesics will require deep knowledge of the underlying pain signaling mechanisms that are activated by HNC treatment; however, there has been essentially no research in this area. Anecdotally, some HNC patients report cold sensitivity or pain associated with the development of OM. This observation led to our preliminary studies and the discovery that a critical component of acute orofacial radiation-associated pain is a signaling pathway mediated by neurons that express the TRPM8 (transient receptor potential melastatin family member 8) ion channel. Previously, this pathway has been associated with ����������������cold pain.��������������� We have evidence from mouse models that: ���������������� Oral irradiation causes local release of a neurotrophic factor called artemin (ARTN), ���������������� ARTN binds its receptor (GFR��������3) on free nerve endings in the mouth, and ���������������� GFR��������3 activates TRPM8 in trigeminal sensory neurons, thus resulting in cold sensitivity and pain.

This study addresses two important challenges related to the development of neoantigen vaccine approaches for treating T-cell lymphoma. The first is quantitative: effective vaccination must elicit anti-tumor effectors in numbers equivalent to those generated in natural infections in order to outpace cancer progression. The second challenge is the negative role that the tumor physiologic microenvironment might play in negatively regulating such immune reactivity, leading to vaccine failure. Peripheral T-cell lymphomas (PTCL) are a group of heterogeneous, clinically aggressive malignancies that are refractory to traditional chemotherapy agents and associated with a poor outcome. Survival with standardof-care CHOP therapy is <8 months.(1, 2) The persistence of a tiny population of chemoresistant malignant cells ������������������ called minimal residual disease, or MRD ������������������ demonstrable by sensitive molecular assays during treatment is thought to be the source of relapse and treatment failure. New chemotherapy agents and small molecule inhibitors have entered the picture, yet outcomes remain stubbornly inferior to those with B-cell tumors.(3, 4) Eradicating MRD via immunotherapy to improve PTCL outcomes and effect cures is a tantalizing possibility. However, trials in PTCL using monoclonal antibodies (Abs) or chimeric antigen receptor T cells targeting various T-cell surface markers have only had limited successes.(5) In solid tumors, immune checkpoint blockade has been shown to elicit T-cell responses directed against tumor-specific neoantigens, which can exert clinically meaningful effects.(6) Recently, neoantigen vaccination in melanoma has also been shown to be an effective, direct means to raise such anti-tumor immune responses.(7, 8) In spite of the strong rationale and some success in treating solid tumors, though, targeting neoantigens to eradicate MRD in PTCL is not well-explored. As non-germline-encoded proteins, neoantigens are attractive mmunotherapy targets because the corresponding (cognate) T-cell repertoire has not been subject to central deletional tolerance, and high-avidity (strongly reactive) effectors are recruitable. Despite this advantage, clinical responses to immune checkpoint blockade are not universal, and success rates vary. In fact, for most tumor types, responding patients are the minority. One mechanism underlying these differential responses is neoantigen availability, which reflects the somatic mutation rate of the tumor; lymphomas, for example, usually have few mutations and are immunologically ����������������cold���������������. Another important cause for the failure of neoantigen-targeted immunotherapy is that T cells in the tumor physiologic microenvironment must operate under harsh, unfavorable conditions, characterized by hypoxia, acidosis and lactate accumulation, which can inhibit effector functions.(9, 10). In PTCL, the situation is potentially more dire, since malignant lymphocytes occupy secondary lymphoid tissues, competing for and depleting nutrients needed by anti-tumor T cells for optimal activation and proliferation.(11) Robust antigen-driven T-cell expansion is vital for effective immunity, and increasingly, it������������������s been recognized that T-cell quantity, not just quality, is a critical determinant of the success or failure of cancer vaccines. Objective responses against tumors are only produced with a massive expansion of the cognate T-cell population to levels rivaling those achieved in viral infections. Characterizing the lymphoid microenvironment in PTCL to determine its ability to support the high metabolic demands of such T-cell proliferation is essential for developing effective strategies for neoantigen vaccination. Our preliminary data examining MRD in canine PTCL, a model of human PTCL, shows that patients always have readily detectable circulating cancer clones, despite induction of complete remission (CR). Our hypothesis is that these malignant cells, trafficking through and proliferating in lymph nodes, continue to cause significant metabolic derangements hostile to T-cell responses to vaccinat

Gliomas are common and devastating intracranial tumors in both dogs and humans, and share high conserved clinical behavior and histologic appearance. Despite decades of therapeutic advances, these tumors remain almost uniformly fatal. Diagnosis currently requires histopathologic evaluation ofy on tumor tissue, a tissue sample, which is difficult challenging and risky to obtain in dogsboth species. There is growing interest in the use of minimally invasive ���������������liquid biopsy������������������ techniques for molecular detection of tumor-associated genomic abnormalities in malignant cells within circulating blood. Recent studies have suggested that these abnormalities may be more readily detectable in cerebrospinal fluid (CSF), which would greatly reduce the risks, costs and distress of obtaining a diagnosis. Furthermore, this approach has tremendous potential to define histologic subtype, grade and likely prognosis, and to identify optimal therapeutic approaches and actionable molecular targets. We have identified several molecular signatures that are common to both canine and human patients with intracranial tumors. We propose here to evaluate the use of cell-free tumor DNA within cerebrospinal fluid (CSF) as a diagnostic clinically predictive marker (����������������liquid biopsy���������������) for canine gliomas, leveraging the many factors that allow these studies to be performed more readily in dogs than in people.

Genotyping is a powerful tool when applied to parentage and individualization analyses. Establishing a single nucleotide polymorphism (SNP) based genotyping system allows many key advantages over existing methods, with future proofing arguably being at the forefront. SNPs often allow for more robust and reproducible genotyping compared to simple tandem repeat (STR) typing, with current platforms allowing the simultaneous evaluation of substantially more markers in a single test. Unlike STR markers, which are typically neutral and not associated with specific traits, SNP markers may be associated with specific inherited traits. In addition to providing a robust means to individualize a dog and provide parentage information, the added value of a SNP-based platform is the opportunity to simultaneously capture such associations, providing a wealth of health information about each dog for little to no added cost. With extensive experience in both STR and SNP based testing in dogs, as well as integration of both types of testing, we propose to help facilitate the migration of the AKC������������������s DNA database and current STR testing methodology to a robust, SNP-based system. The scope of this work will include guidance in 1) selection of the most appropriate set of SNPs to meet the AKC������������������s needs for parentage and individualization testing, and 2) identification of an appropriate platform for these SNPs to be used for the AKC������������������s commercial operation. In addition, we will provide assistance in the selection of additional markers for a variety of purposes including those associated with specific traits.

Prostate cancer (PC) remains the second leading cause of human cancer deaths in the United States and carries a similarly grave prognosis in dogs. The domestic dog develops spontaneous prostate cancer with many characteristics similar to castration-resistant prostate cancer (CRPC) in men, suggesting that canine PC is an important animal model. Epigenetic alterations are hallmarks of prostate cancer, with the number of modified genes increasing with disease severity. Targeted molecular therapy toward hypo- or hypermethylated genes show promise for individualized treatment, but hundreds of genes have been identified as putative targets. Identification of genes affected in both dog PC and human CRPC will aid in determination of the most significant abnormalities, and could result in improved diagnostics and treatment. The hypothesis for this study is that epigenetic profiles and gene expression signatures are conserved between a subset of canine prostate cancer and human castration-resistant prostate cancer. To address this hypothesis, DNA methylation of 96 genes in canine prostate cancer will be assessed and compared with published data for CRPC in men. Gene expression profiles for the same 96 genes will be evaluated and correlated to DNA methylation. Methylation and gene expression changes will also be compared to exome sequencing data to determine whether a methylation signature coexists with causal mutations.