Canine PHA-stimulated adherent cell enhance interferon-gamma production and proliferation of autologous peripheral blood mononuclear cells

Dendritic cells are specialized antigen‐presenting cells with immuno‐modulating functions that are attractive for clinical applications for cancer immunotherapy. This study examined immunostimulatory functions of phytohemagglutinin (PHA)‐stimulated adherent cells (PHA‐Ad cells) from peripheral blood mononuclear cells (PBMCs) in dogs. PHA‐Ad cells enhanced interferon‐γ from autologous PBMC in vitro. PHA‐Ad cells also stimulated antigen‐independent proliferation of peripheral blood lymphocytes. These results suggest that PHA‐Ad cells from PBMC possess a stimulatory function to evoke anti‐tumour immunity and that they demonstrate potential for therapeutic applications in dogs.     

Treatment of canine oral papillary squamous cell carcinoma using definitive‐intent radiation as a monotherapy—a case series

Canine oral papillary squamous cell carcinoma (COPSCC) is a rare neoplasm and although locally invasive it carries a favourable prognosis following wide surgical excision. Radiotherapy has been reported to be effective as an adjunct treatment to surgery. However, limited information is available on the role of radiotherapy as single treatment. This single‐institution retrospective study describes a series of 10 dogs diagnosed with macroscopic COPSCC that were treated with definitive‐intent radiotherapy (DRT) as a monotherapy. These dogs had a median age of 4 years (range: 0.4‐9.6 years). The tumour was located in the rostral oral cavity in all cases with a median tumour size of 2.5 cm (range: 0.8‐6.8 cm). No local or distant metastases were identified. All dogs were treated with electron beam DRT (>32Gy, 10‐16 daily fractions of 3.2Gy). The median follow‐up time was 961 days (range: 333‐3.498 days) with nine dogs achieving a complete response and one dog a partial response. The dog with the partial response developed disease progression at 228 days after initiation of radiotherapy. Two dogs died from non‐tumour‐related causes. The remaining seven dogs were still alive and in complete remission at the time of last follow‐up. Median progression‐free survival time and median survival time were not reached. DRT was generally well tolerated, but all dogs experienced self‐limiting acute radiation mucositis (grade 2‐3) and/or dermatitis (grade 1). No late radiation toxicity was observed. Macroscopic COPSCC appears to be a radiosensitive tumour that can be successfully treated with DRT eliminating the need for aggressive surgery in advanced cases.     

Alterations in activated T‐cell cytokine expression in healthy dogs over the initial 7 days of twice daily dosing with oral cyclosporine

Cyclosporine is a powerful T‐cell inhibitor used in the treatment of immune‐mediated and inflammatory diseases in the dog. There is limited information on how to best monitor patients on cyclosporine therapy. Currently, pharmacokinetic and pharmacodynamic assays are available. Pharmacokinetic assays that measure the concentration of cyclosporine in the blood are used to assess if an appropriate drug concentration has been achieved; however, target blood drug concentrations have not been shown to reliably correlate with suppression of T‐cell function in the dog. In human transplant recipients, therapeutic drug monitoring has shifted to include pharmacodynamic‐based monitoring. Our laboratory has validated a RT‐qPCR assay to measure the pharmacodynamic effects of cyclosporine in the dog. In this study, activated T‐cell expression of IL‐2 and IFN‐γ was measured using RT‐qPCR daily for 7 consecutive days in 8 healthy Walker hounds receiving oral cyclosporine at a dosage of 10 mg/kg every 12 hr. Cytokine production was found to be markedly decreased within 24 hr after the initiation of cyclosporine and remained significantly decreased for the duration of the project. Based on these results, cyclosporine causes a rapid drop in T‐cell cytokine production that is sustained with continued dosing in healthy dogs. Although performed in healthy dogs, this study demonstrated a marked decrease in cytokine suppression within 24 hr of drug administration, suggesting that pharmacodynamic monitoring of cyclosporine's effects on T cells could be considered within several days of commencing therapy in dogs suffering from life‐threatening immune‐mediated disorders.     

Efficacy and safety of electrochemotherapy combined with peritumoral IL‐12 gene electrotransfer of canine mast cell tumours

Electrochemotherapy combined with peritumoral interleukin‐12 (IL‐12) gene electrotransfer was used for treatment of mast cell tumours in 18 client‐owned dogs. Local tumour control, recurrence rate, as well as safety of combined therapy were evaluated. One month after the therapy, no side effects were recorded and good local tumour control was observed with high complete responses rate which even increased during the observation period to 72%. IL‐12 gene electrotransfer resulted in 78% of patients with detectable serum IFN‐γ and/or IL‐12 levels. In the treated tumours vascular changes as well as minimal T‐lymphocytes infiltration was observed. After 1 week, the plasmid DNA was not detected intra‐ or peritumorally and no horizontal gene transfer was observed. In summary, our study demonstrates high antitumour efficacy of electrochemotherapy combined with IL‐12 electrotransfer, which also prevented recurrences or distant metastases, as well as its safety and feasibility in treatment of canine mast cell tumours.     

Oesophageal leiomyosarcoma in dogs: surgical management and clinical outcome of four cases

Oesophageal leiomyosarcoma has yet to be reported in dogs. This retrospective case series describes the case management and clinical outcome of four dogs with oesophageal leiomyosarcoma treated by marginal excision alone. Histological features used to determine tumour grade included capsular invasion, percent necrosis, pleomorphism and mitotic rate. All tumours were designated grade 1 leiomyosarcoma. Excision of all grossly evident tumour tissue was achieved in two of the four cases; however, histopathologic evaluation showed tumour cells at the surgical margins in one of these two cases. Two dogs had grossly incomplete excision. Two dogs died from unrelated conditions, one 3 years and 5.5 months after surgery, the other at 65 days. One dog had persistent mega‐oesophagus and was lost to follow‐up 388 days after surgery and one dog is still alive (last follow‐up 405 days after surgery). Despite large tumour size and incomplete excision, surgical removal of low‐grade leiomyosarcomas can result in long‐term resolution of clinical signs.     

Vinorelbine rescue therapy for dogs with primary urinary bladder carcinoma

The goal of this study was to evaluate the anti‐tumour activity and toxicoses of vinorelbine as a palliative rescue therapy for dogs with primary urinary bladder carcinoma. Thirteen dogs refractory to prior chemotherapeutics and one dog naïve to chemotherapeutic treatment were enrolled. Vinorelbine (15 mg m^?2 IV) was administered intravenously along with concurrent oral anti‐inflammatory drugs, if tolerated. A median of six doses of vinorelbine (range: 1–16) was administered. Two dogs (14%) had partial responses, and eight (57%) experienced stable disease. Subjective improvement in clinical signs was noted in 11 dogs (78%). Adverse events were mild and primarily haematological in nature. Median time to progression was 93 days (range: 20–239 days). Median survival time for all dogs was 187 days; median survival for 13 pre‐treated dogs was 207 days. Vinorelbine may have utility in the management of canine primary urinary bladder carcinoma and should be evaluated in a prospective study.     

Acanthomatous ameloblastoma in dogs treated with intralesional bleomycin

Acanthomatous ameloblastoma (AA) is a benign gingival tumour that often invades bone. This retrospective study evaluated the efficacy of intralesional (IL) bleomycin as a treatment for AA. Six dogs received weekly or bimonthly IL bleomycin injections (dose range, 10–20 U m^?2). A seventh dog presented with advanced, nonresectable AA was treated palliatively. One to sixteen treatments were administered (median, 5). Six of the seven dogs had a complete response within 4 months from initial IL injection (median, 1.5 months), whereas the palliative case had approximately 25% decrease in tumour volume 14 days from initial injection. Local recurrence was not observed during the study period, with a median follow‐up time of 842 days. Adverse effects were limited to wound formation with bone exposure (n = 4), mild tissue reactions (n = 3), local swelling (n = 2) and local infection (n = 1). The conclusions of this study show IL bleomycin is an effective treatment for canines with AA.     

FC‐36 The use of immunostimulatory bacterial DNA sequences in allergen‐specific immunotherapy of canine atopic dermatitis

The purpose of this study was to evaluate a combination of immunostimulatory bacterial DNA sequences and allergen‐specific immunotherapy for the treatment of canine atopic dermatitis. Seven dogs with nonseasonal atopic dermatitis diagnosed by history, clinical signs and exclusion of differential diagnoses were included. All dogs had been on allergen‐specific immunotherapy for at least 12 months with incomplete responses, were on additional antipruritic therapy and showed residual pruritus. Pruritus was marked by the owner on a visual analogue scale, lesions were determined by a clinician using the Canine Atopic Dermatitis Extent and Severity Index (CADESI), and concurrent medications were recorded before entering the study and after 14 weeks of treatment. Peripheral blood mononuclear cells were isolated and cultured; canine cytokine message for IFNγ, IL‐4, TNF and IL‐10 was quantitated using RT‐PCR. A mixture of allergen extract and liposome‐DNA complexes was injected intradermally at the beginning of the study and after 2, 4, 6, 10 and 14 weeks. CADESI, pruritus and medication scores, and cytokine messages at the beginning and end of the study were compared with a paired t‐test. There were significant improvements in pruritus scores (P = 0.0277). Reductions in medication scores and CADESI were not statistically significant. IL‐4 production decreased significantly (P = 0.0428); decreases in other cytokines were not significant. Although the number of dogs in this pilot study was small, the results warrant further investigation of a combination of immunostimulatory bacterial DNA sequences and allergen‐specific immunotherapy for the treatment of canine atopic dermatitis. Funding: Self‐funded.     

Tumour angiogenesis,anti‐angiogenic therapy and chemotherapeutic resistance

In order for a tumour to continue to grow and disseminate, it must acquire a new blood supply. Neovascularisation can be enacted by a number of different mechanisms. This dependence of tumour progression on an augmented vascular supply has been exploited by the development of anti‐angiogenic drugs, which are designed to inhibit new blood vessel formation or disrupt existing tumour‐associated vasculature, both leading to ischaemic–hypoxic tumour cell death. However, the clinical benefits of these therapeutic approaches are frequently variable and often transient, the neoplasm sometimes being able to use other neovascularisation mechanisms to maintain its blood supply and thus evade the current anti‐angiogenic therapy. Tumours may also develop a more malignant phenotype following this treatment. Clinical outcomes may be improved by simultaneously inhibiting different angiogenic pathways, abetted by more effective drug delivery regimens such as metronomic chemotherapy and the concurrent use of other antitumour modalities.     

Antitumour effects of Liporaxel (oral paclitaxel) for canine melanoma in a mouse xenograft model

Paclitaxel, a member of the taxane family, exhibits antitumour effects by targeting the microtubules in cancer cells. Recently, oral paclitaxel has been developed to overcome the side effects of intravenous paclitaxel administration in human patients. The objective of this study was to investigate the antitumour effects of oral paclitaxel in vitro and in vivo. Three weeks after inoculation, oral paclitaxel (25 and 50 mg/kg) or saline was administered every week for three consecutive weeks. To explore the underlying mechanism, tumour angiogenesis was examined by immunohistochemistry with an anti‐CD31 antibody. Tumour cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick‐End Labeling assay, and cell cycle arrest was confirmed by western blot analysis. Oral paclitaxel treatment of canine melanoma cells exerted mediated antiproliferative effects and mediated cell cycle arrest in vitro. In animal experiments, after oral paclitaxel administration, the average tumour size decreased to approximately 30% of that in the control. Histologically, oral paclitaxel showed anti‐angiogenic effects and induced the apoptosis in tumour tissues. Oral paclitaxel also downregulated the intratumoural expression of cyclin D1 and inhibited cell proliferation. The study findings support potential application of oral paclitaxel as a novel chemotherapeutic strategy to treat canine melanoma. This is the first study to investigate the potential of oral paclitaxel as a therapeutic drug against canine tumours.     

Multiple metastases of thyroid cancer in the cranium and pituitary gland in two dogs

Two dogs, a 14-year-old, female American Eskimo dog and a 14-year-old, male Maltese dog, were presented with thalamic syndromes, including lowered levels of consciousness, poor postural responses and presence of masses in the neck region. In both dogs, magnetic resonance imaging revealed multiple masses inside the cranium, including the pituitary gland. One dog died from status epilepticus two days after magnetic resonance imaging and the other died two months after magnetic resonance imaging from respiratory failure. These dogs were histopathologically diagnosed with multiple metastases of thyroid cancer occurring inside the cranium, including the pituitary gland. To the authors' knowledge, this is the first time this tumour pattern has been reported in dogs, but it is possible that it is not uncommon.     

Treatment of five haemodynamically stable dogs with immune‐mediated thrombocytopenia using mycophenolate mofetil as single agent

Five dogs were presumptively diagnosed with immune‐mediated thrombocytopenia. As they had all been chronically treated with non‐steroidal anti‐inflammatory drugs, administration of immunosuppressive doses of corticosteroids was considered contraindicated. Non‐steroidal anti‐inflammatory drugs were temporarily discontinued in all the dogs and mycophenolate mofetil was introduced as first‐line single immunomodulatory therapy. This treatment protocol resulted in complete remission of immune‐mediated thrombocytopenia in all the dogs, and mycophenolate mofetil was discontinued after several months of therapy in four of the five dogs with no relapses, even when non‐steroidal anti‐inflammatory drug administration was resumed. The remaining dog required continued mycophenolate mofetil therapy to avoid relapse. One dog experienced diarrhoea, and another dog had diarrhoea and decreased appetite .     

Efficacy of doxorubicin‐based chemotherapy for non‐resectable canine subcutaneous haemangiosarcoma

Eighteen dogs with measurable subcutaneous haemangiosarcoma (SQHSA) were treated with doxorubicin‐based chemotherapy. Response assessment was evaluated and compared using World Health Organization (WHO), Response Evaluation Criteria in Solid Tumours (RECIST) and tumour volume criteria. The overall response rate for all dogs was 38.8% using WHO criteria, 38.8% using RECIST criteria and 44% using tumour volume criteria. One dog had a complete response. The median response duration for all dogs was 53 days (range 13–190 days). Four dogs had complete surgical excision after neoadjuvant chemotherapy. The median progression‐free interval for dogs with complete surgical excision after neoadjuvant chemotherapy was significantly longer than those not having surgical excision (207 days versus 83 days, respectively) (P = 0.003). No significant difference in metastasis‐free interval or survival time was found between the groups. Doxorubicin‐based chemotherapy appears to be effective for non‐resectable canine SQHSA, although the response duration is relatively short.     

Postsurgical intra-incisional 5-fluorouracil in dogs with incompletely resected, extremity malignant spindle cell tumours: a pilot study

The safety and efficacy of intra‐incisional 5‐fluorouracil (5‐FU) in the management of incompletely resected malignant spindle cell tumours of extremities was evaluated in six dogs. After marginal surgery, the dogs underwent weekly intra‐incisional 5‐FU for a minimum of six cycles. Treatment was well tolerated by all dogs, with no systemic adverse effects and only one episode of local cutaneous hyperpigmentation, which completely and spontaneously resolved. Median follow‐up for all the dogs was 546 days (mean 619; range 297–1207). At the date of analysis, four dogs were still alive with no evidence of local recurrence, and two dogs had died as a result of their disease. The cause of death was development of distant metastases in one dog and tumour regrowth in the other. Despite the small sample size, this study documents that intra‐incisional 5‐FU chemotherapy is a safe and efficacious adjuvant treatment in the case of incompletely resected malignant spindle cell tumours in dogs and that long disease control can be achieved.     

Plasma biomarkers profile of female dogs with mammary carcinoma and its association with clinical and pathological features

The immunological biomarkers profiles were evaluated using Luminex as putative measures to monitor canine mammary carcinomas (MCs). Forty female dogs were categorized into benign mixed tumour (MC‐BMT = 28) and mammary carcinoma (MC=12). The ascendant biomarker signatures were used to compare the groups. For example, a higher frequency of MC‐BMT animals producing IL‐6, CXCL‐8 and CXCL‐10 was observed, whereas for the MC group IL‐2 and CXCL‐8 were detected. MC‐BMT animals without metastasis had an increase in the levels of IL‐2, CXCL‐8, CXCL‐10, IL‐6, TNF‐α, IL‐15 and a decrease in IL‐10 and CXCL‐8. MC‐BMT animals with metastasis showed only an increase in CXCL‐10 and a decrease in IL‐18. After comparing the ascendant signatures following the presence of metastasis in both groups, a higher frequency of dogs exhibiting IL‐10 production was observed. Pearson correlation (P = 0.0273) and receiver operating characteristic (ROC) curve analysis revealed that this pattern was associated with worse outcome and lower survival rates in MC animals.     

Age‐associated changes to pathogen‐associated molecular pattern‐induced inflammatory mediator production in dogs

Objective – To determine whether older dogs will have a more pronounced pro‐inflammatory response and blunted anti‐inflammatory response to pathogen‐associated molecular patterns (PAMPs) compared with younger dogs. Design – Prospective. Setting – University teaching hospital. Animals – Thirty‐eight privately owned sexually altered dogs of various ages. Interventions – Blood was collected for HCT, WBC count, plasma biochemical analysis, and whole blood culture. Whole blood was stimulated with lipopolysaccharide (LPS) or, lipoteichoic acid or, peptidoglycan or, addition of phosphate‐buffered saline. Tumor necrosis factor (TNF), interleukin (IL)‐6, and IL‐10 production from whole blood were compared among young, middle aged, and geriatric dogs. Measurements and Main Results – LPS, lipoteichoic acid, and peptidoglycan stimulated significant TNF, IL‐6, and IL‐10 production from canine whole blood compared with phosphate‐buffered saline. Whole blood from geriatric dogs had a blunted IL‐10 response to LPS stimulation and middle‐aged dogs had increased LPS‐induced TNF production compared with the other groups. Conclusion – PAMPs from gram‐positive and gram‐negative bacteria stimulate TNF, IL‐6, and IL‐10 production from canine whole blood. The inflammatory mediator response to PAMPs from gram‐negative bacteria alters with age and may be one factor contributing to mortality in geriatric dogs with sepsis.     

Evaluation of pancreatic forceps biopsy by laparoscopy in healthy beagles

The effects of laparoscopic biopsies were determined in four healthy laboratory beagles. Biopsies were taken from the pancreas of three dogs and from the peripancreatic fat of one dog. Clinical examinations and blood sampling for hematologic and biochemical tests were performed before laparoscopy and weekly throughout each dog's participation in the study (7 or 21 days). No clinical signs of pancreatitis were observed, and hematologic and biochemical parameters remained within normal limits in three dogs. One dog exhibited a transient increase in trypsinlike immunoreactivity, amylase, and lipase. Minor adhesions between the pancreas, small intestine, and peritoneum were observed macroscopically in this dog. Histologically, granulation tissue and a mild nonsuppurative inflammation in the pancreas were present. No abnormal changes were seen macroscopically or histologically in the other two dogs for which pancreatic biopsies were performed. Thus, laparoscopy appears to be safe, with neither permanent abnormalities in blood parameters nor changes in clinical health occurring during or after the procedure in healthy beagles.     

Renal osteosarcoma in a dog

A seven-and-a-half-year-old dog presented with anorexia, lethargy and haematurla. A 1.8 kg abdominal mass was excised and determined to be a primary renal osteosarcoma. Haematuria was observed five months after surgery and the tumour was radiographically determined to have recurred locally. The dog was euthanased 12 days later due to refractory pain and anorexia. Although osteosarcomas are expected to develop distant metastases, this dog was euthanased due to clinical evidence of local tumour recurrence. Haematuria was an indication both of initial tumour development and later recurrence.     

The clinical and histopathological effects of prednisone on acute radiation-induced dermatitis in dogs: a placebo-controlled, randomized, double-blind, prospective clinical trial

This study evaluated and compared the clinical and histopathological effects of prednisone on acute radiation-induced dermatitis (ARID) in dogs treated with 48 Gray of fractionated irradiation targeted to the skin surface. The study was designed as a double-blind, randomized, placebo-controlled prospective clinical trial. Twenty-two otherwise healthy companion dogs completed the clinical study. Three dogs were excluded from complete histopathological analysis because the owner declined one (one dog) or both (two dogs) biopsies. The study duration for each dog was 36 days from the start of radiation therapy (RT) to the first re-examination post RT. Dogs were treated with either oral prednisone at 0.5 mg kg(-1) or sugar pill, daily. All dogs received 48 Gray of fractionated, standardized RT, beginning 2 weeks after tumour excision. Acute Radiation Morbidity Scores, Cutaneous Toxicity Extent and Severity scores, digital images, and impression cytology were carried out on days 1, 8, 15, 22 and 36. Four-millimetre skin specimens from days 15 (RT-11) and 36 (2 weeks after the last RT dose) were scored by a pathologist and a dermatologist, blind to specimen identity. A one-way analysis of variance for longitudinal data was used to compare scores between groups. Spearman's rho correlation coefficient was used to measure strength of association between clinical and histopathology scores (HPS). There was no significant difference in CUTES, AMS or HPS scores between groups. There was a strong correlation between clinical and HPS scores. Prednisone did not decrease ARID severity clinically or histopathologically.     

Piroxicam and carboplatin as a combination treatment of canine oral non-tonsillar squamous cell carcinoma: a pilot study and a literature review of a canine model of human head and neck squamous cell carcinoma

Results of the treatment with a combination of carboplatin and piroxicam in seven dogs with advanced non‐tonsillar oral squamous cell carcinoma (SCC) were retrospectively analysed. This multi‐agent protocol was well tolerated by all dogs and resulted in a complete regression of the tumour without additional surgery in four of seven patients. Additional surgery was necessary to remove a metastatic lymph node in one dog and residual tumour in a second dog, which achieved a partial response following medical therapy. Median follow‐up for all the dogs was 534 days, while the time‐to‐recurrence, time‐to‐progression and overall survival for this group of patients have not yet been reached. Our study, although limited in number of animals, suggests that this multiagent approach is a useful treatment option for oral non‐tonsillarSCC in dogs and warrants wider application.