Funded Research

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02428:
Identifying the Disease-Defining Autoantibodies in Canine Addison's Disease

Principal Investigator(s): Steven Friedenberg, DVM, PhD
Research Institution: University of Minnesota

Grant Amount: $181,864
Start Date: 3/1/2018 - End Date: 8/31/2022

Progress Report: Mid-Year 4
Report Due: 8/31/2021 - Report Received: 8/16/2021
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:

Addison's disease is a common and life-threatening disorder in dogs in which the body's immune system destroys the outer layer of the adrenal glands. The adrenal glands produce hormones that are critical for energy metabolism, immune system function, intestinal health, and kidney function. Symptoms of Addison's disease can mimic other conditions, and as a result, many dogs remain undiagnosed for years. About one-third of dogs with Addison's disease are diagnosed only after suffering an acute adrenal crisis, which can cause a wide range of complications that require emergency stabilization and hospitalization. Today, there is no way to predict which dogs will develop Addison's disease before they become sick. If such a test were available, veterinarians would be able to evaluate high-risk dogs before they show signs, helping to prevent disease-related complications and potentially enabling earlier treatment. In this study, the investigator will use a novel approach combining gene and protein sequencing to identify the antibodies that target the adrenal glands in Standard Poodles, Portuguese Water Dogs, and English Cocker Spaniels with Addison's disease. These antibodies are produced by the immune system before the onset of clinical signs. The ability to identify these antibodies would therefore provide a test for early diagnosis. This research will contribute to progress in developing an important clinical test for Addison's disease that can help improve the lives of the many dogs at high risk of developing this life-threatening condition.

Publication(s)

None at this time. However, we recently submitted a review paper on the use of autoantibody testing in veterinary medicine to the Journal of Veterinary Internal Medicine in August 2021. We plan on publishing our findings from this study specifically once we have finalized our experiments for Aims 1 and 2.

Presentations:

I have presented very preliminary findings from this study at the AKC-CHF canine health meeting in August 2019, a VetVine seminar in November 2019, at a webinar for the Western Australia Labradoodle Club of America in October 2020, at an Embark canine health conference in February 2021, and at a webinar for the Twin Cities Portuguese Water Dog Club in February 2021. I am also planning on doing a live webinar/Q&A session for members of the CARES Facebook group in mid-September; CARES is an online organization dedicated to helping owners become more knowledgeable about canine Addison’s disease.

Report to Grant Sponsor from Investigator:

The goal of this project is to identify autoantibodies that are present in the blood of dogs who are newly diagnosed with Addison’s disease in three breeds: Standard Poodles, Portuguese Water Dogs, and English Cocker Spaniels. To accomplish these goals, we have been focusing on (1) collecting blood samples from dogs across all three target breeds, and (2) employing methods that allow us to detect these autoantibodies.

In terms of collecting blood samples, during the first 3 years of this project we have collected all the samples required from Standard Poodles and Portuguese Water Dogs, and most of the samples required for English Cocker Spaniels. We are continuing to actively recruit newly diagnosed dogs across all three breeds through many online channels.

More recently, we have used these samples to detect the presence of autoantibodies in newly diagnosed dogs using a technique called two-dimensional Western blotting. Our findings show that there are autoantibodies that are consistently present against adrenocortical proteins in dogs with a new diagnosis of Addison’s disease. We have also shown that the specific proteins targeted by these autoantibodies may be different by breed or by dog.

Currently, we are focused on the next phase of our work which is to identify which specific proteins are targeted by these autoantibodies. We are genetically engineering three candidate proteins in our laboratory at present, and at the moment we are working through some challenges related to protein purification. Once we have made and isolated these proteins, we will test the serum from affected and unaffected dogs for reactivity against these proteins. We hope that these tests of reactivity will help us determine the predominant target of autoantibodies in canine Addison’s disease for each breed. This will then set us on a path to developing a robust immunologic test to predict which dogs are at highest risk of developing the disease.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02534:
Clinical Trial for Evaluation of Propranolol and Doxorubicin in the Treatment of Canine Hemangiosarcoma

Principal Investigator(s): Erin Dickerson, PhD
Research Institution: University of Minnesota

Grant Amount: $334,306
Start Date: 7/1/2019 - End Date: 6/30/2022

Progress Report: End-Year 2
Report Due: 6/30/2021 - Report Received: 6/29/2021
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:

Canine hemangiosarcoma is a largely incurable cancer in dogs, and treatment approaches to improve outcomes have remained relatively stagnant over the past few decades. Treatment remains a challenge partly because the cancer is frequently detected at an advanced stage and because these tumors are often resistant to chemotherapies. Recently published reports showed that propranolol, a drug used to treat heart disease in humans and dogs, substantially increased the survival time of human angiosarcoma patients when used in combination with standard of care treatments. Propranolol was also shown to sensitize hemangiosarcoma cells to doxorubicin, providing a more effective way to kill tumor cells. Because angiosarcoma is strikingly similar to canine hemangiosarcoma, this multi-institutional clinical trial has been designed to determine the efficacy of propranolol in dogs with hemangiosarcoma when used in combination with surgery and chemotherapy. The main goal of the study is to establish whether propranolol in combination with doxorubicin following surgery improves outcomes for dogs when compared to the use of chemotherapy and surgery alone. The investigators will also evaluate the plasma concentrations of propranolol achieved during dosing to assess whether the levels of propranolol correlate to survival times. If successful, the findings from this approach will be rapidly conveyed to the veterinary community, and the guidelines provided to clinicians for the use of propranolol and doxorubicin for the treatment of canine hemangiosarcoma.

Publication(s)

None at this time. The clinical trial is ongoing.

Presentations:

An overview of the study was presented at the 12th Biennial AKC Canine Health Foundation National Parent Club Canine Health Conference (NPCCHC) held August 9-11, 2019 in St. Louis, MO.

Report to Grant Sponsor from Investigator:

During the first 24 months of the trial, we have made progress toward our objectives. The project goals have not been modified.

Our overall objective is to determine a clinically optimal dose and estimate the efficacy of propranolol in dogs with hemangiosarcoma when given as an adjunct to chemotherapy. Specifically:

Objective 1: We will confirm the tolerability and estimate the clinical benefit of propranolol in combination with doxorubicin.

Objective 2: We will assess levels of propranolol in the bloodstream after long-term administration to dogs with hemangiosarcoma to determine if there is a correlation between drug levels in blood and treatment effect. We will also determine if propranolol alters the blood levels (exposure) of doxorubicin in dogs receiving propranolol and compare these levels to those found in the published literature for dogs receiving doxorubicin. Collection of these data will allow us to better understand how these drugs may be working together.

We opened the trial on July 1, 2019. As of June 27, 2021, we have enrolled 14 dogs in the study and no dose limiting toxicities have been observed. Based on these results, we are continuing to enroll dogs at the highest dose of propranolol (1.3 mg/kg) being tested.

Propranolol and doxorubicin levels in the blood from eight of the dogs have been analyzed, and the analyses of samples from the others five dogs are pending. Samples from a sixth dog (dog #14 enrolled in the study) were collected just prior to the submission of our report, and the samples will be sent for analysis within the next 7-14 days.

Currently five dogs enrolled in the study are alive while nine dogs have succumbed to their disease.

Enrollment of dogs into the study was severely delayed by approximately six months due to the COVID-19 pandemic. Enrollment was halted at all three study sites (University of Minnesota, University of Pennsylvania, Purdue University) in early to mid-March. Although enrollment resumed at all three sites in late June/early July, we were unable to enroll another dog until September 2020. The pace of enrollment has continued to increase since this time, likely due to the easing and/or lifting of travel restrictions related to COVID-19 throughout the country.

Our plans are to continue to screen and enroll dogs into the study with the goal of enrolling the remaining six dogs by December 31, 2021. We also plan to complete the analysis of drug levels (propranolol and doxorubicin) in the blood samples. Due to delays related to the pandemic, we plan to request an extension of the study in order to provide a complete follow up (up to one year) for all of the dogs enrolled.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02800:
Defining the Effect of Genotype, Breed and Age on the Risk of Developing Canine Degenerative Myelopathy and Investigating the Molecular Mechanisms Underlying That Risk

Principal Investigator(s): Gary Johnson DVM PhD and Joan Coates DVM MS
Research Institution: University of Missouri

Grant Amount: $108,000
Start Date: 4/1/2020 - End Date: 9/30/2021

Progress Report: End-Year 1
Report Due: 3/31/2021 - Report Received: 3/1/2021
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:

Canine degenerative myelopathy (DM) is a progressive and inevitably fatal neurological disease affecting members of different dog breeds and mixes. It is an inherited disease with an age-related penetrance. The risk of developing the disease when dogs are homozygous for the causal SOD1 variant allele is currently unknown but of great concern to dog breeders and owners. The proposed research will further define the risk for developing DM in genetically at-risk dogs with a health survey distributed to dog owners whose dogs have been tested for the risk factor allele. This work will also examine the molecular mechanisms responsible for disease onset and spread by comparing single-nucleus RNA expression patterns in specific cell types in dorsal root ganglia from normal dogs and from affected dogs at various stages of the disease.

Publication(s)

Dr. Natalie Villani is a neurology resident and graduate student. The [sic] many of the experiments in Objective 1 are part of her thesis project. She is currently drafting a manuscript which we expect to submit for publication in the near future.

Presentations:

Dr. Joan Coates is scheduled to present a lecture about DM at the 2021 AKC Canine Health Foundation National Parent Club Canine Health Conference. She intends to include much of the information from this [sic] projects at that time.

Report to Grant Sponsor from Investigator:

This project has two objectives. The first objective is to generate and interpret empirical evidence about the likelihood that dogs will develop DM, if and when they reach old age. To do this, we sent invitations to the owners of nearly 10,000 dogs to provide us with follow up clinical information about their dogs via an online survey. To be considered for participation in this survey, the dogs had to meet two criteria. First, the dogs should have been DNA-tested for DM before they were 5 years old. When tested, these dogs were too young to show signs of DM. Thus, their owners’ decision to test could not have been biased by prior knowledge of the dogs DM status. Second, the dogs should have been 8 years old or older when surveyed. Thus, the dogs would be old enough to exhibit the clinical signs of early-onset DM.

We received an approximately 25% response to our survey request. After review, the clinical records for 2,143 dogs were eligible for the final analysis. We found that dogs that with homozygous “at risk” DM-test result, began to develop a DM-like disease by 7 years of age. By 12 years of age, over 50% of them developed a DM-like disease and over 60% of them developed a DM-like disease as they got older. This confirmed an earlier conclusion reported in 2014 from a similar study with a cohort of 512 dogs. For the first time, we showed empirical evidence that heterozygous dogs with the “carrier” DM test result had an increased risk of developing DM starting before they reached 12 years of age. Nonetheless, fewer than 5% of these dogs developed a DM-like disease by 14 years of age. Thus, we have not modified our conclusion that, in general, offspring resulting from matings involving at least one homozygous SOD1:c.118G allele dog are very unlikely to develop DM. Nonetheless, the risk “carrier” dogs developing DM was not the same in all breeds and we may need to modify our recommendations to the breeders of certain breeds at higher risk.

We found that the risks for developing DM in female dogs was almost identical to the risks for developing DM in male dogs.

For the first time, we showed empirical evidence that a dog’s breed influences the age at onset of DM. There were sufficient numbers of survey records to statistically compare ages at onset of DM for six dog breeds: Bernese Mountain Dog, Boxer, Chesapeake Bay Retriever, German Shepherd Dog, Pembroke Welsh Corgi and Rhodesian Ridgeback. We found that the Rhodesian Ridgebacks and the Bernese Mountain Dogs had the earliest onsets, while Pembroke Welsh Corgis had the latest onsets.

The second objective was to study stored tissue, collected from owner-requested euthanasias of DM-affected and DM-free dogs, with the goal of learning about molecular mechanisms underlying start and progression of DM. We focused on dorsal root ganglia since these anatomical structures contain the neurons that provide the brain with sensory information necessary for normal gait and abnormal hind-limb gait is the first clinical sign of DM. We are using a technology known as single-nucleus RNA sequence analysis. This technology provides an indication of the patterns of genes that are turned on in the individual nuclei from cells in a tissue sample. This is our first experience with this technology and progress has been slow due to technical problems and laboratory shut downs because of the COVID-19 pandemic. Nonetheless, our latest efforts show promise and we expect progress to accelerate as we continue the study. Our hope is that our experiments will lead to a better understanding of DM-disease mechanisms, thereby identifying druggable targets leading to the development of therapies that delay the onset or slow the development of DM.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02519:
Prevalence of Bartonella spp. Infection in Dogs with Cardiac and Splenic Hemangiosarcomas Within and Between Geographic Locations

Principal Investigator(s): Edward Breitschwerdt, DVM
Research Institution: North Carolina State University

Grant Amount: $219,026
Start Date: 2/1/2018 - End Date: 1/31/2022

Progress Report: End-Year 3
Report Due: 1/31/2021 - Report Received: 2/1/2021
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:

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 are often ambiguous. While some splenic tumors are benign, approximately two-thirds are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs in many large dog breeds, including mixed breeds. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for both cardiac and splenic HSA remain unclear, confounding development of preventative strategies. The investigators recently 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) and 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. The investigators will perform 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.

Publication(s)

Lashnits, E., Neupane, P., Bradley, J. M., Richardson, T., Thomas, R., Linder, K. E., Breen, M., Maggi, R. G., & Breitschwerdt, E. B. (2020). Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States. PLOS ONE, 15(1), e0227234. https://doi.org/10.1371/journal.pone.0227234

Maggi RG, Richardson T, Breitschwerdt EB, Miller JC. Development and validation of a droplet digital PCR assay for the detection and quantification of 2 Bartonella species within human clinical samples. J Microbiol Methods. In Press.

Neupane, P., Sevala, S., Balakrishnan, N., Marr, H., Wilson, J., Maggi, R., Birkenheuer, A., Lappin, M., Chomel, B., & Breitschwerdt, E. B. (2020). Validation of Bartonella henselae Western Immunoblotting for Serodiagnosis of Bartonelloses in Dogs. Journal of Clinical Microbiology. https://doi.org/10.1128/JCM.01335-19

Presentations:

Lashnits E, Abstract oral presentation: Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma. American College of Veterinary Medicine Annual Forum, Phoenix, AZ, June 12-15, 2018.

Breitschwerdt EB. The genus Bartonella and vasoproliferative cancers in dogs and humans. AKC Canine Health Foundation National Parent Club Canine Health Conference in St. Louis, MO August 9-11, 2019.

Report to Grant Sponsor from Investigator:

We are on track to accomplish all of our aims for this study. We were able to obtain the initial set of samples on April 26, 2018 so we had a short delay in starting this study. We have now completed all Year I study aims, with the exception of immunohistochemistry and FISH localization of Bartonella organisms within various cell types. An unanticipated complication arose that the mouse monoclonal antibody was no longer being made commercially. B. henselae specific FISH probes have been designed and validation of FISH probes are in-progress. IHC is also in-progress. All qPCR and ddPCR have been completed at this time and samples are waiting for FISH and IHC analysis.

We have published a manuscript to the Journal of Clinical Microbiology, representing additional research from our AKC-CHF study #02287, which allowed us to define the Western Blotting (WB) criteria for serodiagnosis of bartonellosis in dogs. That work required additional time and research effort to validate WB testing. We are very excited with the qPCR and ddPCR results obtained from the fresh frozen hemangiosarcoma tissues provided by the NIH-CCOGC. The results strongly support a role for Bartonella spp. in the etiopathogenesis of hemangiosarcoma in dogs. The regional study should provide additional insight as to the issue of potential causation. All three of the regions have identified, collected and shipped all necessary samples from their region. These samples have been tested by ddPCR, which has required months of validation. Validation of the ddPCR have been published in the Journal of Microbiological Methods. Because of the limitations on research activities at NCSU during the SARS-CoV2 pandemic, the testing and analysis of the study samples have been delayed. The NCSU College of Veterinary Medicine and the Intracellular Pathogens Research Laboratory have had suspended operations since March 2020. As of July 1, research operations are at limited capacity. Samples will continue to be processed, tested and analyzed as soon as possible given the constraints remaining in place to ensure staff safety.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02519:
Prevalence of Bartonella spp. Infection in Dogs with Cardiac and Splenic Hemangiosarcomas Within and Between Geographic Locations

Principal Investigator(s): Edward Breitschwerdt, DVM
Research Institution: North Carolina State University

Grant Amount: $219,026
Start Date: 2/1/2018 - End Date: 1/31/2021

Progress Report: Mid-Year 3
Report Due: 7/31/2020 - Report Received: 8/3/2020
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:

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 are often ambiguous. While some splenic tumors are benign, approximately two-thirds are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs in many large dog breeds, including mixed breeds. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for both cardiac and splenic HSA remain unclear, confounding development of preventative strategies. The investigators recently 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) and 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. The investigators will perform 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.

Publication(s)

Lashnits, E., Neupane, P., Bradley, J. M., Richardson, T., Thomas, R., Linder, K. E., Breen, M., Maggi, R. G., & Breitschwerdt, E. B. (2020). Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States. PLOS ONE, 15(1), e0227234. https://doi.org/10.1371/journal.pone.0227234

Maggi RG, Richardson T, Breitschwerdt EB, Miller JC. Development and validation of a droplet digital PCR assay for the detection and quantification of 2 Bartonella species within human clinical samples. J Microbiol Methods. In Press.

Presentations:

Lashnits E, Abstract oral presentation: Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma. American College of Veterinary Medicine Annual Forum, Phoenix, AZ, June 12-15, 2018.

Breitschwerdt EB. The genus Bartonella and vasoproliferative cancers in dogs and humans. AKC Canine Health Foundation National Parent Club Canine Health Conference in St. Louis, MO August 9-11, 2019.

Report to Grant Sponsor from Investigator:

We are on track to accomplish all of our aims for this study. We were able to obtain the initial set of samples on April 26, 2018 so we had a short delay in starting this study. We have now completed all Year I study aims, with the exception of immunohistochemistry and FISH localization of Bartonella organisms within various cell types. An unanticipated complication arose that the mouse monoclonal antibody was no longer being made commercially. B. henselae specific FISH probes have been designed and validation of FISH probes are in-progress. IHC is also in-progress. All qPCR and ddPCR have been completed at this time and samples are waiting for FISH and IHC analysis.

As reported above, we have published a manuscript to the Journal of Clinical Microbiology, representing additional research from our AKC-CHF study #02287, which allowed us to define the Western Blotting (WB) criteria for serodiagnosis of bartonellosis in dogs. That work required additional time and research effort to validate WB testing. We are very excited with the qPCR and ddPCR results obtained from the fresh frozen hemangiosarcoma tissues provided by the NIH-CCOGC. The results strongly support a role for Bartonella spp. in the etiopathogenesis of hemangiosarcoma in dogs. The regional study should provide additional insight as to the issue of potential causation. All three of the regions have identified, collected and shipped all necessary samples from their region. These samples have been tested by ddPCR, which has required months of validation. Validation of ddPCR is “In Press” in the Journal of Microbiological Methods. Because of the limitations on research activities at NCSU during the SARS-CoV2 pandemic, the testing and analysis of the study samples have been delayed. The NCSU College of Veterinary Medicine and the Intracellular Pathogens Research Laboratory have had suspended operations since March 2020. As of July 1, research operations are at limited capacity. Samples will continue to be processed, tested and analyzed as soon as possible given the constraints remaining in place to ensure staff safety. Even though we have had the down time we are still proposing to have this project complete by the end of the study on 1/31/2021.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02800:
Defining the Effect of Genotype, Breed and Age on the Risk of Developing Canine Degenerative Myelopathy and Investigating the Molecular Mechanisms Underlying That Risk

Principal Investigator(s): Gary S. Johnson, DVM, PhD and Joan Coates, DVM, MS
Research Institution: University of Missouri

Grant Amount: $15,000
Start Date: 4/1/2020 - End Date: 3/31/2021

Original Project Description:
Canine degenerative myelopathy (DM) is a progressive and inevitably fatal neurological disease affecting members of different dog breeds and mixes. It is an inherited disease with an age-related penetrance. The risk of developing the disease when dogs are homozygous for the causal SOD1 variant allele is currently unknown but of great concern to dog breeders and owners. The proposed research will further define the risk for developing DM in genetically at-risk dogs with a health survey distributed to dog owners whose dogs have been tested for the risk factor allele. This work will also examine the molecular mechanisms responsible for disease onset and spread by comparing single-nucleus RNA expression patterns in specific cell types in dorsal root ganglia from normal dogs and from affected dogs at various stages of the disease.

Publication(s)
None at this time.

Related Grants
00934: Investigation of Inherited Diseases of the German Shepherd Dog Using the SNP Array
00902-A: Sequence Analysis of DLA-DRB1 in German Shepherd Dogs Having Degenerative Myelopathy

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02519:
Prevalence of Bartonella spp. Infection in Dogs with Cardiac and Splenic Hemangiosarcomas Within and Between Geographic Locations

Principal Investigator(s): Edward Breitschwerdt, DVM
Research Institution: North Carolina State University

Grant Amount: $219,026
Start Date: 2/1/2018 - End Date: 1/31/2021

Progress Report: End-Year 2
Report Due: 1/31/2020 - Report Received: 2/1/2020
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
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 are often ambiguous. While some splenic tumors are benign, approximately two-thirds are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs in many large dog breeds, including mixed breeds. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for both cardiac and splenic HSA remain unclear, confounding development of preventative strategies. The investigators recently 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) and 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. The investigators will perform 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.

Publications:
Lashnits, E., Neupane, P., Bradley, J. M., Richardson, T., Thomas, R., Linder, K. E., Breen, M., Maggi, R. G., & Breitschwerdt, E. B. (2020). Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States. PLOS ONE, 15(1), e0227234. https://doi.org/10.1371/journal.pone.0227234

Presentations:
Lashnits E, Abstract oral presentation: Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma. American College of Veterinary Medicine Annual Forum, Phoenix, AZ, June 12-15, 2018.

Breitschwerdt EB. The genus Bartonella and vasoproliferative cancers in dogs and humans. AKC Canine Health Foundation National Parent Club Canine Health Conference in St. Louis, MO August 9-11, 2019.

Report to Grant Sponsor from Investigator:
We are on track to accomplish all of our aims for this study. We were able to obtain the initial set of samples on April 26, 2018 so we had a short delay in starting this study. We have now completed all Year I study aims, with the exception of immunohistochemistry and FISH localization of Bartonella organisms within various cell types. We have published a manuscript to the Journal of Clinical Microbiology, representing additional research from our AKC-CHF study #02287, which allowed us to define the Western Blotting (WB) criteria for serodiagnosis of bartonellosis in dogs. That work required additional time and research effort to validate WB testing. We are very excited with the qPCR and ddPCR results obtained from the fresh frozen hemangiosarcoma tissues provided by the NIH-CCOGC. The results strongly support a role for Bartonella spp. in the etiopathogenesis of hemangiosarcoma in dogs. The regional study should provide additional insight as to the issue of potential causation. All three of the regions have identified, collected and shipped all necessary samples from their region. These samples will be tested by ddPCR, which has required months of validation, over the next six months.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH REPORT PROGRESS SUMMARY

Grant 02519:
Prevalence of Bartonella spp. Infection in Dogs with Cardiac and Splenic Hemangiosarcomas within and between Geographic Locations

Principal Investigator(s): Edward B Breitschwerdt, DVM; Matthew Breen, PhD
Research Institution:North Carolina State University
Sponsors: American Bloodhound Club, American Boxer Charitable Foundation, American Bullmastiff Association, American Pointer Club, Australian Shepherd Health & Genetics Institute, Clumber Spaniel Health Foundation, French Bulldog Club of America, Gordon Setter Club of America, Jeffrey Pepper, Leonberger Health Foundation International, Portuguese Water Dog Foundation, Inc., Saluki Health Research, Inc., Soft Coated Wheaten Terrier Club of America Endowment, Inc., Vizsla Club of America Welfare Foundation, Weimaraner Foundation Fund Corporation
Breeds: All Dogs
Research Program Area: Oncology - Hemangiosarcoma

Grant Amount: $219,026
Start Date: 2/1/2018 - End Date: 1/31/2021

Abstract
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 are often ambiguous. While some splenic tumors are benign, approximately two-thirds are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs in many large dog breeds, including mixed breeds. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for both cardiac and splenic HSA remain unclear, confounding development of preventative strategies. The investigators recently 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) and 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. The investigators will perform 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.
(The content of this report is not confidential and may be used in communications with your organization.)

Publications:
Lashnits, E., Neupane, P., Bradley, J. M., Richardson, T., Thomas, R., Linder, K. E., Breen, M., Maggi, R. G., & Breitschwerdt, E. B. (2020). Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States. PLOS ONE, 15(1), e0227234. https://doi.org/10.1371/journal.pone.0227234

Related Grants:
2025: Growth Signaling Pathways in the Pathogenesis and Treatment of Canine Cancer
00593B: Mapping Genes Associated with Canine Hemangiosarcoma

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02534:
Clinical Trial for Evaluation of Propranolol and Doxorubicin in the Treatment of Canine Hemangiosarcoma

Principal Investigator(s): Erin Dickerson, PhD and Brian Husbands, DVM
Research Institution: University of Minnesota
Co-Investigators: David R. Brown, PhD; University of Minnesota, Michael O. Childress, DVM, MS; Purdue University, Jennifer Mahoney, DVM and Pascale Salah; University of Pennsylvania

Grant Amount: $310,970
Start Date: 7/1/2019 - End Date: 6/30/2022

Progress Report: Mid-Year 1
Report Due: 12/31/2019 - Report Received: 12/22/2019
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
Canine hemangiosarcoma is a largely incurable cancer in dogs, and treatment approaches to improve outcomes have remained relatively stagnant over the past few decades. Treatment remains a challenge partly because the cancer is frequently detected at an advanced stage and because these tumors are often resistant to chemotherA1PIes. Recently published reports showed that propranolol, a drug used to treat heart disease in humans and dogs, substantially increased the survival time of human angiosarcoma patients when used in combination with standard of care treatments. Propranolol was also shown to sensitize hemangiosarcoma cells to doxorubicin, providing a more effective way to kill tumor cells. Because angiosarcoma is strikingly similar to canine hemangiosarcoma, this multi-institutional clinical trial has been designed to determine the efficacy of propranolol in dogs with hemangiosarcoma when used in combination with surgery and chemotherapy. The main goal of the study is to establish whether propranolol in combination with doxorubicin following surgery improves outcomes for dogs when compared to the use of chemotherapy and surgery alone. The investigators will also evaluate the plasma concentrations of propranolol achieved during dosing to assess whether the levels of propranolol correlate to survival times. If successful, the findings from this approach will be rA1PIdly conveyed to the veterinary community, and the guidelines provided to clinicians for the use of propranolol and doxorubicin for the treatment of canine hemangiosarcoma.

Publications:
None at this time.

Presentations:
A description of the study design was presented at the 2019 AKC CHF National Parent Club Canine Health Conference, August 9-11, St. Louis, Missouri.

Report to Grant Sponsor from Investigator:

During the 6 months that the project has been active, we have made progress toward our objectives. Based on our experiences during this time, we have made some minor adjustments to the day-to-day running of the clinical trial and reporting of results. The project goals have not been modified.

Our overall objective is to determine a clinically optimal dose and estimate the efficacy of propranolol in dogs with hemangiosarcoma when given as an adjunct to chemotherapy. Specifically:

  • Objective 1: We will confirm the tolerability and estimate the clinical benefit of propranolol in combination with doxorubicin.
  • Objective 2: We will assess the circulating drug levels of propranolol after long-term administration to dogs with hemangiosarcoma to determine if there is a correlation between plasma drug concentration and treatment effect. We will also determine if propranolol alters the plasma levels (exposure) of doxorubicin in dogs receiving propranolol and compare these levels to those found in the published literature for dogs receiving doxorubicin. Collection of these data will allow us to better understand how these drugs may be working together.

We opened the trial on July 1, 2019, and as of December 15, 2019, we have screened 10 dogs and enrolled three dogs in the study. Dogs that were not enrolled did not meet enrollment criteria (e.g. evidence of metastatic disease) or the owner declined enrollment. The first dog was enrolled in mid-September, the second in October, and the third dog in November.

Overall, propranolol has been well-tolerated by all of the dogs; no dose limiting toxicities were noted in the three dogs enrolled. One dog did have gastrointestinal upset that was attributed to the chemotherapy (doxorubicin) used in the study. Propranolol and doxorubicin levels in the plasma from two dogs has been analyzed, and analysis of samples from the third dog is pending. Our plans are to continue to screen and enroll dogs in the study with the goal of enrolling another 8-10 dogs within the next 6 months. We also plan to complete the initial analysis of drug levels in the plasma samples during this time.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02510-T:
Identification of Novel Synthetic Lethal Partners to Optimize PI3K Targeted TherA1PIes in Canine Hemangiosarcoma

Principal Investigator: Cheryl London, DVM, PhD
Research Institution: Tufts University School of Medicine

Grant Amount: $168,857
Start Date: 3/1/2018 - End Date: 2/28/2021

Progress Report: Mid-Year 2
Report Due: 8/31/2019 - Report Received: 9/1/2019
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
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 are often ambiguous. While some splenic tumors are benign, approximately two-thirds are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs in many large dog breeds, including mixed breeds. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for both cardiac and splenic HSA remain unclear, confounding development of preventative strategies. The investigators recently 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) and 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. The investigators will perform 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.

Publications:

  • Gutwillig, Megan. January 2019. The role of PI3K-β and PI3K-δ in canine hemangiosarcoma and human angiosarcoma (oral presentation). Genetics Seminar Series at Tufts University.
  • Gutwillig, Megan. April 2019. The role of PI3K-β and PI3K-δ in canine hemangiosarcoma and human angiosarcoma (poster presentation). Charleton Research Symposium at the Sackler School.
  • Gutwillig, Megan. June 2019. The role of PI3K-β and PI3K-δ in canine hemangiosarcoma and human angiosarcoma (poster presentation). Genetics Program Retreat.

Report to Grant Sponsor from Investigator:
Hemangiosarcoma (HSA) is a cancer of the cells lining the blood vessels that is very aggressive and has nearly always spread by the time it is diagnosed. HSA accounts for 5-7% of all cancers in dogs resulting in approximately 25-50,000 new cases per year. The treatment of choice is surgical removal followed by chemotherapy administration. Unfortunately, despite aggressive therapy, the majority of dogs diagnosed succumb to their disease within 6-8 months. Virtually no improvements in outcome for affected dogs have occurred in the past 30 years despite multiple clinical trials/efforts. More recently a new therapy has been developed targeting two receptors on HSA cells. However, the majority of dogs still died, by 10-12 months. The molecular pathway known as the PI3K/AKT/mTOR pathway has previously been implicated as a key driver of several cancers including HSA. Indeed, inhibitors of PI3K/AKT/mTOR pathway have some activity against HSA cell lines in the laboratory. The purpose of this study is to fully characterize the expression and function of PI3K in canine HSA tumor cell lines and tumor samples to identify new ways to block this pathway using a combination of small molecule inhibitors that are most effective at killing tumors cells. Over the past year we have characterized the expression of the 4 isoforms that make up PI3K family in HSA cell lines, have characterized sensitivities of the lines to individual isoform inhibitors, and have generated cell lines deficient in two of the isoforms. We have found that in some of the lines loss of the PI3K β isoform causes the cells to reduced migration suggesting that this isoform may play a role in tumor spread. Megan Gutwillig, a combined DVM/MS student, finished her MS work and has returned to complete the last 3 years of her veterinary training. A new student will begin working on the project in the lab this fall.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02519:
Prevalence of Bartonella spp. Infection in Dogs with Cardiac and Splenic Hemangiosarcomas Within and Between Geographic Locations

Principal Investigator: Edward Breitschwerdt DVM
Research Institution: North Carolina State University

Grant Amount: $219,026
Start Date: 2/1/2018 - End Date: 1/31/2021

Progress Report: End-Year 1
Report Due: 7/31/2019 - Report Received: 8/1/2019
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
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 are often ambiguous. While some splenic tumors are benign, approximately two-thirds are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs in many large dog breeds, including mixed breeds. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for both cardiac and splenic HSA remain unclear, confounding development of preventative strategies. The investigators recently 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) and 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. The investigators will perform 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.

Publications:

  • Lashnits E, Neupane P, Bradley JM, Richardson T, Thomas R, Linder KE, Breen M, Maggi RG, Breitschwerdt EB. Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma. Manuscript in preparation for submission to PLoS One

Presentations:

  • Lashnits E, Abstract oral presentation: Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma. American College of Veterinary Medicine Annual Forum, Phoenix, AZ, June 12-15, 2018.
  • Breitschwerdt EB. The genus Bartonella and vasoproliferative cancers in dogs and humans. To be presented at the 12th Biennial AKC Canine Health Foundation National Parent Club Canine Health Conference in St. Louis, MO August 9-11, 2019.

Report to Grant Sponsor from Investigator:
We are on track to accomplish all of our aims for this study. We were able to obtain the initial set of samples on April 26, 2018 so we had a short delay in starting this study. We have now completed all Year I study aims, with the exception of immunohistochemistry and FISH localization of Bartonella organisms within various cell types. We have submitted a manuscript to the Journal of Clinical Microbiology, representing additional research from our AKC-CHF study #02287, which allowed us to define the Western Blotting (WB) criteria for serodiagnosis of bartonellosis in dogs. That work required additional time and research effort to validate WB testing. We are very excited with the qPCR and ddPCR results obtained from the fresh frozen hemangiosarcoma tissues provided by the NIH-CCOGC. The results strongly support a role for Bartonella spp. in the etiopathogensis of hemangiosarcoma in dogs. The regional study should provide additional insight as to the issue of potential causation. Two regions have identified, collected and shipped all necessary samples from their region. The other region is in the process of collecting their samples. These samples will be tested by ddPCR, which has required months of validation, over the next six months.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02519:
Prevalence of Bartonella spp. Infection in Dogs with Cardiac and Splenic Hemangiosarcomas Within and Between Geographic Locations

Principal Investigator: Edward Breitschwerdt, DVM
Research Institution: North Carolina State University

Grant Amount: $219,026
Start Date: 2/1/2018 - End Date: 1/31/2021

Progress Report: End-Year 1
Report Received: ??
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
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 are often ambiguous. While some splenic tumors are benign, approximately two-thirds are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs in many large dog breeds, including mixed breeds. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for both cardiac and splenic HSA remain unclear, confounding development of preventative strategies. The investigators recently 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) and 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. The investigators will perform 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.

Publications: Not at this time.

Presentations: Not at this time.

Report to Grant Sponsor from Investigator:
We are on track to accomplish all of our aims for this study. We were able to obtain the initial set of samples on April 26, 2018 so we had a short delay in starting the study. We have now received all samples for phase one, categorized, prepped, extracted and tested via qPCR the obtained DNA. A research abstract of the qPCR results has been submitted for the 2019 Annual ACVIM Forum. That abstract is currently under review. We will be publishing additional research from our AKC-CHF study #02287, which will define the criteria for serodiagnosis of Bartonellosis in dogs by Western Blotting (WB). Due to a number of unanticipated problems, that work required additional time and research effort to validate criteria for interpretation of Bartonella spp. WB testing. We have processed all samples for IFA and WB in this study as well as performed qPCR on all EDTA and tissue samples. We have contacted the collaborating pathologists in the three designated regions. They have defined specific criteria for inclusion into the study. The pathologists have started identifying and collecting samples for testing during year II of the grant. One region has identified, collected and shipped all necessary samples from their region. The other regions will have theirs collected within the next month. These samples will be tested over the next twelve months.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02510-T:
Identification of Novel Synthetic Lethal Partners to Optimize PI3K Targeted TherA1PIes in Canine Hemangiosarcoma

Principal Investigator: Cheryl London, DVM, PhD
Research Institution: Tufts University School of Medicine

Grant Amount: $168,857
Start Date: 3/1/2018 - End Date: 2/28/2021

Progress Report: End-Year 1
Report Due: 2/28/2019 - Report Received: 3/23/2019
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
Hemangiosarcoma (HSA) is a cancer of the cells lining the blood vessels that is very aggressive and has nearly always spread by the time it is diagnosed. HSA accounts for 5-7% of all cancers in dogs resulting in approximately 25,000-50,000 new cases per year. The treatment of choice is surgical removal followed by chemotherapy administration. Unfortunately, despite aggressive therapy, the majority of dogs diagnosed succumb to their disease within 6-8 months. Virtually no improvements in outcome for affected dogs have occurred in the past 30 years despite multiple clinical trials and research efforts. More recently a new therapy has been developed targeting two receptors on HSA cells. However, the majority of dogs still died by 10-12 months after treatment. The molecular pathway known as the PI3K/AKT/mTOR pathway has previously been implicated in the pathogenesis of many forms of cancer including HSA. Indeed, inhibitors of PI3K/AKT/mTOR pathway have some activity against HSA cell lines in the laboratory. The investigators have generated data showing that a subset of canine HSA tumors possess genetic mutations in PI3K that are known to activate the pathway in cancer cells. In this study, they will fully characterize the expression and function of PI3K in canine HSA tumor cell lines and tumor samples. This information will then be leveraged to identify new ways to block the PI3K/AKT/mTOR pathway using a combination of small molecule inhibitors that are most effective at killing tumor cells. These data will then be used to design future clinical trials in dogs with HSA.

Publications:
Due to the early stage of testing, we have no publication.

Presentations:
Graduate Student Seminar Series at the Sackler School; presentation by Megan Gutwillig as part of her graduate training.

Report to Grant Sponsor from Investigator:
The purpose of this study is to fully characterize the expression and function of PI3K in canine HSA tumor cell lines and tumor samples to identify new ways to block this pathway using a combination of small molecule inhibitors that are most effective at killing tumors cells. Over the past 6 months we have characterized the expression of the 4 isoforms that make up PI3K family in HSA cell lines, and have evaluated the effectiveness of a variety of small molecule inhibitors against cell lines targeting either individual or all PI3K isoforms. We have also generated cell lines deficient in two of the isoforms and are beginning to analyze the effects on the cells. Lastly, we have extended our collaboration with the Broad Institute to more completely explore the genetic targets in HSA and develop techniques for minimally invasive detection of HSA (i.e., a genetic screening test) using a new technique called the blood biopsy. Over the next 6 month we should be able to finish generating the cell lines and then begin the identification of potentially new targets for therapy.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02519:
Prevalence of Bartonella spp. Infection in Dogs with Cardiac and Splenic Hemangiosarcomas Within and Between Geographic Locations

Principal Investigator: Edward Breitschwerdt, DVM & Matthew Breen, PhD
Research Institution: North Carolina State University Office of Sponsored Programs

Grant Amount: $219,026
Start Date: 2/1/2018 - End Date: 1/31/2020

Progress Report: Mid-Year 1
Report Due: 7/31/2018 - Report Received: 8/2/2018
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
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 are often ambiguous. While some splenic tumors are benign, approximately two-thirds are highly malignant and carry a poor prognosis. Hemangiosarcoma (HSA) accounts for the majority of canine malignant splenic tumors and occurs in many large dog breeds, including mixed breeds. A less common site of HSA localization is the heart (cardiac HSA). Risk factors for both cardiac and splenic HSA remain unclear, confounding development of preventative strategies. The investigators recently 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) and 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. The investigators will perform 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.

Publications:
Due to the early stage of testing, we have no publication.

Presentations:
Due to the early stage of testing, we have made no presentations.

Report to Grant Sponsor from Investigator:
We are on track to accomplish all of our aims for this study. We were able to obtain the initial set of samples on April 26, 2018 so we had a short delay in starting the study. We have now received all samples, categorized and prepped them for further testing. We will be publishing additional research from our AKC-CHF study #02287, which will define the criteria for serodiagnosis of Bartonellosis in dogs by Western Blotting (WB). That work has required additional time and research effort to validate WB testing. Once completed, we will process the serum samples from this for WB by December 2018. We have been in touch with the pathologists in the three geographically different regions of the United States so that they can define inclusion criteria and start locating the retrospective samples that they will submit to us in January of 2019 to test.

AKC-CHF

AKC Canine Health Foundation, Inc.
8051 Arco Corporate Drive • Raleigh NC 27617
888-682-9696 • www.akcchf.org

RESEARCH PROGRESS REPORT SUMMARY

Grant 02217:
A Novel Mechanism to Regulate the Growth of Canine Hemangiosarcoma

Principal Investigator: Erin Dickerson, PhD
Research Institution: University of Minnesota

Grant Amount: $86,206
Start Date: 1/1/2016 - End Date: 6/30/2018

Progress Report: FINAL
Report Due: 6/30/2018 - Report Received: 7/16/2018
(The content of this report is not confidential and may be used in communications with your organization.)

Original Project Description:
Hemangiosarcoma is an extremely aggressive cancer that is rA1PIdly fatal in dogs. While the lifetime risk is alarmingly high for some breeds such as Golden Retrievers and German Shepherd Dogs, the disease does not discriminate, and it can strike any dog at any time. Despite considerable efforts by veterinarians and scientists to find effective treatments, the outcome for dogs with hemangiosarcoma has changed very little over the past few decades. Recent evidence provides essential clues into how these tumors grow and progress, generating new ideas for treatment approaches. Such new evidence suggests that hemangiosarcoma cells rely on the metabolism of lipids or fatty acids to supply energy for tissue invasion or continued tumor growth. To obtain these lipids, hemangiosarcomas may take over the metabolic machinery of neighboring cells, forcing them to produce nutrients for the tumor cells to help them proliferate and grow. This study will verify that tumor cells rely on lipid metabolism for growth, and determine if tumor cells alter the metabolism of fat cells to obtain cellular nutrients and accelerate tumor cell lipid metabolism. Identifying and exploiting a novel mechanism that may disrupt this process by inhibiting the interactions between tumor cells and cells in the tumor environment will speed clinical investigations, and ultimately lead to improved outcomes for dogs with this devastating disease.

Publications:
We have not yet published any of our findings. We anticipate that the following data will be part of a paper that is currently in preparation, and we plan to submit this paper in 2018:

  1. Immunohistochemistry of -AR and tyrosine hydroxylase expression in primary hemangiosarcomas.
  2. Expression of -ARs and the catecholamine enzymes in hemangiosarcoma cell lines.
  3. Expression norepinephrine and dopamine in hemangiosarcoma cell lines.
  4. Treatment of hemangiosarcoma and angiosarcoma cell lines with doxorubicin leads to an increase in TH expression, as well as an increase in the synthesis of dopamine and norepinephrine.

We also anticipate the publication of a second paper in 2019 describing the majority of the findings described in this report.

Presentations:
I attended a Keystone Conference on tumor metabolism in cancer cells in Whistler Canada in March 2017. A PhD student in my lab (Derek Korpela, DVM) also attended this meeting, and he presented some of the work described here. Dr. Korpela and I also attended the annual Veterinary Cancer Society meeting, held in Portland, OR, October 2017. Both abstracts were chosen for oral presentation. I presented some of this work as a State of the Art presentation at the conference; Dr. Korpela presented some of his work showing the metabolic impact of propranolol and its enantiomers on hemangiosarcoma cell metabolism. Funding support by the AKC Canine Health Foundation was acknowledged for all presentations.

Report to Grant Sponsor from Investigator:
Hemangiosarcoma is an incurable cancer that is almost uniformly fatal. The tumors often grow quickly and spread rA1PIdly, with half of all dogs dying within six months of diagnosis, even with treatment. Because the prognosis has not changed over several decades, a better understanding of the disease is needed to develop new treatment approaches. We have found that hemangiosarcoma cells appear to rely on the metabolism of lipids to supply some of the energy and essential building blocks needed for tumor growth. We also found that propranolol, a common drug used to treat heart disease in both dogs and people, limits the uptake of lipids into cells and blocks the cell’s ability to process these compounds. Cancer cells have been shown to impose a self-serving metabolic program on normal cells by forcing normal cells to supply nutrients, such as sugars and lipids, to the tumor. Recent studies have shown that cells like adipocytes (fat cells) can be remodeled by tumor cells to help create a niche favoring tumor growth. Because propranolol can block the use of lipids by tumor cells, propranolol may be able to reverse the cancer-imposed metabolic reprogramming on adipocytes or other normal cells, limiting tumor growth. For this study, we sought to: 1) characterize the lipid metabolic program(s) in hemangiosarcoma cells and determine if the use of lipids by these cells could be blocked by propranolol; 2) determine if hemangiosarcoma cells alter the metabolic program(s) of adipocytes; and 3) whether these changes in adipocytes enhanced the tumor cell growth programs and the invasive nature of hemangiosarcomas. We found that propranolol inhibited key metabolic processes in hemangiosarcoma cells, including the uptake and processing of lipids. We also found that hemangiosarcoma cells reprogrammed normal adipocytes in a way that may force the adipocytes to produce nutrients for hemangiosarcoma cells to help them proliferate and grow. Parallel studies supported this idea by showing that adipocytes accelerated metabolic growth programs in hemangiosarcoma cells and enhanced programs favoring more aggressive disease. Future studies will be directed toward further assessing the metabolic programs of hemangiosarcomas and determining whether drugs like propranolol can be used to prevent the manipulation of adipocytes by tumor cells and reduce tumor growth and invasion.

Genetic Testing for PLN-Associated Variant Genes

After years of research supported by hundreds of Wheatens and their owners and breeders, Dr. Meryl Littman and Dr. Paula Henthorn at the University of Pennsylvania School of Veterinary Medicine (Penn Vet) identified mutations associated with PLN in two genes. A cheek swab test to determine an individual dog’s DNA status was introduced in May 2012.
Read more about testing your Wheaten on the SCWTCA website ...

Statement on Genetic Testing for PLN-Associated Variant Genes

Responsible Soft Coated Wheaten Terrier breeders and stud dog owners breed for type, temperament, soundness and health. The recent introduction of the test for PLN-associated variant genes gives us an additional tool to use in the health assessment of breeding stock. Using this test should give Wheaten breeders the ability to reduce the incidence of dogs with these variant genes slowly and carefully while still maintaining genetic diversity.

The Soft Coated Wheaten Terrier Club of America, Inc., and the SCWTCA Endowment, Inc., endorse testing for PLN-associated variant genes by breeders and stud dog owners as one tool in the assessment of breeding stock.

We strongly encourage all owners to become familiar with the test and to understand the meaning of the results and their use in breeding. Resources are available in the health section of the SCWTCA website at www.scwtca.org/health. SCWTCA neither requires nor prohibits any form of breeding beyond what is currently in the Code of Ethics.

All Wheaten owners should remember that screening for health goes beyond performing this test. Members of SCWTCA are reminded of the requirements of the Code of Ethics in regard to other testing and breeding requirements.

Launch of the Soft Coated Wheaten Terrier Database

After a number of years of development, the SCWTCA Endowment, Inc., is excited to announce the launch of the Soft Coated Wheaten Terrier Database at http://www.scwtdb.org.

This database was originally developed by the Berner-Garde Foundation 30 years ago and is in use by several other breeds in addition to Wheatens. Starting with San Jeffries’ database of 46,000 dogs, a team of volunteer data operators has added dogs so we are debuting with records on nearly 60,000 Wheatens from around the world.

In furtherance of the Endowment’s mission, we believe the detailed collection of health and pedigree information in the database will help to identify, track, and reduce the incidence of health problems in the SCWT. Wheaten owners, breeders, and researchers can be used it to assist with decisions about the care and welfare of their dogs to make breeding decisions. We expect veterinarians and veterinary researchers working with Wheatens will find it a valuable data source.

The integrity of the information in the database is vital to its purpose. Much of the data comes from voluntary submissions from owners, which is verified before being published. Publicly available information, such as records from kennel clubs and OFA is also included as verified.

The best way to appreciate the potential of the Database is to use it! It is accessible to all at www.scwtdb.org once you’ve read and accepted the policies. Start by looking up your own dogs and seeing what the Database can do: from producing pedigrees to searching for dogs meeting specific criteria. (While the Database is easy to use, you’ll also find a link to a User’s Guide on the home page.) This will show you how you can help by submitting information on your own dogs to make the Database more robust. There are forms for sending information to add; and you can upload photos of your dog, both formal show shots and informal candids.

Despite the care our operators take, errors are, of course, inevitable. If you find a problem, please report it at comments@scwtdb.org.