Auranofin improves overall survival when combined with standard of care in a pilot study involving dogs with osteosarcoma

Osteosarcoma is the most common paediatric primary bone malignancy. The major cause of death in osteosarcoma is drug‐resistant pulmonary metastasis. Previous studies have shown that thioredoxin reductase 2 is a driver of metastasis in osteosarcoma and can be inhibited by auranofin (AF). Moreover, studies have shown that AF significantly reduces pulmonary metastases in xenotransplant models. Here, we describe a phase I/II study of AF in canine osteosarcoma, a well‐recognized spontaneous model of human osteosarcoma. We performed a single‐arm multicentre pilot study of AF in combination with standard of care (SOC) (amputation + carboplatin). We recruited 40 dogs to the trial and used a historical SOC‐only control group (n = 26). Dogs >15 kg received 9 mg AF q3d PO and dogs <15 kg received 6 mg q3d. Follow‐up occurred over at least a 3‐year period. Auranofin plus SOC improved overall survival (OS) (P = .036) in all dogs treated. The improved outcome was attributable entirely to improved OS in male dogs (P = .009). At the time of writing, 10 dogs (25%) survive without measurable disease in the treatment group with survival times ranging between 806 and 1525 days. Our study shows that AF improves OS in male dogs when combined with SOC. Our findings have translational relevance for the management of canine and human osteosarcoma. Our data justify a larger multicentre phase 2 trial in dogs and a phase I/II trial in human patients with refractory disease at the time of initial surgery.     

Investigating the in vitro interactive effects of ionizing radiation and pamidronate in a canine osteosarcoma cell line

Introduction: Dogs with osteosarcoma suffer intense bone pain. Conventional palliative treatment options include ionizing radiation with or without systemic chemotherapy. Intravenous pamidronate is a first‐line therapeutic agent in people with painful skeletal metastases and is currently being evaluated for the management of osteolytic pain associated with canine osteosarcoma. The theoretical combination of radiation and pamidronate for managing bone pain appears to be a rational treatment option, however, in vitro experiments demonstrating additive or supraadditve cytotoxic effects of these two modalities would further support the clinical institution of this novel combination therapy. The purpose of this study was to evaluate the in vitro cytotoxicity of ionizing radiation and pamidronate in a canine OSA cell line and to characterize the interaction between these two therapeutic modalities when used in combination. Methods: The canine osteosarcoma cell line, HMPOS, was subjected independently to varying doses of ionizing radiation (100 cGy, 300 cGy, 400 cGy, 600 cGy, 800 cGy, or 1000 cGy) or different concentrations of pamidronate (10, 25, 50, 66, 75, 100, 150, 200, and 250 μM). Cells were harvested following 48 hours of incubation and cell viability was assessed by flow cytometric analysis. Dose‐response curves were generated, and a theoretical ED₅₀ for each treatment modality calculated. An ED₅₀ isobologram was created to determine if different combinations of ionizing radiation and pamidronate (isoboles) would demonstrate subadditive, additive, or supraadditve cytotoxic effects. Results: The ED₅₀ of ionizing radiation and pamidronate were 614 cGy and 65.3 μM, respectively. Of the six different isobole combinations evaluated, three produced supraadditve effects, one produced a subadditive effect, and two produced additive effects. Conclusions: Both ionizing radiation and pamidronate independently exert in vitro cytotoxic effects. Positive interactive effects can be generated, but only with specific isobole combinations. The results from this in vitro study support the use of specific combinations of ionizing radiation and pamidronate in order to maximize the cytotoxic properties of both treatment modalities.     

The future of canine glaucoma therapy

Canine glaucoma is a group of disorders that are generally associated with increased intraocular pressure (IOP) resulting in a characteristic optic neuropathy. Glaucoma is a leading cause of irreversible vision loss in dogs and may be either primary or secondary. Despite the growing spectrum of medical and surgical therapies, there is no cure, and many affected dogs go blind. Often eyes are enucleated because of painfully high, uncontrollable IOP. While progressive vision loss due to primary glaucoma is considered preventable in some humans, this is mostly not true for dogs. There is an urgent need for more effective, affordable treatment options. Because newly developed glaucoma medications are emerging at a very slow rate and may not be effective in dogs, work toward improving surgical options may be the most rewarding approach in the near term. This Viewpoint Article summarizes the discussions and recommended research strategies of both a Think Tank and a Consortium focused on the development of more effective therapies for canine glaucoma; both were organized and funded by the American College of Veterinary Ophthalmologists Vision for Animals Foundation (ACVO‐VAF). The recommendations consist of (a) better understanding of disease mechanisms, (b) early glaucoma diagnosis and disease staging, (c) optimization of IOP‐lowering medical treatment, (d) new surgical therapies to control IOP, and (e) novel treatment strategies, such as gene and stem cell therapies, neuroprotection, and neuroregeneration. In order to address these needs, increases in research funding specifically focused on canine glaucoma are necessary.     

Wnt5a expression in canine osteosarcoma

Osteosarcoma is the most common primary malignancy of bone in dogs and is associated with poor long‐term outcomes due to its highly metastatic nature. A better understanding of the signalling pathways and proteins involved with osteosarcoma pathogenesis may aid in improved outcomes through the use of targeted therapies. The Wnt5a protein, a ligand for the non‐canonical Wnt signalling pathway, is implicated in mediating the aggressiveness of cancer cell lines, including those of human osteosarcoma origin. Given the close relationship between human and canine osteosarcoma, the primary goal of this study was to characterize Wnt5a expression in canine osteosarcoma. Second, if Wnt5a expression was present in canine osteosarcoma, the study aimed to determine any potential association with clinical outcome and clinical variables in similarly treated osteosarcoma‐bearing dogs. Wnt5a expression was present in 26 of the 48 (54%) cases of canine osteosarcoma. Wnt5a expression was not associated with progression‐free survival (P = 0.4) or overall survival (P = 0.1).     

Update on the biology and management of canine osteosarcoma.

Osteosarcoma (OSA) is the most common primary bone tumor diagnosed in dogs. Our understanding of the risk factors and genetic changes in canine OSA patients is growing, but specific, innovative therapeutic strategies are slow in coming. Appendicular skeletal osteosarcoma, the most frequent form of this disease, is typically seen in large to giant breeds, with males being overrepresented in most reports. Axial skeletal OSA is less common than appendicular OSA, but the biologic behavior of the disease is equally aggressive in all skeletal sites except for the mandible. Though the current standard of care for dogs with osteosarcoma remains surgical resection of the affected site, followed by chemotherapy with either a platinum- (cisplatin or carboplatin) or doxorubicin-based protocol, novel therapies are being actively investigated.     

The future of immunotherapy for canine atopic dermatitis: a review

Allergen specific immunotherapy (ASIT) is a foundation treatment for canine atopic dermatitis (CAD), though few critical studies have documented its effectiveness as a disease‐modifying treatment in dogs. The mechanisms by which ASIT works in dogs have not been elucidated, although they are likely to parallel those known for humans. Current ASIT approaches in CAD focus on either subcutaneous or sublingual administration. Greater knowledge of major allergens in dogs, ideal dosage regimes and details of allergen admixture are likely to lead to better efficacy in CAD. Evaluation of biomarkers for successful therapy may also be of benefit. Potentially important advances in human medicine, that have yet to be explored in dogs, include use of modified allergen preparations such as allergoids, recombinant major allergens or allergen peptides; modification with adjuvants; or packaging of the above in virus‐like particles. Co‐administration of immunomodulators such as CpG oligodeoxynucleotides or specific monoclonal antibodies might direct the immune response in the desired direction while calming the “cytokine storm” of active disease. Initial trials of alternative routes of administration such as intralymphatic immunotherapy have yielded exciting results in humans, and continuing study in dogs is underway. Progress in ASIT of human food allergy may provide clues that will assist with improved diagnosis and patient management of CAD. Importantly, further study must be undertaken to clarify the conditions under which ASIT is a valuable treatment modality for dogs.     

Cytosine arabinoside in addition to VCAA‐based protocols for the treatment of canine lymphoma with bone marrow involvement: does it make the difference?

Cytosine arabinoside (ara‐C) is a component of many protocols for the treatment of acute leukaemia and non‐Hodgkin lymphomas in humans. The aim of the study was to prospectively evaluate the efficacy of ara‐C in a myeloablative regimen in a cohort of canine lymphomas with bone marrow involvement. Seventeen dogs were enrolled. Eight were treated with a VCAA‐based protocol (Group 1) and nine with the same regimen added with ara‐C (Group 2). Ara‐C was administered on a 5‐day schedule as an i.v. continuous infusion at the dose of 150 mg m^?2 per day for five consecutive days. During treatment complete remission (CR) was achieved in two dogs in Group 1 and in eight dogs in Group 2. CR rate was significantly higher in Group 2 (P < 0.01). Median survival was 72.5 days (range 6–174) in Group 1 and 243 days (range 73–635) in Group 2. Survival was significantly longer in Group 2 (P < 0.001). Both protocols were well tolerated, with a low incidence of adverse events. Ara‐C added to a VCAA‐based protocol appears to be safe and beneficial in dogs with stage V lymphoma. Incorporation of the nucleoside analogue might be crucial for the development of future therapeutic strategies in dogs.     

Production and characterization of monoclonal antibodies specific for canine CD138 (syndecan‐1) for nuclear medicine preclinical trials on spontaneous tumours

We isolated 11 antibodies specific for canine CD138 (cCD138) to validate the interest of CD138 antigen targeting in dogs with spontaneous mammary carcinoma. The affinity of the monoclonal antibodies in the nanomolar range is suitable for immunohistochemistry and nuclear medicine applications. Four distinct epitopes were recognized on cCD138 by this panel of antibodies. CD138 expression in canine healthy tissues is comparable to that reported in humans. CD138 is frequently expressed in canine mammary carcinomas corresponding to the human triple negative breast cancer subtype, with cytoplasmic and membranous expression. In canine diffuse large B‐cell lymphoma, CD138 expression is associated with the ‘non‐germinal center’ phenotype corresponding to the most aggressive subtype in humans. This homology of CD138 expression between dogs and humans confirms the relevance of tumour‐bearing dogs as spontaneous models for nuclear medicine applications, especially for the evaluation of new tumour targeting strategies for diagnosis by phenotypic imaging and radio‐immunotherapy.     

Curative-intent radiation therapy as a treatment modality for appendicular and axial osteosarcoma: a preliminary retrospective evaluation of 14 dogs with the disease

Canine osteosarcoma is a common bone malignancy associated with aggressive local disease and rapid metastasis. Current local therapeutic modalities do not provide curative‐intent options for dogs with significant orthopaedic or neurologic disease, dogs which are denied amputation or dogs with non‐resectable lesions. The goals of this retrospective study included the evaluation of local control, survival, and time to the development of metastases in 14 dogs treated with curative‐intent radiation therapy and chemotherapy. Median local disease control was 202 days (79–777). Median survival was 209 days (79–781). Median time to metastasis was 314 days (7–645). No significant correlation was found between the outcome and pre‐treatment alkaline phosphatase levels, radiographic appearance, tumour site, radiation dose or chemotherapeutics administered. In these dogs, full‐course radiation therapy in conjunction with chemotherapy was not found to yield equivalent results to the standard of care options.     

The use of canine models of inherited retinal degeneration to test novel therapeutic approaches

Inherited retinal degenerations (RDs) are a common cause of blindness in dogs and in humans. Over the past two decades numerous genes causally associated with these diseases have been identified and several canine models have been used to improve our understanding of the molecular mechanisms of RDs, as well as to test the proof of principle and safety of novel therapies. This review briefly summarizes the drug delivery approaches and therapeutic strategies that have been and are currently tested in dogs, with a particular emphasis on corrective gene therapy, and retinal neuroprotection.     

Association of macrophage and lymphocyte infiltration with outcome in canine osteosarcoma

Immunotherapeutic strategies have shown promise for the treatment of canine osteosarcoma (cOSA). Very little is known about the immune microenvironment within cOSA, however, limiting our ability to identify potential immune targets and biomarkers of therapeutic response. We therefore prospectively assessed the disease‐free interval (DFI) and overall survival time (ST) of 30 dogs with cOSA treated with amputation and six doses of adjuvant carboplatin. We then quantified lymphocytic (CD3+, FOXP3+) and macrophage (CD204+) infiltrates within the primary tumours of this cohort using immunohistochemistry, and evaluated their association with outcome. Overall, the median DFI and ST were 392 and 455 days, respectively. The median number of CD3+ and FOXP3+ infiltrates were 45.8 cells/mm² (4.6‐607.6 cells/mm²) and 8.5 mm² (0‐163.1 cells/mm²), respectively. The median area of CD204+ macrophages was 4.7% (1.3%‐23.3%), and dogs with tumours containing greater than 4.7% CD204+ macrophages experienced a significantly longer DFI (P = 0.016). Interestingly, a significantly lower percentage of CD204+ macrophages was detected in cOSA arising from the proximal humerus compared to other appendicular bone locations (P = 0.016). Lymphocytic infiltrates did not appear to correlate with outcome in cOSA. Overall, our findings suggest that macrophages may play a role in inhibiting cOSA progression, as has been suggested in human osteosarcoma.     

Inflammatory bowel disease versus chronic enteropathy in dogs: are they one and the same?

The aim of this review is to discuss why “chronic enteropathy” might be a better term than “inflammatory bowel disease” in dogs, because the treatment and outcome of the disease is very different from that of inflammatory bowel disease in humans. The effect of food, antibiotics and immunosuppressant drugs on chronic enteropathy will be reviewed. New treatments under investigation will also be introduced. Although there are several studies evaluating treatment of chronic enteropathy in dogs, the quality and quantity of evidence supporting individual therapies remains scarce and more work is needed to improve management of this disease. Finally, new findings about dogs with chronic enteropathy complicated by protein‐losing enteropathy will be discussed. Although prognosis for these dogs is poor, recent data might help improve their treatment.     

Pharmacokinetics of single-dose intravenous levetiracetam administration in normal dogs

Objective: To determine plasma pharmacokinetics of levetiracetam after a single intravenous dose (60 mg/kg) in normal dogs using a high‐performance liquid chromatography assay validated for canine plasma. Design: Pharmacokinetic study. Setting: A university‐based canine research facility. Animals: Six healthy adult dogs. Interventions: Intravenous drug administration, multiple blood sample procurement. Measurements and main results: There were no obvious adverse effects associated with the intravenous (IV) bolus administration of levetiracetam in any of the dogs. Plasma levetiracetam concentrations remained above or within the reported therapeutic range for humans (5–45 μg/mL) for all dogs, for all time periods evaluated. Mean and median (in parentheses) values for pharmacokinetic parameters included the following: maximum plasma concentration, 254 μg/mL (254 μg/mL); half‐life, 4.0 hours (4.0 hours); volume of distribution at steady state, 0.48 L/kg (0.48 L/kg); clearance, 1.4 mL/kg/min (1.5 mL/kg/min); and median residence time, 6.0 hours (6.0 hours). Conclusions: In normal dogs, a 60 mg/kg IV bolus dose of levetiracetam is well tolerated and achieves plasma drug concentrations within or above the therapeutic range reported for humans for at least 8 hours after administration. Based on the favorable pharmacokinetics and tolerability demonstrated for IV levetiracetam in this study, in addition to previously demonstrated efficacy of oral levetiracetam, IV levetiracetam may be a useful treatment option for emergency management of canine seizure activity.     

Prostate cancer in dogs: comparative and clinical aspects

The canine prostate gland shares many morphological and functional similarities with the human prostate and dogs are the only other large mammals that commonly develop spontaneous prostate cancer. However, the incidence of prostate cancer is much lower in dogs and the precise cell of origin is not known. Dogs with prostate cancer usually present with advanced disease that does not respond to androgen deprivation therapy. Similar to humans, affected dogs often develop osteoblastic bone metastases in the pelvis and/or lumbar spine with associated pain and neurological deficits. Other clinical signs include weight loss, lethargy, and abnormal urination and/or defecation. Surgery, chemotherapy, and radiation have been used to treat dogs with prostate cancer, but success has been limited by the location and aggressive nature of the disease. It is evident that better methods of early detection and more effective therapies are needed for prostate cancer in dogs and advanced prostate carcinoma in men. Dogs with naturally-occurring prostate cancer are relevant models for the disease in humans and pre-clinical studies of new diagnostics and therapies in dogs may benefit both humans and dogs with prostate cancer.     

Carboplatin versus alternating carboplatin and doxorubicin for the adjuvant treatment of canine appendicular osteosarcoma: a randomized,phase III trial

Despite numerous published studies describing adjuvant chemotherapy for canine appendicular osteosarcoma, there is no consensus as to the optimal chemotherapy protocol. The purpose of this study was to determine whether either of two protocols would be associated with longer disease‐free interval (DFI) in dogs with appendicular osteosarcoma following amputation. Dogs with histologically confirmed appendicular osteosarcoma that were free of gross metastases and underwent amputation were eligible for enrollment. Dogs were randomized to receive either six doses of carboplatin or three doses each of carboplatin and doxorubicin on an alternating schedule. Fifty dogs were included. Dogs receiving carboplatin alone had a significantly longer DFI (425 versus 135 days) than dogs receiving alternating carboplatin and doxorubicin (P = 0.04). Toxicity was similar between groups. These results suggest that six doses of carboplatin may be associated superior DFI when compared to six total doses of carboplatin and doxorubicin.     

Safety evaluation of the canine osteosarcoma vaccine,live Listeria vector

Canine osteosarcoma (OSA) is an aggressive bone tumour in dogs. Standard‐of‐care treatment typically results in relatively short survival times; thus, alternative treatments are needed to confer a survival advantage. It has been shown that OSA is an immunogenic tumour, suggesting that immune modulation may result in superior outcomes. A cryopreserved, Listeria‐based OSA vaccine was recently developed and an initial study in dogs reported prolonged survival for patients receiving the vaccine in conjunction with standard‐of‐care. The goal of the current observational study was to report on the safety of the lyophilized formulation of this vaccine (the canine OSA vaccine, live Listeria vector [COV‐LLV]) in a group of dogs previously diagnosed with OSA. Forty‐nine (49) dogs received the COV‐LLV and were included for analysis. Adverse events (AEs) noted during and after vaccinations were recorded. The AEs observed were typically mild and self‐limiting, with nausea, lethargy and fever being most common. Four dogs (8%) cultured positive for Listeria (three infections including an amputation site abscess, septic stifle joint and bacterial cystitis; and one dog whose lungs cultured Listeria‐positive on necropsy within 24 hours of COV‐LLV administration). These cases join the previously reported Listeria‐positive thoracic abscess that developed in a canine following use of COV‐LLV. Although uncommon, it is important to realize this clinically significant AE is possible in patients treated with live therapeutic Listeria vaccines. As Listeria is zoonotic, caution is required not only for the patient receiving the vaccine, but also for the health care workers and family caring for the patient.     

Telomerase activity in canine osteosarcoma

Appendicular osteosarcoma (OSA) is the most common primary bone tumour in dogs, and the prognosis with standard of care therapy of amputation and adjunctive chemotherapy is generally poor, with median survival times of 1 year. The ability of neoplastic cells to maintain their telomere length, by either telomerase activity or alternate methods, is an important step in tumour development and malignancy. The purpose of this study was to determine the presence of telomerase activity in canine OSA. To evaluate the frequency of alternative lengthening of telomeres in canine OSA, we have used the telomeric repeat amplification protocol in five canine cell lines and in six samples taken from clinical patients at the time of amputation. Our results reveal the presence of telomerase activity in 100% of canine OSA cell lines and 83% of clinical samples evaluated. This is in contrast to human OSA where 25–40% expression levels of telomerase are reported. Importantly, our results not only suggest that canine OSA may serve as a good model for aggressive telomerase‐positive forms of human OSA but also that antitelomerase therapy strategies for treatment of canine OSA may be more successful than in the treatment of majority of human patients with OSA.     

Immune regulation of canine tumour and macrophage PD‐L1 expression

Expression of programmed cell death receptor ligand 1 (PD‐L1) on tumor cells has been associated with immune escape in human and murine cancers, but little is known regarding the immune regulation of PD‐L1 expression by tumor cells and tumor‐infiltrating macrophages in dogs. Therefore, 14 canine tumor cell lines, as well as primary cultures of canine monocytes and macrophages, were evaluated for constitutive PD‐L1 expression and for responsiveness to immune stimuli. We found that PD‐L1 was expressed constitutively on all canine tumor cell lines evaluated, although the levels of basal expression were very variable. Significant upregulation of PD‐L1 expression by all tumor cell lines was observed following IFN‐γ exposure and by exposure to a TLR3 ligand. Canine monocytes and monocyte‐derived macrophages did not express PD‐L1 constitutively, but did significantly upregulate expression following treatment with IFN‐γ. These findings suggest that most canine tumors express PD‐L1 constitutively and that both innate and adaptive immune stimuli can further upregulate PD‐L1 expression. Therefore the upregulation of PD‐L1 expression by tumor cells and by tumor‐infiltrating macrophages in response to cytokines such as IFN‐γ may represent an important mechanism of tumor‐mediated T‐cell suppression in dogs as well as in humans.