Paolo Buracco

European consensus document on mast cell tumours in dogs and cats

In preparing this document the Authors aimed to pool current information on canine and feline mast cell disease. The information was gathered from international studies and a emphasis was placed on material and opinion with a strong evidence base. We intend it to form the basis of our understanding in this disease at the current time and we anticipate that it will be particularly useful for the general practitioner. It should be emphasized that the authors are presenting this work from a European perspective.

A Mouse Model of Pulmonary Metastasis from Spontaneous Osteosarcoma Monitored In Vivo by Luciferase Imaging

Background Osteosarcoma (OSA) is lethal when metastatic after chemotherapy and/or surgical treatment. Thus animal models are necessary to study the OSA metastatic spread and to validate novel therapies able to control the systemic disease. We report the development of a syngeneic (Balb/c) murine OSA model, using a cell line derived from a spontaneous murine tumor. Methodology The tumorigenic and metastatic ability of OSA cell lines were assayed after orthotopic injection in mice distal femur. Expression profiling was carried out to characterize the parental and metastatic cell lines. Cells from metastases were propagated and engineered to express Luciferase, in order to follow metastases in vivo. Principal Findings Luciferase bioluminescence allowed to monitor the primary tumor growth and revealed the appearance of spontaneous pulmonary metastases. In vivo assays showed that metastasis is a stable property of metastatic OSA cell lines after both propagation in culture and luciferase trasduction. When compared to parental cell line, both unmodified and genetically marked metastatic cells, showed comparable and stable differential expression of the enpp4, pfn2 and prkcd genes, already associated to the metastatic phenotype in human cancer. Conclusions This OSA animal model faithfully recapitulates some of the most important features of the human malignancy, such as lung metastatization. Moreover, the non-invasive imaging allows monitoring the tumor progression in living mice. A great asset of this model is the metastatic phenotype, which is a stable property, not modifiable after genetic manipulation.

Adrenalectomy in dogs with adrenal gland tumors: 52 cases (2002–2008)

OBJECTIVE To evaluate predictors of survival time in dogs undergoing adrenalectomy and identify risk factors associated with adrenal gland tumor metastasis and vein thrombosis. DESIGN Retrospective case series. ANIMALS 52 dogs with primary adrenal gland tumors. PROCEDURES Medical records were reviewed. Signalment, tumor features, and information from surgical procedures were evaluated to identify factors predictive of overall survival time, which was defined as the time from surgery until death. The association between metastasis or vein thrombosis and tumor type, size, and site (right or left adrenal gland) was investigated. RESULTS On the basis of results of univariate analysis, survival time was significantly shorter for dogs with adenocarcinoma, tumor major axis length ≥ 5 cm, metastasis, and vein thrombosis and when adrenalectomy was combined with an additional abdominal surgical intervention. On multivariate analysis, survival time was significantly shorter for dogs with an adrenal gland tumor with major axis length ≥ 5 cm and for dogs with metastasis or vein thrombosis. Significant associations were found between metastasis and adenocarcinoma and between vein thrombosis and tumors with major axis length ≥ 5 cm. CONCLUSIONS AND CLINICAL RELEVANCE Dogs with an adrenal gland tumor with major axis length ≥ 5 cm, documented metastasis, or vein thrombosis had a poorer prognosis. Metastasis was more frequent in dogs with adenocarcinoma and vein thrombosis when tumors were ≥ 5 cm in length.

Marginal Excision of Low‐Grade Spindle Cell Sarcoma of Canine Extremities: 35 Dogs (1996–2006)

OBJECTIVE To evaluate recurrence rate and disease-free interval (DFI) of dogs with low-grade soft tissue spindle cell sarcoma of the extremities treated by marginal excision. STUDY DESIGN Retrospective study. ANIMALS Dogs (n=35) with soft tissue low-grade spindle cell sarcoma. METHODS Medical records were reviewed and dogs that had marginal surgical resection of low-grade soft tissue spindle cell sarcoma at or distal to elbow and stifle were included. RESULTS Histopathologic margins were dirty (12 dogs), clean but close (12), and clean (11). Follow-up after surgery occurred from 210 to 2202 days (minimum, 180 days). Local recurrence and metastatic rates were 10.8% and 0%, respectively. Median DFI and survival time were not reached, because <50% of dogs died of disease-related events. Mean DFI and mean survival time were 697.8 days (95% CI: 559.7-836 days) and 703.5 days (95% CI: 566.6-840.5 days), respectively. There were no significant differences among survival functions stratified by histologic margins. CONCLUSION Marginal surgical excision without adjuvant treatment of low-grade soft tissue spindle cell sarcoma of the extremities results in a low local recurrence rate. CLINICAL RELEVANCE Low-grade spindle cell sarcomas located at or distal to the elbow and stifle joints can be excised without need for wide or radical surgery.

Expression of Ki67, BCL-2, and COX-2 in Canine Cutaneous Mast Cell Tumors Association With Grading and Prognosis

The expression of Ki67, BCL-2, and COX-2 was investigated in 53 canine cutaneous mast cell tumors (MCTs) by immunohistochemistry and quantitative real time polymerase chain reaction (qPCR) to evaluate their prognostic significance and the association with the histologic grading and the mitotic index (MI). MCTs were graded according to the Patnaik grading system and the novel 2-tier grading system proposed by Kiupel. The numbers of mitotic figures/10 high-power fields (MI) were counted. Both grading systems were significantly associated with prognosis. The Patnaik grading was of limited prognostic value for grade 2 MCTs, with 23% being associated with mortality. The concordance among pathologists was strongly improved by the application of the 2-tier grading system, and 71% of high-grade MCTs were associated with a high mortality rate. MI and Ki67 protein expression were significantly associated with grading and survival. No significant association between BCL-2 protein expression and either grading system or health status was observed. BCL-2 mRNA expression was significantly higher in grade 2 than in grade 1 MCTs, while no statistically significant differences were detected between low- and high-grade MCTs. The increased BCL-2 mRNA level was significantly associated with increased mortality rate. The COX-2 protein expression was detected in 78% of the MCTs investigated. However, neither association with the tumor grade nor with the health status was observed. COX-2 mRNA was significantly up-regulated in MCTs compared to surgical margins and control skin tissue, but it was neither associated with tumor grade nor with survival.

met oncogene activation qualifies spontaneous canine osteosarcoma as a suitable pre-clinical model of human osteosarcoma†

The Met receptor tyrosine kinase (RTK) is aberrantly expressed in human osteosarcoma and is an attractive molecular target for cancer therapy. We studied spontaneous canine osteosarcoma (OSA) as a potential pre‐clinical model for evaluation of Met‐targeted therapies. The canine MET oncogene exhibits 90% homology compared with human MET, indicating that cross‐species functional studies are a viable strategy. Expression and activation of the canine Met receptor were studied utilizing immunohistochemical techniques in 39 samples of canine osteosarcoma, including 35 primary tumours and four metastases. Although the Met RTK is barely detectable in primary culture of canine osteoblasts, high expression of Met protein was observed in 80% of canine osteosarcoma samples acquired from various breeds. Met protein overexpression was also concordant with its activation as indicated by phosphorylation of critical tyrosine residues. In addition, Met was expressed and constitutively activated in canine osteosarcoma cell lines. OSA cells expressing high levels of Met demonstrated activation of downstream transducers, elevated spontaneous motility, and invasiveness which were impaired by both a small molecule inhibitor of Met catalytic activity (PHA‐665752) and met‐specific, stable RNA interference obtained by means of lentiviral vector. Similar to observations in human OSA, these data suggest that Met is commonly overexpressed and activated in canine OSA and that inhibition of Met impairs the invasive and motogenic properties of canine OSA cells. These data implicate Met as a potentially important factor for canine OSA progression and indicate that it represents a viable model to study Met‐targeted therapies. Copyright

CSPG4-Specific Immunity and Survival Prolongation in Dogs with Oral Malignant Melanoma Immunized with Human CSPG4 DNA

Purpose: Due to the many similarities with its human counterpart, canine malignant melanoma (cMM) is a valuable model in which to assess the efficacy of novel therapeutic strategies. The model is herein used to evaluate the immunogenicity, safety, and therapeutic efficacy of a human chondroitin sulfate proteoglycan-4 (hCSPG4) DNA-based vaccine. The fact that homology between hCSPG4 and cCSPG4 amino-acidic sequences stands at more than 80% provides the rationale for using an hCSPG4 DNA vaccine in the cMM model. Experimental Design: Dogs with stage II–III surgically resected CSPG4-positive oral MM were subjected to monthly intramuscular plasmid administration, which was followed immediately by electroporation (electrovaccination) for at least 6, and up to 20, months. The immunogenicity, safety, and therapeutic efficacy of the vaccine have been evaluated. Results: hCSPG4 electrovaccination caused no clinically relevant local or systemic side effects and resulted in significantly longer overall and disease-free survival times in 14 vaccinated dogs as compared with 13 nonvaccinated controls. All vaccinated dogs developed antibodies against both hCSPG4 and cCSPG4. Seven vaccinated dogs were also tested for a cCSPG4-specific T-cell response and only two gave a detectable interferon (IFN)γ response. Conclusion: Xenogeneic electrovaccination against CSPG4 is able to overcome host unresponsiveness to the “self” antigen and seems to be effective in treating cMM, laying the foundation for its translation to a human clinical setting. Clin Cancer Res; 20(14); 3753–62. ©2014 AACR.

PDGFs and PDGFRs in canine osteosarcoma: new targets for innovative therapeutic strategies in comparative oncology.

Platelet derived growth factor receptor (PDGFR)α and PDGFRβ are tyrosine kinase receptors that are overexpressed in 70-80% of human osteosarcomas (OSAs) and may be suitable therapeutic targets for specific kinase inhibitors (TKIs). Canine OSA shows histopathological and clinical features similar to human OSA, and is considered an excellent model in comparative oncology. This study investigated PDGF-A, PDGF-B, PDGFRα and PDGFRβ expression in 33 canine OSA samples by immunohistochemistry and in seven primary canine OSA cell lines by Western blot and quantitative PCR analysis. Immunohistochemical data showed that PDGF-A and PDGF-B are expressed in 42% and 60% of the OSAs analysed, respectively, while PDGFRα and PDGFRβ were expressed in 78% and 81% of cases, respectively. Quantitative PCR data showed that all canine OSA cell lines overexpressed PDGFRα, while 6/7 overexpressed PDGFRβ and PDGF-A relative to a normal osteoblastic cell line. Moreover, in vitro treatment with a specific PDGFR inhibitor, AG1296, caused a dose- and time-dependent decrease in AKT phosphorylation. Collectively, these data show that PDGFRs/PDGFs are co-expressed in canine osteosarcomas, which suggests that an autocrine and/or paracrine loop is involved and that they play an important role in the aetiology of OSA. PDGFRs may be suitable targets for the treatment of canine OSA with a specific TKI.

Double-J ureteral stenting in nine cats with ureteral obstruction.

Ureteral stenting is a common practice in human medicine and has recently been reported in dogs and cats to provide urinary diversion for ureteral obstructions caused by ureteroliths, strictures, neoplasia, and in an attempt to prevent postoperative complications following ureteral anastomosis. The aim of this report is to describe a surgical technique of ureteral stenting and the follow-up and complications in nine cats. Number 3 French double-J catheters were used during open surgery for ureterotomy/ureterolith removal in eight cats and for segmental ureterectomy/end-to-end anastomosis in one cat for a localized benign stricture. Neoureterocystostomy was necessary in eight of the cats. Uroperitoneum did not occur. Stents were still in place in 7/9 animals after 357-1,565 days (median 1,277 days). A minor complication (stent migration) occurred in one cat, but stent removal was not required. Major complications were encrustation and persistent stranguria (in one cat each), requiring stent removal at 90 and 123 days, respectively. The first cat had a new stent inserted but was euthanased 3 months later for progressive renal failure. Despite the small number of cats, both the outcome and long-term stent tolerance observed in most cases suggest that ureteral stenting is a safe, adjunctive measure to ureteral surgery, mainly for concomitant ureteral and renal pelvic stones to prevent further obstruction and avoid pyelotomy/nephrotomy. However, smaller stents should be used to decrease the need for ureteral surgery.

Bone invading NSCLC cells produce IL-7: mice model and human histologic data

BackgroundBone metastases are a common and dismal consequence of lung cancer that is a leading cause of death. The role of IL-7 in promoting bone metastases has been previously investigated in NSCLC, but many aspects remain to be disclosed. To further study IL-7 function in bone metastasis, we developed a human-in-mice model of bone aggression by NSCLC and analyzed human bone metastasis biopsies.MethodsWe used NOD/SCID mice implanted with human bone. After bone engraftment, two groups of mice were injected subcutaneously with A549, a human NSCLC cell line, either close or at the contralateral flank to the human bone implant, while a third control group did not receive cancer cells. Tumor and bone vitality and IL-7 expression were assessed in implanted bone, affected or not by A549. Serum IL-7 levels were evaluated by ELISA. IL-7 immunohistochemistry was performed on 10 human bone NSCLC metastasis biopsies for comparison.ResultsAt 12 weeks after bone implant, we observed osteogenic activity and neovascularization, confirming bone vitality. Tumor aggressive cells implanted close to human bone invaded the bone tissue. The bone-aggressive cancer cells were positive for IL-7 staining both in the mice model and in human biopsies. Higher IL-7 serum levels were found in mice injected with A549 cells close to the bone implant compared to mice injected with A549 cells in the flank opposite to the bone implant.ConclusionsWe demonstrated that bone-invading cells express and produce IL-7, which is known to promote osteoclast activation and osteolytic lesions. Tumor-bone interaction increases IL-7 production, with an increase in IL-7 serum levels. The presented mice model of bone invasion by contiguous tumor is suitable to study bone-tumor cell interaction. IL-7 plays a role in the first steps of metastatic process.