Characterization of β-catenin expression in canine osteosarcoma

Osteosarcoma (OSA) is the most frequently occurring malignant primary bone tumour in dogs and children and arises from cells of the osteoblast lineage. Inappropriate Wnt signalling activity has been implicated in human OSA. Altered expression of β-catenin, an integral member of the Wnt signalling pathway, has been associated with numerous human cancers, including OSA. In this study, 30 of the 37 primary canine OSA tissues and 2 of the 3 metastatic OSAs were positive for β-catenin expression as determined by immunohistochemistry, whereas 2 normal bones stained negative for β-catenin. No mutations were identified in exon 3 of β-catenin in the three OSA cases in which DNA sequencing was performed. Finally, there was no relationship between β-catenin expression and overall survival time or disease-free interval. Our results indicate β-catenin is frequently expressed within the cytoplasm of neoplastic cells in canine OSA but contains no detectable mutations in exon 3, similar to human OSA.     

Canine mammary gland tumours; a histological continuum from benign to malignant; clinical and histopathological evidence*

This study describes the clinical and histopathological findings in dogs with mammary gland tumours, and compares the histopathological and clinical evidence consistent with progression from benign to malignant to human breast cancer epidemiology. Clinical and histopathological data on 90 female dogs with 236 tumours was included. Dogs with malignant tumours were significantly older than dogs with benign tumours (9.5 versus 8.5 years), P = 0.009. Malignant tumours were significantly larger than benign tumours (4.7 versus 2.1 cm), P = 0.0002. Sixty‐six percent had more than one tumour, and evidence of histological progression was noted with increasing tumour size. Dogs with malignant tumours were significantly more likely to develop new primary tumours than dogs with benign tumours, P = 0.015. These findings suggest that canine mammary tumours progress from benign to malignant; malignant tumours may be the end stage of a histological continuum with clinical and histopathological similarities to human breast carcinogenesis.     

Doxorubicin chemotherapy for presumptive cardiac hemangiosarcoma in dogs†

Sixty‐four dogs were treated with single‐agent doxorubicin (DOX) for presumptive cardiac hemangiosarcoma (cHSA). The objective response rate (CR + PR) was 41%, and the biologic response rate (CR + PR + SD), or clinical benefit, was 68%. The median progression‐free survival (PFS) for treated dogs was 66 days. The median survival time (MST) for this group was 116 days and was significantly improved compared to a MST of 12 days for untreated control dogs (P = 0.0001). Biologic response was significantly associated with improved PFS (P < 0.0001) and OS (P < 0.0001). Univariate analysis identified larger tumour size as a variable negatively associated with PFS. The high rate of clinical benefit and improved MST suggest that DOX has activity in canine cHSA.     

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.     

Canine osteosarcoma cell lines from patients with differing serum alkaline phosphatase concentrations display no behavioural differences in vitro

Osteosarcoma is an aggressive malignancy and represents the most frequent primary bone malignancy of dogs and humans. Prognostic factors reported for osteosarcoma include tumour size, presence of metastatic disease and serum alkaline phosphatase (ALP) concentration at the time of diagnosis. To date, there have been no studies to determine whether the behaviour of osteosarcoma cells differ based on serum ALP concentration. Here, we report on the generation of six canine osteosarcoma cell lines from osteosarcoma‐bearing dogs with differences in serum ALP concentration. To determine whether in vitro behaviour differs between primary osteosarcoma cell lines generated from patients with normal or increased serum ALP, assays were performed to evaluate proliferation, migration, invasion and chemosensitivity. There were no significant differences in cell proliferation, migration, invasion or chemosensitivity between cell lines associated with normal or increased serum ALP concentration.     

Osteosarcoma tissues and cell lines from patients with differing serum alkaline phosphatase concentrations display minimal differences in gene expression patterns

Serum alkaline phosphatase (ALP) concentration is a prognostic factor for osteosarcoma in multiple studies, although its biological significance remains incompletely understood. To determine whether gene expression patterns differed in osteosarcoma from patients with differing serum ALP concentrations, microarray analysis was performed on 18 primary osteosarcoma samples and six osteosarcoma cell lines from dogs with normal and increased serum ALP concentration. No differences in gene expression patterns were noted between tumours or cell lines with differing serum ALP concentration using a gene‐specific two‐sample t‐test. Using a more sensitive empirical Bayes procedure, defective in cullin neddylation 1 domain containing 1 (DCUN1D1) was increased in both the tissue and cell lines of the normal ALP group. Using quantitative PCR (qPCR), differences in DCUN1D1 expression between the two groups failed to reach significance. The homogeneity of gene expression patterns of osteosarcoma associated differing serum ALP concentrations are consistent with previous studies suggesting serum ALP concentration is not associated with intrinsic differences of osteosarcoma cells.     

Association between weight change during initial chemotherapy and clinical outcome in dogs with multicentric lymphoma

The majority of the known prognostic factors in dogs with lymphoma have been evaluated before treatment commences or at the time of diagnosis. Prognostic factors evaluated during the initial phase of treatment are less described but may provide important clinical information. In this retrospective study, 82 canine lymphoma patients were categorized according to the weight change between diagnosis and after 5 weeks of chemotherapy. Dogs that gained greater than 5% or lost greater than 5% of initial body weight were categorized as increased‐ or decreased‐weight groups, respectively. Those in which weight changed less than 5% were categorized as the maintained‐weight group. The median progression‐free survival (PFS) in the increased‐weight group, maintained‐weight group and decreased‐weight group was 226, 256 and 129 days, respectively. The decreased‐weight group had significantly shorter PFS than the increased and maintained groups (P = .023, P = .003, respectively). The median survival time (ST) in the increased‐weight group, maintained‐weight group and decreased‐weight group was 320, 339 and 222 days, respectively. There was no significant difference in ST among the three groups (P = .128). In Cox‐regression results, weight change group and initial body weight were significant risk factors associated to PFS (P = .007, P = .001, respectively) while only patient's initial body weight was a significant risk factor to ST (P = .013). In conclusion, evaluation of initial body weight and weight changes over time can provide valuable information regarding PFS and ST in dogs with multicentric lymphoma.     

The Immunotherapy of Canine Osteosarcoma: A Historical and Systematic Review

Osteosarcoma is a malignant mesenchymal neoplasm that accounts for the majority of primary bone tumors in dogs and shares biological and clinical similarities with osteosarcoma in humans. Despite dose intensification with conventional cytotoxic therapies, survival times for dogs and humans diagnosed with high‐grade osteosarcoma have not changed in the past 20 years, with the principal cause of mortality being the development of pulmonary metastases. Given the therapeutic plateau reached for delaying metastatic progression with cytotoxic agents, exploration of alterative adjuvant therapies for improving management of osteosarcoma micrometastases is clinically justified. Evidence suggests that osteosarcoma is an immunogenic tumor, and development of immunotherapies for the treatment of microscopic lung metastases might improve long‐term outcomes. In this review, the history and foundational knowledge of immune interactions to canine osteosarcoma are highlighted. In parallel, immunotherapeutic strategies that have been explored for the treatment of canine osteosarcoma are summarized. With a greater understanding and awareness for how the immune system might be redirected toward combating osteosarcoma metastases, the rational development of diverse immune strategies for managing osteosarcoma holds substantial promise for transforming the therapeutic landscape and improving disease management in both dogs and human beings.     

Prognostic significance of hypermethylation of death‐associated protein kinase (DAPK) gene CpG island in dogs with high‐grade B‐cell lymphoma

Death‐associated protein kinase (DAPK) is a serine/threonine kinase and a tumour suppressor gene. Diffuse large B‐cell lymphomas with inactivated DAPK through hypermethylation of a CpG island is known to result in a biologically aggressive phenotype in humans. This retrospective study was carried out to analyse the prognostic significance of DAPK CpG island hypermethylation in canine lymphoma. We hypothesized that DAPK CpG island hypermethylation can be a negative prognostic indicator in dogs with nodal high‐grade B‐cell lymphoma. Forty‐seven dogs with high‐grade B‐cell lymphoma, according to the updated Kiel classification, were evaluated after being treated with a CHOP (vincristine, cyclophosphamide, doxorubicin and prednisolone)‐based chemotherapy protocol. The methylation status of the DAPK CpG island was examined by methylation‐specific PCR. Progression‐free survival (PFS) and overall survival (OS) were compared using the Kaplan‐Meier analysis and log‐rank test. The cox proportional hazard regression model was used to evaluate the effect of multiple variables. Hypermethylation of the DAPK CpG island was detected in 21 of the 47 dogs. The PFS and OS in dogs with the hypermethylation (median: 220 and 266 days, respectively) were significantly shorter than those of dogs without hypermethylation (median: 301 and 412 days, respectively) (PFS, P = .036; OS, P = .007). In the multivariate analysis, hypermethylation of the DAPK CpG island remained an independent prognostic factor in predicting shortened PFS (P = .047) and OS (P = .021) as well as clinical substage b. Overall, hypermethylation of the DAPK CpG island was a negative prognostic factor in canine high‐grade B‐cell lymphoma.     

Immune dysregulation and osteosarcoma: Staphylococcus aureus downregulates TGF‐β and heightens the inflammatory signature in human and canine macrophages suppressed by osteosarcoma

Since William Coley utilized bacterial immunotherapy to treat sarcomas in the late 19th century, an association between infection and improved survival has been reported for human and canine osteosarcoma patients. One of the reasons for this improved survival is likely a reactivation of the host immune system towards an inflammatory anti‐tumour response, and one of the key players is the macrophage. Yet, despite their importance, the response of macrophages to infectious agents in the context of osteosarcoma has not been thoroughly evaluated. The aim of this study was to evaluate how in vitro exposure to a bacterial agent (Staphylococcus aureus) influenced canine and human macrophage differentiation in the presence of osteosarcoma. Our hypothesis was that S. aureus would, in the presence of osteosarcoma, induce a macrophage phenotype with significantly increased inflammatory signatures. Consistent with our hypothesis, human macrophages co‐cultured with osteosarcoma and S. aureus exhibited increased IFN‐γ, TNF‐α and IL‐12p70 cytokine secretion, decreased TGF‐β cytokine secretion and increased mRNA expression of TNF‐α when compared with macrophages co‐cultured with osteosarcoma and to macrophages cultured alone. Canine macrophages similarly exhibited increased IFN‐γ and TNF‐α cytokine secretion, decreased TGF‐β cytokine secretion, increased mRNA expression of TNF‐α and increased surface receptor expression of CD80 when co‐cultured with osteosarcoma and S. aureus. Collectively, the findings of this study suggest that infection upregulates the inflammatory immune response to counteract osteosarcoma‐induced immune suppression. This work informs a potential therapeutic strategy to optimize inflammatory stimuli for triggering an anti‐osteosarcoma macrophage response.     

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild‐type p53

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.     

PCNA and grade in 13 canine oral squamous cell carcinomas: association with prognosis

This study evaluated the prognosis factors of age, tumour size, anatomic location, histological grade and proliferating cell nuclear antigen (PCNA) expression in 13 dogs with oral squamous cell carcinoma (OSCC) with bone invasion and without signs of lymph node or distant metastasis. All animals were treated with radical excision performed with at least 1 cm margin, based on computed tomography images. In the 2‐year follow‐up, median disease‐free survival was 138 days for dogs with grade 3 tumours and was not reached for those with grade 2 tumours. Grade 3 tumours and PCNA labelling index ≥65% were related with a shorter disease‐free survival time and consequently poor prognosis (p = 0.003 and p = 0.034, respectively). Mean PCNA labelling index was significantly higher in recurrent cases (p = 0.011). Histological grade and PCNA expression may be important prognosis factors in canine OSCC.     

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.     

Adjuvant therapy with carboplatin and pamidronate for canine appendicular osteosarcoma

Amputation and chemotherapy are the mainstay of treatment for canine appendicular osteosarcoma (OSA). In vitro studies have demonstrated anti‐tumour activity of pamidronate against canine OSA. The purpose of this study was to assess the safety of adding pamidronate to standard post‐operative carboplatin chemotherapy in 17 dogs with appendicular OSA treated with limb amputation. Median disease‐free interval (DFI) and median survival time (MST) were evaluated as secondary endpoints. Incidence of side effects and treatment outcomes were compared to 14 contemporary control patients treated with carboplatin alone. There were no identified side effects to the pamidronate treatment. The median DFI for the study group was 185 days compared to 172 days for the control group (P = 0.90). The MST of the study group was 311 days compared to 294 days for the control group (P = 0.89). Addition of pamidronate to carboplatin chemotherapy for the treatment of canine appendicular OSA is safe and does not impair efficacy of standard carboplatin treatment.     

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.     

Clinical and histopathological features of pemphigus foliaceus with and without eosinophilic infiltrates: a retrospective evaluation of 40 dogs

The importance of cellular infiltrates in tissues has been investigated as a diagnostic tool, mechanism of pathogenesis, and prognostic indicator in certain human diseases. Eosinophils, in particular, have a distinct role in the development of cutaneous lesions in human autoimmune diseases. Identification of an eosinophilic infiltrate can aid the diagnosis of immunobullous disease in the early stages of the disease process. In canine pemphigus foliaceus, eosinophils are present to a variable degree within lesional tissue. This study retrospectively evaluated 40 dogs with pemphigus foliaceus, and examined clinical and histologic features and final outcomes in cases with and without eosinophilic infiltrates. Twenty‐five of 40 dogs (63%) had an eosinophilic infiltrate in either the pustules/crust, follicular infundibulum or dermis. There was no statistically significant difference in clinical distribution or appearance of dermatological lesions, response to treatment, or disease outcome in dogs with or without an eosinophilic infiltrate. However, dogs with concurrent disease were significantly more likely to have an eosinophilic infiltrate (P = 0.01). Dogs with adverse effects associated with immunosuppressive therapy were significantly more likely to have an eosinophilic infiltrate (P = 0.05). Fifteen of 40 dogs (38%) had a history of allergic disease and a significantly higher proportion of these dogs had an eosinophilic infiltrate (P = 0.04). An eosinophilic infiltrate was found in more than half of the dogs in this study. These findings justify further studies to investigate the role of eosinophils in the pathogenesis, therapy and prognosis in dogs with pemphigus foliaceus.     

Metastatic immune infiltrates correlate with those of the primary tumour in canine osteosarcoma

Our lack of understanding of the immune microenvironment in canine osteosarcoma (cOSA) has limited the identification of potential immunotherapeutic targets. In particular, our ability to utilize readily available tissue from a dog's primary tumour to predict the type and extent of immune response in their pulmonary metastatic lesions is unknown. We, therefore, collected 21 matched pairs of primary tumours and pulmonary metastatic lesions from dogs with OSA and performed immunohistochemistry to quantify T‐lymphocyte (CD3), FOXP3+ cell, B‐lymphocyte (Pax‐5), and CD204+ macrophage infiltration. We found that T‐lymphocytes and FOXP3+ infiltrates in primary tumours positively correlated with that of metastatic lesions (ρ = 0.512, P = 0.038 and ρ = 0.698, P = 0.007, respectively), while a strong trend existed for CD204+ infiltrates (ρ = 0.404, P = 0.087). We also observed T‐ and B‐lymphocytes, and CD204+ macrophages to be significantly higher in a dog's pulmonary metastasis compared to their primary tumour (P = 0.018, P = 0.018, P = 0.016, respectively), while FOXP3+ cells were only significantly higher in metastases when all primary tumour and metastasis lesions were compared without pairing (P = 0.036). Together, these findings suggest that the metastatic immune microenvironment may be influenced by that of the primary cOSA, and that primary tumour immune biomarkers could potentially be applied to predict immunotherapeutic responses in gross metastatic disease. We, therefore, provide a rationale for the treatment of cOSA pulmonary metastases with immunotherapeutics that enhance the anti‐tumour activity of these immune cells, particularly in dogs with moderate to high immune cell infiltration in their primary tumours.