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.     

Biological behaviour of canine mandibular osteosarcoma. A retrospective study of 50 cases (1999–2007)

The purpose of this retrospective study was to describe the biological behaviour of canine mandibular osteosarcoma (OSA) and to examine factors for their impact on metastasis‐free interval (MFI) and survival time (ST). Records from dogs treated with mandibulectomy for OSA (1999–2007) were reviewed. Archived tumour samples were evaluated for mitotic index (MI) and tumour grade. Fifty dogs were included, 21 received chemotherapy. Twenty‐nine dogs (58%) developed metastatic disease. The median MFI was 627 days, and median ST was 525 days. In univariate analysis MI > 40 was prognostic for decreased MFI and ST. Grade also influenced MFI and ST, with 5/21 (24%) dogs with grade II/III tumours metastasis‐free at one year versus 16/22 (72%) dogs with grade I tumours (P = 0.002); and 5/21 (24%) dogs with grade II/III tumours alive versus 17/22 (77%) dogs with grade I tumours (P = 0.001). In multivariate analysis, histological grade and adjuvant chemotherapy were prognostic for MFI and ST.     

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.     

Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (D_m) values ranging from 4.6–15.8 μM and 2.1–5.1 μM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with D_m values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.     

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.     

High COX‐2 expression is associated with increased angiogenesis,proliferation and tumoural inflammatory infiltrate in canine malignant mammary tumours: a multivariate survival study

COX‐2 expression affects mammary tumourigenesis by promoting angiogenesis and cell proliferation, encouraging metastatic spread and tumour‐associated inflammation. Samples of canine mammary tumours (n = 109) were submitted to immunohistochemistry to detect COX‐2, CD31, VEGF, Ki‐67, CD3 and MAC387 expression. Concurrent high expression of COX‐2/CD31, COX‐2/VEGF, COX‐2/Ki‐67, COX‐2/CD3 and COX‐2/MAC was associated with elevated grade of malignancy, presence of intravascular emboli and presence of lymph node metastasis. Tumours with high COX‐2 (P < 0.001) and tumours with concurrent expression of high COX‐2 and high CD31 (P = 0.008); high VEGF (P < 0.001); high Ki‐67 (P < 0.001); high CD3+ T‐lymphocytes (P = 0.002) and elevated MAC387 macrophages (P = 0.024) were associated with shorter overall survival (OS) time. Interestingly the groups with high COX‐2/CD31 and high COX‐2/VEGF retained their significance after multivariate analysis arising as independent predictors of OS. Present data highlight the importance of COX‐2 in canine mammary tumourigenesis.     

Tumour‐associated macrophages are associated with vascular endothelial growth factor expression in canine mammary tumours

Tumour‐associated macrophages (TAMs) have been implicated in carcinogenesis including an important role in angiogenesis. In this study, we describe the relationship between TAMs and angiogenesis in canine mammary tumours (CMT). Formalin‐fixed paraffin‐embedded CMT samples [(n = 128: malignant (n = 97) and benign (n = 31)] were submitted to immunohistochemical staining to detect MAC387, vascular endothelial growth factor VEGF and CD31 expression. A statistical analysis was carried out to assess possible associations with clinicopathological variables and biological markers of tumour angiogenesis. TAMs, detected by MAC387 expression, were significantly associated with malignant CMT (P < 0.001) and VEGF positive tumours (P = 0.002) and also associated with VEGF expression within malignant CMT (P = 0.043). Associations with clinicopathological variables were found between TAMs and the presence of infiltrative growth (P = 0.031), low tubule formation (P = 0.040) and lymph node metastasis (P = 0.016). The results support the hypothesis that TAMs influence angiogenesis in CMT suggesting TAMs may represent a therapeutic target in this disease.     

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.     

Surgery and Doxorubicin in Dogs With Hemangiosarcoma

Forty-six dogs with histologically confirmed hemangiosarcoma of various locations other than skin were used in a prospective study to determine the efficacy of adjuvant doxorubicin (30 mg/m² IV q 3 weeks for 5 treatments) 10 to 14 days after the tumor was partially or completely excised. Analysis of the data included information on variables that were hypothesized to influence response to therapy, disease-free interval (DFI), or survival time (ST). Other information collected included age, gender, breed, weight, prior therapy, type of surgery, location of the primary tumor, presence of metastases, number of doses of doxorubicin, response to doxorubicin therapy (complete or partial response!, and the following histological criteria: overall differentiation, nuclear pleomorphism, percent necrosis, mitotic score, total histological score, and grade. Surgery outcome (complete versus incomplete surgical excision) markedly influenced survival times (P < .001). Twenty percent of the dogs rendered free of disease were alive at 1 year, whereas none of the dogs that had residual tumor after surgery were alive at 1 year. Most of the histological criteria (nuclear pleomorphism, mitotic score, grade, overall differentiation) had marked (P < .05), or close to marked, independent associations with ST for dogs that had complete tumor removal. Results from analysis of DFI were generally similar to those of ST in dogs with complete excision of the tumor. Twenty-seven of the 46 dogs (58.7%) had all clinical evidence of tumor successfully removed. Logistic regression analysis of surgical outcome (ability to remove all visible tumor) suggested that age of the subject was the only factor markedly influencing surgical outcome (P= .017). As age increased, the probability of success increased. Those dogs that had previous treatment for their hemangiosarcoma tended (P= .08) to have a shorter DFI and ST. Therefore, complete removal of all evidence of tumor followed by 5 doses of doxorubicin may be an effective treatment for dogs with hemangiosarcoma. Dogs that had all tumor successfully removed had a mean and median ST of 267 and 172 days, respectively. Dogs with incomplete tumor removal had a mean and median ST of 172 and 60 days, respectively. Similarly, prognostic variables such as the ability to completely excise all evidence of tumor, histological criteria, and age of the patient are potentially important prognostic variables for predicting outcome.     

Characterization of spindle cell component of ferret (Mustela putorius furo) adrenal cortical neoplasms – correlation to clinical parameters and prognosis

Adrenal cortical epithelial tumours are common in ferrets. A variant tumour type with prominent spindle cell proliferation has been identified. We characterized these variant tumours with light microscopy and immunohistochemical analysis and correlate these features to clinical parameters and prognosis. We classified 24 ferret adrenal cortical masses with recognizable spindle cell proliferation obtained from the AMC and AFIP databases, based on percentage of spindle cells present and features of malignancy. These masses were separated into hyperplastic nodules and adenomas (both with 1–24% spindle cells), ‘mixed’ adenomas (≥25% spindle cells), adenocarcinomas (1–24%) and ‘mixed’ adenocarcinomas (≥25% spindle cells). Tumours were evaluated immunohistochemically for smooth muscle actin (SMA) and estrogen receptor (ER) expression. Disease‐free interval (DFI) and survival time (ST) were calculated using Kaplan–Meier product limit method. Of 24 cases of spindle cell variant adrenal tumours, one was a hyperplastic nodule, 10 were adenomas, three were ‘mixed’ adenomas, six were adenocarcinomas and four were ‘mixed’ adenocarcinomas. The proliferative spindle cell cytoplasm was SMA‐positive (smooth muscle myocyte origin). ER positivity, seen in nine of 24 cases, was restricted to adenocarcinomas, ‘mixed’ adenomas and ‘mixed’ adenocarcinomas. DFI and ST were significantly reduced in ‘mixed’ adenocarcinomas or tumours with ER expression. DFI was significantly reduced in tumours with marked smooth muscle. The spindle cell component of these variant adrenal tumours is smooth muscle in origin. The presence of abundant smooth muscle, a more malignant histologic grade (‘mixed’ adenocarcinomas) and ER expression are significantly positively correlated to both decreased DFI and decreased ST.     

FC‐54 Evaluation of IgE‐mediated late‐phase reactions in the skin of normal placebo‐ and prednisolone‐treated dogs: cellular,cytokine and chemokine responses

IgE‐mediated late‐phase reactions can be induced in the skin of normal and atopic dogs by intradermal injections of anti‐IgE antibody. The histology of these reactions is very similar to that of naturally occurring atopic dermatitis. To characterize the cellular, cytokine and chemokine responses in the skin of placebo‐ and prednisolone‐treated dogs, normal beagles received either placebo or 0.5 mg/kg prednisolone twice daily for three days prior to intradermal injection of polyclonal rabbit anti‐canine IgE. Eight‐millimetre punch biopsy skin samples were taken before injection and at the injection sites after 6, 24 and 48 h. Histological and immunohistochemical examination revealed a rapid cellular influx. Eosinophil and neutrophil numbers increased from <1 to 61.4 ± 14.1, and from 7 to 62.2 ± 10.8 cells/mm², respectively, within 6 h after injection, and remained moderately elevated 48 h later. The numbers of CD1c+, CD3+ and CD4+ mononuclear cells were also increased by 6 h. Taqman analysis demonstrated 2.5‐ to 72‐fold increases in mRNA expression for IL‐13, IL‐5, MCP (CCL2), RANTES (CCL5) and TARC (CCL17). Levels of mRNA for IL‐2, IL‐4, IL‐6, and IFNγ remained negligible. Prednisolone administration suppressed the influx of neutrophils and eosinophils, and the expression of IL‐13, CCL2, CCL5 and CCL17 (33, 97, 58, 86, 73 and 90%, respectively), as well as the influx of CD1c+ and CD3+ cells. These data document the cytokine and chemokine response to anti‐IgE injection and demonstrate the anti‐inflammatory effect of prednisolone. Funding: Schering‐Plough Animal Health.     

Association of Blood Monocyte and Lymphocyte Count and Disease‐Free Interval in Dogs with Osteosarcoma

Background: Identification of biomarkers that predict outcomes in dogs with osteosarcoma (OSA) would be valuable to veterinarians and owners. Leukocyte numbers in peripheral blood are associated with outcomes in some types of cancer in humans. Hypothesis/Objectives: We hypothesized that increased numbers of monocytes would be associated with reduced disease‐free interval (DFI) in dogs with OSA. Animals: Medical data from 69 dogs with appendicular OSA treated with amputation and chemotherapy were selected for study. Methods: Retrospective study. Statistical associations were assessed by univariate and multivariate analysis. Information about DFI and leukogram values, tumor location, and serum alkaline phosphatase was abstracted from the medical record. Results: Higher numbers of circulating monocytes (>0.4 × 10³ cells/μL) and lymphocytes (>1.0 × 10³ cells/μL) before treatment were found to be significantly (P < .05) associated with shorter DFI in dogs with OSA. Other parameters associated with poor outcomes were increased alkaline phosphatase, primary tumor location, and age. Conclusion and Clinical Importance: These results indicated that pretreatment evaluation of monocyte and lymphocyte counts provided prognostic information for dogs with appendicular OSA. Notably, most animals in this study had monocyte counts within the normal reference range, indicating that variations within the reference range of leukocyte values might also have prognostic significance.     

Metronomic therapy with cyclophosphamide and piroxicam effectively delays tumor recurrence in dogs with incompletely resected soft tissue sarcomas

Background: Continuous administration of low doses of cyclophosphamide and standard doses of cyclooxygenase‐inhibiting drugs has been shown to suppress tumor angiogenesis, reverse immunosuppression, and deplete regulatory T cells in cancer models. Hypothesis: We hypothesized that continuous treatment with low‐dose cyclophosphamide and full‐dose piroxicam would delay tumor recurrence in dogs with soft tissue sarcomas (STS). Animals: Eighty‐five dogs with incompletely resected STS, 30 treated dogs, and 55 contemporary control dogs. Methods: Treatment outcomes in 85 dogs with incompletely resected STS were evaluated in a retrospective study. Dogs in the treatment group received continuously administered low‐dose cyclophosphamide (10 mg/m²) and standard dose piroxicam (0.3 mg/kg) therapy. Time to local tumor recurrence (disease‐free interval; DFI) was compared between the 30 treated dogs and 55 untreated control dogs matched for age and tumor site and grade. Results: DFI was significantly (P < .0001) prolonged for STS of all sites (trunk and extremity) in treated dogs compared with untreated control dogs. The DFI also was significantly longer in treated dogs when tumor site (trunk and extremity) was compared. Twelve treated dogs (40%) experienced mild toxicity (grade 1 and 2) at some point during treatment and 1 dog developed grade 4 cystitis. Every other day dosing was tolerated better than daily dosing. Conclusions: Metronomic therapy with cyclophosphamide and piroxicam was very effective in preventing tumor recurrence in dogs with incompletely resected STS. These findings suggest that further evaluation of this approach is warranted as adjuvant therapy in dogs with highly metastatic tumors such as osteosarcoma and melanoma.     

Effects of Thymomimetic Drugs and Zinc Supplementation on the Cellular Immune Response in Hydrocortisone‐suppressed Mice

The studies were carried out on Balb/c mice (5–6 weeks of age) exposed to immunosuppression by a single intraperitoneal dose (125 mg/kg) of hydrocortisone. Prior to hydrocortisone injection the mice were treated with diethyldithiocarbamate (DTC) intra‐peritoneally at a dose of 20 mg/kg, five times at 48 h intervals or calf thymus extract (TFX) at a dose of 10 mg/kg, 10 times at 24 h intervals. The two drugs were used per se or in zinc ions interactions, by adding zinc ions (as sulphate salt) to drinking water at a dose of 72 μg/mouse per day. The results obtained in the study show that hydrocortisone injection drastically decreases the number of thymocytes and splenocytes, which is also accompanied by a decreasing weight ratio of the thymus and spleen. The decreasing number of thymic and spleen cells corresponds to a decreasing percentage of CD⁴⁺, CD⁸⁺ and CD¹⁹⁺ splenocytes and double positive CD4⁺CD⁸⁺ thymocytes. Changes in the number of thymic cells affect their activity, which is expressed in a decreased proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohaemagglutinin (PHA). It has also been found that a single hydrocortisone dose decreases interleukin (IL)‐1 production by murine intraperitoneal macrophages stimulated in vitro with lipopolysaccharide (LPS) from Escherichia coli. TFX or DTC counteract hydrocortisone‐induced immunosuppression, which is expressed in partial normalization of the total number of thymic and spleen cells, accelerated regeneration of the two lymphatic organs, shorter suppressive action of hydrocortisone on the percentage of CD⁴⁺, CD⁸⁺ splenocytes and double positive (CD4⁺CD⁸⁺) and CD⁴⁺ thymocytes. Furthermore, total counteraction against the suppressive action of hydrocortisone to proliferative activity of thymocytes stimulated in vitro with Con A and PHA was observed. TFX administered prior to hydrocortisone injection partially prevented the suppressive action of the drug on IL‐1 production by intraperitoneal macrophages, but such an effect was not observed with DTC. The immunorestorative effect of TFX and DTC was augmented by zinc supplementation. The results obtained in the study show that neither TFX nor DTC administration per se and in interaction with zinc supplementation were able to change the suppressive effect of hydrocortisone on the percentage of B splenocytes (CD19⁺ cells).     

Serum vascular endothelial growth factor concentrations and postsurgical outcome in dogs with osteosarcoma

Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor, playing putative roles in both tumour growth and metastasis. The purpose of this study was to correlate pretreatment serum concentrations of VEGF in dogs with osteosarcoma (OSA) with disease‐free interval (DFI) and overall survival (OS). Additionally, the effect of serum from dogs with OSA on ex vivo canine endothelial cell (EC) growth was determined. Pretreatment platelet‐corrected serum VEGF levels correlated significantly with DFI. No other examined variable predicted outcome. The ability of sera from dogs with OSA to stimulate canine EC proliferation did not correlate with VEGF concentration or outcome. These data support a role for VEGF in the development or progression of metastatic disease in dogs with OSA. The VEGF concentration in tested sera was not a major determinant of ex vivo canine EC proliferation in this study.     

Adjuvant carboplatin and gemcitabine combination chemotherapy postamputation in canine appendicular osteosarcoma

Background: Appendicular osteosarcoma (OSA), the most common bone tumor in dogs, is typically treated by amputation and adjuvant chemotherapy. Despite numerous efforts, the median survival time (MST) for dogs receiving a platinum compound, doxorubicin, or a combination of these remains at 8–12 months. Evidence from studies in mice suggests that gemcitabine has activity against OSA in vivo. Our preliminary work demonstrated that the addition of low‐dosage (10 mM) gemcitabine to carboplatin resulted in synergistic inhibition of OSA cell viability in vitro. Objective: The purpose of the following study was to determine whether the addition of low‐dosage (2 mg/kg) gemcitabine to carboplatin chemotherapy in dogs with OSA after amputation would improve MST over carboplatin monotherapy. Animals: Fifty dogs with histologically confirmed appendicular OSA. Methods: Dogs were treated prospectively with amputation and up to 4 dosages of carboplatin and gemcitabine in combination every 3 weeks. Results: The chemotherapeutic regimen was well tolerated with only 5 episodes of grade 3 or 4 hematologic toxicity. The median disease‐free interval (DFI) was 203 days and the MST was 279 for all dogs in this study. The 1‐ and 2‐year survival rates were 29.5 and 11.3%, respectively. Dogs with proximal humeral OSA had a shorter median DFI (P= .04) compared with dogs with OSA in other locations. Conclusions and Clinical Importance: These results are comparable to those reported for carboplatin monotherapy indicating that the addition of gemcitabine to carboplatin in dogs with appendicular OSA does not appear to improve outcome.     

High COX‐2 expression in canine mast cell tumours is associated with proliferation,angiogenesis and decreased overall survival

COX‐2 overexpression is associated with several hallmarks of carcinogenesis such as proliferation, angiogenesis, invasion and metastasis. Fifty cases of canine mast cell tumours (MCT) were retrospectively evaluated and submitted to immunohistochemistry for COX‐2, CD31, Ki‐67, MAC‐387 and CD3. Furthermore its relationship with clinicopathological variables and overall survival (OS) was analysed. COX‐2 intensity (P = 0.016), but not COX‐2 extension nor score was associated with decreased OS and higher grades of malignancy according to Patnaik (P = 0.002) and Kiupel (P < 0.001) grading systems. Cox‐2 intensity was also associated with higher Ki‐67 scores (P = 0.009), higher mitotic index (P = 0.022) and higher microvascularization density (P = 0.045). No association was observed for COX‐2 intensity and CD3‐T lymphocyte (P = 0.377) and macrophage infiltration (P = 0.261) by MAC‐387 immunollabelling, suggesting an active role of COX‐2 in MCT oncogenesis mainly through proliferation and angiogenesis stimulation making it a potentially clinical relevant prognosis marker and therapeutic target.     

Resection of Pulmonary Metastases in Canine Osteosarcoma: 36 Cases (1983–1992)

Thirty-six dogs underwent pulmonary metastatectomy for osteosarcoma. All patients had been treated for histologically confirmed osteosarcoma of the appendicular skeleton. Treatment for the primary tumor consisted of amputation or a limb sparing procedure in conjunction with adjuvant chemotherapy, local radiation therapy, or both.
Significant factors in determining prognosis included the disease-free interval (DFI) between treatment of the primary tumor and development of pulmonary metastases and the number of metastatic nodules present at surgery. Dogs that developed pulmonary metastases 300 days or more after diagnosis of the primary tumor had a median DFI of 128 days after metastatectomy. Dogs that developed pulmonary metastases fewer than 300 days after diagnosis had a median DFI of 58 days. Dogs with one or two metastatic nodules removed had a median DFI of 95 days, whereas dogs with three or more nodules removed had a median DFI of 53 days. The results of this study indicate that prognostic variables exist for dogs with metastatic pulmonary osteosarcoma and can help predict survival after metastatectomy. These variables are similar to the prognostic variables that have been determined for human patients undergoing pulmonary metastatectomy because of osteosarcoma. Though a controversial procedure, pulmonary metastatectomy seems to be a valid treatment option for selected dogs with metastatic pulmonary osteosarcoma.     

Electrochemotherapy in treatment of canine oral non‐tonsillar squamous cell carcinoma. A case series report

Non‐tonsillar squamous cell carcinoma (ntSCC) is a common and locally aggressive oral tumour in dogs. The treatments of choice are currently surgery and radiotherapy. Electrochemotherapy (ECT) is a local ablative anti‐tumour technique using electric pulses to enhance the intracellular diffusion of cytotoxic drugs. The aim was to retrospectively evaluate the outcome of patients with oral ntSCC treated with ECT. Twelve dogs with ntSCC were retrospectively enrolled. ECT was combined with IV bleomycin (15 000 UI/m²) alone in 11 cases and post‐surgery in 1. Parameters considered were: tumour site and size, electroporation parameters, response rate (complete remission [CR], partial remission [PR]), median survival time (MST), recurrence rate (RR), median disease‐free interval (DFI) and treatment toxicity (6‐point scale). Median tumour size was 1.65 cm (range 0.3‐8.0 cm) and the response rate was 90.9% (10/11; 8 CR and 2 PR). Two dogs underwent a second ECT. MST for dogs dead with tumour (n = 2) was 110 days and for dogs dead without tumour (n = 3) was 831 days. Among five surviving dogs, one experienced tumour recurrence and four were in CR. Results from two dogs were analysed separately. Overall RR was 27.3%. DFI and MST for dogs with recurrence were 50 and 115 days, respectively. Treatment toxicity was very low. We noticed that all dogs with tumours smaller than 1‐2 cm achieved CR without recurrence suggesting a favourable prognosis when using ECT. ECT for canine ntSCC could be considered a valid treatment option especially for smaller tumours, but a larger caseload would be needed to confirm this statement.