Pet Trust Funded Projects – Fall 2012

Dr. Dorothee Bienzle

Improved classification and prognostication of canine leukemia

Leukemia is a cancer of bone marrow cells. There are many different types of cells in the bone marrow that give rise to the cells in blood – red blood cells, white blood cells and platelets. Leukemia evolves from malignant transformation of immature or more mature white blood cells in the bone marrow, which then results in acute myeloid leukemia (AML) or chronic leukemia/myeloproliferative neoplasia (MPN), respectively. There are also some leukemias that cannot be classified. In dogs, AML is more common than MPN or unclassifiable leukemia, and is generally associated with rapid progression and death. MPN has a more slowly progressive course of disease, and unclassified leukemia can follow any course. We have very limited understanding of the causes, categories and prognoses of leukemia in dogs. Currently, most of the time a diagnosis of leukemia is established, the patient is given a very poor prognosis and the animal is euthanized in a matter of days. However, some dogs have been treated with combination chemotherapy and have achieved long-term remission, indicating that leukemia may not be a uniformly fatal cancer. Leukemia is usually diagnosed from microscopic assessment of a bone marrow biopsy. Samples may also be analyzed for expression of specific markers; however, leukemias that appear to be similar may vary a great deal in their response to therapy. In humans, genetic abnormalities such as structural (translocations, inversions) and numerical (duplications, deletions, insertions) alterations are very common and characteristic of certain types of leukemia. Importantly, leukemias with specific genetic abnormalities have unique treatments and prognoses. The technologies to perform comparable genetic investigations in dogs has been established, but has not yet been widely used.

In this proposal we wish to determine associations between specific genomic alterations and the response of canine leukemias to combination chemotherapy. Since all cases will also be typed for antigen expression and genetic abnormalities, we simultaneously will identify genomic factors associated with specific subtypes of leukemia. Information of this nature is expected to dramatically increase our understanding of leukemia, a cancer currently poorly understood and difficult to treat in dogs. We anticipate this study will highlight genetic changes in canine leukemia that inform the biology of the cancer, and thus lead to the development of more specific therapies for this group of cancers.

Dr. Byram Bridle

Assessment of the potential to treat canine lymphoma with an oncolytic vaccine

Lymphoma, the most common blood cancer in dogs, responds well to chemotherapy. However, relapse of chemo-resistant disease is common, with a median survival of only 12 months. Improving this outcome will require novel approaches. Two promising new cancer treatments are immunotherapy and oncolytic virotherapy. Immunotherapy harnesses the power of the immune system to destroy cancerous cells. Oncolytic virotherapy uses replicating viruses that infect and kill malignant cells, leaving normal tissues unharmed. Both treatments have the potential to destroy tumour cells anywhere in the body with no or minimal side-effects. We have published a strategy to synergize immunotherapy and oncolytic virotherapy, leading to durable cures in mouse models of cancer. To translate our success into a future canine lymphoma clinical trial, we must conduct preliminary studies to demonstrate safety and efficacy. This proposal has four aims: 1. prove that oncolytic immunotherapy is safe in dogs, 2. show that robust tumour-specific immune responses can be induced, 3. confirm expression of the targeted tumour antigen on canine lymphomas, and 4. show that effector mechanisms mediated by the treatment can kill lymphoma cells. This will provide the scientific rationale for a future clinical dog lymphoma trial. It will also allow us to get a permit for field testing from the Canadian Food Inspection Agency (CFIA), which is required before clinical testing of oncolytic viruses in pets.

Dr. Brenda Coomber

Exploration of the anti-cancer impact of Itraconazole on signaling in canine mast cell cancer cells

Mast cell tumours in dogs are the most common type of skin cancer, and are generally treated with surgery, radiation and/or chemotherapy. While these approaches are frequently effective, some mast cell tumors are very aggressive and require treatment with newly developed therapies like Palladia which kill cancer cells by interfering with cell signalling. However, Palladia does not work for all dogs. New treatments are therefore needed, but these can take years to develop. Another possibility is to use 'old' treatments in new ways. The drug itraconazole (Sporanox) which is used to treat fungal infections in dogs, has recently been shown to have possible anticancer activity. Our study will look at this question by using canine mast cell tumour cell lines we have isolated from surgically removed tumors. These cell lines have cell signalling changes seen in many canine mast cell tumors, and we feel they will be an excellent cell culture system to test whether itraconazole could be an effective cancer therapy for this disease. We will therefore expose these cell lines to itraconazole and other inhibitors like Palladia, and determine how well these treatments lead to changes in cell signalling and cell growth. Based on what we find, in future it may be possible to use this safe and inexpensive drug to treat dogs with aggressive mast cell tumours.

Dr. Brenda Coomber

Using ribosomal RNA disruption as a predictor of early relapse in canine lymphoma

The idea behind this study is to test whether molecular analysis of samples of canine lymphoma can predict how well a particular dog is responding to multi-agent sequential chemotherapy ("CHOP" chemotherapy). Lymphoma is one of the most common types of cancer in dogs, and most dogs with lymphoma are treated with CHOP chemotherapy. About 40% of treated dogs do well on this regime, but the remaining dogs either do not achieve remission, or their remission lasts less than 8 months. We currently have no way of identifying which dogs respond well to CHOP from those that would benefit from a different chemotherapy. For this study, samples will be collected using fine needle aspirate biopsy of enlarged lymph nodes from dogs undergoing CHOP chemotherapy at OVC. These samples will be used for a molecular analysis test that will tell us how each dog's lymphoma is responding, and that test result will eventually be matched with the clinical outcome for each dog. We hope to determine whether this test is good at predicting how well dogs with lymphoma respond to CHOP chemotherapy.

CHOP chemotherapy is usually given in 4 cycles over 4-5 months. Our long-term goal is to be able to use this test to identify those dogs not responding well, early enough in the CHOP cycle so they can be switched to another chemotherapy. Ultimately, this will ensure that more dogs with lymphoma get the best treatments we have available.

Dr. Karol Matthews

Antimicrobial activity of raw honey against common & multi-drug resistant bacteria of wounds in cats & dogs

Many bacteria are resistant to antibiotics. Extensive research in New Zealand [manuka honey] has shown that honey has retained its antimicrobial and healing properties over several thousand years. Animal studies and use in humans and veterinary patients, confirm safety and successful healing properties of honey. The honey from the U of G apiary has been successful in treating wounds in cats and dogs at OVC for several years. To support our clinical observations and encourage a more general use of honey with local availability we must investigate the potency of Ontario honey in killing bacteria. We will test batches of honey collected from several apiaries, throughout the year, against at least nine species of bacteria including those resistant to at least three classes of antibiotics. We will pursue identification of the plants where the bees forged to produce this honey. This information will be shared with the bee industry as production of ‘medicinal’ honey will be an important aspect of supporting the industry and bee population. We anticipate that providing an affordable source of ‘medicinal’ honey, with scientific support of efficacy, will lead to more frequent use of honey in managing wounds and thereby reduce human and veterinary patient suffering, costs, antibiotic use and environmental spread of multi-drug resistant bacteria.

Dr. Michael Meehan

Evaluation of the OVC Pet Loss Support Hotline: Grief & the Human-Animal Bond

The bond or emotional connection that pet owners may have with their pets is increasingly being recognized as a legitimate attachment relationship. It follows that if a pet dies or is euthanized then a pet owner will grieve the loss of that relationship. In addition for some pet owners they may have to face the fact that they are the person responsible for making the decision to end their pet’s life. For some pet owners the decision making process and the grief they experience can be overwhelming, especially when thy may not have emotional support from friends and family. At these times pet owners might need extra emotional support, listening and potentially grief counsel. At these times a pet loss support hotline would be helpful for those people that are experiencing grief and do not feel they have access to extra support. The primary purpose of a pet loss support hotline is to provide emotional support, via telephone, for pet owners who are experiencing grief due to the loss of their pet or who are anticipating the death of their pet. The American Veterinary Medical Association (AVMA) recognizes the benefits of pet loss support hotlines for pet owners as well as for the profession. For pet owners the hotline may help alleviate their grief. For the veterinary profession this service provides a unique insight into the strength of the human-animal bond and the grief process. In addition this service has the potential to provide volunteers, veterinary students and veterinarians with the opportunity to improve their communication skills when talking to clients who are experiencing the grief. The AVMA has written a set of guidelines that states that a pet loss support hotline should evaluate the role and value of the hotline from the caller’s point of view as well as from volunteer’s point of view. In addition the AVMA guidelines stipulate that effective training of the volunteers in effective listening skills is also required. Therefore the main aim of this study is to explore the impact of the Ontario Veterinary College (OVC) pet loss support hotline on callers and volunteers and to develop an effective listening and grief training program for the hotline and the veterinary profession.

Dr. Tony Mutsaers

Quantification of the radiosensitivity & the ability to repaire radiation-induced injury of three common canine tumours

An understanding of the inherent radiosensitivity of tumour cells, as well as their ability to repair radiation-induced injury can be utilized to optimize the radiation treatment protocol of a tumour. The linear quadratic model is used in the radiobiology to determine the radiosensitivity of cells. The radiosensitivity of cells is expressed as the alpha/beta ratio. The majority of human tumours have shown a relatively high alpha/beta ratio, which is best treated with a small dose per treatment of radiation multiple times over an extended period (curative protocol). However, certain cancers, such as malignant melanoma in humans, have been considered to be radioresistant, until it was determined that these tumours had a low alpha/beta ratio and therefore would be better treated with a palliative type protocol that involves fewer treatments but a much higher dosage per treatment.

Palliative protocols have been commonly used in radiation oncology to relieve discomfort. Unfortunately, the doses applied in this setting are usually too low to cure the tumour, as the total dose delivered is limited by the normal tissue toxicity. With the new linear accelerator in the OVC Animal Cancer Centre, we will be able to apply a “curative” dose to tumours using a large fraction per dose protocol (stereotactic radiation). It will be ideal to know which tumour types, and eventually which individual tumours with a certain type, would benefit from this approach, rather than our current standard of care using a more palliative approach.

Canine oral melanomas, mast cell tumours, and anal sac carcinomas are common tumours in dogs, and radiation is a treatment modality commonly utilized in their management. Each of these tumour types are conventionally treated with either a curative protocol (large dose per treatment), but the most efficacious type of protocol for these tumour types is still undefined.

Dr. Tony Mutsaers

The effect of chemotherapy on circulating bone marrow-derived progenitor cell populations in canine cancer patients - a pilot study

The use of chemotherapy has greatly improved our management of cancer, however tumour resistance remains a very significant clinical problem. One possible mechanism contributing to drug resistance is the body’s reaction/response to chemotherapy exposure and how this response may paradoxically promote the growth of cancer cells that survive anti-cancer drug treatment. One example of such a reaction is the bone marrow’s response to chemotherapy. Conventional chemotherapy drugs target rapidly dividing cell populations, so it is common for certain normal cells that experience rapid turnover, such as bone marrow precursor cells and intestinal lining cells, to become damaged along with tumor cell populations. The likelihood of damage to normal cells is a function of dose, with higher doses of chemotherapy more likely to produce these side effects. Release of progenitor cells from the bone marrow can be induced by maximum tolerated dose (MTD) chemotherapy protocols. These cells then enter the blood circulation, home to tumor sites, and contribute to tumour growth through incorporation into the tumour mass and/or local blood vessels. These cells may also alter the tumor microenvironment through their production of growth-promoting cytokines. Using flow cytometry, we can measure subsets of bone marrow cell populations and quantify the number of different cell types present in circulation before and during various forms of cancer treatment – beyond what can be accomplished through routine complete blood count procedures. The surface markers used to detect many of these bone marrow cells have been validated and reported for use in the dog, but serial assessment during routine chemotherapy treatment has not been performed. This study will investigate the detection of bone marrow-derived progenitor cells in circulation during the course of treatment with 3 commonly utilized chemotherapy drugs in veterinary oncology.

Dr. Ameet Singh

Characterization of biofilm production by Staphylococcus pseudintermedius using in-vitro & ex-vivo techniques

Surgical site infections (SSIs) are an increasing cause of illness and death in veterinary patients. As veterinary medicine progresses, the risk of SSIs can actually increase because of more advanced surgical procedures as well as procedures being performed on patients that would previously have been too ill to be surgical candidates. Additionally, the emergence of multi-drug resistant (MDR) bacteria has created significant challenges since these infections are becoming more difficult to treat as they can be recalcitrant to antibiotic therapy. SSIs are of greater importance with implanted biomaterials (e.g. orthopedic bone plates), as was shown in a recent year-long study of SSIs in dogs and cats conducted at the OVCHSC. Of particular concern in dogs and cats has been the emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP), which has recently emerged as the leading cause of SSIs in dogs at the OVCHSC (and internationally). Reasons for the rapid emergence of this highly drug resistant bacterium are not known, but one possible cause is biofilm production. A biofilm is a complex environment consisting of ‘slime’ produced by bacteria. Being encased in a biofilm matrix protects bacteria from the patients’ immune system and the effect of antimicrobials. Aggressive biofilm production has been hypothesized as one of the reasons for the dramatic and widespread emergence of a few successful MRSP clones internationally. Preliminary investigation performed by the authors has revealed that some MRSP isolates from dogs are biofilm producers. The mechanism by which biofilm producing strains of MRSP result in clinical infections in dogs is poorly understood. Further investigation into the mechanism of biofilm formation by MRSP will help to formulate effective therapeutic interventions.

Dr. Adronie Verbrugghe

Vitamin D status in canine cancer patients & the relationship with dietary vitamin D intake

Canine cancer has become more prevalent in the last decades because of increased life expectancy and greater attention to the health of pets. The range of cancers seen in dogs is as diverse as that in human patients, and despite more intensive therapeutic interventions, fatality rates remain unacceptably high in both species. Therefore, more research investigating risk factors, prognostic factors as well as treatment options is definitely warranted. In humans, epidemiological data indicate that a low vitamin D status is associated with an increased risk of a variety of cancers. Also, it has been shown in human cancer patients that vitamin D deficiency may be linked with poor prognosis, as lower serum vitamin D concentrations are related to reduced overall survival. The mechanism by which vitamin D status alters cancer development is not completely uncovered. However, it is known that many cell types have vitamin D receptors and when these receptors are activated by 1,25- dihydroxycholecalciferol (1,25(OH)2D3), the most active metabolite of vitamin D, differentiation is induced and proliferation, invasiveness, angiogenesis, and metastatic potential are inhibited. When vitamin D status is suboptimal, these activities may be impaired, leading to development and/or progression of cancer.

The purpose of this study is to compare the vitamin D status of cancer-bearing dogs with healthy dogs and evaluate the relationship between vitamin D status and dietary vitamin D intake. Also, the prognostic value of vitamin D levels for cancer-bearing dogs will be investigated. The results of this study may eventually lead to new perspectives for cancer prevention and treatment in dogs and perhaps people.

Dr. Scott Weese

Is minocycline a treatment option for multi-drug resistant methicillin-resistant Staphylococcus pseudintermedius (MRSP)

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a multidrug resistant bacterium that has emerged with startling speed in dogs and cats. In the period of only a few years, this bacterium has become a leading cause of opportunistic infections (especially skin infections, ear infections and post-surgical infections) in Ontario and elsewhere, and it currently accounts for significant patient illness. A remarkable aspect of MRSP has been its ability to become resistant to many antibiotics over a short period of time. At this point, it is common to encounter MRSP infections where the bacterium is resistant to virtually all antibiotics, and the choices that may be available are often undesirable because of toxicity, cost, the need for injectable administration or their importance in human medicine.

Doxycycline was a highly effective for treatment for MRSP until recently, but now resistance is now very common. Diagnostic laboratories test staphylococci for susceptibility to doxycycline (and sometimes the older, related drug tetracycline) and it is usually assumed that doxycycline resistance indicates resistance to all of the tetracycline family drugs. However, this is not always true and depends on the gene that is responsible for doxycycline resistance. Evidence from methicillin-resistant S. aureus in humans indicates that a related drug, minocycline, might be effective. Data from Europe indicate that the predominant European MRSP strain in dogs is doxycycline resistant but minocycline susceptible. MRSP has marked differences in regional distribution and data from Ontario (or North America as a whole) are lacking. Given the difficulty in treating MRSP and the potential that minocycline could be an effective option, testing of MRSP isolates from infections from dogs in Ontario is needed.

Dr. Paul Woods

PET-CT investigation of the biological response to toceranib (Palladia) treatment of mast cell tumour in dogs

Mast cell tumours (MCT) are the most common cutaneous tumour in the dog representing 20-25% of all canine skin tumours. Unfortunately, the biologic behavior of MCTs is extremely variable. Surgery and/ or radiation therapy has been the best approach for localized MCT (which in some cases may be curative). However, multiple tumours, recurrence, metastases, or advanced local disease can only be managed by systemic chemotherapy. Glucocorticoids and cytotoxic drugs (i.e. vinblastine, lomustine) have been used. Although remissions may be achieved, cure is unlikely. Recently, a targeted small molecule receptor tyrosine kinase inhibitor (toceranib or Palladia™) has become available to treat canine MCTs particularly those MCT with mutations in c-Kit, the receptor for stem cell factor. Unfortunately, the response to toceranib in dogs with MCT has been mostly based on empiric monitoring (e.g. gross measurement of lymph nodes) rather than more objective evaluation of biological response (e.g. molecular imaging and analysis of targeted pathways). Notably, at present we have no way of reliably predicting which MCTs will respond to toceranib and which ones will not. Molecular imaging is increasingly being used for diagnosis, staging, and evaluation of response in human cancer patients. Positron emission tomography (PET) utilizes a nuclear medicine tracer to target specific mechanisms (metabolism) that differ between normal cells and cancer cells. These changes can be employed to detect cancer often before changes in anatomic structure can be visualized with conventional imaging (X-rays) or advanced imaging (CT, MRI). The addition of computed tomography (CT) adds precise anatomic localization to the functional imaging of PET in a combined device (PET-CT). With their combined advantages, PET-CT may surpass previous conventional diagnostic imaging procedures. More importantly, by performing PET-CT imaging before and after treatment with toceranib, changes in tumour metabolism can be measured long before gross responses (i.e.tumour shrinkage) are detectable. Therefore, this pilot study proposes to evaluate the biological response to toceranib treatment in dogs with mast cell tumour utilizing PET-CT and validating these changes by molecular assays of biopsied tissue. Hence this project is investigating an easier and quicker way to demonstrate when the new novel targeted chemotherapy (toceranib) is of benefit to dogs afflicted with mast cell tumor (MCT). Hence in cases where the MCT is resistant to toceranib, this project will provide veterinarians a new tool to demonstrate lack of efficacy much earlier than monitoring gross tumor response (i.e. tumor size change/RECIST). Therefore, in those resistant cases the health and well-being of the dog will be improved by preventing the dog from suffering needless and fruitless chemotherapy (i.e. reduced discomfort and distress).

Dr. Tony Yu

Measurement of topical antifungal sensitivity for use in the treatment of Malassezia otitis externa

In today’s veterinary practice, ear infections (otitis) are responsible for a large number of presenting complaints in companion animals. Otitis has a multifactorial etiology however, yeast, such as Malassezia spp., plays an important role in ear disease. Recently, multiple cases of Malassezia otitis have been reported in literature that have been non-responsive to topical antifungals used in other cases with great success. This may indicate individual Malassezia strains may demonstrate resistance to our current therapeutics. Without an available laboratory test to determine whether a Malassezia spp. isolate is susceptible or resistant to a certain antifungal, we may be perpetuating resistance with continued treatment of cases with antifungals that the specific isolate may be resistant too. The aim of this study is to identify a more appropriate method of evaluation of antimicrobial sensitivity to otic Malassezia by generating a Topical Otic Panel of Sensitivities to measure Minimum Inhibitory Concentrations to topically delivered antifungals. Culture swabs will be taken from cases of Malassezia otitis in dogs. These isolates will be grown on agar medium and then incubated with serial dilutions of multiple antifungal medications found in widely available veterinary otic preparations used to treat cases of Malassezia otitis. The MIC obtained for the different antifungals can then be compared to concentrations of the antifungals found in the ear medications. Results of this study will more accurately reflect the true resistance patterns of Malassezia in otitis externa and provide more appropriate recommendations for topical treatment of Malassezia Otis. By providing more accurate recommendations, this will improve the veterinary-client relationship and lead to less emotional and financial stress for the pet owner.

$212,105 was awarded in research funds

Educational Awards:

Dr. Alice Defarges - Small Animal Clinical Cases Bank in ARTICULATE

Dr. Scott Weese - Development of infectious disease information sheets for pet owners & veterinarians