Developing a Next Generation Sequencing Diagnostic Platform for Tick-Borne Diseases

Posted on by Janet Boyd

Diagnostic tests based on the detection of DNA from harmful organisms in clinical samples have revolutionized veterinary medicine in the last decades. Currently, diagnostic panels for several vector-borne organisms are available through universities and private labs in the USA and abroad. However, the vast majority of results from sick dogs are negative, which frustrates veterinarians and dog owners trying to reach a definitive diagnosis. These panels are based on the detection of previously known DNA sequences of each pathogen, which limits their ability to detect novel organisms.

In this study, the investigators will adapt high-throughput nextgeneration sequencing (NGS) to the detection of tick-borne bacteria in dog blood in an effort to overcome the limitations of current diagnostics for tick-borne diseases. If successful, increasing the capabilities of NGS to detect infected dogs and to accurately determine which bacteria are responsible for disease will support the development of a better diagnostic tool to simultaneously advance canine and human health. This work expands on Dr. Diniz’s previous CHF-funded study #02292.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 02528

RESEARCHERS

Pedro Diniz, DVM, PhD; Western University of Health Sciences
Amount: $5,000

Measuring Chemotherapy Drug Resistance in Dogs with T-cell Lymphoma

Posted on by PWDF Admin

Quantitative Assessment of Minimal Residual Disease Kinetics during CHOP Chemotherapy of Canine T-cell Lymphoma via Next-Generation TCRVß Sequencing

Human cancer treatment is becoming more and more tailored to each individual case and tumor characteristics. This study will be conducted by researchers at North Carolina State University and will follow a small number of cells that evade chemotherapy agents, providing information on the effectiveness of particular agents against T-cell lymphoma. The data from this study will be used to personalize treatment protocols for dogs on an individual basis in hopes of improving outcomes, survival rates, and quality of life.

Description: Since the use of combination chemotherapy was first reported in 1968, little progress has been made in improving the survival of dogs with T-cell lymphoma. Effectively monitoring chemosensitivity – the number of tumor cells killed by chemotherapy– of individual lymphoma cells exposed to multiple agents over many treatments remains a challenge. Following the small numbers of cells that evade chemotherapy would provide information on the effectiveness of a particular chemotherapy agent.

In this clinical trial, researchers will use state-of-the-art DNA technology to measure changes in this small population of resistant cancerous T-cells in client-owned dogs with lymphoma. Data will be used to personalize treatment protocols for individual dogs in hope of improving survival and quality of life.

Co-sponsored with the Morris Animal Foundation, Grant Number: D16CA-056

RESEARCHERS

Dr. Paul R. Hess, DVM, PhD
North Carolina State University
Amount: $10,000

Filling the Gaps in the Canine Genome

Posted on by PWDF Admin

The sequencing of the genome of man’s best friend in 2005 has provided an invaluable resource to the canine research community, and has reinforced the position of the dog as an important model organism to study human physiology and disease.

Unlike the human and the rodent models (the mouse and the rat), very few dog genes had been sequenced prior to its whole genome sequencing. Consequently, the dog genome has been annotated for its gene content primarily based on mapping the gene-related sequences from the human, the mouse, the rat, and other non-dog species to the dog genome.

While providing the research community with an unprecedentedly large set of dog genes, the definition of DNA sequences as coding sequences (i.e. gene annotation) has substantial errors and is missing in dog-specific information in many aspects. This significantly hinders research in many fields such as disease gene discovery and cancer-causative gene mutation identification, where functional information about a gene is required to make progress. Recently emerged next-generation sequencing (NGS) technologies provide an unprecedented opportunity to correct these errors and to supply the missing information in the current dog gene annotation in a time- and cost-effective fashion.

We propose herein to use state of the art NGS strategies to identify genes/transcripts expressed in major dog tissues and cell types. The valuable data, along with more refined sequence alignment between the dog and other species, will be used to build the most accurate and complete annotation of the dog genome for its gene annotation. The project will significantly facilitate research in areas of canine health most significant to the AKC Canine Health Foundation constituency and lead to important RNA-based (transcriptomic) and protein-based (proteomic) research in the future.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01849

RESEARCHERS

Shaying Zhao, PhD
University of Georgia

Beyond the Genome: The Intersection of Genes and the Environment in Canine Cancer

Posted on by PWDF Admin

Not all genes are active at all times. DNA methylation (the addition of methyl groups to DNA) is one of several mechanisms that cells use to control gene expression. Abnormal patterns of DNA methylation have been observed in human cancer. However, methylation remains an unexplored dimension of canine disease.

This seed grant to Dr. Wayne will allow him to establish the techniques and methodologies necessary to define the pattern of normal variation in methylomes (the genome-wide collection of methylated sites) from an array-based analysis of a variety of domestic dog breeds. Differences in methylation found between breed lineages will be complemented by the study of gene expression to understand how methylation regulates levels of expression.

Upon completion of this study, Dr. Wayne’s laboratory will have proof-of-principle for evaluation of the canine methylome. Ultimately, he intends to establish a public web-based resource to serve as a repository for the dog methylomes. The collection of methylomes they generate will contribute to the growing resources that are available for investigation of disease etiology as well as advancing therapeutic approaches. These data will provide a new resource for understanding how gene regulation through methylation affects phenotype, disease and overall canine health.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01822

RESEARCHERS

Robert K. Wayne, PhD
University of California

Genetic Background and the Angiogenic Phenotype in Cancer

Posted on by PWDF Admin

This project will continue the researchers’ observations on gene appearance profiles in hemangiosarcoma from Golden Retrievers to German Shepherd Dogs and Portuguese Water Dogs, and it also will define how new targeted therapies may effectively control the disease in these and other dog breeds.

Results

Certain dog breeds are prone to develop certain types of cancer; yet, there has been little progress to define genes or other factors that account for this risk. Our recent work on hemangiosarcoma was the first to demonstrate that a dog’s genetic background, defined by “breed,” can influence the profile of genes that are expressed by tumors. Among other important implications, this implies that certain breeds are diagnosed with specific cancers more frequently than others because of the behavior of tumors after they arise, and not simply because they arise more frequently. Specifically, this may apply to the observed predisposition for hemangiosarcoma seen in Golden Retrievers, German Shepherd Dogs, and Portuguese Water Dogs. Here, we continued to test this premise by evaluating genome-wide gene expression profiles in samples from dogs of various breeds.

Our results suggest that, while there are subtle differences that are influenced or modulated differently in tumors from dogs of different breeds, these differences may disappear when tumors are considered in their context as “tissues” that include microenvironment constituents. Rather, there appear to be distinct subtypes of hemangiosarcoma (perhaps with different biological behavior and prognosis?), which might arise from different cells of origin, or more likely, which develop in response to adaptation of the hemangiosarcoma cells to environments that show different patterns of inflammation, angiogenesis, coagulation, and hypoxia, each of which alters not only the predominant or favored differentiation of the tumor cells themselves, but also the way they instruct microenvironment cells to create a favorable niche.

This underscores the importance of looking at these tumors in their context as “new tissues” or “new growths” rather than at the cells in isolation as we work to develop more effective strategies for detection, diagnosis, and therapy. To follow on this premise, we evaluated new therapy approaches that target both tumor and microenvironment compartments. Specifically, one such approach also shows efficacy to kill tumor-initiating cells.

Data funded by this project grant and others allowed us to validate the therapy and move it to the clinic. Angiosarcoma Awareness, Inc. provided the initial funds to support a dose finding and efficacy trial where we will treat ~20 dogs with hemangiosarcoma using a bispecific ligand targeted toxin. We completed production of the molecule under “Good Manufacturing Practices” (i.e., suitable for use in human patients) and enrollment is ongoing. Finally, we identified other potential drugs to treat this disease – or perhaps more likely, the pathways they disrupt as potential targets for development of new therapies.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01131

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Minnesota

Mechanistic Relationship of IL-8 in Cell Proliferation and Survival of Canine Hemangiosarcoma

Posted on by PWDF Admin

Characterize the direct effects of IL-8 on HSA cells.

Results

The hypothesis tested in this project was that interleukin-8 (IL-8) promotes growth and survival of hemangiosarcoma cells. This hypothesis was based on our previous results showing significant enrichment of IL-8 gene expression in hemangiosarcoma cells compared to normal endothelial cells isolated from non-malignant hematomas. Here, we confirmed that IL-8 is constitutively expressed by canine hemangiosarcoma cells in laboratory culture, as well as by primary tumors (fresh frozen samples). However, the levels of IL-8 are moderately variable among tumors.

Hemangiosarcoma cells in culture and primary hemangiosarcoma tumors also express IL-8 receptors (IL-8Rs). The receptors are expressed at comparable levels by virtually all the cultured cells and all the tumors, suggesting changes in expression of the receptor are unlikely to contribute to malignant behavior. We also confirmed that IL-8 binds to IL-8 receptors, and this interaction has functional consequences: IL-8 promotes signal transduction (calcium mobilization) in cultured HSA cells, and when we added IL-8 to cultured cells, they were able to “sense” this IL-8 excess and downregulated the expression of their own IL-8 gene. In contrast, if we blocked the interaction of their own secreted IL-8 with the receptor, they increased the amount of IL-8 gene expression. This is a classic response of compensatory regulation to negative feedback. Expression of a gene whose protein product turns on IL-8 gene expression followed the same pattern. It was downregulated when IL-8 was present in excess and induced when IL-8 was prevented from interacting with its receptor.

Despite its biological activity, IL-8 did not promote growth of hemangiosarcoma cells in culture, and IL-8 blockade did not hinder IL-8 growth in culture. When cells were deprived of nutrients and growth factors, they did not compensate by increasing production of IL-8; instead, IL-8 expression was reduced. And the addition of IL-8 did not prevent these nutrient-deprived cells from dying, and neither did it prevent cells treated with chemotherapeutic drugs from dying. Together, the data suggested that IL-8 did not directly mediate growth or survival of hemangiosarcoma cells in culture, refuting the initial hypothesis.

We then compared the gene expression profiles of cells and tumors that expressed high levels of IL-8 (and thus were adapted to growing in an environment rich in IL-8) with those of cells and tumors that expressed lower levels of IL-8 (adapted to growing in environments with relatively scant IL-8). The data show that cells adapted to high IL-8 environments had gene expression profiles indicative of greater inflammation, coagulation, fibrosis, and angiogenesis. These data suggested that IL-8 could be important to modulate the microenvironment and provide a suitable tumor niche. Experiments from an independent, complementary project funded by the National Canine Cancer Foundation showed that indeed, blocking IL-8 hindered the ability of hemangiosarcoma cells to establish a tumor niche in vivo. Finally, preliminary data suggest that IL-8 also may be necessary to maintain the tumor-initiating populations of canine hemangiosarcoma, by enhancing self-renewal. This hypothesis is under investigation in our newly funded project supported by AKC CHF.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 01429

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Minnesota

Genetic Analysis of Hypoandrenocorticism in Nova Scotia Duck Tolling Retrievers

Posted on by PWDF Admin

Addison’s disease, also known as hypoadrenocorticism, is a deficiency of hormones that are produced by the adrenal glands and help regulate a dog’s metabolism, blood pressure, electrolyte balance and stress response. Though the disease is relatively uncommon in dogs, certain breeds—including Nova Scotia Duck Tolling Retrievers, Bearded Collies, Great Danes, Leonbergers, Portuguese Water Dogs, Standard Poodles and West Highland White Terriers—have a much higher risk than the general dog population.

Results

Researchers identified a region of the genome that is associated with the development of Addison’s disease in Nova Scotia duck tolling retrievers. Additionally, it appears that dogs that are homozygous (both chromosomes carrying the same genes) with respect to this region are at greater risk of developing Addison’s disease, even at a young age (under 2 years). Although additional genes are likely involved, this information is the first step toward understanding the genetics of this disease and developing a genetic test that will help eliminate Addison’s disease through informed breeding practices. This fellowship training grant also provided hands-on training for a veterinarian who is pursuing a research career.

Co-sponsored with the Morris Animal Foundation, Grant Number: D08CA-402

RESEARCHERS

Angela M. Hughes, DVM
University of California at Davis

MicroRNA Profiling and MicroRNA-Based Treatment of Canine Cancers

Posted on by PWDF Admin

The goals of this study are to identify important canine miRNAs that can be used to improve cancer diagnosis and treatment in the dog.

Results

MicroRNAs (miRNAs) are small non-protein coding RNAs that have been implicated in humans as having a fundamental role in cancer initiation and progression. Osteosarcoma (OSA) is the most common bone tumor in dogs, and although breeds such as Rottweilers and Greyhounds are at higher risk for developing OSA, the understanding of its molecular etiology is limited.

In this study, we determined that canine OSA possess a unique miRNA expression signature distinct from that found in normal canine osteoblasts and that these dysregulated miRNAs contribute to OSA pathogenesis and progression. Notably, we identified 84 miRNAs that were expressed significantly higher or lower in OSA cells compared to normal osteoblasts (bone cells). Several of these miRNAs, have previously been identified as having a role in a variety of human cancers, including miR-155 and mir-21. Particularly interesting was the identification of miR-9 as being highly expressed in primary OSA tumors. This miRNA was simultaneously identified by us as being highly expressed in aggressive canine mast cell tumors (primary tumors that metastasized) compared to mast cell tumors that did not metastasize.

We then focused our efforts on understanding the role of miR-9 in canine OSA. To do this we developed methods to alter the expression (levels) of miR-9 in normal osteoblasts and in OSA cells in vitro. We were able to show that by increasing expression of miR-9 in OSA cells that we increased their invasive properties, a key biological feature in the metastatic process. Based on these findings, our laboratory is continuing to explore the consequences and functions of miR-9 dysregulation in OSA and other canine cancers.

This work has resulted in the development by our laboratory of a genetically engineered mouse where miR-9 can be overexpressed in selected specific cell types (e.g. osteoblasts) in vivo. By understanding miR-9 functions in OSA more fully, we should be able to develop strategies for effectively targeting miR-9 therapeutically, for example with “antagomiRs” that have been chemically modified, or by developing nanoparticle antagomiRs to inhibit miR-9 in vivo. These goals will form the basis of our subsequent proposal to the AKC-CHF.

Co-sponsored with the AKC Canine Health Foundation, Grant Number: 00790

RESEARCHERS

William C. Kisseberth, DVM, PhD
Ohio State University

Pooled Association Mapping for Canine Hereditary Disorders

Posted on by PWDF Admin

To develop a system that will map genetic traits causing health problems.

More than 450 canine genetic traits are listed on the Online Mendelian Inheritance in Animals list. These traits affect all body systems in dogs and can cause health problems ranging from mild disease susceptibility to severe illness and death. Researchers will use a genetic tool called the Affymetrix Canine SNP Chip to develop a mapping strategy using pooled DMA samples that will map genes for hereditary canine disorders. This method promises to be 10 times more efficient and cost effective than current methods for analyzing canine inherited traits. Once this method is validated, it will be used to map a series of hereditary retinal disorders affecting multiple breeds.

Co-sponsored with the Morris Animal Foundation, Grant Number: D07CA-085

RESEARCHERS

Gregory M. Acland, BVSc
Cornell University

Dr. Acland is a professor of medical genetics at Cornell University and an adjunct p of ophthalmology at the University of Pennsylvania. He received his veterinary degree from the University and completed a residency and postdoctoral work at the University of Pennsylvania.

Reciprocal Relationship of PTEN and p21 in Canine Cancer

Posted on by PWDF Admin

An estimated one out of every two dogs alive today will get cancer in its lifetime, and as many as 50 percent of those will die from the disease. Despite significant gains in cancer treatment, a thorough understanding of why cancers arise and why they behave as they do is essential to improving prevention and treatment. For this project, researchers will investigate two proteins whose interactions appear to be intimately tied to the behavior of two serious cancers, melanoma and hemangiosarcoma. What they learn may help to test targeted therapies for these cancers and significantly improve the lives of affected dogs.

Results

Cancer is the leading cause of disease-related death in dogs, which is why the Morris Animal Foundation and the Portuguese Water Dog Foundation have invested considerable resources to understand this group of diseases to develop more effective treatments. Cancer happens when genes that control the balance of division and survival cease to function normally in a cell and cause it to become malignant. One of these genes is called PTEN. The protein product of this gene generally restrains cell division, in part by controlling another protein called p21.

Scientists at the University of Minnesota and at the University of California, Davis have found that using compounds to lower the levels of p21 in some tumors decreased the resistance to conventional chemotherapy drugs. They also concluded that chemotherapy resistance is sometimes unrelated to abnormalities of PTEN, while it is often associated with elevations of p21. The results have allowed the investigators to justify efforts to move these compounds to the next step of clinical development.

Co-sponsored with the Morris Animal Foundation, Grant Number: D06CA-065

RESEARCHERS

Jaime F. Modiano, VMD, PhD
University of Minnesota